c© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim10.1002/14356007.a27 443

Vitamins 1

Vitamins

Manfred Eggersdorfer, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 1)

Geo Adam, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 1)

Michael John, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 2)

Wolfgang Hahnlein, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 2)

Ludvik Labler, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 3)

Kai-U. Baldenius, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 4)

Linda von dem Bussche-Hunnefeld, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic ofGermany (Chap. 4)

Eckhard Hilgemann, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 4)

Peter Hoppe, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 4)

Rainer Sturmer, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 4)

Fritz Weber, Munchenstein, Switzerland (Chap. 5)

August Ruttimann, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 5)

Gerard Moine, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 6)

Hans-Peter Hohmann, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 6)

Roland Kurth, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 7)

Joachim Paust, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 7)

Wolfgang Hahnlein, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 7)

Horst Pauling, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 8)

Bernd–Jurgen Weimann, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 8)

Bruno Kaesler, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 9)

Bernd Oster, E. Merck, Darmstadt, Federal Republic of Germany (Chap. 10)

Ulrich Fechtel, E. Merck, Darmstadt, Federal Republic of Germany (Chap. 10)

Klaus Kaiser, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 11)

Bernd de Potzolli, BASF Aktiengesellschaft, Ludwigshafen, Federal Republic of Germany (Chap. 11)

Michael Casutt, E. Merck, Darmstadt, Federal Republic of Germany (Chap. 12)

Thomas Koppe, E. Merck, Darmstadt, Federal Republic of Germany (Chap. 12)

Michael Schwarz, E. Merck, Darmstadt, Federal Republic of Germany (Chap. 12)

Bernd-Jurgen Weimann, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 13)

Urs Hengartner, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 13)

Antoine de Saizieu, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 13)

Christof Wehrli, F. Hoffmann-La Roche Ltd, Basel, Switzerland (Chap. 13)

Rene Blum, Lonza, Basel, Switzerland (Chap. 14)

2 Vitamins

1. Introduction . . . . . . . . . . . . . . 61.1. Definition . . . . . . . . . . . . . . . . 61.2. Substances with Vitamin-Like

Character . . . . . . . . . . . . . . . . 81.3. History . . . . . . . . . . . . . . . . . 81.4. Determination of Requirement . . 81.5. Application and Tolerance . . . . . 81.6. Studies with Vitamins . . . . . . . . 101.7. Use ofVitamins inFood- andFeed-

stuffs . . . . . . . . . . . . . . . . . . . 101.8. Antivitamins . . . . . . . . . . . . . . 121.9. Analysis of Vitamins . . . . . . . . . 121.10. Production . . . . . . . . . . . . . . . 132. Vitamin A (Retinoids) . . . . . . . . 132.1. Introduction . . . . . . . . . . . . . . 132.2. Historical Aspects . . . . . . . . . . 142.3. Physical Properties . . . . . . . . . . 152.4. Chemical Properties . . . . . . . . . 172.5. Occurrence . . . . . . . . . . . . . . . 172.6. Biosynthesis . . . . . . . . . . . . . . 182.7. Production . . . . . . . . . . . . . . . 202.7.1. Isolation and Purification from Nat-

ural Raw Materials . . . . . . . . . . 202.7.2. Industrial Synthesis . . . . . . . . . . 202.8. Metabolism and Physiological

Functions . . . . . . . . . . . . . . . . 282.8.1. Preformed VitaminA . . . . . . . . . 282.8.2. β-Carotene . . . . . . . . . . . . . . . 292.9. Deficiency Symptoms and Re-

quirements . . . . . . . . . . . . . . . 292.10. Analysis and Standardization . . . 302.11. Trade Names and Economic As-

pects . . . . . . . . . . . . . . . . . . . 312.12. Tolerance . . . . . . . . . . . . . . . . 323. Vitamin D . . . . . . . . . . . . . . . . 323.1. Introduction . . . . . . . . . . . . . . 323.2. History . . . . . . . . . . . . . . . . . 323.3. Structure and Nomenclature . . . 333.4. Chemical and Physical Properties 333.5. Biosynthesis and Occurrence . . . 353.5.1. Biosynthesis . . . . . . . . . . . . . . 353.5.2. Occurrence . . . . . . . . . . . . . . . 353.6. Vitamin D Requirements . . . . . . 353.7. Pharmacological Effects and Uses 363.7.1. CalciumHomeostasis and Disorders

of Calcium Metabolism . . . . . . . 363.7.2. Further Activities . . . . . . . . . . . 373.8. Synthesis . . . . . . . . . . . . . . . . 373.8.1. Synthesis of VitaminD3 and D2 . . 373.8.2. Synthesis of Calcitriol . . . . . . . . 393.9. Assays for Vitamin D and Metabo-

lites . . . . . . . . . . . . . . . . . . . . 393.10. Trade Names and Economic As-

pects . . . . . . . . . . . . . . . . . . . 41

4. Vitamin E (Tocopherols, Tocotri-enols) . . . . . . . . . . . . . . . . . . . 41

4.1. Introduction . . . . . . . . . . . . . . 414.2. History . . . . . . . . . . . . . . . . . 424.3. Physical Properties . . . . . . . . . . 434.4. Chemical Properties . . . . . . . . . 434.5. Metabolismand Importance in the

Organism . . . . . . . . . . . . . . . . 434.6. Deficiency, Requirement, and Ap-

plication . . . . . . . . . . . . . . . . . 444.7. Analysis and Standardization . . . 454.8. Occurrence . . . . . . . . . . . . . . . 464.9. Economic Aspects . . . . . . . . . . 464.10. Biosynthesis . . . . . . . . . . . . . . 474.11. Production . . . . . . . . . . . . . . . 474.11.1. Semisynthetic Vitamin E: Isolation

from Plant Oils and Methylation . . 474.11.2. Totally Synthetic Vitamin E: Indus-

trial Synthesis . . . . . . . . . . . . . . 474.11.3. Trimethylhydroquinone . . . . . . . 484.11.4. Phytol and Isophytol . . . . . . . . . 494.11.5. Stereoselective Syntheses of

α-Tocopherol . . . . . . . . . . . . . . 505. Vitamin K . . . . . . . . . . . . . . . 525.1. Introduction; History . . . . . . . . 525.2. Physical Properties . . . . . . . . . . 535.3. Chemical Properties . . . . . . . . . 545.4. Occurrence . . . . . . . . . . . . . . . 555.5. Biosynthesis . . . . . . . . . . . . . . 565.6. Chemical Synthesis . . . . . . . . . 575.7. Analysis . . . . . . . . . . . . . . . . . 605.8. Metabolism . . . . . . . . . . . . . . . 615.9. Importance for the Organism . . . 635.9.1. Function . . . . . . . . . . . . . . . . . 635.9.2. Antagonists and Anticoagulants . . 655.9.3. Relative Activity . . . . . . . . . . . . 665.10. Deficiency Symptoms . . . . . . . . 685.10.1. Causes . . . . . . . . . . . . . . . . . . 685.10.2. Evaluation of VitaminK Status . . . 685.11. Requirement . . . . . . . . . . . . . . 695.12. Application . . . . . . . . . . . . . . . 705.13. Tolerance . . . . . . . . . . . . . . . . 715.14. Trade Names and Economic As-

pects . . . . . . . . . . . . . . . . . . . 716. Vitamin B1 (Thiamin) . . . . . . . 716.1. Structure and Nomenclature . . . 716.2. History . . . . . . . . . . . . . . . . . 726.3. Physical Properties . . . . . . . . . . 726.4. Chemical Properties . . . . . . . . . 736.5. Natural Occurrence and Isolation 736.6. Biosynthesis in Microorganisms . 756.6.1. Biosynthesis of the Pyrimidine

Component in Prokaryotes . . . . . . 76

Vitamins 3

6.6.2. Biosynthesis of the PyrimidineComponent in Eukaryotes . . . . . . 76

6.6.3. Biosynthesis of the Thiazole Com-ponent . . . . . . . . . . . . . . . . . . 76

6.6.4. Biosynthesis of Thiamin Pyrophos-phate . . . . . . . . . . . . . . . . . . . 77

6.6.5. Thiamin Biosynthetic Genes fromE. coli . . . . . . . . . . . . . . . . . . . 79

6.6.6. Mapping and Cloning of ThiaminBiosynthetic Genes from OtherSpecies . . . . . . . . . . . . . . . . . . 79

6.6.7. Regulation of Thiamin Biosynthesisin Prokaryotes . . . . . . . . . . . . . 80

6.6.8. Regulation of Thiamin Biosynthesisin Yeasts . . . . . . . . . . . . . . . . . 80

6.6.9. Thiamin-Overexpressing Microor-ganisms . . . . . . . . . . . . . . . . . 80

6.7. Chemical Synthesis . . . . . . . . . 816.7.1. Condensation of the Pyrimidine and

Thiazole Rings . . . . . . . . . . . . . 816.7.2. Construction of theThiazoleRing on

a Preformed Pyrimidine Portion . . 816.8. Commercial Forms . . . . . . . . . 836.9. Derivatives, Analogues, and An-

timetabolites . . . . . . . . . . . . . . 836.10. Biochemical and Physiological

Functions . . . . . . . . . . . . . . . . 856.10.1. Metabolic Functions in the Organ-

ism . . . . . . . . . . . . . . . . . . . . 856.10.2. Thiamin Requirements and Defi-

ciency . . . . . . . . . . . . . . . . . . 866.11. Analytical Methods . . . . . . . . . 867. Riboflavin . . . . . . . . . . . . . . . . 867.1. Introduction . . . . . . . . . . . . . . 867.2. History . . . . . . . . . . . . . . . . . 877.3. Physical and Chemical Properties 877.4. Occurrence . . . . . . . . . . . . . . . 887.5. Biosynthesis . . . . . . . . . . . . . . 897.6. Production . . . . . . . . . . . . . . . 897.6.1. Syntheses . . . . . . . . . . . . . . . . 897.6.2. Industrial Chemical Production . . . 917.6.3. Industrial Production by Fermenta-

tion . . . . . . . . . . . . . . . . . . . . 927.6.4. Syntheses of FlavinMononucleotide

(FMN) . . . . . . . . . . . . . . . . . . 937.6.5. Syntheses of Flavin Adenine Dinu-

cleotide (FAD) . . . . . . . . . . . . . 937.7. Importance for the Organism . . . 937.8. Requirements, Deficiency Symp-

toms, and Therapeutic Applica-tion . . . . . . . . . . . . . . . . . . . . 94

7.9. Analysis . . . . . . . . . . . . . . . . . 957.10. Economic Aspects . . . . . . . . . . 967.11. Tolerance . . . . . . . . . . . . . . . . 96

8. Vitamin B6 . . . . . . . . . . . . . . . 968.1. Introduction . . . . . . . . . . . . . . 968.2. History . . . . . . . . . . . . . . . . . 968.3. Physical Properties . . . . . . . . . . 978.4. Chemical Reactions . . . . . . . . . 988.5. Occurrence . . . . . . . . . . . . . . . 998.6. Biosynthesis . . . . . . . . . . . . . . 998.7. Production of Vitamin B6 Com-

pounds . . . . . . . . . . . . . . . . . . 998.7.1. Pyridoxine . . . . . . . . . . . . . . . . 998.7.1.1. Oxidative Degradation of Bicyclic

Heterocyclic Compounds . . . . . . 998.7.1.2. Condensation Reactions with

Aliphatic Precursors . . . . . . . . . . 1008.7.1.3. From Furan Compounds . . . . . . . 1018.7.1.4. Diels –Alder Syntheses with Oxa-

zoles . . . . . . . . . . . . . . . . . . . 1018.7.1.5. Cobalt-Catalyzed [2+2+2]-

Cycloaddition Reactions ofAcetylenes and Acetonitrile . . . . . 103

8.7.2. Pyridoxine 5′-Phosphate . . . . . . . 1038.7.3. Pyridoxal . . . . . . . . . . . . . . . . 1038.7.4. Pyridoxal 5′-Phosphate . . . . . . . . 1048.7.5. Pyridoxamine . . . . . . . . . . . . . . 1048.7.6. Pyridoxamine 5′-Phosphate . . . . . 1048.8. Metabolism and Importance for

the Organism . . . . . . . . . . . . . 1048.9. Deficiency Symptoms and Appli-

cations . . . . . . . . . . . . . . . . . . 1058.10. Analysis . . . . . . . . . . . . . . . . . 1068.11. Economic Aspects . . . . . . . . . . 1068.12. Requirements and Tolerance . . . 1069. Vitamin B12 (Cobalamins) . . . . 1079.1. Introduction . . . . . . . . . . . . . . 1079.2. Properties of Vitamin B12 . . . . . 1099.3. Analysis . . . . . . . . . . . . . . . . . 1109.4. Biosynthesis . . . . . . . . . . . . . . 1109.5. Production . . . . . . . . . . . . . . . 1119.5.1. Fermentation . . . . . . . . . . . . . . 1119.5.2. Work-Up . . . . . . . . . . . . . . . . . 1139.5.3. Patents and Scientific Survey . . . . 1139.6. Specifications and Legal Aspects . 1149.7. Economic Aspects . . . . . . . . . . 11410. Vitamin C (l-Ascorbic Acid) . . . 11410.1. Introduction . . . . . . . . . . . . . . 11410.2. History . . . . . . . . . . . . . . . . . 11510.3. Physical and Chemical Properties 11510.4. Analysis . . . . . . . . . . . . . . . . . 11610.5. Occurrence and Sources of Vita-

min C . . . . . . . . . . . . . . . . . . . 11810.6. Biosynthesis . . . . . . . . . . . . . . 11810.7. Manufacture of Vitamin C . . . . . 11810.7.1. Reichstein Synthesis . . . . . . . . . 119

4 Vitamins

10.7.2. IndustrialManufacture by theReich-stein Route . . . . . . . . . . . . . . . 120

10.7.3. Other Methods for Production ofAscorbic Acid . . . . . . . . . . . . . 122

10.8. Absorption and Metabolism . . . . 12510.9. Medical Aspects of Vitamin C . . 12510.10. Industrial Uses . . . . . . . . . . . . 12610.11. Economic Aspects . . . . . . . . . . 12711. Pantothenic Acid . . . . . . . . . . . 12711.1. Introduction . . . . . . . . . . . . . . 12711.2. History . . . . . . . . . . . . . . . . . 12711.3. Physical and Chemical Properties 12711.4. Occurrence . . . . . . . . . . . . . . . 12711.5. Biosynthesis . . . . . . . . . . . . . . 12811.6. Production . . . . . . . . . . . . . . . 12911.7. Metabolism and Importance for

the Organisms; Coenzyme A andits Precursors . . . . . . . . . . . . . 130

11.8. Deficiency Symptoms, Require-ment, and Application . . . . . . . 132

11.9. Analysis . . . . . . . . . . . . . . . . . 13411.10. Economic Aspects . . . . . . . . . . 13412. Biotin . . . . . . . . . . . . . . . . . . 13412.1. Introduction . . . . . . . . . . . . . . 13412.2. History . . . . . . . . . . . . . . . . . 13512.3. Physical and Chemical Properties 13512.4. Occurrence . . . . . . . . . . . . . . . 13512.5. Biosynthesis . . . . . . . . . . . . . . 13612.6. Function as Prosthetic Group . . . 13612.7. Isolation and Production . . . . . . 13812.8. Metabolism and Importance for

the Organism . . . . . . . . . . . . . 13812.9. Deficiency Symptoms, Require-

ment, and Application . . . . . . . 140

12.9.1. Symptoms and Therapy in Humans 14012.9.2. Symptoms and Therapy in Animals 14012.10. Biotin Analogs . . . . . . . . . . . . . 14112.11. Analysis and Standardization . . . 14112.12. Uses and Economic Aspects . . . . 14212.13. Tolerance and Environmental

Protection . . . . . . . . . . . . . . . . 14313. Folic Acid . . . . . . . . . . . . . . . . 14313.1. Introduction . . . . . . . . . . . . . . 14313.2. Historical Notes . . . . . . . . . . . . 14413.3. Properties . . . . . . . . . . . . . . . . 14513.4. Content in Food and Bioavailabil-

ity . . . . . . . . . . . . . . . . . . . . . 14613.5. Biosynthesis . . . . . . . . . . . . . . 14613.6. Chemical Synthesis . . . . . . . . . 14713.7. Metabolism and Biochemical

Functions . . . . . . . . . . . . . . . . 14813.8. Nutritional Requirements and

Medical Use . . . . . . . . . . . . . . 14913.9. Analysis . . . . . . . . . . . . . . . . . 15013.10. Economic Aspects . . . . . . . . . . 15014. Niacin (Nicotinic Acid, Nicoti-

namide) . . . . . . . . . . . . . . . . . 15014.1. Introduction . . . . . . . . . . . . . . 15014.2. Physical and Chemical Properties 15114.3. Biochemical Functions . . . . . . . 15114.4. Production . . . . . . . . . . . . . . . 15214.5. Quality Specifications . . . . . . . . 15314.6. Analysis . . . . . . . . . . . . . . . . . 15314.7. Uses . . . . . . . . . . . . . . . . . . . 15314.8. Economic Aspects . . . . . . . . . . 15514.9. Toxicology . . . . . . . . . . . . . . . 15615. References . . . . . . . . . . . . . . . 156

1. Introduction

1.1. Definition [1]

Vitamins are essential, organic compoundswhich are either not synthesized in the humanand animal organism or formed only in insuffi-cient amounts. Therefore, theymust be regularlyconsumed with the diet either as such or as aprecursor (provitamin) that can be converted tothe vitamin in the body. A typical representativeof the provitamins is β-carotene, which is splitinto twomolecules of vitaminA in the organism.The metabolic functions of vitamins are mainlycatalytic or regulatory. In contrast to energy-providing nutrients, vitamins are required onlyin exceptionally small amounts. An inadequatesupply of a vitamin leads to typical pathological

deficiency symptoms which can be remedied byintake of the lacking vitamin.

Vitamins are classified not chemically butby their activity. The historical distinction bet-ween fat- and water-soluble vitamins has beenretained to this day [2] because the solutionprop-erties are important not only for the occurrence,but also for the behavior of vitamins in the organ-ism (resorption, transport, excretory pathways,and storage).

However, it is not the case that fat-solublevitamins are stored much more effectively thanwater-soluble vitamins. The assumption that adeficiency of fat-soluble vitamins occurs after amuch longer period than in the case of water-soluble vitamins (higher reserve capacity of li-posoluble substances) is incorrect. In fact, thereserve capacity depends on the special prop-

Vitamins 5

erties of the particular vitamin, irrespective ofdifferences in solubility [3].

In the case of vitaminB12, for instance, thereserve capacity amounts to three to five years,but for thiamine it is only two weeks. AlthoughvitaminK is fat-soluble, deficiency of this vita-min occurs shortly after the interruption of foodintake and suppression of enteral synthesis. Con-sequently, the solubility properties of the indi-vidual vitamins only provide vague informationabout the occurrence and stimulation of absorp-tion by lipids in the food and bile acids.

Table 1. Active forms and functions of vitamins

Vitamin Active form Function [4]

VitaminA (11-Z )-retinal involvement in visionVitaminD regulation of calcium

and phosphatemetabolism

Vitamin E intracellularantioxidant

VitaminK biosynthesis ofprothrombin

VitaminB1 thiamin pyrophosphate(TPP),

decarboxylation ofα-oxo acids, transferof aldehyde

thiamin triphosphate(TTP)

groups, phosphatedonor

VitaminB2 flavinmononucleotide(FMN),

hydrogen or electrontransport inenergy-releasing

flavinadeninedinucleotide (FAD),

metabolism

coenzymes offlavoproteins

Niacin nicotinamide adeninedinucleotide

hydrogen or electrontransport

(NAD), nicotinamideadeninedinucleotide phosphate(NADP)

Pantothenic acid coenzyme A (CoA) transfer of acetylgroups

VitaminB6 pyridoxal-5-phosphate transfer of aminogroups

Biotin biocytin transfer of carboxylgroups

Folic acid tetrahydrofolic acid transfer of formylgroups

VitaminB12 coenzyme B12 1,2-hydrogen shiftVitaminC involvement in

hydroxylation,ultracellularantioxidant

On the basis of the above definition, thirteencompounds or groups of compounds have beenclassified as vitamins for humans (Table 1):Fat-soluble vitaminsVitamin A (retinols)Vitamin D (calciferols)

Vitamin E (tocopherols, tocotrienols)Vitamin K (phylloquinone)

Water-soluble vitaminsVitamin B1 (thiamin)Vitamin B2 (riboflavin)Vitamin B6 (pyridoxal group)Vitamin B12 (cobalamins)Vitamin C (l-ascorbic acid)Pantothenic acidBiotinFolic acidNiacin

Other compounds exist that are also impor-tant for the organism and cannot always be pro-duced in sufficient quantities. Hence, an alimen-tary additional requirement can arise under cer-tain conditions. These are known as pseudo-vitamins.

1.2. Substances with Vitamin-LikeCharacter

In the past, many authors regarded the es-sential fatty acids (“vitamin F”), lipoic acid,ubiquinones, choline, myoinositol, “vitaminU”(S-methylmethionine), and some other sub-stances and substance mixtures as vitamins.Some characteristics of these substances arelisted in Table 2.

1.3. History

The term “vitamins” was introduced by Funk[5] in 1911. Searching for the causes of beriberi,a nervous disease often encountered in EastAsia, Eijkman showed in 1896 [6] that polishedrice caused this disease and that it could be curedby consumption of rice bran. In 1911, Funk iso-lated a substance in the form of water-solublecrystals from rice bran. This substance was re-sponsible for curing beriberi and was named“beriberi vitamin”.

The designation “vitamin” was accepted forall the essential nutritional factors discoveredin the following period, although it was soonrecognized that not all these substances areamines.According to aproposalmadebyDrum-mond [7] in 1920, the beriberi vitamin was des-ignated vitaminB, the fat-soluble antixeroph-thalmia factor was named vitaminA, and the

6 Vitamins

Table 2. Compounds with vitamin or coenzyme character

antiscurvy factor was named vitaminC. All thevitamins discovered later were included in thisclassification.

A survey of the discovery, isolation, and as-signment of the chemical structure of the indi-vidual vitamins is presented in Table 3.

As mentioned above, the discovery of the vi-tamins was due to the fact that a deficiency can

severely damage the organism. Therefore, thesubstances that should be regarded as vitaminsare all known today. It is unlikely that new foodconstituents which fully correspond to the defi-nition of vitaminswith regard to their occurrenceand action will be discovered.

Nevertheless, the “history of vitamins” hasstill not come to an end. A constantly increasing

Vitamins 7

Table 3. Discovery of the vitamins

Vitamin First isolation Discovery Isolation Structural elucidation

VitaminA fish liver oil 1909 1931 1931VitaminD fish liver oil 1918 1932 1936Vitamin E wheat germ oil 1922 1936 1938VitaminK alfalfa 1929 1939 1939VitaminB1 rice 1897 1911 1936VitaminB2 eggs 1920 1933 1935VitaminB6 rice 1934 1938 1938VitaminB12 liver 1926 1948 1956Niacin liver 1936 1935 1937Folic acid liver 1941 1941 1946Panthothenic acid liver 1931 1938 1040Biotin liver 1931 1935 1042VitaminC lemons 1912 1928 1933

number of publications deal with the preventiveeffects of vitamins on vascular diseases and var-ious types of cancer (see Section 1.6).

1.4. Determination of Requirement

According to the definition of the term vita-min, an inadequate supply of vitamins causes acertain deficiency (hypovitaminosis or avitami-nosis).

In the case of avitaminosis, a minimumamount of vitamin is required to remedy theseconditions. However, this amount does not cor-respond to the true requirement. No exact datacan be given for this requirement because itvaries from individual to individual and stronglydepends on the physical condition of the particu-lar person. In stress situations (e.g., illness, preg-nancy, and physical exertion), the requirementis considerably higher. Even when in human ex-periments the selected group of persons is wellbalanced in this respect, substantial variationsremain, which are mainly due to the difficultyof producing the same state of deficiency at thestart of the investigation and in evaluating theoften complex deficiency symptoms.

For this reason, it is meaningful to stipu-late the “desirable supply” of a vitamin (recom-mended dietary intake =RDI or recommendeddietary allowance =RDA) instead of the exactrequirement. Experience shows that full healthand productivity are observed at these levels ofintake. RDA values are regularly issued by theU.S. Food andNutritionBoard, theU.K.Depart-ment of Health and Social Security, WHO, andby the German Society for Nutrition (DGE) [8].

A compilation issued in 1991 is listed in Table 4.

1.5. Application and Tolerance

Vitamins are used as single preparations or asmultivitamin preparations in the treatment ofhypovitaminosis and avitaminosis and in theprophylaxis of these conditions.

Recently, much attention has been paid to themegadosing of vitamins, i.e., the controlled ad-ministration of vitamins in doses which exceedby far the physiological intake.

The following applications can be differenti-ated:

1) Combating deficiency diseases, e.g., mega-doses of vitaminK for infants and small chil-dren in developing countries

2) Prophylactic or therapeutic applications inthe case of diseases which can cause vitamindeficiencya) VitaminC for coldsb) Fat-soluble vitamins in chronic distur-

bances of fat absorption (chronic Pauls re-action, chronic cholesterase)

c) Megadoses of thiamin for alcoholics3) Alleviation of vitamin metabolic diseases

a) Megadoses of vitaminD in cases ofchronic kidney insufficiency

b) Megadoses of biotin in cases of multiplecarboxylase deficiency

A survey of the use of megadoses of vitaminsin prophylaxis and therapy is given in [9].

Hyperdosing can occur in the case of the fat-soluble vitamins A and D because they can be

8 VitaminsTa

ble

4.Recom

mendedvitamin

intake

perday

Group

VitaminA,

Vit-

Vit-

VitaminK,

Thiam

ine,

Ribofl

avine,

Niacin,

VitaminB6,

Folic

acid,

Vit-

Vit-

mgRE

aam

inD,

aminE,

mg

mg

mg

mgNE

cmg

µgam

inB12,

aminC,

mf

µgmgTE

bm

fm

fm

fm

fm

fd

eµg

mg

Infa

nts

0–4months

0.5

103

50.3

0.3

50.3

400.5

404–12

months

0.6

104

100.4

0.5

60.6

8040

0.8

50C

hild

ren

1–4years

0.6

56

150.7

0.8

90.9

120

601.0

554–7years

0.7

58

201.0

1.1

121.2

160

801.5

607–10

years

0.8

59

301.1

1.2

131.4

200

100

1.8

6510

–13

years

0.9

0.9

510

4040

1.2

1.2

1.4

1.3

1514

1.6

1.5

240

120

2.0

7013

–15

years

1.1

1.0

512

5050

1.4

1.2

1.5

1.4

1715

1.8

1.6

300

150

3.0

75You

thsan

dad

ults

15–19

years

1.1

0.9

512

7060

1.6

1.3

1.8

1.7

2016

2.1

1.8

300

150

3.0

7519

–25

years

1.0

0.8

512

7060

1.4

1.2

1.7

1.5

1815

1.8

1.6

300

150

3.0

7525

–51

years

1.0

0.8

512

7060

1.6

1.3

1.8

1.7

1815

1.8

1.6

300

150

3.0

7551

–65

years

1.0

0.8

512

8065

1.3

1.1

1.7

1.5

1815

1.8

1.6

300

150

3.0

7565

years

1.0

0.8

512

8065

1.3

1.1

1.7

1.5

1815

1.8

1.6

300

150

3.0

75andolder

Pre

gnan

tw

omen

1.1

1014

651.5

1.8

172.6

600

300

3.5

100

Nur

sing

mot

hers

1.8

1017

651.7

1.7

202.2

450

225

4.0

125

a1mgretin

olequivalent=6mgall-trans-

β-carotin=12

mgotherprovitaminAcarotin

oide

=1.15

mgall-trans-retin

ylacetate=1.83

mgall-trans-retin

ylpalm

itate.

b1mg(RRR)-

α-tocopherolequivalent=

1.1mgRRR-α

-tocopherylacetate=2mg(RRR)-

β-tocopherol=

4mg(RRR)-

γ-tocopherol=

100mg(RRR)-

δ-tocopherol=

3.3mg

(RRR)-

α-tocotrienol=1.49

mgall-rac-

α-tocopherylacetate.

c1mgniacin

equivalent=60

mgtryptophan.

dCalculatedon

thebasisof

“totalfolate”(sum

offolate-activecompounds

innorm

alfood).

eFo

lateequivalent

orfree

folic

acid

(pteroyl

monoglutamate).

Vitamins 9

stored and can be eliminated only by conversionto excretable substances. To prevent toxic sideeffects, controlled administration is necessary.

1.6. Studies with Vitamins

The effects of overdoses of vitamins (vitamin C,E, β-carotene) in the prevention of cancer andcoronary heart diseases are being studied in aseries of investigations.

A summary of the studies on the effects ofantioxidative vitamins is given in Table 5.

An evaluation of these studies shows that theintake of certain vitamin/carotene combinationshas a prophylactic effect. Further information onthese results is given in [10–12].

1.7. Use of Vitamins in Food- andFeedstuffs

For humans, the most important sources of vi-tamins are plant and animal foods. In the in-dustrial countries, the vitamin requirement canbe completely covered by the food consumed.Nevertheless, the danger of a suboptimal sup-ply exists in large segments of the population ofthese countries. According to the nutritional re-port of the Deutsche Gesellschaft fur Ernahrung(GermanSociety forNutrition), the supply of thevitamins B1, B2, and B6 is regarded as criticalin Germany [13].

The reasons for this are the one-side prefer-ence for certain foods and a steady transitionfrom fresh, home cooked meals to the consump-tion of canned foods and mass catering. Theindustrial production of foods, which requirespreservation in most cases to give long shelflives, results in a considerable loss of vitaminsdue to the effect of heat, air, and light. To com-pensate for these losses, fortification of basicfoods (flour, bread, and cakes with B complexvitamins; margarine and milk products with vi-tamins A, E, and D) diet foods (e.g., baby foodscontaining all the vitamins) and fruit juices (vi-taminC, etc.) is gaining importance. For legalaspects, see [14].

In the feed industry, vitamins are used on alarge scale as additives in animal feeding to en-sure an optimal supply.

Breedingdiseases, fertility losses, andperfor-mance changes of all types are avoided by theselective feeding of vitamins, improving prof-itability in animal husbandry. This applies es-pecially to today’s intensive farming of poultry,fish, and pigs and to calf breedingwithmilk sub-stitute.

In Germany, the type and quantity of feed ad-ditives that are mixed with the different kinds offeedstuffs are stipulated by the feed law [15].

1.8. Antivitamins

Antivitamins are compounds that reduce or re-verse in a specific manner the effect of vita-mins. A distinction ismade between compoundswhich destroy or capture the vitamin, and sub-stances which compete with the vitamin for thebinding site on the receptor (competitive inhibi-tion).

Examples of the first class of compounds are,e.g., thiaminase, which cleaves the vitamin thi-amine, and avidin, which forms a complex withbiotin, making it unavailable for the body.

Antivitamins can be used to produce definedvitamin deficiency diseases, allowing the bio-chemical background of vitamin activity to bestudied.

Some antivitamins are therapeutically ap-plied, e.g., folic acid antagonists in the treatmentof certain forms of leukemia and vitaminK an-tagonists as inhibitors of clotting.

1.9. Analysis of Vitamins

Methods of determination for vitamins whichare based on the specific properties of vitaminsinclude biological,microbiological, and enzymeactivation processes.

Biological methods are mostly growth testswith experimental animals fed vitamin deficientdiets. They were important especially for thechecking the progress of enrichment during theisolation of vitamins. Today, these processes areused only for special purposes because they aredifficult and tedious to conduct and evaluate and,consequently, inaccurate.

Microbiological methods are based on thefact that various vitamins are growth factorsfor certain microorganisms. The photometri-callymeasurable turbidity of the culturemedium

10 Vitamins

Table 5. Summary of studies on the effects of antioxidative vitamins

Period Vitamin Participants Country Results

1970 – 1980 β-carotene 3000 men USA the men who died of cancerduring thestudy had 20% lessβ-carotene in theblood than control persons

1980 – 1990 β-carotene, vitamins E, C 2000 nurses recovered fromcardiac infarct or angina

USA antioxidant vitamins reducethe risk of cardiac infarct by33% and stroke by 71%

1985 – 1991 β-carotene, vitamin E 29 000 smokers Finland no preventive effect wasdetected during the study

1985 – 1991 β-carotene, vitamin E, Se 30 000 smokers China vitamins reduce risk ofcancer

1991 – 1992 β-carotene, vitamin E 1400 persons: 50% with acutecardiac infarct, 50% controlpersons

Europe infarct patients have a lowerβ-carotene and vitamin Estatus

1988 – 1992 β-carotene, vitamins E, C 900 patients with removedadenomas

USA the treatment does notinfluence the probability offormation of new adenomas

1989 – 1994 β-carotene 400 albinos with skin diseases Tanzania β-carotene reduces thedevelopment of skin cancer

1990 β-carotene, vitamins E, C 500 men UK low plasma concentrationscorrelate with high risk ofangina

1986 – 1990 vitamin E 40 000 men USA vitamin E reduces the risk ofsevere heart diseases by 37%

1980 – 1988 vitamin E/multi-vitaminpreparation

37 000 women USA vitamin E reduces the risk ofcoronary diseases by 40%

is a measure of the vitamin concentration. Mi-crobiological methods are characterized by highspecificity and accuracy. They are used for thedetermination of vitamins in biological materialand in pharmaceutical preparations (e.g., vita-mins of the B group).

For clinical investigations (e.g.,measurementof the vitamin level in the blood and urine), en-zyme activation is commonly used because of itshigh specificity. It is based on the addition of thespecific apoenzyme of the vitamin (coenzyme)to be determined and subsequent measurementof the activity of the holoenzyme.

The chemical and physical methods of deter-mination used correspond to the normalmethodsfor the analysis of organic substances. Modernchromatographic processes are particularly im-portant.

In the case of vitamin preparations, the con-centrations given refer to the quantity byweight.Only for vitamins A, D, and E, it is customaryto state the international units as well.

1.10. Production

Vitamins are produced on an industrial scale bychemical synthesis or partial synthesis, by fer-

mentation, or by extraction from natural mate-rial, whereby chemical synthesis is the dominantmethod.

The production processes used commerciallyare summarized in Table 6.

Table 6. Typical processes for the commercial production ofindividual vitamins∗Vitamin Synthesis Ferment- Isolation

ation

VitaminA +◦

+

VitaminB1 + +VitaminB2 +

◦VitaminB6 + +VitaminB12 +VitaminC +VitaminD3 + +Vitamin E + +VitaminK + +Biotin +

◦Folic acid +

◦Niacin +Pantothenic acid +

◦∗+ Commercially used; ◦ commercially possible.

Many vitamins have become large-scaleproducts because of the constantly increasing

Vitamins 11

demand. The economic importance of the vi-tamins has increased to the same extent. Atpresent, the worldwide market value for vita-mins as a bulk product is ca. 26×109¤/a. Interms of value, the feed industry accounts for ca.50%, the pharmaceutical industry for ca. 30%,and the food industry for ca. 20%.

2. Vitamin A (Retinoids)

2.1. Introduction

In the broadest sense, the term vitamin A refersto a group of monocyclic diterpenes with sim-ilar biological activity. Since the term vita-minA is frequently linked to vitaminA alco-hol [(all-E )-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraen-1-ol, retinol,vitaminA1], and a large number of other natu-ral and synthetic vitaminA derivatives exist, thedesignation retinoids is often employed [31].

In addition to retinol (1), there are retinoidsthat can be distinguished by their side chainand their ring system. These include retinal(2), retinoic acid (3), retinyl phosphate (4),the 3-dehydro compounds (5), (6), (7), andretroretinoids (8), (9), (10).

VitaminA is found only in animal tissue,whereas the carotenoids known as provitaminA

are found in the plant world. The latter are ox-idatively degraded to vitaminA compounds inthe animal organism.

The important provitaminsA are β-carotene(11), α-carotene (12), γ-carotene (13), β-apo-8′-carotinal (14), β-carotene-5,6-epoxide (15),cryptoxanthin (16), and echinenone (17).

ProvitaminA activity requires at least onecyclohexene or 5,6-epoxycyclohexane ring andone polyene chain equivalent with a chain lengthcorresponding to that of vitaminA.

2.2. Historical Aspects

In 1876, Snell showed that cod-liver oil couldcure night blindness and xerophthalmia [32].

12 Vitamins

In 1880, Lunin found that the growth and vi-tal functions of experimental animals could notbe maintained with carbohydrates, fats, and pro-teins alone, but that the addition of small quan-tities of milk powder was also required [33].

In 1909, Stepp extracted vital fat-solublesubstances frommilk [34]. In 1916,McCollummade a distinction between “fat-soluble A” and“water-soluble B” [35]. Later, the fat-solubleagent was called “vitaminA” on the proposal ofDrummond [36]. (The name “vitamin” had al-ready been suggested by Funk in 1912 when heisolated the anti-beriberi agent thiamin [37]).

Progress in the concentration of retinol (1)was made only after its sensitivity to oxygenand higher temperatures was recognized and theconnection between the growth promoting ef-fect and the color intensity in the Carr – Pricetest was discovered [38]. In 1931, Karrer iso-lated almost pure retinol (1) from the oil of a typeofmackerel (Scom-bresox saurus) [39]. The firstcrystalline preparations were made in 1937 byHolmes and Corbet [40] and in 1942 by Bax-ter and Robeson [41], who also succeeded inisolating crystalline (13Z )-retinol in 1946 [42].The compound 3-dehydroretinol (4) was foundin the liver oil of freshwater fish in 1937 [43], iso-lated in 1948 [44], and obtained in the crystallineform in 1962 [45]. As early as 1831,Wacken-roder [46] succeeded in isolating the orange-yellow pigment of the carrot (Daucus carota)which he called carotene. The wide distributionof carotene in the plant world was described byArnaud in 1887 [47]. Itwas only after improvedisolation methods became available that the pro-duction of pure preparations was possible. Inthe course of this work, Willstatter in 1907was able to establish the empirical formula ofcarotene [48], which was thought to be a homo-geneous compound.

In 1931 Kuhn, Karrer et al. succeeded inisolating optically active α-carotene (8) by frac-tionated crystallization or chromatography oncalcium hydroxide [49]. Of importance for thestructural elucidation of the carotenoids was thediscovery that the chromophoric group consistsof conjugated olefinic double bonds [50]. Fromthe amount of hydrogen absorbed in catalytichydrogenation, it was calculated that carotenehas 11 and the isomeric lycopene has 13 dou-ble bonds [51]. In 1929, the provitaminA ac-tivity of carotene was unambiguously demon-

strated in animal experiments and itwas simulta-neously shown that the other carotenoids knownat that time were completely inactive in this re-spect [52]. Thus, further experiments for eluci-dating the structures of carotene and vitaminAwere closely connected. Important progress indetermining the structure was made bymeans ofoxi-dative degradation with potassium perman-ganate, chromic acid, and ozone to give charac-teristic fragments [53]. One of the first correctsuggestions for the structure of retinol (1) wasmade by Karrer who had shortly before deter-mined the structure of β-carotene [54]. The con-stitution of 3-dehydroretinol (4) was long dis-puted and finally established with its synthesisin 1952 [55].

In 1937,Karrer obtainedβ-apo-8′-carotinalby oxidative degradation of β-carotene withpotassium permanganate [56]. The compoundβ-apo-8′-carotinoic acid ((14a) with X=OH)was isolated from egg yolk after feeding layinghens with 14 [57].

2.3. Physical Properties

The melting points and the UV spectroscopicdata of vitaminA compounds and their most im-portant derivatives are presented in Table 7.

Retinol (1), retinal (2), and the dehydro com-pounds 5 and 6 are readily soluble at room tem-perature in fats, oils, and common organic sol-vents. In contrast, retinoic acid (3) is appreciablysoluble only in chlorinated hydrocarbons.

High-vacuum distillation can be used for thepurification of retinol (1), its esters, and retinal(2) (retinol at 125 ◦C, retinyl acetate at 132 ◦Cand 400 Pa). The double bonds in the side chainalso allow the existence of Z isomers, someof which occur in nature. Thus, (all-E )-retinolis frequently accompanied by the (13Z isomer(18). The compound (11Z )-retinal (19) is of im-portance as the prosthetic group of the proteinopsin [58].

Vitamins 13

Table 7. Characteristic data of vitaminA compounds and their most important derivatives∗

Compound mp, ◦C UV absorption (in Ethanol) Biological Activity

λ, nm ε1 %1 cm in %∗∗

Retinol 62 – 64 325 1835 100(13Z )-Retinol 58 – 60 328 1686 75Retinal 61 – 62 381 1530 91(11Z )-Retinal 64 – 65 254, 290, 376 614, 412, 878 48Retinoic acid 179 – 180 350 1510(13Z )-Retinoic acid 174 – 175 354 1325Retinyl acetate 57 – 60 326 1550Retinyl palmitate 28 – 29 327 9753-Dehydroretinol 63 – 65 276, 286, 350 555, 715, 1455 403-Dehydroretinal 78 – 79 (314), 401 395, 14703-Dehydroretinoic acid 183 – 184 (305), 370 443, 1395

∗ The values refer to the (all-E ) isomers unless otherwise noted.∗∗ Curative growth test in rats.

In comparison with the other stereoisomersof retinoic acid, (9Z )-retinoic acid (20) exhibitsthe highest binding affinity for the nuclear re-ceptor RXR α [59], resulting in an altered geneexpression as a consequence of direct ligand in-teraction.

With four double bonds in the side chain ofvitaminA, 16 stereoisomeric forms (E andZ iso-mers) are theoretically possible. In the individualisomers, the steric effects allow a distinction tobe made with regard to the stability of hinderedand unhindered cis forms. While the trisubsti-tuted double bonds (9 and 13) of the side chainare quite stable in the (Z ) configuration, the di-substituted bonds (7 and 11) rearrange readily tothe (E ) form (21) [60].

In the case of unhindered cis double bonds,the 1,4 steric interaction of two H atoms isweak, but strong interaction occurs between a H

atom and a methyl group or between twomethylgroups in hindered cis double bonds. Therefore,facile isomerization from the hindered cis formsto the trans forms is observed.

Thus, the four stable retinal isomers (all-E;9Z; 13Z; 9Z ,13Z ) are well known. Of the re-maining 12 isomers, however, only four of thelabile forms (7Z; 11Z; 7Z ,9Z; 11Z ,13Z ) areknown [61]. Other labile Z forms have been syn-thesized under special conditions [62,63].

X-ray analyses of retinol (1), its esters [64],and retinal (2) [65] assign a 6,7-s-cis conforma-tion to the molecule in the crystal lattice.

Two different crystal modifications ofretinoic acid have been determined [66]. In ad-dition to the stable 6,7-s-cis form (22, triclinicmodification), the labile 6,7-s-trans form (23,monoclinic modification) is also known.

In the all-trans forms, the polyene chain hasa saber-like curvature which results from the in-teraction of the methyl group in the side chainwith the hydrogen on the next carbon atom butone.

The provitaminsA have colors which varyfrom bright yellow to red, due to the system ofconjugated double bonds.

With increasingnumbers of double bonds andwith the introduction of carbonyl groups conju-gated to the double bond system, the maxima in

14 Vitamins

the UV and visible region shift to longer wave-lengths.

The melting points, UV spectroscopic data,and biological activity of the most importantprovitaminsA are listed in Table 8.

Both α- and β-carotene (12 and 11) are read-ily soluble in chlorinated hydrocarbons and incarbon disulfide, and poorly soluble in ether,lower alcohols, and in aromatic or aliphatic hy-drocarbons. In comparisonwith the carotene hy-drocarbons (11) and (12), the β-apocarotenoidsare in general more readily soluble. Data onthe solubility of (all-E )-β-carotene (11) ing/100mL at 20 ◦C follow:

Water 0.0Ethanol < 0.01Fats and oils 0.05 – 0.08Orange oil 0.2 – 1Cyclohexane ca. 0.1Ether ca. 0.1Acetone ca. 0.1Chloroform ca. 3

The provitaminsA occur in fresh plant tissuein the (all-E ) form. During drying and storage,however, partial conversion to the Z forms takesplace. Heating in solution or in the presence ofiodine causes isomerization. This process pro-duces all-E compounds as well as 9Z and 13Zforms as the main components. In comparisonwith the all-E compounds, the Z isomers have alower extinction and biological activity [67].

Table 9. Typical reactions of retinoids. In principle, they also applyto the 3-dehydro forms.

Retinoids Reagents Products

Retinol manganese dioxide retinal (2) [68,69]acetic anhydride (Ac2O) retinyl acetatepalmitoyl chloride retinyl palmitatemaleic anhydride Diels –Alder adductssinglet oxygen 5,8-peroxide (24) [70]peroxy acids 5,6-epoxide (25) [71]ozone geronic acidpotassium permanganate acetic acid

Retinal sodium borohydride retinol [72]silver oxide retinoic acid [73]manganese oxide 4-oxoretinal [74]

Retinoic acid alcohols retinoic estersglucuronic acid glucuronideslithium aluminumhydride

retinol [75]

2.4. Chemical Properties

Retinol (1) and its esters are readily oxidized byatmospheric oxygen and other oxidizing agents,

especially in the presence of light or at highertemperatures. The addition of antioxidants (e.g.,α-tocopherol) or storage in the cold with the ex-clusion of light and oxygen ensures a certain sta-bility. Some typical reactions of vitaminA arelisted in Table 9.

In all these reactions, the stereochemistry ofthe polyene side chain remains unchanged pro-vided light and oxygen are excluded.

VitaminA is resistant to bases, but retinol (1)and its esters undergo rearrangement and elimi-nation reactions in the presence of acids. Diluteacids cause the conversion of retinol (1) to an-hydroretinol (10).

In dichloromethane, retinyl acetate is con-verted in the presence of hydrogen bromide toretroretinyl acetate (9)within seconds. The treat-ment of vitaminA compounds with halogenat-ing agents (e.g.,N-bromosuccinimide) results inthe preferential halogenation of the methylenegroup at position 4. Subsequent base-catalyzedelimination of hydrogen halide provides ac-cess to the 3-dehydroretinoids (5, 6, 7), which,however, have no significant vitaminA activ-ity. Strong Lewis acids react with vitaminAto produce characteristically colored complexes(e.g., blue color of complex with SbCl3 in theCarr – Price reaction), which form the basis ofanalytical identification [76]. With dienophiles(e.g., maleic anhydride), retinol (1) and its estersundergo typical [4 + 2] cycloaddition reactions.The 11,13-di-trans configuration reacts prefer-entially, allowing easy differentiation and sepa-ration of the all-trans and 9-cis compounds fromthe slower reacting 13-cis, 11-cis, 11,13-di-cis,and 9,13-di-cis stereoisomers [82].

The provitaminsA are also sensitive to oxida-tion. The degradation of β-carotene with potas-sium permanganate leads to β-apo-12′- and β-apo-8′-carotinal (14) [77]. Chromic acid cleavesthe ring double bond with formation of open-chain diketones [78]. Peroxy acids preferentiallygive 5,6-epoxides, which rearrange to 5,8-di-

Vitamins 15

Table 8. Characteristic data of the most important provitaminsA [(all-E) isomers]

Compound mp, ◦C UV absorption (in Ethanol) Biological Activity

λ, nm ε1 %1 cm in %∗

β-Carotene 182 – 183 456, 484 2500, 2200 100(Cyclohexane)

(R)-(+)- α-Carotene 187 – 188 422, 446, 474 1790, 2720, 2500 54(Hexane)

β-Apo-8′-carotinal 138 – 139 461, 488 2640, 2165 (Cyclohexane) 72β-Apo-8′-carotinoic acidethyl ester

137 – 138 449, 475 2550, 2140 (Cyclohexane) 25, 78

∗ Curative growth test in rats.

hydrofuran derivatives (26) under acid catalysis[79].

The reaction of provitaminA with N-bromosuccinimide or iodine preferentially leadsto 4,4′-dihalo compounds [80], which allow thesynthesis of 4,4′-dioxo-β-carotene (canthaxan-thin) by continued oxidation [81].

The E,Z isomeric vitaminA compounds dif-fer considerably in their biological activity [83].The highest activity is exhibited by (all-E )-retinol and its esters (see Table 7). Industrially,retinyl acetate is isomerized either photochemi-cally [84] or in the presence of catalytic amountsof iodine or palladium compounds [85]. The all-E form, which predominates at equilibrium, canbe separated by crystallization from hexane ormethanol.

2.5. Occurrence

Retinol (1) occurs exclusively in animal organs,especially in the liver. Generally, it is esterifiedwith higher fatty acids (e.g., palmitic acid). Rel-atively high concentrations are found in milk,eggs, and blood plasma.

VitaminA is largely formed by degradationof carotenoids ingested with food. Carotenoidsare synthesized only by higher plants and mi-croorganisms. After consumption carotenoids,also known as provitaminsA, are enzymaticallyconverted to vitaminA, especially in the cells ofthe intestinal mucosa [86]. The compounds α-and β-carotene (12 and 11) are permanent com-panions of chlorophyll. High concentrations arefound especially in paprika, carrots, and in palmoil (400 – 800 ppm) [87], which is used to coloredible fats. The β-apo-carotinals mainly occurin citrus fruits. However, their concentration istoo low to permit economic commercial isola-tion [88].

2.6. Biosynthesis

In the animal organism, vitaminA compoundsoccur as metabolites of the provitaminsA, espe-cially of β-carotene (11).

The C40 carotenoids are tetraterpenes. Theyare biosynthesized according to the princi-ple valid for all terpenes [89] (see Scheme 1;→Terpenes, Chap. 1.3.). The C5 structural unit1-isopentenyl pyrophosphate (28) is made fromthree molecules of acetyl coenzyme A viamevalonic acid (27). Isomerization of 28 givesthe more reactive 3,3-dimethylallyl pyrophos-phate (29). The linkage of these two compo-nents starts a chain of alkylation steps whichleads to the C20 intermediate geranylgeranyl py-rophosphate (30) and is interrupted by dimeriza-tion to give (15Z )- or (all-E )-phytoene ((32a)and (32b)), the first C40 carotenoid. The in-termediate in this reaction is prephytoene py-rophosphate (31). Dehydrogenation of 32 giveslycopene (33) via phytofluene, ζ-carotene, andneurosporin. From lycopene, α- and β-carotene(12 and 11) are formed by stepwise cyclization.

The subtle stereochemical problems involvedin the individual steps have been elucidated bythe use ofmevalonic acid (27), stereospecificallylabelled at the prochiral centers C-2, C-4, and C-5 (27′). Thus, it is known that the anhydrodecar-boxylase step 27 → 28 proceeds as a trans elimi-nation. In the isomerase reaction 28 → 29, a pro-ton is added at the re,re side of the double bond,followed by eliminaton of the 2-pro-R proton of28 (H4S of 27′).

The prenyltransferase steps 29 →30 also pro-ceed with elimination of the 2-pro-R proton(Scheme 1) of 28 and with inversion at C-1of the allylpyrophosphate components (C-5 of27′).An enzymatic addition – eliminationmech-anism leads to E double bonds.

16 Vitamins

In the linkage of two molecules of ger-anylgeranyl pyrophosphate (30) to form (15Z )-phytoene (32a), the 5-pro-S hydrogen atoms ofmevalonic acid are lost. In the dehydrogenation32 → 33, double bonds in the E configurationare introduced stepwise with the elimination ofone 2-pro-S and one 5-pro-R hydrogen atom ofmevalonic acid (27′).

The cyclization 33 → 11 or 12 is startedby,re,re addition of a proton to C-3 of the ter-minal double bond (C-4 of 27′) and intramolec-ular alkylation of the 6,7 double bond. Cleavageof a proton from C-6 and C-4 (C-4 and C-2 of27′) results in the parallel formation of the α-and β-ionone rings. The methyl group from C-2of mevalonic acid (27′) occupies the pro-S po-sition.

From a stereochemical standpoint, the reac-tion sequence can be represented by the labellingscheme 27′ → 30′ → 33′ → 12′ (Scheme 2).

The conversion of the provitaminsA to vi-taminA compounds takes place in the intesti-nal mucosa. In this process, α- and β-carotene(12 and 11) are cleaved by oxygen in the pres-ence of carotene 15,15′-dioxygenase into twomolecules of retinal (2) [90]. Similar enzymesalso catalyze the oxidative degradation of β-apocarotinals [91]. Retinal (2) is predominantlyreduced to retinol (1) and partly oxidized toretinoic acid (3).

After transfer into the intestinal cells, retinol(1) is esterified with higher fatty acids and re-leased into the lymph in this form [92]. Afterthe direct vitamin requirement of the organismis covered, the retinyl esters are stored in theliver [93]. Free retinol (1) predominates in theblood stream; a specific protein is responsiblefor transport [94].

2.7. Production

2.7.1. Isolation and Purification fromNatural Raw Materials

Owing to the sensitivity of retinol (1) to air,higher temperatures, and acids, it must be iso-lated under especially mild conditions (ex-traction with organic solvents under protectivegases, e.g., CO2, N2). Since retinol (1) and itsesters occur in the free form in tissues and or-gans, the first concentration step can be extrac-

tion with heptane. For retinal (2), however, spe-cial isolation methods are required because it iscovalently bound to primary amino groups in theform of a Schiff’s base [95].

In general, vitaminA is isolated by liquid –liquid extraction,molecular distillation [96], andHPLC [63]. Commercial isolation of retinol (1)from natural raw materials starts with fish oilsand is conducted in six steps: (1) accumulationof crude vitaminAby theSolexol countercurrentextraction process [97], (2) short-path distilla-tion of the retinyl esters (200 ◦C, 400 Pa), (3)ester saponification (KOH, 60 – 70 ◦C, 30min,N2), (4) extraction of retinol (1) with ether,(5) short-path distillation of retinol (1) (120 ◦C,400 Pa), and (6) final purification by crystalliza-tion from ethyl formate (−35 ◦C).

With the development of efficient processesfor the synthesis of vitaminA on a commercialscale, isolation from natural raw materials haslost importance.

2.7.2. Industrial Synthesis

Since retinol (1) was first synthesized by Kuhn[98], numerous strategies for its synthesis havebeendeveloped.VitaminA is formedby the link-age of various cyclic components with the cor-responding open-chain compounds [99,100]:C10 +C10 [101],C11 +C9 [102],C13 +C7 [103],C14 +C6, C15 +C5, C16 +C4, C18 +C2, andC18 +C1 +C1.

The key building block of all industrial vita-minA syntheses is β-ionone (34), which can bemade from citral (35) [104] and acetone or from

Vitamins 17

Scheme 1.

Scheme 2.

18 Vitamins

dehydrolinalool (36) [105] and diketene [106]or isopropenyl methyl ether [107] via pseu-doionone (37) [108].

Synthesis of the C20 Skeleton of VitaminsA. C15 +C5 (Sumitomo) [109]. Knoevenagelcondensation of β-ionylidene acetaldehyde (38)[110] with (E )-3-methyl-2-butenoic ester us-ing potassium amide in ammonia produces (all-E )-retinoic ester (39). Reduction with lithiumaluminum hydride and subsequent acetylationyields retinyl acetate (40).

C16 +C4 (DPI, Glaxo) [111]. The reactionof β-ionone (34) with 2-propinyl chloride in thepresence of zinc yields β-propargyl-β-ionone(β-C16 alkinol) (41). Grignard reactionwith 4,4-dimethoxy-2-butanone (42), followed by partialhydrogenation and hydrolysis of the acetal aswell as elimination of water, gives retinal. Reti-nal is then converted to retinyl acetate (40) byreduction and subsequent acetylation.C18 +C2 (Philips) [112]. β-Ionone (34) and

the homologousβ-C18 ketone (43) are convertedtoβ-ionylidene acetaldehyde (38) and retinal (2)by condensation with cyanoacetic ester and re-duction of the nitriles 44 and 45 with diisobut-yl aluminum hydride. The β-C18 ketone (43) ismade from β-ionylidene acetaldehyde (38) bycondensation with acetone.

C18 +C1 +C1 (AEC) [113]. Starting with β-ionone (34) and β-C18 ketone (43), a stepwiseconversion to β-ionylidene acetaldehyde (38)and retinal (2) is carried out. Condensation ofthe ketones 34 and 43 with formic ester givesthe β-C14 and β-C19 enolates, which yield thehydroxyacetals 48 and 49 after acetal formation(46 or 47) and Grignard reaction. Subsequentacetal hydrolysis and water elimination give thefinal products of the reaction sequence 38 and 2.C14 +C6 (Hoffmann LaRoche) [114]. β-

Ionone (34) is converted to the β-C14 alde-hyde (50) in a Darzens glycidic ester synthe-sis [115,116]. Subsequent Grignard reactionwith 3-methyl-2-penten-4-in-1-ol(51) yields theC20alkynediol (52).

(All-E)-retinyl acetate (40) is obtained afterpartial hydrogenation of the triple bond to givethe (11Z , 13Z ) compound 53, acetylation of the

Vitamins 19

primary hydroxyl group, and subsequent acid-catalyzed dehydration with isomerization of thedouble bonds into the E configuration.

TheC6 structural unit 51 ismade frommethylvinyl ketone by ethynylation and alkyl rear-rangement of the tertiary alkyne carbinol 54[117].

C15 +C5 (BASF) [118] (Scheme 3). Thesynthesis of the C20 skeleton is achievedby using the Wittig reaction [119]. Alkyli-denetriarylphosphanes are obtained fromalkyltriarylphosphonium salts by treatment withbase [120–122]. The formation of olefins withsimultaneous release of triarylphosphine oxideis achieved by condensation of these alkylidenetriarylphosphanes with carbonyl compounds[123].

The C15 phosphonium salt 56 can be madefrom β-ionone (34) in two different ways. β-Vinylionol (55) is obtained by ethynylation andsubsequent partial hydrogenation [124] or byvinylation [125]. Treatment with triphenylphos-phine and acid yields the C15 phosphonium salt56 [126], the key building block for the synthesisof retinoids. Retinyl acetate (40) is prepared byWittig reaction with the C5 aldehyde 57, whichis accessible by various routes [127,128], e.g.,by rhodium-catalyzed hydroformylation of 1,2-diacetoxy-3-butene (58) [129]. Coupling of 56with 57 is carried out in organic solvents suchas alcohols or DMF, or in aqueous solution withmild bases (e.g., alkali metal carbonate or am-monia) [130]. In this way, a mixture of the dou-ble bond isomers 11Z and all-E is obtained ina yield of ca. 90%. The labile 11Z isomers canbe converted to the all-E form in various ways[131].

If theC15 phosphoniumsalt 56 is reactedwiththe alternative C5 building blocks 59 [132], 60[133], or 61 [134], retinal (2), retinoic acid (3),or (13Z )-retinoic acid (62) is obtained [if nec-essary after hydrolysis and isomerization of the(11Z ) form].C15 +C5 (Rhone-Poulenc) [135] (Scheme

4). This synthesis of vitaminA is based on a two-step olefin synthesis [136] using Julia sulfonechemistry. Allyl phenyl sulfones are metalatedin theα-position and alkylatedwith alkyl halide.The double bond is formed by elimination ofbenzenesulfinic acid. The coupling and elimi-nation steps both require comparatively strongbases such as potassium tert-butoxide or otheralkali metal alkoxides [137].

20 Vitamins

Scheme 3.

Scheme 4.

Scheme 5.

Vitamins 21

As in the BASF process, the C15 buildingblock is obtained from β-vinylionol (55). Thereaction of β-vinylionol with sodium phenylsul-finate yields the C15 sulfone 63 [138].

Subsequent reaction of 63with 1,1-dialkoxy-4-bromo-3-methyl-2-butene (64) [139] or 1-acetoxy-4-chloro-3-methyl-2-butene (65) [140]yields the C20 sulfones 66 or 67 [141]. Elimina-tion of benzenesulfinic acid, followed by acety-lation affords retinyl acetate (40) (if necessaryafter hydrolysis of the acetal group and reduc-tion of the aldehyde [142]). While the C5 build-ing block 64 is obtainable from prenal [143], 65is derived from isoprene [144].

Formation of the polyene skeleton is alsopossible by treating β-vinylionol (55) with γ-halogeno (68), γ-acetoxy (69) or γ-thiophenyldienol ethers (70), catalyzed by Lewis acids[145]. The primary coupling products 71, 72,and 73 can be converted to retinyl acetate (40)via a reaction cascade (Scheme 5). TheC5 build-ing blocks used are obtainable from prenal. Acommercial application of this synthesis has notbeen realized.C10 +C10 (Kuraray, Solvay –Duphar)

[146].On the basis of sulfone chemistry, the syn-thesis of retinoids is achieved by coupling twoC10 building blocks [147]. The common startingmaterial for both compounds is myrcene (74)or linalool (75), which is converted in a mul-tistep sequence to β-cyclogeranyl sulfone (76)and 8-acetoxy-2,6-dimethyl-2,6-octadienal (77)[148]. The β-hydroxysulfone 78 is obtained byα-metalation of the C10 sulfone 76, followedby condensation with the C10 aldehyde acetate77. The β-hydroxysulfone is then converted toretinyl acetate (40) via the derivatives 79, 80[149] (Scheme 6).C13 +C2 +C5 (L’Oreal) [150]. Starting

from β-ionone (34), β-ethynylionol (81) is pro-duced by ethynylation. In a subsequent Grignardreaction, coupling with β-formyl crotonate (60)is carried out, giving the corresponding acety-lene ester 82, which after reduction to the triolis selectively etherified at the primary alcoholgroup.

The resulting ether diol 83 is stereospecif-ically reduced to retinyl ether (84) after partialhydrogenation of the triple bond with a mixtureof titanium trichloride and lithiumaluminumhy-dride.

C13 +C2 +C3 +C2 [151]. Another pathleading to retinal (2) is via the vinylogizationof carbonyl compounds with the formation ofα,β-unsaturated aldehydes and methyl ketones.The building blocks for a synthesis of this typeare 2-lithiovinyloxy(trialkyl)silanes (85) and 2-lithio-1-methylvinyloxy(trialkyl)silanes (86). Ina further development of this reaction principle[152], retinal (2) is obtained with the C5 build-ing blocks 87, 88 or with the C7 building block89 (Scheme 7).

More recently that concepts have been de-veloped for the synthesis of polyenes via Heckcoupling of C –C single bonds [153].

22 Vitamins

Scheme 6.

Scheme 7.

Synthesis of the C40 Skeleton of Provita-mins A. The syntheses of the provitaminsA areclosely related to those of the vitaminA com-pounds.C19 +C2 +C19 (Hoffmann LaRoche) [154].

β-Carotene is produced from the β-C14 alde-hyde 50, an intermediate in the synthesis of vita-minA. 50 is converted to the β-C19 aldehyde 90in two steps: First, with ethyl vinyl and thenwithethyl isopropenyl ether in aMuller – Cunradi re-action. Subsequently, two molecules of 90 arelinked together by acetylene giving the C40 diol91, which after partial hydrogenation and dehy-dration yields β-carotene (11).

Other syntheses of β-carotene (11) aremainly based on the building principlesC15 +C10 +C15 and C20 +C20. While the firstsynthetic sequence comprises the Wittig con-densation between two molecules of C15phosphonium salt 56 and 2,7-dimethyl-2,4,6-octatrien-1,8-dial (92) [155], β-carotene canalso be made by the condensation of retinal (2)with retinyltriphenylphosphonium salt (93), ob-tainable from retinyl acetate (40) [156,157].

The synthesis of β-carotene (11) is also pos-sible by using the Julia reaction [158]. Retinylphenyl sulfone (94) is made by the reactionof retinol (1) with sodium benzenesulfinate. α-

Vitamins 23

Scheme 8.

Metalation with potassium tert-butoxide to givetheC40 sulfone 96 is followed by alkylationwithretinylhalide (95). Subsequent elimination ofsulfinic acid yields β-carotene (11) (Scheme 8).

Anewconcept for the synthesis ofβ-carotene(11) uses the precursors of the vitaminA processof Kuraray (C10 +C10) 79 or 80. After selec-tive oxidation to the corresponding aldehydes97, 98, these compounds are subjected to Wit-tig condensation with retinyltriphenylphospho-nium salt (93) [159]. Subsequent double elimi-nation results in formation of β-carotene (11).

Other processes for the synthesis of β-carotene are based on the linking of two identi-cal C20 components. Both the reductive dimer-ization of retinal in a manner analogous tothe McMurray reaction [160] with lithium alu-

minum hydride and titanium trichloride, as wellas a modified Wittig condensation with ox-idative coupling of retinyltriphenylphosphoni-um hydrogensulfate (93, X =HSO−

4 ) [161] withpotassium carbonate and hydrogen peroxide inaqueous solution yield β-carotene (11).

The coupling of the C20 sulfone 99 with reti-nal (2) and the reaction of two molecules of C15sulfone 63with the C10 dialdehyde 92 have beendescribed in the literature as possible methodsfor the synthesis of β-carotene [162].

24 Vitamins

All syntheses of β-carotene initially producemixtures of E,Z isomers. As a result of the goodcrystallization properties of the all-E form [163],these mixtures can be almost quantitatively iso-merized to the all-E formunder isomerizing con-ditions [164].

Synthesis of the C30 Skeleton of Apoc-arotenoids. The syntheses of β-apo-8′-carotenoids (14), (14a) follow the pathsC19 +C6 +C2 +C3, C15 +C10 +C5, andC20 +C10 [165].C19 +C6 +C2 +C3. Startingwith theC14 al-

dehyde 50, the C19 aldehyde 90 is made viaenol ether condensation, followed by reactionwith 2-methylpent-2-en-4-yne-1-al diethyl ac-etal (100) in a Grignard reaction. After acetalcleavage and dehydration, 15-dehydro-β-apo-12′-carotinal (101) is accessible. Subsequent ac-etal formation, enol ether condensation [166],and hydrolysis yields 15-dehydro-β-apo-10′-carotinal (102). Further enol ether condensa-tion or Wittig reaction with the correspondingC3 building blocks and subsequent partial hy-drogenation allows the synthesis of 14 and 14a[167].C15 +C10 +C5. Wittig condensation of the

C15 phosphonium salt 56 with the C10 dial-dehyde 92 first produces β-apo-12′-carotinal(103), which can be condensed to 14 or14a byfurther treatment with C5 alkylidenephosphanes104, 105 [168].C20 +C10. Wittig condensation of cyclic

C20 components with open-chain C10 build-ing blocks allows the reaction of both retinal(2) with the corresponding C10 alkylidenephos-phane (106 with Y=OR or O) [169] as wellas retinyl-(triphenyl)phosphonium salt (93) withC10 aldehydes (107 with X=H or OR) [170](Scheme 9). As with β-carotene, isolation of the

all-E isomer exploits its good crystallizability,which allows almost quantitative isolation un-der isomerization conditions [171].

2.8. Metabolism and PhysiologicalFunctions

2.8.1. Preformed Vitamin A

About 90% of the vitaminA stored in the liveris in the form of retinyl palmitate. Free retinol

Vitamins 25

Scheme 9.

is esterified here. The liver has a high storagecapacity for retinyl palmitate.

Mobilization from the liver occurs as re-quired. Retinyl palmitate is enzymatically hy-drolyzed and the resulting retinol is released intothe blood plasma by means of a specific retinolbinding protein (RBP) and transported to the tar-get cell [172].

At the receptor of the target cell, retinol ispassed on to a cellular RBP (CRBP). As re-quired, retinol can now be used as such, metab-olized to retinoic acid, or reesterified to retinylpalmitate, which can be stored in the cell for ashort time and used to bridge a temporary deficit.

VitaminA plays a number of roles in the or-ganism, the most important being:

1) Growth, development, and differentiation ofepithelial tissue. In these processes, retinoicacid is the active compound. Retinol only re-presents the precursor of retinoic acid. Themechanisms of action in this area are ex-tremely complex and not yet fully elucidated.

2) Reproduction. VitaminA is essential for thereproduction of higher animals. In men andmale animals, it is required in the form ofretinoic acid for testosterone synthesis, andas retinol for themaintenance of spermatoge-nesis [173]. In women and female animals,retinol is substantially involved in the healthydevelopment of the placenta and the fetus.

3) Vision. In the retina, retinol (1) is dehydro-genated and isomerized to (11Z )-retinal (19)which condenses with the lysine residue ofthe protein opsin to give the aldimine, form-ing the light receptor rhodopsin. The ac-tion of light causes conversion of the chro-mophore from the 11Z to the all-E form

(bathorhodopsin, lumirhodopsin). After achange in the conformation of the proteinpart (metarhodopsin), retinal (2) is releasedhydrolytically. This affects ion transport andmembrane potentials, resulting in the genera-tion of an optic nerve impulse. VitaminA de-ficiency causes night blindness (nyctalopia).

In the purple membrane of Halobacteriumhalobium, (13Z )-retinal is bound to a proteinas a Schiff’s base (bacteriorhodopsin) [174]. Onillumination, light energy can be converted tochemical energy (“proton pump”, ATP synthe-sis) with the isomerization of the chromophoreto the all-E form. Furthermore, vitaminA playsa role in the important metabolic step of glyco-protein synthesis [175] and is involved in main-tenance of the immune system [176,177].

2.8.2. β-Carotene

β-Carotene is resorbed from plant food in thesmall intestine to an extent of 30 – 60%, depend-ing on the added fat content [178]. In intesti-nal cells, part of the β-carotene is homolyticallycleaved to retinal by the enzyme 15,15′-carotenedioxygenase. Retinal is, in turn, reversibly re-duced to retinol by retinal reductase and, to asmaller extent, irreversibly oxidized to retinoicacid [179].

Apart from homolytic cleavage, heterolyticcleavage of β-carotene also occurs, produc-ing various apo-carotinals. These substances arealso converted to retinal, depending on the chainlength.

That part of β-carotene that is not metabo-lized in the intestinalmucosa is transported to thetarget organs in lipoproteins, especially LDL,

26 Vitamins

and in erythrocytes and leukocytes. Here, furtherconversion occurs to retinol and retinoic acid viaretinal or apo-carotinals. From the standpoint ofthe physiology of nutrition, it is assumed thatthe conversion of β-carotene to vitaminA is ca.15% [180]. The conversion decreases with in-creasing amount of β-carotene absorbed [181]and depends on the individual vitaminA sta-tus [182]. The activity of 15,15′-carotene dioxy-genase appears to decrease with an increasingsupply of vitaminA. Consequently, it is con-trolled by the supply of vitaminA. For this rea-son, hypervitaminosis A cannot result from β-carotene.

The β-carotene that is not metabolized to vi-taminA can be resorbed unchanged by humansand has its own biological functions, indepen-dent of its provitaminA properties.

Like the other carotenoids, β-carotene is re-garded as a natural physiological antioxidant. Asa result of its ability to deactivate singlet oxygen[181] and to interrupt oxidative radical chain re-actions (e.g., lipid peroxidations in cell mem-branes) [182], β-carotene has a protective func-tion in cells and, therefore, has a preventive ef-fect on a series of radical-induced degenerativediseases.

The following applications are known or arebeing discussed:

1) Protection of the skin from light and treat-ment of solar dermatosis and pigmentanomalies [183,184]

2) Prevention of various types of cancer, espe-cially lung cancer [185]

3) Prevention of atherosclerosis [186]4) Boosting of the immune functions [187]5) Prevention of certain eye diseases such as

cataract and macula degeneration [187]

2.9. Deficiency Symptoms andRequirements

Deficiency Symptoms. VitaminA defi-ciency causes numerous symptoms such as dis-turbances of vision (night blindness), damage tothe skin and mucous membranes, atrophies, andgrowth retardation.

Even if the exogenous supply of vitaminA isadequate, deficiency symptoms can arise due to

a deficit of carotene 15,15′-dioxygenase and car-rier protein [188], caused by insufficient proteinsupply (hypovitaminosis A syndrome in under-nourished persons).

Requirements. In modern animal hus-bandry, green fodder is being increasingly re-placed by ready-to-use feed. Since this industri-ally produced feed contains little provitaminA,it is enriched with vitaminA (especially retinylacetate).

The vitaminA requirement for the mainte-nance and performance (growth, eggs, milk) ofanimals is largely dependent on the body weightand is in the range of 50 I.U. per kilogram ofbody weight per day. For optimal utilization offodder (fattening) and optimal milking or layingcapacity, daily doses of 100 – 200 I.U. per kg ofbody weight are recommended for working an-imals. Independent of its importance as provita-minA, β-carotene has a specific influence on thefertility of cattle (progesterone synthesis) andother animals [189].

German recommendations for the vitaminAsupply of working and domestic animals [in I.U.retinol per kg of dry feed or per animal (∗) perday] are as follows:

Piglets 10 000 – 16 000Porkers 5 000 – 10 000Milk cows∗ 80 000 – 120 000Horses∗ 30 000 – 60 000Chicks, feedercocks 8 000 – 12 000Layers, turkeys 8 000 – 12 000Dogs 8 000 – 12 000Cats 15 000 – 25 000Fish 8 000 – 12 000Beef cows∗ 40 000 – 70 000

For humans, the daily doses recommendedby the German Society for Nutrition (DGE) areshown in Table 10.

2.10. Analysis and Standardization

Vitamin A. An unequivocal characterizationof vitaminA can be achieved by its UV spec-tra, by color reactions, and from its biologi-cal activity. For purity testing, chromatographicmethods (SFC, HPLC) have become the meth-ods of choice and are essential for quantificationand identification. Spectrophotometric determi-nation is meaningful only at relatively high vita-minA concentrations [190] because even small

Vitamins 27

amounts of interfering impurities result in con-siderable errors. In this method, the UV absorp-tion is measured and has a maximum between325 and 328 nm depending on the solvent.

In colorimetric determination, the intensity ofthe blue color ismeasured at 620 nm after the ad-dition of antimony(III) or antimony(V) chloridein trichloromethane (Carr – Price reaction). Cis,trans isomers give equally intense colors. Othercharacteristic color reactions with vitaminA areexhibited by concentrated sulfuric acid, tin chlo-ride, arsenic trichloride, and boron trifluoride.The mechanism of these reactions has been de-scribed [191].

Although the colorimetric method is moresensitive than the spectrophotometric method, itis somewhat less accurate since the colors faderelatively quickly. Exact determination of vita-minA is possible byfluorometry, a very sensitiveand specific detection method [192,193]. How-ever, carotenoids and tocopherol [194] interferewith the analysis. The fluorimetric test is moreeffective in the presence of holo-retinol bindingprotein (holo-RBP) [195] because retinol boundto apo-RBP (i.e., holo-RBP) fluoresces 14 timesmore intensively than free retinol dissolved ina hydrocarbon. Holo-RBP can easily be sepa-rated electrophoretically in polyacrylamide geland purified. Fluorimetric determination can becarried out directly on the electrogram.

Table 10. VitaminA requirement of humans in retinol equivalents

Age Retinol, mg RE∗m m/f f

0 – 4 months 0.54 – 12 months 0.61 – 4 years 0.64 – 7 years 0.77 – 10 years 0.810 – 13 years 0.9 0.913 – 15 years 1.1 1.015 – 19 years 1.1 0.919 – 25 years 1.0 0.825 – 51 years 1.0 0.851 – 65 years 1.0 0.8> 65 years 1.0 0.8Pregnant women 1.1Nursing women 1.8

∗ 1mg Retinol Equivalent = 6mgall-trans-β-carotene = 12mg other provitaminA carotenoids.mg Retinol Equivalent is the official unit of vitaminA activity.1mg RE corresponds to 3333 I.U.

Biological methods [196] are based on thecurative and prophylactic growth test in the rat,

the colpokeratosis test in the rat and mouse, theantiestrus test, the liver storage test in the rat,and the curative xerophthalmia test. These testsare laborious and are mainly used to check theaccuracy of new methods for the determinationof active substances and to determine the totalconcentration of active vitaminA compounds.

Today, the separation of retinoids of the samepolarity (e.g., cis, trans isomers) is exclusivelyconducted with high-pressure liquid chromato-graphy (HPLC). Detection systems range fromsimple UV detection to electrochemical pro-cesses [197] and laser photoionization [198,199].

Provitamin A. For the determination ofprovitaminsA, spectrophotometric measure-ment of the absorptionmaximum is themost im-portant method. If necessary, the substances arefirst purifiedonbasic or neutral alumina, calciumoxide, magnesium oxide, or silica gel. Separa-tion of structural isomers (cis, trans) is achievedby using HPLC [200].

2.11. Trade Names and EconomicAspects

Retinol (1) is sold almost exclusively in the formof the more stable esters (mainly as retinyl ac-etate, also as retinyl palmitate and propionate).About 80% of the production is used in animalfeed, the remainder in the food and pharmaceu-tical sectors.

For practical reasons, various preparationsare made and mostly stabilized with antioxi-dants: solutions in fats and oils (food sector, es-pecially margarine); liquid, water-miscible dis-persions containing emulsifiers (pharmaceuticalsector, animal feed); and, above all, dry powderswhich contain the agent embedded in a matrixof gelatin and/or polysaccharides (especially an-imal feed, and specialities in the food and phar-maceutical sectors).

At present, world production is ca. 2700 t/a.The producers areHoffmann LaRoche (Switzer-land and United States), BASF (Germany) andRhone-Poulenc (France) as well as smaller pro-ducers in India, China, and the CIS. The mar-ket price is 200 – 250DM/kg (calculated on thebasis of 100% active agent) depending on thepreparation.

28 Vitamins

Retinoic acid (for acne) and (13Z )-retinoicacid (for psoriasis) have attained importance indermatology.

The economic importance of the carotenoids,especially of β-carotene, has increased signifi-cantly in the past years and is still increasing.

In the form of oily dispersions, β-caroteneis used to color margarine and other high-fatfoods. As an active agent (see Section 2.8.2.)or as provitaminA, these dispersions are beingincreasingly filled into soft gelatin capsules.

Water-dispersible dry powders are used tocolor beverages, pudding powders, and manyother foods. They are also employed as provi-taminA in vitamin drinks or tablets.

In the animal feed sector, other carotenoids,e.g., β-apo-8′-carotinoic acid ethyl ester or can-thaxanthin, are used as a dry powder to pigmentthe skin and egg yolk of poultry. Carotenoidsidentical to the naturally occurring substancesare being increasingly used to pigment salmonand trout in fish farming.

About 400 t/a of carotenoids are synthesized(calculated as pure compounds). Theprice is bet-ween 800 and 2000DM/kg (calculated on thebasis of 100% purity). Producers are HoffmannLaRoche and BASF.

2.12. Tolerance

Excessive feeding of vitaminAcompounds (sin-gle doses of ca. 2×106 I.U. or repeated admin-istration of 0.2×106 I.U. and more to adults)causes severe damage (hypervitaminosis A).Typical signs in experimental animals are skele-tal fractures and bleeding. The molecular causefor these symptoms is an increase in the mem-brane permeability in lysosomeswith the releaseof proteolytic enzymes (cathepsinD) [201]. Thisis produced by vitaminA that is not bound to acarrier protein.

High doses of provitaminA compounds donot cause hypervitaminosis A.

3. Vitamin D

3.1. Introduction

The group of vitaminsD (calciferols) are com-pounds which play a central role in the mainte-nance (homeostasis) of calcium and phosphorus

concentration in the body fluids of vertebrates,including humans, by controlling intestinal cal-cium absorption and calcium mobilization ofthe bones. According to present knowledge, thevitaminsD are biologically inert, and beforethey can fulfil their physiological role they mustbe converted by two subsequent enzymatic hy-droxylations in the liver and the kidney to thebiologically active vitaminD derivative.

A selection of reviews and books is givenin general references [202–210]. An excellentoverview of major developments in particularvitaminD research frontiers is offered by theProceedings of VitaminD Workshops which areorganized every three years [211–213].

3.2. History

Rickets, a disease of sucklings and children, isa serious disorder in bone development due to adisturbed calcium and phosphorus metabolismas a consequence of vitaminD deficiency. Rick-ets became a serious health problem with indus-trialization in geographical areas of low-incidentsunlight in northern Europe and North Amer-ica, as well as with the accompanying advancesin urbanization of society. A relation betweenexposure to sunlight and its preventive actiontowards rickets was already recognized in theearly 1800s (Sniadecki). In 1919 Melanbysucceeded in producing rickets experimentallyin dogs and cured the disease by adding codliver oil, a known popular remedy for the pre-vention of rickets, to their food. Shortly af-terwards, Huldshinski demonstrated that ex-posure of rachitic children to sunlight or ar-tificial UV light also cures the disease. Foodand vegetable oils exposed to UV light exhib-ited the same therapeutic effect as cod liver oil.The component of the vegetable oil activatedby UV light was identified as a chromophorictrace impurity accompanying the plant sterolsin the nonsaponifiable fraction and inseparablefrom the parent sterol even by repeated crystal-lization. Analogous impurity was also detectedin the crude animal sterol cholesterol. The ab-sorption spectra of the impurities mentioned, re-sembled that of ergosterol, an already known

Vitamins 29

∆5,7-sterol isolated from yeast. Indeed, ergos-terol when irradiated yielded a mixture of prod-ucts possessing high antirachitic activity, fromwhich two independent teams (Webster,Win-daus) succeeded, in the early 1930s, in isolat-ing the active principle: first in impure (vita-minD1 or calciferol) and later in pure crystallineform, named it vitaminD2 (ergocalciferol), andelucidated its structure by means of classi-cal chemical degradation reactions (Windaus,Heilbron). VitaminD3 (cholecalciferol), theactive principle isolated from cod liver oil(Brockmann) was prepared by irradiation of 7-dehydrocholesterol (cholesta-5,7-diene-3β-ol),a synthetic analogue of ergosterol (and accom-panying impurity of crude cholesterol), and itsstructure (Windaus, Heilbron) was verifiedby total synthesis (Inhoffen). Further vita-minsD, products of irradiation of 7-dehydroderivatives of 22,23-dihydroergosterol (D4), β-sitosterol (D5), stigmasterol (D6) and campes-terol (D7), were reported, the three last namedpossessing only low activity. The complex pho-tochemistry was elucidated by Havinga andVelluz who recognized previtaminD as thekey product of photolysis of steroidal 5,7-dienes(provitaminsD) and as the intermediate of allsubsequent transformations.Havinga correctedfurther the erroneous assumption of Windaus,who took vitaminD for the end product of thephotolytic sequence, and identified it as a prod-uct of a thermal isomerization.

The years 1966 – 1971 brought a dramatic re-vival to the field of vitaminD due to the pioneerinvestigations of the three teams of DeLuca,Kodicek, and Norman. By using synthetic, la-belled vitaminD3 the named authors proved thatin warmblooded animals it is converted by step-wise enzymatic hydroxylations and that one ofthe resultingmetabolites, calcitriol (1α,25-dihy-droxycholecalciferol) is the true highly activeprinciple which performs all the functions as-cribed previously to vitaminD3. Calcitriol andother metabolites were isolated by means of ad-vanced experimental techniques, and their struc-ture was elucidated from their chemical andspectrometric properties and verified by synthe-sis. As under normal physiological conditions,humans and animals produce sufficient amountsof vitaminD3 in the skin, the latter should not,strictly speaking, be classified as a vitamin, incontrast to vitaminD2 which is available only

from the diet. (A vitamin is a dietary componentessential for health which the body is not ableto synthesize.) However, the use of the designa-tion vitaminD has been preserved as a generalterm for compounds exhibiting qualitatively thebiological activity of calciferols and for termssuch as vitaminD activity, vitaminD deficiency,etc. The name vitaminD3 or vitaminD2 is stillused as a synonymfor cholecalciferol or ergocal-ciferol, respectively. The active metabolite cal-citriol, in view of its synthesis in the organismand its function, must, per definitionem, be clas-sified as a hormone, and its precursor vitaminD3as a prohormone.

3.3. Structure and Nomenclature

ThevitaminsDbelong chemically to the steroids[214] in which the B-ring of the steroid nucleusis cleaved between carbon atoms 9 and 10 (9,10-secosteroids). X-Ray analysis and NMR data[215–217] indicate that vitaminD2 and D3 andtheir derivatives exist in the crystal and predom-inantly in solution not as the cisoid, steroid-likeform (1a, 1b) – though according to computercalculations, this is the lowest energy confor-mation [218] – but as the coplanar conformer 2(Fig. 1). (In 2 the A-ring is rotated through ca.180◦ around the C6 –C7 linkage, giving a tran-soid diene system extending from C5 to C8.) Inaddition, 2 is an approximately equimolar mix-ture of two A-ring chair conformations [219,220].

To avoid a certain confusion in the steroidnomenclature arising with the transoid con-formation 2 for which the descriptors α andβ do not apply, IUPAC [221] has recom-mended designating the chiral centres in theA-ring consequently as R and S. Neverthe-less, systematic steroid nomenclature, as wellas that of Chemical Abstracts, still use thetraditional steroid notation derived from thecisoid form 1. Thus vitaminD3 (cholecalcif-erol, calciol) in IUPAC notation is (3S,5Z ,7E )-9, 10-seco-5,7,10(19)-cholestatriene-3-ol (2a),and in systematic nomenclature (5Z ,7E )-9,10-secocholesta-5,7,10(19)-triene-3β-ol. Anal-ogously, vitaminD2 (ergocalciferol, ercal-ciol) (2b), which differs from vitaminD3in the side chain, is (3S,5Z ,7E,22E )-9,10-seco-5,7,10(19),22-ergostatetraene-3-ol or

30 Vitamins

Figure 1. Structure of vitamin D2 and D3 and derivatives

(5Z ,7E,22E )-9,10-secoergosta-5,7,10(19),22-tetraene-3β-ol. The abbreviated form of re-presentation of the hydroxylated vitaminDmetabolites such as 25-OH-D3 for 25-hy-droxycholecalciferol (3a) or 1,25-(OH)2-D3for calcitriol (4a), though discouraged [221], isgenerally used throughout the literature. Sub-script ciphers attached to trivial names (e.g.,tachysterol3 or lumisterol3) correspond to thatof the particular vitaminD.

3.4. Chemical and Physical Properties

The vitaminsD, their metabolites, and ana-logues containing the characteristic 5,7,10(19)-triene system are highly unstable towards light,atmospheric oxygen, and acids; they are sta-ble towards alkalies if protected from oxy-gen and light. Even in the crystalline state thevitaminsD contain a certain amount of iso-meric previtaminD with which they are ina temperature-dependent equilibrium. Prepara-tions should therefore be kept protected fromlight and at low temperatures (+4◦C or less)under inert gas. Heating to 180 ◦C leads toirreversible B-ring closure yielding pyro- andisopyrocalciferol (9α, 10α- or 9β,10β-cholesta-5,7-diene-3β-ol, respectively). Contrary to theprovitamins (7-dehydrosterols), the vitaminsDfail to give a precipitate with digitonin in etha-nolic solution.

Vitamin D3 (cholecalciferol, calciol)[67-97-0] (2a), C27H44O, Mr 384.6, color-less crystals, mp 84 – 85 ◦C (acetone), [α]D+84 ◦ (acetone), UV (ethanol): λmax 265 nm(ε 18 200) [222], NMR: [223], MS: [224], 13CNMR: [223,225].

Vitamin D2 (ergocalciferol, ercalciol)[50-14-6] (2b), C28H44O, Mr 396.6, color-less prisms, mp 114.5 – 117 ◦C (acetone), [α]D+102.5◦ (ethanol); UV (ethanol): λmax 265 nm(ε 19 200) [222], NMR: [226], MS [227].

Previtamin D3 [(6Z )-tacalciol], (3S,5Z ,7E)-9,10-secocholesta-5 (10),6,8-triene-3-ol, (6Z )-9, 10-seco-5 (10),6,8-cholestatriene-3β-ol[1173-13-3] (12a), C27H44O, Mr 384.6, oily,UV (ethanol): λmax 262 nm (ε 9000).

25-Hydroxycholecalciferol monohy-drate (calcidiol, calcifediol), (3S,5Z ,7E )-9,10-secocholesta-5,7,10(19)-triene-3,25-diol,(5Z ,7E )-9,10-seco-5,7,10(19)-cholestatriene-3β,25-diol [19356-17-3] (3a), C27H44O2 ·H2O,Mr 418.6, mp 81 – 83 ◦C (aq. acetone), [α]D+77◦ (trichloromethane), UV (ethanol): λmax265 nm (ε 18 000), NMR: [228,229],MS: [229],13C NMR: [225].

1α,25-Dihydroxycholecalciferol (cal-citriol), (1S,3R,5Z ,7E )-9,10-secocholesta-5,7,10(19)-triene-1,3,25-triol, (5Z ,7E )-9,10-seco-5,7,10(19)-cholestatriene-1α,3β,25-triol

Vitamins 31

[32222-06-3] (4a), C27H44O3,Mr 416.6, whitesolid, mp 115 – 116 ◦C (methyl formate) [230],[α]D +45◦ (ethanol), UV (ethanol): λmax265 nm (ε 16 600), IR: [231], NMR: [232], MS:[232], 13C NMR: [225].

24R,25-Dihydroxycholecalciferol (secal-ciferol), (3S,24R,5Z ,7E)-9,10-secocholesta-5,7,10(19)-triene-3β,24,25-triol, (24R,5Z ,7E )-9,10-secocholesta-5,7,10(19)-triene-3β-24,25-triol [55721-11-4] (5a), C27H44O3, Mr 416.6,white crystals, mp 136 – 137 ◦C (methyl for-mate), [α]D +113◦ (ethanol), UV (ethanol):λmax 265 nm, NMR: [233], MS: [234].

1α-Hydroxycholecalciferol (alfacalcidiol)(1S, 3R,5Z ,7E )-9,10-secocholesta-5,7,10(19)-triene-1,3-diol, (5Z ,7E )-9,10-seco-5,7,10(19)-cholestatriene-1α,3β-diol [41294-56-8] (6a),C27H44O2, Mr 400.6, white crystals, mp 138 –139.5 ◦C (methyl formate), [α]D +29◦ (di-oxane), UV (ethanol): λmax 264 nm (ε 17 600),IR: [235], NMR: [235], MS: [235].

Calcipotriol (1S,3R,24S,5Z ,7E,22E )-24-cyclopropyl-9,10-seco-5,7,10(19),22-cholatetraene-1,3,24-triol, (24S,5Z ,7E,22E )-24-cyclopropyl-9,10-seco-5,7,10(19),22-cholatetraene-1α,3β,24-triol [112965-21-6](4c), C27H40O3, Mr 412.6, crystals, mp 166 –168 ◦C (methyl formate), UV (ethanol): λmax264 nm (ε 17 200), NMR: [236], MS: [236].

3.5. Biosynthesis and Occurrence

3.5.1. Biosynthesis

The natural source of vitaminD3 in warm-blooded animals and humans is the transforma-tion of provitaminD3 (7-dehydrocholesterol), aproduct of cholesterol biosynthesis, in the skin[210] when exposed to the ultraviolet compo-nent of sunlight (λ= 290 – 315 nm, UV-B radi-ation). The light penetrates into the epidermisand converts provitaminD3 photochemically toprevitaminD3, and this isomerizes at body tem-perature to vitaminD3. The photolysis is veryefficient [206], though liable to seasonal and cli-matic variations. Pigmentation and aging signif-icantly decrease the capacity of human skin toproduce vitaminD3.

The vitaminD3 produced either endoge-nously or obtained dietarily enters the lymphaticsystem bound to the specific vitaminD bind-ing protein (DBP) and is transferred first tothe liver and hydroxylated there at C25 to thebiologically inactive 25-hydroxycholecalciferol(calcifediol, 25-OH-D3) (3a), the DBP-bound,major circulating form of vitaminD3, and thentransferred to the kidney, the site of furtherhydroxylation. Renal hydroxylation is strictlycontrolled by serum calcium level: a decreasebelow the physiological concentration (86 –105µg/mL) stimulates production of parathy-roid hormone (parathormone, PTH) [237] in theparathyroid gland, which activates renal 25-OH-D3-1α-hydroxylase. The enzyme converts 25-OH-D3 into the 1α-hydroxylated highly activehormone calcitriol (4a), which carries out all theknown functions ascribed to the vitaminD3. Theresulting normalization of calcium in serum sup-presses PTH production, and calcitriol deacti-vates the 1α-hydroxylase by feed-back control.The deactivation is associated with the activa-tion of renal 25-OH-D3-24-hydroxylase, whichstarts to convert 25-OH-D3 into 24R,25-dihy-droxycholecalciferol [24R,25-(OH)2-D3] (5a).The physiological role of this metabolite, ifany, is still the subject of controversial reports.Both metabolites are subjected to a complexcatabolic process which proceeds via additionalhydroxylations up to the final cleavage of theside chain [203,206,238]. Should calcium con-centration rise above normal, the thyroid glandstarts to secrete the hormone calcitonin (CT)[239], a counterregulator of PTH, which blocksmobilization of calcium from the bone and stim-ulates excretion of calcium in the kidney. Vi-taminD2 (2b), the source of which is the diet,is metabolized in mammals and humans anal-ogously to vitaminD3, to give 25-OH-D2 (3b)and 1α, 25-(OH)2-D2 (4b).

3.5.2. Occurrence

Fish oils contain considerable amounts of vita-minD3, accompanied by small amounts of vi-taminD2. Lesser concentrations of vitaminD3are found in egg yolk, mammalian and humanbreast milk (as sulfate), invertebrates, andmush-rooms.Natural sources of vitaminD are summa-rized in [240]. Generally, vegetables and fruits

32 Vitamins

do not contain vitaminD. The glycoside of cal-citriol was detected in the leaves of calcinogenicplants, e.g., Solanum malacoxylon [241]. Thecutaneous photosynthesis of vitaminD3 in fishis questionable because of the low transmittanceofwater forUV light. Themode of accumulationof large hepatic stores of vitaminD3, the phys-iological significance of which is poorly under-stood, is most probably a result of concentra-tion through food chains starting from zoo- andphytoplankton. Hypothetical, nonphotochemi-cal enzymic mechanisms of vitaminD3 synthe-sis in fish and some terrestrial subterranean ornocturnal herbivores have been discussed [242].

3.6. Vitamin D Requirements

The daily vitaminD requirements in humanadults probably do not exceed 100 – 200 I.U.(1mg= 40 000 I.U.); this can readily be obtainedfrom the dietary sources (eggs, cheese, liver, oilyfish) and exposure to sunlight. For children upto the age of six, the recommended allowanceis 400 I.U./d; this dose may represent the up-per limit of vitaminD required [203,243]. In-take of vitaminD in excess of 1000 I.U. per dayis potentially dangerous to normal children andadults and must be avoided. VitaminD intoxica-tion is believed to be the result of high circulating25-OH-D3 levels that, mimicking 1,25-(OH)2-D3 on the receptor, lead to dangerous hypercal-cemia, which may result in irreversible soft tis-sue calcifications (kidney, lungs, heart, pancreas,aorta). Food is supplemented in many countriesby vitaminD3 or D2 (milk, margarine); the dailyportion of a certain food contains usually onethird or one half of the total vitamin requirementand the vitamin amount in a definite portion offood is declared on the package. The vitaminizedfoodproducts are subject to control by state insti-tutions. In veterinary nutrition, food fortificationwith vitaminD is important for the normal de-velopment of young animals and for husbandryof cattle, swine, and poultry. The vitaminD re-quirements are given in I.U. either per animal orkilogram body weight and day or per kilogramdry foodper day [244,245]. It has, however, to beemphasized that the data related to requirementlevels depend on a number of different factorsand should be considered as guideline recom-

mendations only and that generally valid dataare not obtainable.

3.7. Pharmacological Effects and Uses

Calcitriol is a hormone which functions at thelevel of the target cell nucleus in a fashion anal-ogous to that of steroid hormones. The actions ofcalcitriol aremediated by a specific 1,25-(OH)2-D3 receptor molecule which belongs to the su-perfamily of protein receptors for steroid hor-mones [246,247]. The calcitriol – receptor com-plex is transported to the nucleus and stimulatesthe transcription of the gene coding for proteinsthat modify the functions of the target cell. Cal-citriol exerts its main task, the control of calciummetabolism [248], on the classic target tissues,i.e., intenstine, bone, and kidney. As, however,specific 1,25-OH-D3 receptor molecules havebeen identified in a series of other tissues aswell,this indicates far broader calcitriol activities thanformerly assumed. Some biological responsesof calcitriol are generated by nongenomic path-ways and two different forms of the receptorswere postulated for genomic and nongenomicmechanisms [249]. The steroid-like cisoid con-formation 1 is suggested to be responsible forthe nongenomic function [249].

3.7.1. Calcium Homeostasis and Disordersof Calcium Metabolism

The most obvious effect of calcitriol is normalbone development by maintenance of plasmacalcium and phosphorus concentrations at nor-mal levels. This mechanism is carried out byactivation of intestinal calcium and phosphorusabsorption, improvement of renal calcium reab-sorption, and stimulation of calcium mobiliza-tion from the bone. The most important indi-cation in human medicine in this field is theprevention and healing of vitaminD deficiencyin growing children (rickets) and in adults (os-teomalacia). Patients suffering from renal fail-ure and anephric patients on dialysis produceeither insufficient amounts of calcitriol or failto produce it at all, and as a result exhibit lowintestinal calcium absorption, low plasma cal-cium concentration (hypocalcemia), and sec-ondary hyperparathyroidism (overproduction of

Vitamins 33

PTH), the latter being the major factor in the de-velopment of renal osteodystrophy. Oral dosesof calcitriol or its synthetic analogue 1α-hy-droxycholecalciferol (6a) – 1α-OH-D3, beforeit acts, is hydroxylated at C25 in the liver –normalize the hypocalcemia and the PTH con-centration in the serum. The therapy requiresmedical surveillance and thorough control ofserum calcium concentrations. Postmenopausaland age-related (senile) osteoporosis, the mostfrequent metabolic bone disease seen in medi-cal practice, is a failure of calcium absorptionwhich results in a shift from dietary sources toskeletal calciummobilization accompanied by acontinuous loss of bone substance. Clinical trialsusing calcitriol for treatment of postmenopausalwomen have been reported [250,251] but theapplication of calcitriol for treatment of senileosteoporosis is still the subject of controversy.Calcitriol has been reported to support increasedegg production and egg shell breaking strength,and recommended for prevention and treatmentof parturient paresis (milk fever), a hypocal-cemic disorder of dairy cows. Substantial eco-nomic losses in farming have been caused by se-vere calcinoses of grazing animals after intake ofleaves of calcinogenic plants containg calcitriolin glycosidic form [241]. VitaminD2 and 1α,25-(OH)2-D2 are approximately equally as activein humans and mammals as the correspondingvitaminD3 derivatives but are substantially lessactive in avian species; the discrimination is dueto the vitaminD transport protein, which has lowaffinity towards vitaminD2 derivatives [252].

3.7.2. Further Activities

Nuclear 1,25-(OH)2-D3 receptors have beendemonstrated in tissues not primarily related tomineral homeostasis, such as the skin, parathy-roid cells, pancreas, heart muscle, placenta, andothers. Further, a substantial fraction of all ma-lignant cells contain significant amounts of thereceptor. Calcitriol has been shown to influencethe proliferation and differentiation of severaltissues and may also have immunoregulatoryproperties [253]. Calcitriol-receptor complexsuppresses the gene of the PTH in the parathy-roid gland [254], stimulates insulin secretionin the pancreas [255,256], inhibits leukemiccell growth and is an effective antiproliferative

agent with potential use in cancer therapy [257].Similar effects have been demonstrated on hu-man epidermal cells, where calcitriol inhibitsproliferation and induces terminal differentia-tion of keratinocytes; this has been used fortreatment of psoriasis (hyperproliferation epi-dermal disorder). The antiproliferative and dif-ferentiation inducing effects of calcitriol aredose dependent and require supraphysiologicdoses. The obvious clinical limitation, the poten-tially serious hypercalcemic side effect of doseshigher than a few micrograms per day, ques-tions a long-term therapeutic use and calls foranalogues having diminished calcemic activity.Analogues have been synthesized that possesshigh proliferation/differentiation activity com-bined with a significantly lower calcemic ef-fect [236,258] and the structure – function re-lationship has been discussed [259]. Two syn-thetic derivatives, calcipotriol (4c) and tacalci-tol [(5Z ,7E,24R)-9,10-secocholesta-5,7,10(19)-triene-1α,3β,24-triol], have been introduced tothe market for topical treatment of psoriasis.

3.8. Synthesis

3.8.1. Synthesis of Vitamin D3 and D2

On an industrial scale, the vitaminsD3 aremanufactured by chemical synthesis only. Thefish oils, the natural source of vitamin D3,are only used for preparation of vitamin con-centrates. Starting materials for vitaminsDare 7-dehydrosterols (∆5,7-sterols). VitaminD2(2b) is prepared from the natural substanceergosterol (11b); its synthetic analogue, 7-dehydrocholesterol (11a) is the starting materialfor vitaminD3 (2a).

The synthesis of 7-dehydrocholesterol(Fig. 2) starts from commercially available cho-lesterol (7a), the hydroxy group of which isprotected as acetate or benzoate (8a). Allylicbromination to the 7-bromo derivative (9a) anddehydrobromination to the ∆5,7 ester (10a)followed by saponification gives the free 7-dehydrocholesterol (11a).

The photolysis of the 5,7-diene system ofeither 7-dehydrocholesterol (11a) or ergosterol(11b) (for details see → Photochemistry,Chap. 5.2.) is carried out by irradiation with ul-traviolet light of a mercury lamp in an inert sol-

34 Vitamins

Figure 2. Synthesis of vitaminD3 and D2Ac=CH3CO, Bz =C6H5COa, b: R1 see Figure 1

vent [260] yielding previtaminD ((12a), (12b))by reversible fission of the cyclohexadiene sys-tem in theB-ringbetweenC9 andC10. Theprevi-taminD is in an equilibrium with its ring reclo-sure photoproducts – the starting diene ((11a),

(11b)) and its 9β,10α-isomer lumisterol ((13a),(13b)) – and with the product of E/Z photoi-somerization of the 6,7 double bond, tachys-terol ((14a), (14b)). The ratio of products andthe predominating component in the reaction

Vitamins 35

mixture depends on the wavelength of the UVlight used [261]. To avoid the slow, irreversibleformation of over-irradiation products, photol-ysis is discontinued after max. 40% turnoverof the starting diene. The subsequent step, thethermal isomerization of previtaminD into vita-minD (12a, 12b → 2a, 2b), is a reversible sig-matropic 1,7-hydrogen shift from C19 to C9,performed by heating the solution of the photo-products at 80 ◦C to a vitamin – previtamin equi-librium (ratio ca. 4 : 1). The reaction solutionis then concentrated and treated with methanolto recover the sparingly soluble starting ∆5,7-diene. Tachysterol is removed as a Diels –Alderadduct with maleic anhydride. The resulting so-lution is evaporated to give crude vitaminD (vi-taminD resin) which either is used directly forveterinary purposes or purified to crystalline vi-taminD3 or D2 via an ester that crystallizes well[262] (3,5-dinitrobenzoate or butanoate), subse-quent saponification, and final crystallization.

The introduction of triplet sensitizers that in-fluence the E/Z isomerization of the 6,7 doublebond by reconverting the undesirable side prod-uct tachysterol to previtamin (14 →12), broughtdecisive progress in the photochemical transfor-mation of 7-dehydrosterols and a very efficientoptimization of the previtaminD yield. In prac-tice, the 5,7-diene is irradiated as above, thenthe sensitizer is added and irradiation is contin-ued to transform the tachysterol almost quanti-tatively to previtamin (double irradiation tech-nique) [263].

3.8.2. Synthesis of Calcitriol

The classical pathway startswith the synthesis of25-hydroxycholesterol (15) from an appropriatesteroid derivative by degradation and resynthe-sis of the side-chain [238,264,265]. Themethodof choice for introduction of the 1α-hydroxygroup in the A-ring of 25-hydroxy-cholesterol(15) is the three-step procedure of Barton(15 → 16 → 17 → 20) [266] which in a mod-ified form [267] was applied for the industrialproduction of 1α,25-dihydroxycholesterol (20)(Fig. 3). The chemical introduction of the 1α-hydroxyl group was replaced later by an eco-nomic and for industrial purpose adapted [268]fermentative one-step 1α-hydroxylation [269]using dehydroepiandrosterone (18), a commer-

cially available compound prepared by micro-biological degradation of cheap plant sterols, asstarting material. The synthesis of the side chainin the 1α-androstane derivative 19 gives 1α,25-dihydroxycholesterol (20) [270], followed bythe conversion to calcitriol analogous to the syn-thesis of vitaminD (Fig. 2) using the double-irradiation technique [271]. Calcitriol is isolatedby preparativeHPLC and crystallized. The strat-egy of both the above-mentioned synthetic path-ways is based on multistep manipulations of in-tact steroid ring systems, and the final conver-sion of the resultingly expensive 1α,25-dihy-droxycholesterol to the seco-triene system viathe 5,7-diene is realized by a sequence of reac-tions notorious for their moderate yields. A con-ceptually new alternative is the synthesis start-ing from the commercially available bulk prod-uct ergocalciferol (vitaminD2) (2b) [228]. Thepathway (Fig. 4) involves protection of the trienesystem as sulfur dioxide adduct 21, cleavage andresynthesis of the side chain and chemical in-troduction of the 1α-hydroxy group (23 → 29)[228]. An alternative synthesis of calcitriol [230,272] and calcipotriol [236] is based on a simi-lar strategy. A promising approach, the directfermentative 1α- and 25-hydroxylation of vita-minD3 to calcitriol [273] has been reported. Thetotal synthesis of calcitriol or other vitaminDmetabolites and analogues [264,274] has not yetbeen realized on an industrial scale.

3.9. Assays for Vitamin D andMetabolites

Assays for vitaminD and metabolites have beenreviewed [275]. The official method for deter-mination of vitaminD in feedstuffs, tissues, andbody fluids in the years 1922 – 1958 was therat line test, a bioassay based on curative andpreventive effects in rachitic or normal animals,kept on a rachitogenic diet containing varyingamounts of vitaminD. The measure of healingof the rickets induced was evaluated after silver-nitrate staining of the split tibias. The method issensitive (25µg vitaminD2 or D3) but time con-suming (7 – 14 d assay period) and nonspecific.An alternative to the line test, a subsequentlyadopted chemical method, is based on spectro-metric estimation of the red coloration of vita-minD with antimony trichloride at 500 nm. The

36 Vitamins

Figure 3. Synthesis of 1α,25-dihydroxycholesterolDDQ=2,3-Dichloro-5,6-dicyano-1,4-benzoquinone

Figure 4. Synthesis of calcitriol from ergocalciferolTES=Et3Si, Ts = p-CH3C6H4SO21) SO2 (liquid); 2) Et3SiCl, imidazole, CH2Cl2; 3) O3/NaBH4, 4) TsCl, pyridine; 5) NaHCO3, EtOH; 6) SeO2, N-methyl-morpholine N-oxide, CH2Cl2/MeOH; 7) hν, anthracene; 8) BrMgCH2CH2C(CH3)OTES, Cu(I), THF; 9) Bu4NF, THF

Vitamins 37

method was designed for estimations in phar-maceutical preparations containing larger quan-tities of vitaminD (> 250µg) [276]. Officialmethods for vitaminD estimation are describedin detail in [277].

Given the knowledge of vitaminDmetabolism and the low concentrations ofthe metabolites in the plasma or serum (25-OH-D3: 30 ng/mL, 1,25-(OH)2-D3: 30 – 40 pg/mL,24,25-(OH)2-D3: 0.5 –2.0 ng/mL), two ap-proaches were developed for the measurementof metabolites in plasma and tissues in vitrousing 3H-radiolabeled vitaminD derivatives:competitive protein-binding analysis (CPB) andradioimmunoassay (RIA). The CPB techniquetakes advantage of the high affinity for vitaminDmetabolites of proteins occurring in serum (vi-taminD-binding protein) or in intestinal targetcells (cytoreceptors). For RIA, the vitaminDmetabolites being themselves not antigenic,must be chemically modified to haptens whichare then covalently linked to a protein molecule[mostly bovine serum albumin (BSA)]. The vi-taminD derivative –BSA complex then consti-tutes the antigen which, injected into an animal,induces antibody formation. Both assays involvecompetition between a radiolabeled metaboliteand the vitaminD derivative of interest for avail-able sites on the binding protein. However, thelow specificity of both the binding protein andthe antibody and, furthermore, the presence ofinterfering substances in serum make prepu-rification and separation of the metabolites byextraction and chromatographic techniques in-dispensable. Significant advance in the isolationtechniques has been the introduction of solid-phase extraction and “phase-switching” [278,279]. The introduction of high performanceliquid chromatography (HPLC) rendered sep-aration and isolation of individual metabolitespossible either for the assays mentioned aboveor for the direct measurement of HPLC peakareas using UV detection (for vitaminD and25-OH-D only) or the highly sensitive fluoro-metric assay [280]. A combination of gas chro-matography and mass spectrometry (GC –MS)[279] has also been introduced in this field. Theaccuracy and precision of differentassays forvitaminD and metabolites have been comparedand discussed [281].

3.10. Trade Names and EconomicAspects

The pharmaceutical industry offers vitaminD3,and to a lesser extent vitaminD2, in pure crys-talline form or as a solution in vegetable oil(1 000 000 I.U./g) for preparation of pharmaceu-ticals and for food supplementation. The major-ity of vitaminD3 produced is the crude prod-uct, so-called vitaminD3 resin, an intermediatefor manufacturing dispersions in plant proteinand dextrin matrix (about 500 000 I.U./g) usedfor fortification of animal feeds. VitaminD2 andD3 are available in numerous drug products[282], inmultivitamin specialities, and in fish oilconcentrates. The main bulk producers of vita-minD are Solvay-Duphar (TheNetherlands, D3,D2), Hoffmann-La Roche (Switzerland, D3),BASF (Germany, D3), Synthesia (Czech Re-public, D2). Annual worldwide vitaminD3 pro-duction amounts to ca. 1.5×1015 I.U.; the pro-duction of calcitriol, the daily dose of which is0.25µg only, does not exceed several hundredsof grams. Trade names and the producers of syn-thetic metabolites and analogues [282] are sum-marized in Table 11. Commercially available tri-tiated derivatives for assays are available fromAmersham (UnitedKingdom) andNewEnglandNuclear Corp. (United States).

4. Vitamin E (Tocopherols,Tocotrienols)

4.1. Introduction

The eight naturally occurring substances withvitamin E activity are all derivatives of 6-chromanol. They belong to two groups of com-pounds. The first group (1a–1d) is derived fromtocol (1 with R1 =R2 =R3 =H), a substancewhich has a saturated C16 isoprenoid side chainand has not yet been found in nature.

The second group (2a–2d) consists of deriva-tives of tocotrienol, which has a triply unsatu-rated C16 side chain (at position 3′, 7′, and 11′).The same α, β, γ, and δ structures are foundas in the tocopherol group (1a–1d).

38 Vitamins

Table 11. Trade names and producers of synthetic vitaminD metabolites and analogues

Abbreviation Generic Name Trade Name Producer

25-OH-D3 calcifediol Dedrogyl Roussel-Uclaf (France)Calderol Upjohn (United States)Hidroferol Juventus (Spain)

25-OH-5E-D3 5,6-trans-25-hydroxy-cholecalciferol Delakmin Roussel-Uclaf (France)

1α,25-(OH)2-D3 calcitriol Rocaltrol Hoffmann-La Roche(Switzerland, United States)

Calcijex Abbott (United States)1α-OH-D3 alfacalcidiol Alfarol Chugai (Japan)

Dediol Leo (Denmark)Eenalfadrie Teva (Israel)Einsalpha Thomae (Germany)Odinal Hemofarm (Former Yugoslavia)OneAlpha Leo (Denmark)Vetalpha Crown Chem. (United Kingdom)

calcipotriol Daivonex Leo (Denmark)Dovonex Leo (United Kingdom)

1α,24R-(OH)2-D3 tacalcitol Bonealfa Oint. Teijin (Japan)dihydrotachysterol A.T. 10 Bayer (Germany)

Calcamin Wander (Switzerland)DHT Roxane (United States)Dihydral Duphar (Belgium)Dygratyl Ferrosan (Denmark)Hytakerol Sterling (United States)Tachyrol Duphar (Denmark)Tachystin Ankerwerk (Germany)

Of the above eight compounds, α-tocopherol(1a), 2,5,7,8-tetramethyl-2-(4′,8′,12′-trimeth-yltridecyl)-6-chromanol, has the highest bio-logical vitamin E activity and, therefore, thegreatest industrial and economic importance.It is the predominant tocopherol in human andanimal tissue.

The tocopherols (1) have three chiral centers,at positions 2, 4′, and 8′. Until now, only toco-pherols having the R configuration at all threechiral centers have been found in nature.

The correct denomination for naturally oc-curring α-tocopherol (1a) is (2R,4′R,8′R)-α-tocopherol [59-02-9] or briefly (R,R,R)-α-tocopherol. Totally synthetic α-tocopherol,which is racemic with respect to each stereocen-ter, should be denominated (2RS,4′RS,8′RS )-α-tocopherol [10191-41-0], or briefly (all-rac)-α-tocopherol. The denominations d- and dl-α-tocopherol, which have been used frequently forthe (R,R,R)-enantiomer and the mixture of alleight stereoisomers, respectively, should not beused anymore.

The tocotrienols (2) have the (2R,3′E,7′E)configuration.

Vitamins 39

4.2. History

While studying the connection between fertilityand nutrition, Evans and Bishop discovered in1922 [291] that the absence of a substance inthe diet of female rats led to death of the fetusand its resorption. This substance was first des-ignatedFactor X and later vitamin E [292]. In the1920s and 1930s, it was observed that apart fromthe damage done to the reproductive system inmale and female rats, a diet in which vitamin Ehad been oxidatively destroyed by iron(III) chlo-ride produced encephalomalacia in chickens andmuscular dystrophy in guinea pigs and rabbits[293–295].

In 1936, Evans isolated from wheat-germoil an alcohol which was given the name α-tocopherol (Greek: tokos, childbirth; phero, tobear) [296].

In 1937, two additional substances were iso-lated via crystallized allophanates and namedβ- and γ-tocopherol; they have lower biologicalactivity than α-tocopherol [297]. δ-Tocopherolwas discovered later [298].

In 1938, Fernholz elucidated the structureofα-tocopherol based on the results of pyrolyticand oxidative degradation [299]. In the sameyear, Karrer et al. succeeded in synthesizingα-tocopherol from trimethylhydroquinone andphytyl bromide [300].

The structures ofα-, β-, γ-, and δ-tocotrienolwere elucidated in the 1960s [301]. The first syn-thesis of the tocotrienols was achieved by Isleret al. in 1963 [302]. This was followed in 1976by the synthesis of α-tocotrienol in the natural2R form [303].

4.3. Physical Properties

The melting point of natural α-tocopherollies between 2.5 and 3.5 ◦C and that of γ-tocopherol between −3 and −2 ◦C [304]. Themelting points of various esters, e.g., acetate[305], allophanate [297], p-phenylazobenzoate[306], p-nitrophenylurethane [297], and 2,4-dinitrobenzoate [300], can be used to determinethe purity.

The optical rotation of the natural tocopherolsis very small and solvent dependent. The specificoptical rotations [α]25546 of some stereoisomericα-tocopherols and their derivatives are listed in

Table 12 and those of the natural tocopherols aregiven in the following:

α-Tocopherol +0.32 (ethanol) −3.0 (benzene)β-Tocopherol +2.9 (ethanol) +0.9 (benzene)γ-Tocopherol +2.2 (ethanol) −2.4 (benzene)δ-Tocopherol +3.4 (ethanol) +1.1 (benzene)

Tocopherols are light yellow viscous oils atroom temperature. They are readily soluble inlipophilic solvents and ethanol, but practicallyinsoluble in water.

The UV spectra of the tocopherols and to-cotrienols in ethanol have a maximum at 292 –298 nm (α-tocopherol: 292 nm). Acetylation ofthe phenolic hydroxyl group shifts this max-imum to 276 – 285 nm (α-tocopherol acetate:284 nm) [309].

Nuclear resonance spectroscopy [283,312,313], IR spectroscopy [310,311], and massspectroscopy [314,315] are used to differenti-ate differently substituted tocopherols and to-cotrienols.

4.4. Chemical Properties

The tocopherols (1) are gradually oxidized byatmospheric oxygen, producing a reddish color.In the absence of oxygen, they are resistant toalkali and heat up to 200 ◦C, and can be vacuumdistilled without decomposition.

Tocopherol esters are resistant to atmosphericoxygen at ambient temperature.

Other chemical properties of the tocopherolsare determined by:

1) The free phenolic hydroxyl group, which canbe esterified [316] or etherified

2) A free position in the aromatic ring (exceptin α-tocopherol and α-tocotrienol) which isavailable for hydroxymethylation, chlorome-thylation, nitrosation, diazotization, etc.

3) Ready oxidizability (as the monoether of ahydroquinone) [317]

Tocopherol exerts an antioxidative effect, ter-minating radical chain reactions. This is due tothe ready release of a hydrogen atom with theformation of the relatively inert tocopheroxyradical [318–320]. Tocopherol can be regen-erated from the tocopheroxy radical in vivoby ascorbate or coenzymeQ10 – hydroquinone[319,320]. More recent studies show that α-tocopherol is a much more potent antioxidant

40 Vitamins

Table 12. Specific optical rotation of stereoisomeric α-tocopherols [284,306,308]

Stereoisomer Free α-tocopherol, [α]25D(ethanol)

p-Phenylazobenzoate,[α]25600 (CHCl3)

Prod. of oxidation withK3Fe(CN)6, [α]25D(isooctane)

Acetate, [α]25D(ethanol) [307]

2R,4′R,8′R +0.75◦ +7.07◦ +26◦ +3.3◦

2RS,4′R,8′R +0.38◦ 0◦ 0◦

2S,4′R,8′R +0.19◦ −7.64◦ −25.8◦ −2.1◦

2R,4′RS,8′RS +6.96◦ +25.8◦ +1.9◦

2S,4′RS,8′RS −0.14◦ −7.47◦ −23.6◦

than γ-tocopherol and the other homologueswith a lower degree of methylation [321].

4.5. Metabolism and Importance in theOrganism

Vitamin E is themost important lipid-soluble bi-ological anitoxidant [322]. It is localized in cel-lular and subcellular membranes which are richin polyene fatty acids. Since it reacts with freeradicals faster than polyene fatty acids, it inter-rupts the chain reactionof lipid oxidation.There-fore, vitamin E is essential for themorphologicalintegrity and functionality of the membranes ofall cells [323].

The antioxidative effect in vitro is not iden-tical with the biological activity: γ-tocopherolhas about half the antioxidative effect of α-tocopherol but only about one-tenth of its bio-logical activity [324]. This discrepancy appearsto be due to differences in the lipoprotein-boundtransport of tocopherols in the plasma.

In the course of fat digestion, vitamin E isabsorbed in the small intestine [286,325]. Bileacids and pancreatic lipase are required for ab-sorption. In the case of malabsorption of lipids,the risk of vitamin E deficiency exists. Trans-port from the intestine to the systemic circula-tion proceeds via the chylomicrons of the lymph.Vitamin E is transported in the plasma boundto lipoproteins (mainly low density lipopro-teins, LDL) [324]. The normal plasma level is10µg/mL.

The functions of vitamin Eare revealed by thepathological results of its deficiency (see Sec-tion 4.6) [286]. Epidemiological studies on hu-mans have shown that a high vitamin E intake isassociated with a lower risk of atherosclerosisand coronary heart disease [326]. Prophylacti-cally, vitamin E appears to be of importance incell ageing, in diseases caused by the involve-

ment of free radicals (e.g., ventricle bleedingin premature infants, senile cataract, coronaryheart disease, and other ischemic reperfusiondamage), and in cancer of various organs [327,328]. Vitamin E stimulates cellular and humoralimmunity [329].

Vitamin E is practically nontoxic, excess vi-tamin being eliminated via the bile.

4.6. Deficiency, Requirement, andApplication

Vitamin E deficiency can occur due to an inad-equate supply of the vitamin and lipid malab-sorption. A lack of selenium and a high intakeof polyene fatty acids enhance this deficiency.In animals, vitamin E deficiencies mainly af-fect themuscles, nervous system, cardiovascularsystem, formation of blood, and reproduction:

1) Muscles: The most frequent and economi-cally most important manifestation of vita-min E deficiency in farm animals is degen-eration of skeletal muscles (“white muscledisease,” nutritionalmuscular dystrophy).Asa rule, newborn and young animals (youngchicks, lambs, piglets, calves, foals) are af-fected because their vitamin E supplies arelow. Adequate vitamin E supply to the damprevents this condition, and a combined in-jection of vitamin E and selenium is thera-peutically effective.

2) Nervous system: In young chicks vitamin Edeficiency leads to encephalomalacia, a fataldisease of the cerebellum which is caused byedema and necrosis.

3) Reproduction: In the female rat, vitamin Edeficiency results in fetal death and resorp-tion of the fetus. The classical biotest forthe determination of the activity of vitamin Epreparations is based on this manifestation.In the male, testicular function is disturbed

Vitamins 41

by irreversible degeneration of the germ cells(rat, rabbit, dog, monkey, pig).

4) Cardiovascular system: Piglets develop mul-berry heart disease, an acute and usually fatalcardiomyopathy.

Clinical vitamin E deficiency in humans israre [324]. This disease is usually associatedwith prolonged deficiency and lipid malabsorp-tion. In rare cases, an inborn deficiency ofplasma lipoproteins (abetalipoproteinemia) isthe cause [324]. Neurological disorders such ascerebellar ataxia, a disturbed sense of position,loss of reflexes, and loss of the sensation of vi-bration are the main symptoms. They are causedby degenerative changes in the large motoneu-ron axons and in the myelin sheath [330].

In animal feeding, the following vitamin Eadditions are recommended (per animal andday): dairy cows 200mg, young cattle 40 –70mg, beef cows 60 – 100mg, and horses600mg.

The following additions are recommendedper kilogram of complete feed: laying hens20mg, growing/finishing broilers 40mg,turkeys 45mg, piglets 45mg, growing/finishingpigs 30mg.

The recommended supply of vitamin E in thehuman diet in Germany is listed in Table 13.

Table 13. Recommended levels of vitamin E in the diet inmilligrams tocopherol equivalent∗ per day as stipulated by theDeutsche Gesellschaft fur Ernahrung (German Society forNutrition) 1985

RDI

Infants up to 2 months 3Infants 3 to 11 months 4Children, 1 to 3 years 5Children, 4 to 6 years 7Children, 7 to 9 years 8Children, 10 to 12 years 10Adolescents (15 years up) 12Adults, 19 to 65 years 12Pregnant women 14Nursing mothers 17

∗ 1mg (R,R,R)-α-tocopherol equivalent = 1.1mg(R,R,R)-α-tocopheryl acetate = 1.49mg (all-rac)-α-tocopherylacetate = 2.0mg (R,R,R)-β-tocopherol = 4mg(R,R,R)-γ-tocopherol = 1.49 I.U.

4.7. Analysis and Standardization

Determination. Apart from photometricmethods, chromatographic methods are primar-ily employed for analysis of vitamin E today.

An older method used to determine vita-min E in biological material is the Emmerie –Engel reaction, which is based on the col-orimetric measurement of colored Fe(II) com-plexes with bipyridine [331] or 4,7-diphenyl-1,10-phenanthroline [332]. Spectrofluorometryis a very sensitive analytical method for free andesterified tocopherols. For instance, this methodis used for vitamin E determinations in the spinalcord and in erythrocytes [333]. Photometric de-termination of tocopherol with the 2,2-diphenyl-1-picrylhydrazyl radical is also possible [334].

Of the chromatographic methods, gas chro-matography (GC) and high performance liquidchromatography (HPLC) allow the most accu-rate determinations [335]. HPLC permits the si-multaneous determination of vitamin E homo-logues [336], even directly in plasma and tissuesamples [337].

The measurement of the optical rotation ofK3Fe(CN)6 oxidation products can be used todifferentiate the individual stereoisomers of α-tocopherol (see Table 12). However, only theconfiguration at C-2 can be determined withreasonable reliability. The diastereomers canbe differentiated by gas chromatography [338]and 13C NMR spectroscopy [339]. All eightstereoisomers of (all-rac)-α-tocopherol can beseparated by using a combination of chiralHPLC and GC [340].

Standardization of Biological Activity.The relative biological activity of vitamin Eis expressed in International Units (I.U.),1 I.U. corresponding to the activity of 1mg ofracemic (2RS,4′RS,8′RS)-α-tocopherol acetate[7695-91-2]. The relative biological activitieswere mainly derived from fetus resorption testson rats. They have been established by the Na-tional Formulary as follows [341]:

1mg (2RS,4′RS,8′RS )-α-tocopherol acetate= 1.00 I.U.

1mg (2RS,4′RS,8′RS )-α-tocopherol = 1.10 I.U.1mg (2R,4′R,8′R)-α-tocopherol acetate

= 1.36 I.U.1mg (2R,4′R,8′R)-α-tocopherol = 1.49 I.U.

According to [342], 2R,4′R,8′R-α-tocopherol has a higher biological activity thanthat established by the National Formulary. Asfar as farm animals and humans are concerned,

42 Vitamins

the biological activity of the different forms ofvitamin E is a controversial subject.

The relative biological activity of the eightα-tocopherol stereoisomers, determined by fe-tus resorption tests in rats [343] is as follows:

2R,4′R,8′R 1002R,4′R,8′S 902R,4′S,8′S 732R,4′S,8′R 572S,4′S,8′S 602S,4′R,8′R 312S,4′S,8′R 212S,4′R,8′S 37

The R configuration at C-2 of the chromanering seems to be important for high biologicalactivity.

Biotests. Data on the relative biological ac-tivities of the tocopherol stereoisomers are ob-tained in animal experiments. The biological ac-tivity is determined by the ability of the test sub-stances to prevent or remedy symptoms of vita-min E deficiency in comparison with a standardsubstance. The following test systems (bioas-says) are preferentially used:

1) The fetus resorption test [344] in pregnantrats with vitamin E deficiency is the classicalmethod for the determination of the biologi-cal activity of tocopherols.

2) The erythrocyte hemolysis test in the rat[345] is an in vitro process which measuresthe protective function of vitamin E againstperoxide-induced hemolysis of erythrocytes.This test is also applied to humans [346].

3) In the muscular dystrophy test in the rat, rab-bit, and chicken, muscular damage due tovitamin E deficiency is measured either di-rectly [347] or via the determination of en-zyme activities, e.g., plasma pyruvate kinaseactivity [348] as a measure of the course ofmuscular dystrophy.

4.8. Occurrence

All eight tocopherol (1a–1d) and tocotrienol ho-mologues (2a–2d) occur in nature. Apprecia-ble amounts are contained, above all, in plantoils. The highest concentration by far is foundin wheat germ oil (ca. 0.2%) [349]. Consider-able amounts are also contained in soybean, sun-flower, rape, corn, and peanut oil.

A survey of the total concentration of toco-pherols in foods (in milligrams per 100 g) is pro-vided in the following:

Wheat germ oil 215.4Soybean oil 14.6Sunflower oil 55.8Peanut oil 17.2Olive oil 12.0Cocoa butter 1.0Coconut fat 0.8Pollard 2.4Corn 2.0Wheat 1.4Barley 0.2Oats 1.5Soybeans 1.2Beans (white) 2.1Asparagus 1.5Tomatoes 0.9Carrots 0.6Beans (green) 0.3Garlic 0.1Cauliflower 0.1Walnut 20.0Hazelnut 26.0Peanut 9.0Chestnut 1.2Coconut 1.0

The concentrations of vitamin E in animalfoods are much lower than those in plant prod-ucts. The concentration is subject to major fluc-tuations caused by the seasonal concentrationvariations in green forage plants.

The concentration of various tocopherol andtocotrienol isomers in foods and the effect ofprocessing and storage on the tocopherol con-tent are described in [350].

4.9. Economic Aspects

Animal feeds represent the most important mar-ket for vitamin E. Of the world production, ca.75% is added to industrially produced feed mixfor animals. The main product is a 50% adsor-bate of tocopherol acetate on silica gel. Formu-lations dispersible in cold water also exist.

The pharmaceutical, food, and cosmetic sec-tors account for ca. 25% of world consumption.Special formulations are used here, e.g., sprayformulated dry powders or adsorbates. The com-pound mainly used in the encapsulation of vi-tamin E is 98% (all-rac)-α-tocopherol acetate.(All-rac)-α-tocopherol is also employed to pre-vent oxidation processes in oils and fats.

Vitamins 43

While totally synthetic (all-rac)-α-tocopherol is represented in all areas of ap-plication, the use of semisynthetic (R,R,R)-α-tocopherol from natural sources is almost ex-clusively restricted to the pharmaceutical, food,and cosmetic industries.

The most important producers of (all-rac)-α-tocopherol acetate are Hoffmann-La Roche(Switzerland) which has plants in Switzer-land and the United States, and BASF (Ger-many), with plants in Germany and the UnitedStates. Other producers are Rhone-Poulenc(France) and Eisai (Japan). Vitamin E from nat-ural sources is produced by ADM and Henkelin the United States. Smaller amounts are alsoproduced in Japan.

In 1994, ca. 20 000 t of synthetic vitamin Eand ca. 2000 t of vitamin E from natural sources(calculated as 100% product) were consumedby the world market. Regions of high demandare the United States and Western Europe. Ow-ing to consumer habits, demand in the humansector in the United States is more pronouncedthan in other regions of the world.

4.10. Biosynthesis

Tocopherols are synthesized by higher plantsand algae. The precursor of the aromatic nucleusof the tocopherols is shikimic acid (3) which isconverted to homogentisic acid (4) [351]. Thissynthesis can proceed in plants, algae, and bac-teria, but not in the animal organism.

Starting with homogentisic acid (4), there aretwo pathways for the biogenesis of vitamin E(Scheme 10).

In the “tocopherol pathway,” [352] homogen-tisic acid (4) reacts with phytyl pyrophosphatewith elimination of CO2 to give the interme-diate 6 [353]. The “tocotrienol pathway” startswith the condensation of homogentisic acid (4)with geranylgeranyl pyrophosphate to give theintermediate 5. Enzymatic cyclization of 5 or6 yields δ-tocotrienol or δ-tocopherol, respec-tively. Some of the enzymes involved in thesesynthetic pathways have been isolated [354].

The S-methyl group of S-adenosylmethio-nine methylates δ-tocotrienol to give β-, γ-, andα-tocotrienol, and δ-tocopherol to give the cor-responding tocopherols [355]. In some organ-isms, the second methyl group in the aromatic

ring is introduced before cyclization. The en-zyme responsible for subsequent cyclization toγ-tocopherol has been isolated from algae [356],and the cyclizationmechanism elucidated [357].

The hydrogen source for the conversion oftocotrienols to the corresponding tocopherols isNADPH [358].

4.11. Production

4.11.1. Semisynthetic Vitamin E: Isolationfrom Plant Oils and Methylation

The most important natural sources of vita-min E are plant oils and fats. The deodoriza-tion of edible oils [359] yields as a byproductdeodorizer sludges which contain tocopherolsin economically interesting concentrations (3 –15%) [360]. The deodorizer sludge from alka-line chemical refining of soybean oil, for in-stance, contains ca. 10% of tocopherols, 10 –20% of free sterols and their fatty acid esters,40 – 50% of free fatty acids, triglycerides, andhydrocarbons (e.g., squalene).

Various processes are available for the pu-rification of tocopherols. The fatty acids can beextracted [361] and separated in the formof spar-ingly soluble calcium salts [362]. Alternatively,they can be eliminated by distillation, preferablyafter esterification [363]. The sterols

can be separated by treatment with hydrogenchloride [364] or CaCl2 [365], by crystallization[361,366], or by extraction [367]. Alternatively,the free sterols can be esterified with the excessfatty acids, and the tocopherols can be separatedfrom the high-boiling sterol fatty acid esters bydistillation [368].

After removal of the free fatty acids, the to-copherols can be concentrated by adsorption onbasic ion exchangers.

The amount of α-tocopherol and, conse-quently, the specific vitamin E activity in themixture of tocopherols isolated from soybean oilare low (high proportion of δ- andγ-tocopherol).

To improve the natural mixed tocopherols,methyl groups are substsituted for the aro-matic hydrogens. As a rule, halogen-, nitrogen-,or oxygen-substituted methyl groups are intro-duced and the substituents subsequently elim-inated by catalytic hydrogenation or reduction

44 Vitamins

Scheme 10. Biosynthesis of tocotrienols and tocopherols

with zinc/hydrochloric acid. Hydroxymethyla-tion with formaldehyde under acidic [370], al-kaline [371], or neutral [372] conditions isthe method of choice. Other processes arechloromethylation with formaldehyde and HCl[373] or POCl3 [374]; formylation with zinccyanide/ HCl, chloroform/alkali, or hexameth-ylenetetramine [375]; and Mannich amino-methylation [376].

4.11.2. Totally Synthetic Vitamin E:Industrial Synthesis

The industrial total synthesis of α-tocopherol(1a) is based on the condensation of 2,3,6-tri-

methylhydroquinone [700-13-0] (7) with phytol(8), phytyl halides, or preferentially isophytol(9).

Lewis and Bronsted acids [306], especiallyzinc chloride with a mineral acid [377], serve ascatalysts for condensation. Boron trifluoride andAlCl3 [378], Fe/HCl [379], trifluoracetic acid[380], and boric acid/carboxylic acid [381] arealso good catalysts. Awide range of solvents canbe used, e.g., ethyl acetate, acetic acid, (chlori-nated) hydrocarbons or supercritical CO2 [382].

When synthetic phytol (8) or isophytol (9) isused, the product consists of equal amounts ofall eight α-tocopherol stereoisomers and is thusdenominated (all-rac)-α-tocopherol.

The majority of synthetic α-tocopherol istreated with acetic anhydride to give the acetate,which is stable on storage. In addition, the succi-nate (monoester of succinic acid) [383] and thenicotinate are produced commercially. Purifica-tion of the crude product is possible at the freetocopherol or acetate stage by short-path dis-tillation under high vacuum or extraction withsupercritical CO2 [384].

Vitamins 45

4.11.3. Trimethylhydroquinone

The starting material in the most important in-dustrial syntheses of trimethylhydroquinone (7)is 2,3,6-trimethylphenol [2416-94-6] (11) [385],which is oxidized by oxygen or peroxides togive trimethylquinone (13) [935-92-2]. The cat-alysts mainly used are salcomin [386], copperchloride with diverse cocatalysts [387], or het-eropoly acids [388]. Trimethylhydroquinone (7)is finally obtained by the catalytic hydrogenationof trimethylquinone (13) [389].

The intermediate 2,3,6-trimethylphenol (11)is produced from m-cresol (12) by catalyticalkylation with methanol [390].

Another important large-scale process for thesynthesis of trimethylphenol (11) is the con-densation of diethyl ketone with crotonaldehydeto yield trimethylcyclohexenone [20030-30-2](10) [391] or diethyl ketone with methylvinyl ketone to give trimethylcyclohexenone[20030-29-9] (10′) [392], followed by catalyticdehydrogenation of trimethylcyclohexenone to2,3,6-trimethylphenol (11).

Still another synthesis of trimeth-ylhydroquinone starts with mesitol (14) [393],which is

oxidized with oxygen [394] or hypochloriteor chlorine/water [395] to give trimethylquinol(15). Compound 15 is then rearranged bytreatment with alkali [396] to yield trimeth-ylhydroquinone (7). This pathwaywas long usedby Rhone-Poulenc, but has now been replaced

by oxidation of 2,3,6-trimethylphenol (11) withoxygen because oxidation with chlorine causeswastewater problems [397].

Trimethylhydroquinone (7) can also be madefrom the inexpensive compound isophoron (16).Oxidation to trimethylquinone (13) can be per-formed directly [398] or via the intermediate2,3,5-trimethylphenol (11′), which gives 7 onhydrogenation [399]. Oxidation of 16 to ox-oisophoron (17), followed by isomerization pro-duces trimethylhydroquinone (7) directly [400].These processes are, however, not used commer-cially.

4.11.4. Phytol and Isophytol

The key sequence in the formation of the vita-min E side chain is a series of C2 and C3 chainextensions, starting with a ketone.

The C2 extension of the ketone pro-ceeds via vinylation with vinyl Grignard orby ethynylation with subsequent partial hy-drogenation. The allyl alcohol thus formed

46 Vitamins

is extended to give the γ,δ-unsaturated ke-tone (18) by means of acid catalyzed ketalformation/elimination/rearrangement with theC3 building block 2-methoxypropene (Saucy –Marbet reaction [401]) or by transesterifica-tion/rearrangement with methyl acetoacetate(Carroll reaction [402]). The ketone (18) canbe subjected to the same sequence again. TheSaucy –Marbet reaction can also be appliedto tertiary propargyl alcohols, forming the β-ketoallene (18′). In this case, partial hydrogen-ation after ethynylation is not required [403].

In this procedure, acetone is first con-verted to methyl heptenone [110-93-0] (18,R =Me), and then via linalool [78-70-6] (19)to geranylacetone [689-67-8] (18, R = prenyl),nerolidol [1119-38-6] (20) and farnesylace-tone [1117-52-8] (18, R = geranyl), or their hy-drogenation products. Hexahydrofarnesylace-tone [502-69-2] is converted to isophytol (9),which can be isomerized to phytol (8) by acid-catalyzed rearrangement.

One of the four isophytol producers (Ku-raray) makes methylheptenone (18, R =Me)from prenyl chloride and acetone [404]. In thelate 1980s, Rhone-Poulenc changed its pro-cess, basing it on the inexpensive raw ma-terial myrcene (21) [123-35-3] [397], whichyields geranylacetone (18, R = prenyl) in a rhodi-um-catalyzed reaction with methyl acetoacetate[405]. This compound is then further processedin the conventional way.

4.11.5. Stereoselective Syntheses ofα-Tocopherol

In 1938, the first synthesis of (2RS,4′R,8′R)-α-tocopherol (1a) was conducted byKarrer et al.by the condensation of trimethylhydroquinone(7) and phytyl bromide (made from natural phy-tol) with zinc chloride [300].

This reaction was frequently modified [306].Most of the α-tocopherol syntheses proceed viaacid-catalyzed condensation of hydroquinonederivatives with phytol or isophytol derivatives.Tocopherols produced in these syntheses are al-ways racemic at the C-2 center of the chromanering.

The first synthesis of (2R,4′R,8′R)-α-tocopherol (1a) was conducted by Mayeret al [308] in conjunction with the elu-cidation of the absolute configuration. Inthis process, trimethylhydroquinone (7) was

Vitamins 47

converted to (2R,4′R,8′R)-tocopherol viachromanyl(ethinylcarboxylic acid) (22) –racemate separation was achieved via the qui-nine salt – and via the chromane carbaldehyde(23). The optically active side chain originatedfrom natural phytol (8), which was converted tothe corresponding Wittig reagent (Scheme 11).

Several modern total syntheses of(2R,4′R,8′R)-α-tocopherol were described inthe late 1970s and early 1980s [406,407]. Theseprocesses depended on the separation of racemicmixtures for the stereoselective formation of thechromane skeleton.

According to more recent results, the qua-ternary center at C-2 can be made by asym-metric synthesis, which uses either chiral aux-iliaries in stoichiometric amounts [408,409](Scheme 12) or enantioselective catalytic pro-cesses [409,410] (Scheme 13).

Enzymatic methods using tocopherol cy-clases have also been successfully employed togenerate the C-2 asymmetric center [356,357,411]. The γ-tocopherol initially obtained canbe converted to the α-derivative by methylation(see Section 4.11.1).

In the mid-1980s [412], an asymmetric cat-alytic hydrogenation process was developed forthe synthesis of the enantiomerically pure sidechain (Scheme 14).

This method has greatly improved the syn-thetic availability of the side chain with regardto both the number of reaction steps and the to-tal yield. The enantiomeric excess achieved is98.4%. Nevertheless, compared with the avail-ability of natural vitamin E, the effort involved inthe commercial synthesis of (R,R,R)-tocopherolmakes it uneconomical.

5. Vitamin K

5.1. Introduction; History

In 1929, while studying the metabolism of cho-lesterol in chickens fed a diet extractedwith non-

polar solvents, Dam observed bleeding in vari-ous organs and tissues and a prolonged bloodclotting time [413]. The symptoms could be pre-vented or cured by the feeding of plant or animalfeedstuffs. Thus,Dam had discovered a new, es-sential, fat-soluble factor that controls the bloodprothrombin level, which is important for bloodclotting. In 1935, he suggested calling this fac-tor vitaminK. At that time, the letter K was notonly the next free letter for the designation of anexisting or postulated vitamin, but K is also thefirst letter of the word for coagulation in Germanand the Scandinavian languages.

At the same time, Almquist found that anantihemorrhagic factor is also formed by bac-teria in decaying feedstuffs (e.g., fish meal)[414]. In 1939/40, isolation, structural elucida-tion, and synthesis in various laboratories (Dam,Almquist, Karrer, Doisy, Fieser; see [413,414]) showed surprisingly that two differentforms of vitaminK occur in nature. They dif-fer in the length and degree of saturation ofthe isoprenoid side chain linked to 2-methyl-1,4-naphthoquinone at C-3. VitaminK1 fromplants has a mono-unsaturated C20 side chain,and bacterial vitaminK2 has a polyunsaturatedside chain of different length.

It was shown in 1974/75 that vitaminK func-tions as a cofactor for the γ-carboxylation ofspecific glutamic acid residues of the precursorprotein of prothrombin, converting the proteinto biologically active prothrombin [415].

The termvitaminK is nowused as the genericname for 2-methyl-1,4-naphthoquinone and allderivatives that qualitatively exhibit the biologi-cal activity of phylloquinone (vitaminK1) [416].The following names have been recommendedby IUPAC for the individual vitaminsK [417]:Phylloquinone, abbreviated K, previ-

ously vitaminK1, for 2-methyl-3-phytyl-1,4-naphthoquinone. The U.S. Pharmacopoeia usesthe name phytonadione [418]. The synonymphytomenadione is occasionally found in the

48 Vitamins

Scheme 11. Synthesis of (R,R,R)-α-tocopherol (1a)

Scheme 12. Asymmetric chromanol synthesis with chiral auxiliary

Scheme 13. Asymmetric synthesis of (R,R,R)-α-tocopherolPTC= phase transfer catalyst

literature. Natural phylloquinone [84-80-0] (1)has the configuration 2′E,7′R,11′R [419].

Menaquinone-n (2), abbreviated MK-n, pre-viously vitaminK2, for 2-methyl-3-polyprenyl-

1,4-naphthoquinone, n being the number ofprenyl residues.

For instance, the compound origi-nally isolated from rotting fish meal andnamed vitaminK2 was later identified asmenaquinone-7 (MK-7) [2124-57-4] (2-methyl-3-farnesylgeranylgeranyl-1,4-naphthoquinone)[419]. ForMK-7, the older literature contains thedesignation vitaminK2(35) or vitaminK2(35),which is no longer used.Menaquinones found innature have side chains of 4 – 13 prenyl residuesand usually exhibit the all-trans configuration.

Vitamins 49

Scheme 14. Synthesis of R,R side chain by catalytic hydrogenation

However, menaquinones with the cis configura-tion and partially saturated side chains also exist[420].Menadione [58-27-5] (3), previously vita-

minK3, for 2-methyl-1,4-naphthoquinone. Itdoes not occur in nature but can be convertedto menaquinone-4 in the organism [421].

5.2. Physical Properties

All the vitaminsK are lipophilic compoundswhich are readily soluble in ether, petroleumether, benzene, hexane, and acetone, poorly sol-uble in methanol and ethanol, and insoluble inwater. Due to the naphthoquinone chromophore,they have characteristic UV maxima (Table 14).

Table 14. UV maxima and extinctions (E1 %1cm) of phylloquinone

and menaquinone-7 (n = 7) in petroleum ether [419]

Phylloquinone Menaquinone-7

243 (395) 243 (278)249 (420) 248 (295)261 (390) 261 (266)270 (392) 270 (267)325 (68) 325 – 328 (48)

The infrared spectrum of vitaminK1 and ofall the K2 vitamins shows intensive bands at1660, 1618, 1300, and at 700 cm−1, character-istic of the naphthoquinone ring [422].

In the 1960s, the 1HNMR spectra were usedfor the determination of the configuration ofthe trisubstituted double bond in the side chainof vitaminK1 [CH3-C(3′): trans: 1.78 ppm, cis:1.68 ppm] [423].

Vitamin K1, phylloquinone 1 [422] is a vis-cous yellow oil at room temperature. It can becrystallized out at low temperatures in acetone orethanol, mp −20 ◦C, bp 140 – 145 ◦C at 133 Pa,[α]20D −0.3◦ (dioxane), C31H46O2, Mr 450.71,and 0.967 g/cm3. It decomposes at normalpressure at 100 – 120 ◦C, but can be subjectedto high vacuum distillation without appreciabledecomposition. The redoxpotentials are 0.005V(25 ◦C, 80% ethanol, 0.02 molar in NaOAc and0.02 molar in AcOH) and 0.363V (20 ◦C, 95%ethanol, 0.2N in HCl and 0.02 molar in LiCl).

Vitamins K2, menaquinone-n 2. A vita-minK2 (menaquinone-7; 2, n = 7)withmp 54 ◦Cwas first isolated in the pure form from rot-ting fish meal by Doisy in 1939 [424]. Mostof the K2 vitamins are light yellow, crystallinecompounds. The melting points and the extinc-tions (E1 %

1cm) at the UV maximum (248 nm) ofmenaquinone-n (2, n = 1 – 10) are presented inTable 15.

50 Vitamins

Table 15. Melting points and UV absorptions at 248 nm E1 %1cm of

menaquinones-n (n = 1 – 10) in petroleum ether [422]

n E1 %1cm (248 nm) mp, ◦C

1 7842 617 533 496 354 440 355 363 396 320 507 295 548 268 569 246 56 – 5710 224 62

The extinction E1 %1cm of the K2 vitamins

decreases continuously with increasing chainlength. In the structural elucidation, a compari-son of these extinction values provided impor-tant information about the number of isopreneunits in the side chain.

5.3. Chemical Properties [419]

General Aspects. The vitaminsK are rela-tively heat resistant (≤100 ◦C) and stable in air.However, they are very sensitive to alkali andlight. Therefore, they should always be protectedfrom light.

Color Reactions. With sodium ethoxide, thevitaminsK give a violet-blue color which, withtime, changes to red and subsequently to brown(Dam–Karrer test) [425]. An intense blue coloris also obtained with sodium diethyldithiocarb-amate [426]. A stable orange color is producedby the reaction of the vitaminsK with 5-imino-3-thioxo-1,2,4-dithiazolidine (Schilling –Damtest) [427].

Oxidation/Reduction. The structural eluci-dation of the vitaminsK is based primarilyon the oxidative degradation of the quinonesand their hydroquinone diacetates. Chromicacid degradation of vitaminK1 (1) yields amixture of phthalic acid (4) and 2-methyl-1,4-naphthoquinone-3-acetic acid (5). Reduc-tive acetylation of 1 gives dihydrovitamin K1diacetate [604-87-5] (6) which yields a mix-ture of diacetoxyaldehyde 7 and 6,10,14-tri-methyl-2-pentadecanone (8) after ozonization(Scheme 15). On ozonolysis, dihydrovitaminK2diacetates (9) give the same diacetoxyaldehyde7, variable amounts of levulinic aldehyde (10)

(depending on the chain length), and one equiv-alent of acetone (11) (Scheme 15).

Treatment of vitamin K1 (1) with H2O2 inEtOH in the presence of Na2CO3 produces vi-taminK1 2,3-epoxide [25486-55-9] (12), whichcan be reduced to dihydrovitaminK1 [572-96-3](13) with sulfurous acid. Reduction of vita-minK1 with sulfurous acid or with H2/Lindlarcatalyst also yields dihydrovitamin K1 (13),which is, in turn, convertible to vitaminK1 byreaction with oxidizing agents.

Table 16. Phylloquinone content of some vegetables∗

Vegetable Phylloquinone content, µg/100 gfresh weight

According According toto [428] [429]

Kale 724 817Spinach 415 385Purslane 381Brussel sprouts 175Outer leaves 400 475Sprouts 177

Broccoli 147 205Cos lettuce 120 123White cabbage 55Outer leaves 137Inner leaves 83

Cress 88Beans 46 53Peas 39 33Cauliflower 27 25Red cabbage 19 57Cucumber 15 22Leek 10 18Tomato 6 6Carrot 5 11

∗Determined by high performance liquid chromatography(HPLC).

Vitamins 51

Scheme 15. Oxidation/reduction of vitamin K1

Formation of Chromanol and Chromenol.Chromanols and chromenols regained impor-tance after it was shown that the correspond-ing 6-phosphate derivatives of these compoundsappear as intermediates in oxidative phos-phorylation in cells.

Chemical conversion of vitaminK1 (1) tochromanol (14) is achieved with tin(II) chloride.Cyclization to chromenol (15) is possible withpyridine or with sodium hydride.

5.4. Occurrence

Phylloquinone is widely distributed in higherplants and in green and blue algae. It is lo-calized mainly in the chloroplasts [420]. Thus,green leafy vegetables contain relatively highamounts of phylloquinone (Table 16). The phyl-loquinone content is not considerably altered bycooking [429]. Comparatively little phylloqui-none is present in fruit.

The vitamin K1 content of some fruits (inµg/100 g fresh weight) is as follows [430]:

52 Vitamins

Avocados 20Apples 5Peaches 5Bananas 1Oranges 1Pears 1Pumpkins 1

Plant oils contain greatly varying amounts ofvitamin K1 (in µg/100 g) [431]:

Soybeans 193Rapeseed 141Olive 55.5Sesame 15.5Walnut 15Safflower 9.1Sunflower 9Almond 6.7Corn 2.9Peanut 0.6

The phylloquinone concentration in plantscan vary depending on genetic factors or on thesite and degree of ripeness [432].

There is little information available on the vi-taminK content of milk and meat products (Ta-ble 17). Apart from phylloquinone, the liver alsocontains long-chainmenaquinones in concentra-tions several times higher than vitaminK1. Rel-atively large amounts of menaquinones, espe-cially of MK-9, are contained in cheese [434].

Table 17. VitaminK content of milk and meat products a [430,433]

Foods VitaminK content,µg/100 g

Cow’s milk 0.5 – 1 b

Cheese 10 – 50 c

Butter 10 c

Skeletal meats <1 – 5 b

Liver 20 – 100 c

a Determined by HPLC.b Phylloquinone.c Phylloquinone and menaquinones.

Menaquinones occur in certain, predomi-nantly gram-positive bacteria. Other bacteriacontain ubiquinones (16) with chain lengths of6 – 10 prenyl residues instead of menaquinones.Some bacteria exhibit both quinones, usuallywith the same chain length. Among the mena-quinones, MK-7 to MK-9 predominate. Thosefound in minor amounts areMK-4 toMK-6, andMK-10 toMK-13 [420]. In humans, some anaer-obic intestinal bacteria produce mainly MK-6to MK-13; MK-8 to MK-10 with two saturatedisoprene units in the side chain (17) was also

detected [435]. The menaquinones are local-ized in the cytoplasmic membranes of the bac-teria. Their concentrations are in the range 0.6 –1.7mg/kg of bacterial dry weight [436].

5.5. Biosynthesis

Both plants [437] and bacteria [438] use thesame biosynthetic pathway for the forma-tion of the naphthoquinone ring of vitaminK(Scheme 16). Shikimic acid (18) is made fromthe precursors erythrose 4-phosphate and phos-phoenolpyruvic acid. After phosphorylationand reaction with phosphoenolpyruvic acid,shikimic acid is converted via chorismic acid(19) to isochorismic acid (20). In a complexreaction of the 2-succinylbenzoic acid syn-thase system [439], 2-oxoglutaric acid is decar-boxylated in the presence of thiamine diphos-phate (TPP), forming a succinylsemialdehyde-TPP carbanion (21). This substance then reactswith 20 yielding 2-succinyl-6-hydroxy-2,4-cy-clohexadiene-1-carboxylic acid (22) after elim-ination of pyruvic acid.Aromatization of 22 pro-duces 2-succinylbenzoic acid (23), which is con-verted to 1,4-dihydroxy-2-naphthoic acid (25)after activation via a coenzyme A ester 24 and asecond aromatization step.

In the further biosynthesis of the vitaminsK(Scheme 17), e.g., in the formation of bacterialmenaquinones, 25 is condensed with polyprenyldiphosphate (26) to give 2-polyprenyl-1,4-naphthoquinol (27) on decarboxylation [436].The methyl group of (S )-adenosyl-l-methio-nine is transferred to 27, producing 2-methyl-3-polyprenyl-1,4-naphthoquinol (28), whichyields menaquinone-n (2) on oxidation. The for-

Vitamins 53

Scheme 16. Biosynthesis of the naphthoquinone ring of vitaminK

mation of phylloquinone (1) proceeds analo-gously. In the same reaction sequence, phytyldiphosphate (29) is incorporated instead of thepolyprenyl side chain. In the chloroplasts ofplants, phytyl diphosphate ismade fromgeranyl-geranyl diphosphate by hydrogenation. How-ever, phytylation and methylation of the corre-sponding vitaminK1 precursors occur in sep-arate membrane systems of the chloroplasts[440]. The synthesis of polyprenyl disphos-phate starts with condensation of 1-isopentenyldiphosphate, which is formed from meval-onic acid, with the isomeric 3,3-dimethylallyldiphosphate to give geranyl diphosphate. Ger-anyl diphosphate is then extended by furthercoupling reactions with 1-isopentenyl diphos-phate until the appropriate number of isopreneunits in the side chain are obtained [420].

5.6. Chemical Synthesis

In 1939, the first syntheses of vitaminK1 werepublished almost simultaneously by four groups[441] in connection with the structural elucida-tion. The starting materials were menadione (3)

or menadiol (30) as the aromatic component andnatural phytol (31) or one of its derivatives.

In the Fieser synthesis [443], which was thepreferred method for a long time, an excess ofmenadiol [481-85-6] (30) was condensed withnatural phytol [150-86-7] (31) in dioxane at75 ◦C in the presence of oxalic acid as cata-lyst. The excess menadiol was subsequently ex-tractedwith dilute alkali and the alkylation prod-uct 13 was precipitated with petroleum ether.

54 Vitamins

Scheme 17. Biosynthesis of vitaminsK

Oxidation of this waxy precipitate with silveroxide gave vitaminK1 (1) in a total yield of ca.30% based on phytol (31).

A disadvantage of this synthesis is theFriedel – Crafts alkylation, which does not pro-ceed regioselectively with regard to positions 2and 3 of menadiol. In addition to the desiredalkylation product 13, almost equal amounts ofthe isomer 32 are produced. Furthermore, theacidic alkylation conditions cause partial iso-merization of the trisubstituted double bond inthe side chain. Under these conditions, phyl-lochromanol (14), a cyclization product of 13,and phytadienes of type 33 are also formed.

With regard to commercial synthesis, abreakthrough was achieved in the 1950s whenIsler et al. [445] at Hoffmann-LaRoche andHirschmann et al. [446] at Merck found that

monoacylated menadiols, e.g., the monoacetate35a or themonobenzoate 35b, could be advanta-geously used in the alkylation step (Scheme 18).Thus, the undesired alkylation in position 2 ofmenadiol (30) is prevented. Moreover, it wasfound that boron trifluoride etherate is a muchbetter catalyst than anhydrous oxalic acid andthat natural phytol (31) can be replaced by iso-phytol [505-32-8] (36), which is easy to synthe-size.

There are various processes available for theproduction ofmenadione (3). Themost commonis the oxidation of 2-methylnaphthalene with,e.g., CrO3 or H2O2 in acetic acid or Na2Cr2O7in sulfuric acid [447].

Starting with menadione (3), the monoben-zoate 35b can be produced in three steps: cat-alytic reduction of menadione (3), e.g., in eth-anol or methanol, followed by acylation withtwo equivalents of benzoyl chloride to givethe dibenzoate 34b, and partial saponification,e.g., with KOH or NaOH, to produce the de-sired monobenzoate 35b. Menadiol monoac-etate (35a) can be produced in an analogousmanner and used for the synthesis of vitaminK1(38).

In the Isler – Lindlar method [445], excessmenadiol monobenzoate (35b) is condensedwith isophytol (36) in dibutyl ether at 85 ◦C in

Vitamins 55

the presence of boron trifluoride etherate as cat-alyst. Unreacted monobenzoate 35b is precipi-tated with petroleum ether, filtered off, and reco-vered. After evaporation of the filtrate, the alkyl-ation product 37b is obtained as an E/Z mix-ture (E/Z 70 : 30). The E form can be enrichedby recrystallization (e.g., from methanol/etha-nol; USP requirement: ≤20% of Z form in thefinal product vitaminK1). 37b can be obtainedin the practically pure E form by repeated re-crystallization.

Scheme 18. Synthesis of vitaminK1

The E-enriched alkylation product 37b (E/Z9 : 1) is saponifiedwith potassiumhydroxide andoxidized to vitaminK1 (38) with oxygen. In thismethod, E/Z-vitaminK1 (38) is produced as ayellow oil in a yield of ca. 60 – 70% based onisophytol (36).

For the synthesis of isophytol, see 4.11.4.The fact that only E-vitaminK1 exhibits K-

activity in biological tests led to intensive workon this synthesis in the 1970s and 1980s, withthe aim of synthesizing pure E-vitaminK1.

Many multistage syntheses with organome-tallic reagents, e.g., Grignard reagents [448];zinc reagents [449]; aryl [450] and vinylcuprates [451]; trialkyltin compounds [452]; π-allylnickel [453]; phythaloylcobalt [454]; andchromium carbene complexes [455], have beenpublished and patented. These and other syn-thetic possibilities are discussed in a review ar-ticle [451].

In all these variants, a disadvantage is theuse of stoichiometric amounts of organometallicreagents which must be recovered for economi-cal and ecological reasons.

A potentially economic, short synthesis of vi-taminK1 and vitamins K2 has been patented byF. Hoffmann-LaRoche [451,456] (Scheme 19).

56 Vitamins

Menadione (3) is first subjected to a Diels –Alder reaction with cyclopentadiene, produc-ing the crystalline endo-Diels –Alder adduct[97804-50-7] 39 in 93% yield. Subsequently,the proton α to the carbonyl group can be ab-stracted with a strong base (e.g., potassium tert-butoxide) and the resulting enolate alkylatedwith phytyl bromide (40) (E/Z ≥98 : 2). Thealkylation product 41 is obtained in a yield of≥90%. As a result of the basic alkylation con-ditions, the trans geometry of the double bondin phytyl bromide [76524-59-9] (40) (≥98%E )is completely retained during alkylation.

The alkylation product 41 [97804-51-8] isthermally unstable and undergoes a retro-Diels –Alder reaction. On heating in toluene (110 ◦C),it decomposes rapidly into vitaminK1 (38)(≥98% E ) and cyclopentadiene. The yield ofvitaminK1 is practically quantitative. The cy-clopentadiene can be recovered by distillationand reused.

The vitaminsK2 (menaquinones) (2) andubiquinones (16) can be synthesized accordingto the same scheme.

A patent application for a similar syntheticroute to vitaminsK and analogous compoundshas been filed by Eisai Chemical Co. [457]. In-stead of menadione (3), naphthoquinone (42) isused as starting material.

Analogously, the isoprenoid side chain [e.g.,phytyl bromide (40)] and then the methyl grou-pare introduced via the cyclopentadiene adduct43. The intermediate 41 [451,456] describedabove is obtained via compound 44 and givesthe desired quinone [e.g., vitaminK1 (38)] afterelimination of cyclopentadiene.

5.7. Analysis

Physicochemical Methods. As a result ofits high selectivity and sensitivity, high perfor-mance liquid chromatography (HPLC) is themethod of choice for the determination of phyl-loquinone and menaquinones in the blood, tis-sues, milk, and in foods. Various procedures forextraction and preliminary purification, internalstandardization, HPLC conditions (with normalor reversed phase) and possibilities for UV, elec-trochemical, and fluorescence detection (bothafter electrochemical or chemical reduction andafter photochemical decomposition) of the vita-

minsK have been described [458]. The detec-tion limits for phylloquinone are in the range25 –500 pg, depending on the detection methodemployed [459]. Similar values that vary accord-ing to the length of the side chain apply to themenaquinones [460]. HPLC methods are alsoavailable for the determination of menadioneand water-soluble derivatives (see Section 5.12)in feedstuffs, premixes, and in vitamin concen-trates [458,461].

Alternative methods are thin layer chromat-ography (TLC) [462], high performance thinlayer chromatography (HPTLC), and gas chro-matography (GC) [458]. The spectrophotomet-ric, fluorometric, and colorimetric methods pre-viously used without chromatographic purifi-cation of the samples to be analyzed have thedisadvantage of low sensitivity and specificity.They are unable to distinguish between phyllo-quinone and menaquinones [436,462]. The de-tection limits are ca. 25µg/mL for phylloqui-none and menadione.

Biological methods measure the biologicalactivity of a specific vitaminK compound or de-tect vitaminK activity in a sample under anal-ysis. It is not possible to differentiate betweendefinite forms of vitaminK in the sample. Thebiological methods are based on the measure-ment of the prolonged blood clotting time (pro-thrombin time) in vitaminK deficient animals(chicks, rats) and its normalization after the ad-ministration of the sample exhibiting antihem-orrhagic activity [463]. The bioavailability andbiopotency of formulated vitaminK compoundsare tested mainly in the prophylactic test, i.e.,after intermixing with vitaminK deficient feedand feeding for a period of 24 d [464]. On theother hand, the vitaminK activity of pure sub-stances is determined after a single dose in thecurative test [465]. The biological determina-tion of vitaminK can also be conducted in an-imals that have prolonged blood clotting timescausedby the feedingof anticoagulants (seeSec-tion 5.9.2). Strictly speaking, however, this testdoes not measure the reversal of vitaminK de-ficiency, but the competition between the vita-minK compound to be tested and the antagonistused.

Vitamins 57

Scheme 19. Synthesis of vitaminsK (Hoffmann-LaRoche)

5.8. Metabolism

Alkylation of Menadione. Menadione (vi-taminK3) itself has no antihemorrhagic activ-ity. However, it can be prenylated to activemenaquinone-4 in the liver and in other animaltissues [421].

Intestinal bacteria can form menadione byeliminating the phytyl side chain of phylloqui-none. After absorption, menadione is convertedtoMK-4 in the animal organism [433]. It appearspossible that dealkylation of phylloquinone and,consequently, conversion of vitaminK1 to MK-4 can occur in animal tissues as well [466,467].

Absorption. Phylloquinone and menaqui-nones present in food are absorbed in the proxi-mal small intestine. Sufficient amounts of bile

and pancreatic juice enzymes are required inthe intestinal lumen so that the vitaminsK canbe emulsified in the mixed micelles consistingof bile salts, fatty acids, and monoglycerides,before they are absorbed by the intestinal mu-cosa cells [468]. A saturable, energy-dependentmechanism that cannot be influenced by themenaquinones seems to be responsible for theabsorption of phylloquinone [433]. In the caseof themenaquinones produced by intestinal bac-teria, absorption occurs in the large intestine bypassive diffusion [434]. In the intestinal cells, theabsorbed vitaminsK are incorporated into chy-lomicrons and transported to the liver via thelymph. The degree of absorption for vitaminKin the small intestine is 40 – 70% [469].

The absorption of menadione occurs in thedistal small and large intestine by a passive,

58 Vitamins

energy-independent process. It enters the bloodstream directly from the intestine [470].

Distribution. A part of the vitaminK trans-ported to the liver is incorporated into VLDL(very low density lipoproteins), released intothe blood stream, and transferred via LDL (lowdensity lipoproteins) to other organs [436] (e.g.,lungs, kidneys, adrenals, bone marrow, lymphnodes [421], testicles, and skin [471]). The partremaining in the liver, which is the main site ofactivity of vitaminsK, is mainly taken up by themitochondria and the microsomes [433].

The distribution of the various forms of vi-taminK in the organism varies greatly. Phyllo-quinone is only moderately retained in the liver.It has a half life of ca. 17 h in rat liver [433]. Inhuman liver, the average concentration of phyl-loquinone is 5.5 ng/g (range: 1.1 – 21.3 ng/g for32 individuals tested: N = 32) [472]. In contrastto phylloquinone, the more lipophilic menaqui-nones have a longer retention time or slowerturnover in the liver [434], which can lead toa certain accumulation (Table 18). The mena-quinones account on an average for 92mol% ofthe total vitaminK content of the adult humanliver (individual variations of 75 –97%) [472].With the exception of horse liver, in which onlyphylloquinone could be detected, considerableamounts of menaquinones are also present in theliver of other animal species. Bovine liver con-tains mainlyMK-10 toMK-12, while in dog andpig liver MK-8 to MK-10 predominate [436].Chicken liver contains much more MK-4 thanphylloquinone [466]. In rats, relatively high lev-els of MK-4 are found in the pancreas and sali-vary gland [467].

Compared to liver, the ratio of phylloqui-none to menaquinones in human blood plasmais substantially shifted (Table 18). The mena-quinones exceed the amount of phylloquinoneonly by a factor of 1.2 – 2.3, and very long chainmenaquinones (MK-9 to MK-13) are no longerfound. This is another indication of a slower hep-atic turnover of the highly lipophilic menaqui-nones and their greater affinity for intracellu-lar membranes. The average phylloquinone con-centration in the plasma is 0.485± 0.314 ng/mL(N = 332) [475].

Breast milk can contain low concentrationsof MK-5 to MK-7, but the main form of vi-taminK present is phylloquinone [459,476].

Its concentration fluctuates between 1.1 and6.5 ng/mL (average 2.1 ng/mL). Cow’s milkhas a much higher phylloquinone content: 10 –20 ng/mL [436].

In the organism, menadione is distributedover many tissues, but very rapidly eliminated.Only a small part of menadione is converted tomenaquinone-4 [421].

Degradation and Excretion. In studies onthe catabolism of phylloquinone (38) in hu-mans and laboratory animals, the followingmetabolites were identified [477]: 2-methyl-3-(7′-carboxy-3′,7′-dimethyl-2′-heptenyl)-1,4-naphtho- quinone (46), 2-methyl-3-(5′-carboxy-3′-methyl-2′-pentenyl)-1,4-naphthoquinone(47), and 2-methyl-3-(3′-carboxy-3′-methyl-propyl)-1,4-naph-thoquinone (48). They areproduced from 38 by ω-oxidation to the cor-responding carboxylic acid 45, followed by

Vitamins 59

Table 18. Concentration of vitaminK in human liver and blood plasma (ng/g liver or ng/mL plasma)

VitaminK Liver, Plasma

form N = 7 N = 22 N = 11[473] [473] [474]

K1 12.6± 1.9 0.59± 0.06 0.51± 0.37MK-4 0.4± 0.2 0.05± 0.01MK-6 0.05± 0.01MK-7 21.8± 7.8 0.57± 0.08 0.29± 0.18MK-8 6.3± 1.2 0.08± 0.05 0.54± 0.32MK-9 1.5± 0.5MK-10 62.6± 8.0MK-11 89.0± 13.4MK-12 13.3± 2.4MK-13 5.0± 1.4

repeated β-oxidation of the side chain. Afterreduction to 1,4-naphthohydroquinone and con-junction with glucuronic acid, the metabolitesare excreted in the urine as mono- or diglu-curonides. A part of the less polar

metabolites is also excreted in the conjugatedform via the bile and feces [478].Menaquinones(like ubiquinones (16) and tocopherols) are de-graded in the same manner.

Menadione is also excreted in the urine inthe conjugated form as the phosphate, sulfate,or glucuronide of menadiol (30).

5.9. Importance for the Organism

5.9.1. Function

VitaminK plays a vital role as a cofactor inthe γ-carboxylation of specific glutamic acidresidues near the amino terminal end of sevenprotein precursors of the blood clotting sys-tem (see also→Blood, Chap. 2.2.2.) [479]. Thispost-translational modification of inactive pre-proteins produces active coagulation proteins(Table 19) with 10 – 13 γ-carboxyglutamic acidresidues per molecule. In the presence of cal-cium ions, these residues allow the proteins tobind to phospholipid membrane surfaces [436].The classical vitaminK dependent factors II,VII, IX, X, and protein C can thereby per-form their specific functions (Table 19) withinthe complex blood clotting system. The overallfunction of this system ismaintenance of normalhemostasis, i.e., the absence of hemorrhagic orthrombic events. In principle the reactions of theblood clotting system proceed in such a man-ner that in conjunction with additional compo-

nents of an activation complex, an activated clot-ting factor converts an inactive clotting factorto an active proteolytic enzyme. This enzyme,in turn, acts as a proteinase in the next stepof the blood clotting cascade. The last activa-tion step involves the conversion of prothrom-bin (factor II) to thrombin, which finally causesthe formation of fibrin from its precursor fibrino-gen, producing a stable fibrin clot. The coagu-lation proteins are synthesized in the liver andthen released into the blood. In vitaminK defi-ciency, the blood clotting proteins are produced,but they remain inactive because of insufficientor lacking γ-carboxylation of the glutamic acidresidues [469].

The form of vitaminK that is “active” asa cofactor for the γ-glutamyl carboxylase lo-cated in the microsomes (E1 in Scheme 20) is1,4-dihydrovitaminK (50). Simultaneous withthe carboxylation of a protein-bound glutamylresidue (51) to a γ-carboxyglutamyl residue(52), 1,4-dihydrovitaminK is oxidized to the2,3-epoxide of vitaminK (53) [481]. It is as-sumed that carboxylation and epoxidation arecatalyzed by the same enzyme [434]. The ep-oxide 53 is then reduced to vitaminK (49) by adithiol-dependent vitaminK epoxide reductase(E2). Another dithiol-dependent vitaminK re-ductase (E3) reforms vitaminK hydroquinone(50). It is possible that the reduction (E3) of49 to 50 is also carried out by vitaminK ep-oxide reductase (E2). However, vitaminK canalso be reduced to the quinol 50 by a NAD(P)H-dependent vitaminK reductase (E4). The activ-ity of the dithiol-dependent reductases (E2 andE3) is inhibited by anticoagulants (A) (see Sec-tion 5.9.2).

60 Vitamins

Table 19. VitaminK dependent proteins of the blood clotting system [480]

Protein Function Molecular mass Concentration in humanblood, ng/mL

Factor VII (proconvertin) activation of factor X to X a 48 000 0.6Factor IX (PTC∗) activation of factor X to X a 57 000 5Factor X (Stuart – Prower Factor) as factor X a: conversion of factor II to

thrombin55 000 10

Factor II (prothrombin) precursor of thrombin 72 000 100Protein C inhibition of coagulation 62 000 4 – 5Protein S cofactor of protein C 80 000 25Protein Z still unknown 55 000 <1∗∗∗ Plasma thromboplastin component.∗∗According to [436].

Scheme 20. Reactions of the vitaminK cycleR = phytyl (phylloquinone) or geranylgeranyl (MK-4); 1E1 = γ-glutamyl carboxylase; E2 = vitamin K epoxide reductase;E3 = dithiol-dependent vitaminK reductase; E4 =NAD(P)H-dependent vitaminK reductase; D-(SH)2 = reduced dithiol; D-S2 = oxidized dithiol (functional dithiols: dithiothreitol and thioredoxin); NAD(P)H= reduced nicotinamide adenine dinu-cleotide (phosphate); NAD(P)+ = oxidized nicotinamide adenine dinucleotide (phosphate); A = inhibition by anticoagulants

This vitaminK (or vitaminK epoxide) cy-cle acts very effectively. It is assumed thatone molecule of vitaminK is recycled sev-eral thousand times before it is convertedto inactive metabolites [482]. Approximately10% of the phylloquinone in the liver is

present as phylloquinone-2,3-epoxide [483].Menaquinones, especially MK-3 andMK-4, arealso active in the vitaminKcycle.However, theiractivity decreases with increasing length of theside chain (see Section 5.9.3). Menadione can-not replace the vitaminsK.

Vitamins 61

After the elucidation of the cofactor role of vi-taminK in the formation of γ-carboxyglutamicacid (Gla) in the vitaminKdependent blood clot-ting factors in the liver, it was observed that thevitaminK cycle is active in other tissues as well(e.g., bone, cartilage, kidney, spleen, and lung).In fact, various organs form Gla-containing pro-teins which are not involved in blood clotting[434,484]. Little is known about the function ofthese proteins. Until now, the bone Gla-protein(osteocalcin) and the matrix Gla-protein havebeen relatively well studied. Osteocalcin is pro-duced by the osteoblasts of bone tissue, approx-imately 30% of this protein being released intothe blood [482]. Osteocalcin, which is subjectto regulation by 1,25-dihydroxyvitaminD3, isquantitatively the most common noncollagenicprotein component of the bone matrix and ac-counts for ca. 2% of the total proteins of thebone. In addition, it is quantitatively the mostcommon vitaminK dependent protein in thebody [436]. It is assumed that theGla-containingpart of osteocalcin plays an important role inmineralization or demineralization of the bone.The matrix Gla-protein, which is formed in a se-ries of organs (especially heart,lung, kidney, andcartilage tissue), is enriched above all in the boneand cartilage. It probably has a mediator func-tion in cell growth and differentiation [485].

These examples show that vitaminK not onlyplays a specific role in blood clotting, but this vi-tamin apparently has a wider biological signif-icance (see Section 5.10.1) which is connectedwith the strong calcium-bindingproperties of thenegatively chargedGla residues in vitaminKde-pendent proteins.

In plants, phylloquinone acts as an electroncarrier in the chloroplasts. In the plasma mem-brane of bacteria, menaquinones are involved asredox substances in electron transport and ox-idative phosphorylation [486].

5.9.2. Antagonists and Anticoagulants

Around 1930, a hemorrhagic disease was ob-served in cattle in Canada and the UnitedStates and was put down to spoilt sweet cloverhay fodder. In 1941, the substance responsi-ble for this disease was identified by isola-tion and structural elucidation as 3,3′-methy-lene-bis-(4-hydroxycoumarin), usually called

dicumarol (54) [433]. This led to the synthe-sis of other derivatives of 4-hydroxycoumarinwhich can be used in the treatment of throm-boembolic diseases and for the preventionof thrombus formation in vessels. Activeoral anticoagulants are, e.g., phenprocumarol[3-(1′-phenylpropyl)-4-hydroxycoumarin](55), warfarin [3-(α-acetonylbenzyl)-4-hy-droxycoumarin] (56), acenocumarol [3-(α-acetonyl-4-nitrobenzyl)-4-hydroxycoumarin](57), and ethyl-biscoumacetate [3,3′-carboxy-methylene-bis-(4-hydroxycoumarin) ethyl es-ter] (58) [487].

The action of all oral anticoagulants is basedon the inhibition (A in Scheme 20) of vitaminKepoxide reductase (E2) and of dithiol-dependentvitaminK reductase (E3) in the vitaminK cy-cle. This leads to an increase in the concentra-tion of vitaminK 2,3-epoxide (53) in the liver,a decrease in the activity of the blood clottingproteins due to lacking γ-carboxyglutamic acidformation and, consequently, to an increase inthe blood clotting time [434]. Since oral antico-agulants inhibit the dithiol-dependent vitaminKreductase (E3) but not the NAD(P)H-dependentreductase (E4), inhibition of the vitaminK cy-cle can be avoided by large doses of phylloqui-none. Under these conditions, however, the 1,4-dihydrophylloquinone (50) formed can only actto a limited extent because the recycling of vita-minK1 epoxide is inhibited or blocked. The re-sult is a relatively high vitaminK epoxide levelin the blood. Oral anticoagulants interfere not

62 Vitamins

only with the vitaminK cycle in the liver, butalso in other organs. A decrease of at least 50%in the blood level of osteocalcin and a reduc-tion of the γ-carboxyglutamic acid content ofosteocalcin by 50% have been observed undertherapy conditions [482].

More effective, extremely lipophilicderivatives of 4-hydroxycoumarin have nowbeen synthesized for use as rodenticides(→Rodenticides, Chap. 5.3.). Examples aredifenacoum (59a) and brodifacoum (59b).These substances have a very long biologicalhalf life of 100 – 200 d. They are potentiallyharmful to humans [434].

A competitive inhibitor of vitaminK is 2-chloro-3-phytyl-1,4-naphthoquinone (60), alsoknown as chloro-K. It inhibits the γ-glutamylcarboxylase reaction (E1 in Scheme 20) bypreventing epoxidation of vitaminK. 2,3,5,6-Tetrachloro-4-pyridinol (61) which is struc-turally related neither to vitaminK nor to 4-hy-droxycoumarin, inhibits the carboxylase appar-ently as a direct antagonist of the vitamin [433,484].

5.9.3. Relative Activity

Studies of the biological activity of vita-minK clearly show that two structural ele-ments are required: a 2-methyl-3-substituted1,4-naphthoquinone ring which can be reducedto hydroquinone, and a side chain at theC-3 atomwhich guarantees binding to or interaction withmicrosomal membranes.

Totally synthetic phylloquinone (38) con-sists of an equimolar mixture of four stereoiso-mers ((62a), (62b),(62c), (62d)). In cura-tive prothrombin-time chick assays (see Sec-tion 5.7), no substantial differences were foundin their antihemorrhagic activity [488] (Ta-ble 20).

Table 20. Antihemorrhagic activity of the stereoisomers ofphylloquinone and of (E, all-rac)-phylloquinone [488]

Compound Configuration Activity, % Confidencelimits62a 2′E,7′R,11′R 100 88 – 116

(standard)62b 2′E,7′R,11′S 93 75 – 11562c 2′E,7′S,11′S 119 110 – 12862d 2′E,7′S,11′R 99 88 – 11138 2′E,7′RS,11′RS 111 100 – 122

(E, all-rac)

Although 62c and (E, all-rac)-phylloquinone(38) have a slightly higher biological activitythan natural phylloquinone (62a = 1), all fourstereoisomers and 38 are to be considered iden-tical with regard to biological activity within thestatistical confidence limits [488]. Totally syn-thetic commercial phylloquinone always con-tains a low proportion (up to 10%) of (Z , all-rac)-phylloquinone (65). However, the antihe-morrhagic activity of 65 is only 0.3 – 1% (Ta-ble 21) and thus, negligible [489].

Phylloquinone-2,3-epoxide (12), the maincomponent of the vitaminK cycle (see Sec-tion 5.9.1), is 1.66 times as active as phyllo-quinone (Table 21). Changes in the side chain,e.g., 6′-hydroxylation (63) or hydrogenation ofthe double bond of the side chain (64), re-sult in a great decrease in the biological ac-tivity. If, in addition, the naphthoquinone ring

Vitamins 63

Table 21. Antihemorrhagic activity of structural analogs of phylloquinone (K1) [465,489]

Compound Structure Activity, % Confidence limits

38 (2′E,7′RS,11′RS )-K1 (standard) 100 87.8 – 116.712 (2′E,7′RS,11′RS )-K1 2,3-epoxide 166 137.3 – 200.263 (2′E,7′RS,11′RS )-6′-hydroxy-K1 20.5 16.1 – 26.064 (3′RS,7′RS,11′RS )-2′,3′-dihydro-K1 6.7 5.1 – 8.865 (2′Z ,7′RS,11′RS )-K1 <1

is also partially hydrogenated to 2′,3′,5,6,7,8-hexahydrophylloquinone (66), the biological ac-tivity is totally lost [465]. Substitution of thephytyl side chain of phylloquinone by an un-branched alkyl side chain (e.g., 2-methyl-3-octadecyl-1,4-naphthoquinone) also causes amajor loss of biological activity [420,421].

The length of the isoprenoid side chain is alsoof importance. In the prothrombin-time assayin vitaminK deficient chicks (Table 22), com-parisons of the antihemorrhagic activity of iso-prenologs of phylloquinone and menaquinones(on a molar basis) showed that a C20 side chain(four isoprene units) is optimal for the phyllo-quinone series. In the case of the menaquinones,MK-5 is 20% more active than phylloquinone,whileMK-4 andMK-6 have the same activity asvitaminK1. The biological activity of the mena-quinones decreases with increasing length of theside chain [487]. Kinetic studies on the cofac-tor activity of the menaquinones, measured asthe reaction rate of partially purified γ-glutamic

acid carboxylase at saturating vitamin concen-trations, confirm that the activity depends on thelength of the side chain (Table 23). In this studyMK-3 had optimal activity, MK-2 and MK-4were almost as active as phylloquinone, whilethe activity steadily decreases from MK-5 toMK-10 [490].

Table 22. Antihemorrhagic activity of isoprenologs ofphylloquinone and menaquinones [487]

Phylloquinone series Menaquinone series

Isoprene Activity, % MK-n Activity,%

units inthe sidechain

2 10 2 153 30 3 404 100∗ 4 1005 80 5 >1206 50 6 100

7 708 689 60

10 25

∗ Standard: (2′E,7′RS,11′RS )-phylloquinone.

Table 23. Activity of menaquinones as a cofactor of vitaminKdependent γ-glutamyl carboxylase from liver. Measurement of thereaction rate at saturating vitamin concentrations (Vsat) [490]

Vitamin Vsat, Vsat, %µmol boundCO2/h

Phylloquinone 25.4 100.0MK-1 4.0 15.9MK-2 24.9 98.1MK-3 30.5 120.0MK-4 24.7 96.7MK-5 22.2 87.1MK-6 19.6 77.0MK-7 18.0 70.7MK-8 15.8 62.0MK-9 14.0 55.8MK-10 13.0 51.2

64 Vitamins

5.10. Deficiency Symptoms

5.10.1. Causes

VitaminK deficiency leads to the secretion ofincompletely carboxylated, biologically less ac-tive or inactive forms of the blood clotting fac-tors II, VII, IX, and X (see Section 5.9.1) and,consequently, to subcutaneous or organ bleed-ing. VitaminK deficiency can result from inad-equate dietary supply or from diseases whichinterfere with the absorption of the vitamin inthe intestine. Examples are pancreatic insuffi-ciency andbiliary obstruction (lack of pancreaticenzymes and bile salts for the solubilization ofvitaminK, see Section 5.8), intestinal diseases(malabsorption of vitamins), or parenteral nutri-tion (if not supplemented with vitaminK) [468].In liver diseases, the protein synthesis of theblood clotting factors can be reduced so that inspite of an adequate supply of vitaminK, therecan be a quantitative lack of circulating bloodclotting factors [480]. As in true vitaminK de-ficiency, therapeutic treatment of patients withanticoagulants produces a qualitative lack of theblood clotting factors II, VII, IX, andX (see Sec-tion 5.9.2). The effect of anticoagulants can bereversed by high doses of vitaminK. Whethera decrease in the synthesis of menaquinones inintestinal bacteria due to antibiotics or sulfon-amides can cause vitaminK deficiency is stillunknown (see Section 5.11).

In healthy adults, the risk of vitaminK defi-ciency is very low under normal nutritional con-ditions. Newborn infants, however, have a rela-tively high risk of developing hemorrhagic dis-ease of the newborn (HDN). If newborn infantsare not prophylactically treated with vitaminK,2.5 – 5 per 1000 infants suffer from HDN [491].This disease is manifested in bleeding (e.g., inthe gastrointestinal tract), which can appear onthe first day (early HDN) or in the first week af-ter birth (classical HDN). In late HDN (2nd to10th week), dangerous and often fatal intracra-nial bleeding occurs [434]. The cause of HDNis a lack of blood clotting factors in the blood ofthe newborn as a result of an inadequate supplyof vitaminK. This is due to the low permeabilityof the placenta for vitaminK, limiting the trans-port of the vitamin from the blood of the motherto the fetus. The lack of an intestinal flora, i.e.,absence of menaquinone production in the new-

born during the first days of life, can also play apart [471]. Another serious factor is that breastmilk is a relatively poor source of vitaminK.Consequently, newborns can cover only about20% of their daily vitaminK requirement [469].The requirement of newborn infants is relativelyhigh because vitaminK is not only needed in theliver for the formation of active blood clottingfactors, but rapidly growing bones also requirea considerable amount of vitaminK for the syn-thesis of active osteocalcin [471]. In many coun-tries, newborns are prophylactically given 1mgof phylloquinone at birth to prevent HDN [492].

It is possible that postmenopausal womenalso bear a risk of becoming deficient in vita-minK. In patients suffering from osteoporosis,a decrease in the plasma concentration of phyllo-quinone of up to 30% of the normal value [493]and insufficient γ-glutamyl carboxylation of thecirculating osteocalcin at normal prothrombinvalues have been observed [482]. Administra-tion of vitaminK to these patients increased notonly the plasma level of osteocalcin and the con-tent of γ-carboxyglutamic acid, but also reducedby 50% the previously increased excretion ofcalcium in the urine [471]. However, the physio-logical significance of these results and the asso-ciation between dietary or subclinical vitaminKdeficiency and the pathogenesis of osteoporosismust be further investigated [494].

With regard to animal nutrition, in particularpigs deficient in vitaminK suffer from subcu-taneous hemorrhage [495]. Shortly after wean-ing, young piglets can develop severe internaland external bleeding, a condition called porcinehemorrhagic syndrome. The causes are an insuf-ficient vitaminK content of the feed and sulfon-amide/antibiotic medication [496].

5.10.2. Evaluation of Vitamin K Status

In principle, two methods are available for theevaluation of vitaminK status in humans andanimals: the quantitative determination of vita-minK in the blood or organs by HPLC (see Sec-tion 5.7) or the mostly indirect detection of pro-thrombin or other vitaminK dependent proteinsin the plasma.

The general blood clotting tests for the de-termination of the prothrombin time [480] havea low sensitivity. They are either more sensi-

Vitamins 65

tive to the concentration of factor VII than toprothrombin or they do not respond to a mildvitaminK deficiency [433,482]. For this rea-son, it is better to determine under-γ-carboxyl-ated prothrombin, i.e., prothrombin containinglittle or no γ-carboxyglutamic acid residues (of-ten called PIVKA, prothrombin induced by vita-minK absence or antagonists). The prothrombintime is measured in one sample after activationof prothrombin with thromboplastin (only com-pletely carboxylated prothrombin is activated)and in another sample, after activation of pro-thrombin with a certain snake venom (whichactivates both normal and under-γ-carboxylatedprothrombin, i.e., total prothrombin). The ratioof the two test results is normally ca. 1, but <1under conditions of vitaminK deficiency [497,498]. PIVKA can also be directly determinedcross-immuno-electrophoretically or by usingmonoclonal or monospecific antibodies [498].There are, however, two limitations on the detec-tion of PIVKA: abnormal prothrombin is a veryheterogeneous molecule and, therefore, it is un-known to what extent various forms of PIVKAreact in the different methods. In addition, ab-normal prothrombin is also found in the plasmaof patients suffering fromhepatic cancer or otherliver diseases [499]. Parallel determinations ofthe vitaminK concentration in the blood and ofintact and/or abnormal prothrombinmay be nec-essary to evaluate the vitaminK status more ac-curately.

In vitaminK deficiency, under-γ-carboxyl-ated osteocalcin has been detected in the plasmaby means of a radio-immunological method[498]. However, further studies are necessaryto clarify whether this parameter is suitable forthe evaluation of vitaminK status or can evenbe used as an early indicator of vitaminK defi-ciency. This also applies to the excretion of γ-carboxyglutamic acid (Gla), which results fromthe degradation of vitamin K dependent proteinsand could serve as an indicator for the assess-ment of vitaminK deficiency. In combinationwith the determination of the plasma vitaminK1level, the measurement of Gla in the urine ap-pears to be useful [475].

5.11. Requirement

The daily vitaminK requirement of a healthyhuman is generally estimated at 1 – 2µg/kg ofbody weight [500]. In 1989, the U.S. NationalResearch Council first published recommendeddietary allowances for vitaminK of 1µg per kgof body weight per day (Table 24) [469]. Forbabies, the recommended daily dose is 5µg ofvitaminK for the first six months and 10µg forthe second sixmonths on condition that newborninfants are prophylactically given at least 1mgof vitaminK at birth to prevent bleeding (seeSection 5.10.1). For pregnant or nursingwomen,an increase in the vitaminK requirement is notconsidered necessary [469].

Table 24. Daily vitaminK intake recommended (1989) by the U.S.National Research Council for man [469]

Category Age, Weight, VitaminK,a kg µg

Small children 0 – 0.5 6 50.5 – 1 9 10

Children 1 – 3 13 154 – 6 20 207 – 10 28 30

Young males 11 – 14 45 45and men 15 – 18 66 65

19 – 24 72 7025 – 50 79 80>51 77 80

Young females 11 – 14 46 45and women 15 – 18 55 55

19 – 24 58 6025 – 50 63 65>51 65 65

The amount of vitaminK in the diet con-sumed daily by a healthy American adult is esti-mated to be 300 – 500µg. This estimate is prob-ably too high [469]. In general, however, it canbe assumed that the quantity of vitaminK in thedaily diet is fully sufficient to cover the require-ment under normal nutritional conditions. How-ever, it is doubtful whether the earlier assump-tion that the menaquinones formed by intesti-nal bacteria contribute partially or completelyto meeting the human vitaminK requirementis correct. Studies on volunteers showed that arestriction of the phylloquinone supply to 2 –5µg/d for a period of two weeks caused a sig-nificant decrease in the blood clotting factor VIIand an increase in the concentration of insuf-ficiently γ-carboxylated (abnormal) prothrom-bin in the plasma (see Section 5.10.2), although

66 Vitamins

the relatively insensitive measurements of theprothrombin time remained in the normal range[501]. In a second study on students, a reduc-tion by 50% of the daily supply of vitaminK1(from 82 to 32 – 40µg/d) for a period of threeweeks resulted in a significant decrease in theserum level of phylloquinone, formation of ab-normal prothrombin, and a significant reductionin the excretion of γ-carboxyglutamic acid inthe urine [497]. These results show that dietaryphylloquinone is very important for humans andthat long-chain bacterial menaquinones are notable to prevent the appearance of vitaminK de-ficiency symptoms. The reasons for this includethe highly lipophilic properties, lower bioavail-ability [499], slower turnover, and lower biolog-ical activity of long-chain menaquinones (seeSection 5.9.3).

The vitaminK requirement of some animalspecies is given in Table 25. The high require-ment of poultry is remarkable and was attributedto insufficient absorption of the vitamin fromtheir short digestive tract. However, more re-cent results have shown that the activity of vita-minK epoxide reductase (see also Section 5.9.1)in chicken liver is ten times lower than in therat. This results in high concentrations of vita-minK 2,3-epoxide in the liver and serum of thechicken [466]. In pigs, an increase in the cal-cium content of the feed results in a higher vi-taminK requirement [502], indicating that thereis a relationship between calcium homeostasisand vitaminK supply.

Table 25. VitaminK requirement of various animal species [433]

Species Requirement, Amount in feed,µg/kg body weight µg/kgper day

Dog 1.25 60Pig 5 50∗Rat (male) 11 – 16 100 – 150Chicken 80 – 120 530Turkey 180 – 270 1200

∗Other authors recommend a concentration of 500 –2000µgvitaminK3/kg feed [495,496].

5.12. Application

Phylloquinone is the preferred formof vitaminKfor clinical application. The main indicationsare:

1) Prophylactic treatment of newborn infants toprevent hemorrhagic disease of the newborn

2) Treatment of vitaminK deficiency causedby disturbances of absorption in variousdiseases (e.g., pancreatic insufficiency, bil-iary obstruction, lipid malabsorption), insuf-ficient alimentary supply, or parenteral nutri-tion

3) Countertreatment of anticoagulant therapy4) Prophylactic administration before opera-

tions in which bleeding can become a prob-lem (e.g., gallbladder operations)

In Japan, menaquinone-4 (menatetrenone) isused for the prevention of hemorrhagic diseaseof the newborn [490].

In animal nutrition (pigs and especially poul-try), water-soluble forms of menadione aremainly used to cover the vitaminK require-ment because phylloquinone is too expensivefor this application. Menadione itself is lesssuitable for this purpose because it is insta-ble in feed and its absorption in the intes-tine depends on the presence of lipids in thefeed [433]. The most common water-solubleforms of menadione (3) are menadione sodiumbisulfite (MSB) (67); menadione sodium bisul-fite complex (MSBC) (68); menadione di-methylpyrimidinol bisulfite, known as mena-dione pyrimidinol bisulfite (MPB) (69) [495];and menadione nicotinamide bisulfite (MNB)(70) [503]. MSBC (68) is still more stable thanMSB (67) and, therefore, is widely used in poul-

Vitamins 67

try feeding. In chickens, all these compounds ex-hibit approximately the same biological activityon a molar basis as phylloquinone [504]. Mena-dione is readily converted to menaquinone-4 inthe body of the chicken.

5.13. Tolerance

Toxic effects of phylloquinone and menaqui-nones are so far unknown. Very rare cases ofcardiopulmonary reactions observed on intra-venous administration of phylloquinone weredue to the use of auxiliary agents for the emulsi-fication of the vitamin [470]. Intramuscular in-jections of water-miscible formulations of phyl-loquinone are unproblematic [495].

Today, parenteral administration of mena-dione or of water-soluble forms such as mena-diol sodium diphosphate (71) is no longeremployed. Menadione can react with freesulfhydryl groups in various tissues, caus-ing hemolytic anemias, hyperbilirubinemia, andkernicterus [433]. This side effect of mena-dione is not connected with its conversion tomenaquinone-4 in the organism, but is due to itschemical properties as a quinone. For this rea-son, phylloquinone (ormenaquinone-4 in Japan)is used exclusively today in the medical treat-ment of hemorrhagic diseases and in the pro-phylactic treatment of newborn infants.

In animal nutrition, menadione and water-soluble forms are well tolerated when adminis-tered orally within the dosage range. While theLD50 for a single parenteral application ofmena-dione is 75 – 200mg/kg of body weight in thechicken, mouse, or rat, the LD50 for a single oraldose is 600 – 800mg/kg of body weight. Thismeans that side effects would only be expectedif the amount of menadione ingested daily withthe feed exceeds the daily requirement by a fac-tor of 1000 [504].

5.14. Trade Names and EconomicAspects

Phylloquinone is available for parenteral ap-plication in the form of colloidal suspensions,emulsions, and aqueous suspensions and for oralapplication in the form of tablets and mixedmicelle solutions. In Japan, preparations con-taining menaquinone-4 are also available. Theproducts sold are, e.g., Konakion, KonakionMM, Mephyton, Aquamephyton, and Kay-two(menatetrenone).

World production of phylloquinone is esti-mated at 3000 – 3500 kg/a. The main producersare Roche, Merck, Eisai, and Nisshin Chemical.

Water-soluble forms of menadione aremainly produced in feed-grade quality as feedadditive for poultry feed and other animal feed(e.g., for pigs). The worldwide demand formenadione derivatives is ca. 500 t/a. The mainproducers are Vanetta and Heterochemical.

6. Vitamin B1 (Thiamin)

6.1. Structure and Nomenclature

VitaminB1 (1a) has the IUPAC– IUB name thi-amin [59-43-8], although the term thiamine isused in many official and commercial docu-ments.

The basic skeleton of thiamin containsa thiazole ring and a pyrimidine ring.The exact chemical nomenclature is 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium chloride,C12H17N4OSCl.

Thiamin is used chiefly in the form of itschloride hydrochloride (1b) [67-03-8] and ni-trate (1c) [532-43-4].

68 Vitamins

6.2. History

The history of the discovery of thiamin is closelyassociated with beriberi, a disease prevalent inmany parts of the world where the diet con-sisted mainly of milled or polished rice. Thefirst “cure” was obtained by Takaki, then sur-geon general of the Japanese Navy, who sug-gested that the patients should reduce their car-bon/nitrogen intake ratio by increasing their pro-tein intake with a “Western-style” diet [506]. Inthe 1890s the Dutch physician Eijkman noticedby chance that hens kept on a diet of dehuskedrice developed a disease very similar to humanberiberi [507], and that adding rice bran to thefeed could prevent it. He thus postulated thatberiberi was caused not by a pathogen or bynoxious agents in the food, but by the lack ofa vitally important food constituent. However,this conclusion was firmly established only in1901 by Grijn who showed that this “polyneu-ritis preventive factor” was destroyed in meat orbran by heat [508].Grijn extracted the factor from rice bran,

showing that it was water soluble. The purefactor was isolated from rice bran by Funk,who obtained a crystalline substance that pre-vented beriberi [509]. Having found that thesubstance had the character of an amine, hecoined the term “vitamin” (vitally importantamine). Pure crystals of thiamin hydrochloridewere isolated from rice bran extracts in 1926by Dutch chemists Jansen and Donath [510]who, however, omitted the sulfur atom in the for-mula they published, delaying elucidation of thechemical structure of vitaminB1 [511]. Thiswasachieved in 1936, independently by Williams[512,513] and Grewe [514], who discoveredthat the molecule could be split quantitatively

by sulfite into a 6-aminopyrimidinesulfonic acid(2b) and a thiazole ring (3a) (see section 6.4).

Confirmation of the structure of thiamin wasobtained by Williams who accomplished thefirst total synthesis by building up each ring in-dependently and then linking the two together[515]. The synthetic product was shown to beidentical to the natural compound by its UVspectrum and biological tests on microorgan-isms and animals. In 1937 Todd and Bergelsynthesized thiamin by a different approach[516,517], soon followed by Andersag andWestphal [518].

Industrial production was started in 1937 byHoffmann-La Roche in Switzerland and Merckin the United States. Demand and productionrose rapidly.

6.3. Physical Properties

Thiamin chloride hydrochloride (1b)C12H18N4OSCl2 is a white crystalline mate-rial, mp 248 – 250 ◦C (decomp.). It crystallizesas colorless monoclinic needles. It has a charac-teristic odor and a slightly bitter taste. It is solu-ble in water (1 g/mL) and glycerin (1 g/18mL),sparingly soluble in alcohol or acetone, and al-most insoluble in other organic solvents. Onexposure to air of average humidity, dry thiaminis converted to the hydrate by water absorption(up to 4wt%). The water can be removed byheating at 100 ◦C, or under vacuum over con-ventional drying agents. If protected from lightand humidity, thiamin is relatively stable againstair oxidation, even on heating.

Thiaminnitrate (1c)C12H17N5O4S is awhitecrystalline material with mp 190 – 200 ◦C (withdecomposition). It shows weak solubility in wa-ter (about 2.7 g per 100mL).

The UV spectrum of thiamin chloride hydro-chloride (1b) in aqueous solution showsmaximaat 234 nm (ε1= 307) and 266 nm (ε1= 277) [519,520]. Its IR spectrum inKBr is reported in [521].

The 1HNMR spectrum of thiamin chloridehydrochloride (1b) in D2O is reported in [522],and the 13C and 15NNMR spectra in [523–525].

Since the mass spectrum of thiamin chloridehydrochloride (1b) shows no molecular ion un-der standard EI conditions, literature refers todata obtained using the fast atom bombardmenttechnique [526–528].

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X-ray crystallographic data [529] andmolec-ular orbital calculations of the electron density[530] have also been published.

6.4. Chemical Properties

Hydrolysis. Under mildly alkaline aqueousconditions (at pH 7.0 or above), thiamin is con-verted to the thiol form (4) (Fig. 5). The rate-limiting step appears to be ring opening [520,531,532].

A 5% aqueous solution of the hydrochloride(1b) has pH ca. 3.5, whereas the correspondingsolution of the nitrate (1c) has pH 6.5 – 7.1.

Under normal conditions, an aqueous solu-tion of thiamin is stable below pH5.5, evento oxidation, but decomposes to (4-amino-2-methyl-5-pyrimidinyl)methanol (2a) [73-67-6]and 4-methyl-5-(2-hydroxyethyl)thiazole (3a)[137-00-8] when heated in a sealed tube at140 ◦C (Fig. 5) [533].

By treatment with sulfite in weakly acidic so-lutions, thiamin chloride (1a) is split into themethanesulfonate derivative (2b) and the thia-zole compound (3a) (Fig. 5) [511,512]. Understrongly acidic conditions, thiamin chloride (1a)is converted to oxythiamin (5), which has no vi-tamin activity (Fig. 5).

Various aspects of the chemistry of thiaminare still under investigation, especially the re-versible opening of the thiazole ring in aqueousalkaline solution [528,534–537].

Intramolecular Aminolysis. Understrongly basic, but anhydrous conditions (2molof sodium ethoxide in ethanol), the 4′-ami-no group of thiamin adds to the thiazole ringto give tricyclic dihydrothiochrome (6), whichthen eliminates a thiolate ion to give the sodiumsalt of the yellow form of thiamin (7) [531,538–540]. Addition of acid converts 7 to thethermodynamically more stable thiol form (4)(Fig. 6).

Oxidation. In the presence of an oxidant,the thiolate (7) is irreversibly converted tothiochrome (8) (Fig. 6). Thiochrome (8) is a yel-low crystalline compound which has been iso-lated from yeast but has no physiological im-portance. In solution it exhibits a strong blue

fluorescence, a property used for the quantita-tive determination of thiamin (see Section 6.11).Reduction of thiochrome (8) converts it back tothiamin, in up to 60% yield.

Ylid Formation. Upon abstraction of the hy-drogen atom from the 2-position of the thiazolering, the ylid of thiamin (9) is formed (Fig. 6),which plays a central role in both the coen-zymatic reaction of vitaminB1 as well as innonenzymatic reactions such as the acyloin con-densation. The ability of thiamin to form anylid can be rationalized in terms of molecu-lar orbital theory and resonance structures [534,541]. There is, however, widespread disagree-ment about whether the 4′-amino group acts asan intramolecular acid or base in either the chem-istry or the enzymology of ylid formation fromthiamin. Isolation of the ylid in the solid stateafter deprotonation of thiamin chloride with oneequivalent of sodium ethoxide has been the sub-ject ofmuch controversy [531,534], the tricyclicstructure (6) being favored over a tetracyclic one,based on NMR studies. Kinetic and thermody-namic studies have been made to interpret thelability of the 2-proton in the thiazole ring ofthiamin [542–544].

The pKa of thiamin in aqueous solution at25 ◦C is estimated at 12.6 for the CH in the thia-zole ring and 4.8 for the NH2 of the pyrimidinemoiety [531,541].

Reduction. Thiamin undergoes reduction ofthe thiazole ring by a variety of reducing agents(lithium aluminum hydride, sodium borohy-dride, etc.) to afford, via the intermediate di-hydrothiamin (10), tetrahydrothiamin (11) [545,546] (Fig. 7).

6.5. Natural Occurrence and Isolation

Thiamin is widespread in nature, but only in rel-atively small quantities [547–549].

Average thiamin concentrations in foodstuffs(in µg/100 g) are as follows:

Wheat germ 2050Brewer’s yeast, dried 1820Soyabeans 1300Dried beans and peas 680

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Figure 5. Reactions of thiamin chloride (1a)

Nuts 300 – 560Rice, brown 300Rice, white 50Potatoes 170Beef heart 600Beef liver 300Pork 600 – 950Milk whey, dried 500Whole milk 30 – 70Eggs 70Fish 50 – 90Vegetables (flower and fruit) 70Vegetables (leaf and stem) 70Vegetables (root and tuber) 60

Thiamin can be found in animal tissue inphosphorylated forms, mainly as the pyrophos-phate (12b) [154-87-0] (see section 6.9), whereit is bound to an enzyme as a protein complex.The heart, liver, kidney, and the brain are or-gans with a high thiamin concentration of ca.100mg/100 g. Normal human blood containsca. 90 ng of thiamin per liter, although thereare strong variations among individuals, with49 ng/L being considered as indicative of a pos-sible deficiency.

In plant products, free thiamin is the mostabundant form. It is found in the pericarp and theseeds of grains, cereal grains, yeast, dried veg-etables, rice, and potatoes. Oils, fats, and highlyprocessed foods such as refined sugars are es-sentially devoid of thiamin.

Although thiamin is widespread in food-stuffs, its concentration in individual foodsvaries widely and is relatively low since consid-erable amounts are destroyed in cooking, eitherbyheat, the presence ofmetals, chlorine inwater,or by reactive organic substances. Therefore, torestore the nutritional quality of processed foodsto the original or acceptable levels, they have tobe supplemented with synthetic thiamin or itsderivatives. Thus in developed countries mostwhite rice and white flour are fortified with vita-minB1. Since extraction of thiamin from naturalsources would not be economically profitable, ithas to be manufactured by chemical synthesis.

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Figure 6. Reactions of thiamin chloride (1a)

Figure 7. Reduction of thiamin

6.6. Biosynthesis in Microorganisms

Thiamin pyrophosphate (12b) is synthesizedin prokaryotic and eukaryotic microorganismsfrom the precursor compounds hydroxymeth-

ylpyrimidine (2a) and hydroxyethylthiazole(3a). The biosynthetic pathways leading to theseprecursor compounds are different in faculta-tive anaerobic bacteria, e.g., Escherichia colior Salmonella typhimurium, and in lower eu-

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karyotes such as Saccharomyces cerevisiae orSchizosaccharomyces pombe. Reviews coveringthiamin biosynthesis up to the mid-1980s canbe found in [550,551]. The latter is restricted toE. coli and S. typhimurium.

6.6.1. Biosynthesis of the PyrimidineComponent in Prokaryotes

One of the key findings revealing the origin ofthe pyrimidine component (2a) of thiamin inprokaryoteswas the observation that certainmu-tants of S. typhimurium defective in one of theearly steps of purine biosynthesis were thiaminauxotroph as well [552–555].

Specifically, mutants defective for conver-sion of phosphoribosyl pyrophosphate (PRPP)to phosphoribosyl aminoimidazole (13) (AIR)(e.g., purF mutants) required thiamin supple-mentation in addition to purine nucleotides forgrowth (Fig. 8). Purine mutants impaired in con-version of AIR (13) to inosine monophosphate(IMP), however,were independent of exogenousthiamin supply. These and other observations[550,551] suggested that purine nucleotide andthiamin biosynthesis use the same route up toproduction of AIR (13). Conversion of AIR (13)to the pyrimidine component (2a) is a complexintramolecular rearrangement, which in E. colidepends on the thiC gene product (see below).

Additional pathways to the pyrimidine com-ponent (2a) in S. typhimurium, independent ofpurF, have been suggested [556–558].

6.6.2. Biosynthesis of the PyrimidineComponent in Eukaryotes

An initial indication that bacteria and yeast em-ploy different biosynthetic pathways to the py-rimidine component (2a) of thiamin was thefinding that in yeast cultivated with 14C-labelledformate the 14C atom was recovered at theC4 position of the pyrimidine component (2a)[559].

In contrast, the pyrimidine component from14C-formate-fed prokaryotes contained the 14Clabel in the C2 position. Feeding of S. cerevisiaewith 14C- or 15N-labelled histidine resulted innonrandomly labelled thiamin [560,561] and in-dicated that N3, C4, and the amino nitrogen N9

of the pyrimidine moiety (2a) were derived as aunit from theN –C –Npart of the imidazole ringof histidine. The amino nitrogen of histidine wasnot incorporated. The source of C2, C5, C6, themethoxy C7, and the methyl C8 atom of the py-rimidine component (2a) was ribose [562–564].A minor pathway to the pyrimidine component(2a) in eukaryotes has been suggested in whichthe single carbon atom of formate is incorpo-rated into the C2 position. This pathway, how-ever, is not identical to the hydroxymethylpyrim-idine biosynthesis pathway in prokaryotes [562].

6.6.3. Biosynthesis of the ThiazoleComponent

Cysteine is the common sulfur source forbiosynthesis of the thiazole component (3c) ofthiamin [565]. In yeasts the N and C2 atoms areobtained from glycine (Fig. 9) [566,567]. In theyeast Candida the source of the C4 to C8 atomsis ribulose-5-P (14) derived from the oxidativebranch of the pentose phosphate pathway [568].In S. cerevisiae the pentose contributing C5 toC8 of the thiazole component (3c) is derivedfrom the oxidative and nonoxidative branchesof the pentose phosphate pathway to a similarextent [569,570].

In facultative anaerobic bacteria such asE. coli andS. typhimurium theC2andNatomsofthe thiazole component (3c) are derived from theα-carbon and nitrogen atom of tyrosine (Fig. 10)[571–573]. The C5 sugar d-1-deoxyxylulose(15), derived from the glycolysis intermedi-ates pyruvate and glyceraldehyde-3-phosphate,serves as donor for the C4 to C8 carbon atomsof the thiazole moiety (3c) [574].

In aerobic bacteria such as B. subtilis the C2and N atoms of the thiazole component (3c) arederived from glycine, as in yeasts [575].

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Figure 8. Biosynthesis of the pyrimidine component in prokaryotes

Figure 9. Biosynthesis of the thiazole component in yeasts

6.6.4. Biosynthesis of ThiaminPyrophosphate

Biosynthesis of thiamin pyrophosphate (12b)from the pyrimidine (2a) and thiazole (3a)precursors (Fig. 11) depends on the activ-ity of five enzymes, four of which are ki-nases. Hydroxymethylpyrimidine kinase andphosphomethylpyrimidine kinase convert py-rimidine component (2a) to the mono- (2c)and diphosphorylated (2d) derivatives, respec-tively. The enzymes have been purified fromyeast [576,577]. Hydroxyethylthiazole kinasecatalyzes phosphorylation of the thiazole com-ponent (3a) to the phosphorylated derivative(3c). An S. cerevisiae mutant defective for thisenzyme grew well on minimal medium with-

out thiamin supplementation [578]. It was con-cluded that in yeast, hydroxyethylthiazole ki-nase is not required for de novo synthesis ofthiamin but seems to be involved in a sal-vage pathway of thiamin biosynthesis using pre-formedhydroxyethylthiazole (3a). The couplingenzyme thiamin phosphate synthase has beenpurified from S. cerevisiae and partially puri-fied from E. coli [579,580]. Hydroxyethylthi-azole kinase and thiamin phosphate synthaseof S. cerevisiae mapped in the same gene, andthe two enzymatic activities could not be sepa-rated during purification to apparent homogene-ity. This finding indicated a bifunctional enzymein the thiamin pyrophosphate biosynthetic path-way of yeast [581].

74 Vitamins

Figure 10. Biosynthesis of the thiazole component in facultative anaerobic bacteria

Figure 11. Biosynthesis of thiamin pyrophosphate

In E. coli, thiamin monophosphate (12a) canbe derived from exogenous thiamin (1a) bymeans of the enzyme thiamin kinase. Conver-sion of thiamin monophosphate to the activecoenzyme thiamin pyrophosphate (12b) is cat-alyzed by thiamin monophosphate kinase. In

yeasts and many other organisms, including hu-mans (for references, see [582]), thiamin py-rophosphate (12b) can be obtained from exoge-nous thiamin (1a) by a one-step phosphorylationreaction catalyzed by thiamin pyrophosphoki-nase.

Vitamins 75

6.6.5. Thiamin Biosynthetic Genes fromE. coli

A number of mutations leading to thiamin aux-otrophy in E. coli (thimutants) mapped at 90′ ofthe E. coli chromosome [583,584]. A 6.5 kilo-base genomic DNA fragment was isolated fromE. coli that complemented all of the mutationsclustered in the 90′ thi locus [585]. Five tightlylinked open-reading frames, designated as thiC,thiE, thiF, thiG and thiH were identified. To eval-uate the function of the genes encoded by theseopen-reading frames mutants were constructed,each of them carrying another defect in one ofthe five open-reading frames. The growth re-quirements of these mutants indicated that thethiC gene product catalyzes an essential stepin biosynthesis of the pyrimidine component(2a) of thiamin from 5-aminoimidazole ribonu-cleotide (13) (AIR) (see Section 6.6.1). The thiE,thiF, thiG and thiH mutants responded to thia-zole (3a), but not to d-1-deoxyxylulose (15), in-dicating that the thiE, thiF, thiG, and thiH genesare involved in synthesis of the thiazole compo-nent (3a) from the precursor compounds tyro-sine, cysteine, and d-1-deoxyxylulose (15) (seeFig. 10).

The thiG gene exhibits significant homologyto the chlN gene of E. coli [586], encoding anenzyme of the molybdopterin biosynthetic path-way [587].Molybdopterin complexedwith aMoatom is the cofactor of Mo-containing enzymes,e.g., respiratory chain nitrate reductase, formatedehydrogenase, or biotin-d-sulfoxide reductase[588, and references therein]. Molybdopterin,like the thiazole component (3a) of thiamin, con-tains a sulfur atom, suggesting a role of the chlNand the thiG gene products in sulfur incorpo-ration into molybdopterin and into the thiazolecomponent (3a), respectively.

The kinases encoded by thiD, thiM, thiL andthiK , which are involved in thiamin pyrophos-phate biosynthesis from the pyrimidine (2a) andthiazole component (3a) or from thiamin (1a)(see Fig. 11), have been mapped on the E. colichromosome [589,590].

The nuvC gene located at 45′ of the E. colichromosome was found to be involved in thi-amin pyrophosphate biosynthesis, as well [591].The nuvC gene product is one of the two en-zymes, designated factors A and C, responsi-ble for modification of the uridine moiety of

certain tRNAs with sulfur. E. coli nuvC mu-tants lacking factor C were found to be thi-amin auxotroph in addition to being unable toproduce 4-thiouridine. The mutations could becomplemented by administration of hydroxy-ethylthiazole (3a). One could speculate that fac-tor C, which transfers the sulfur atom from cys-teine to tRNA uridine to form 4-thiouridine, isalso responsible for delivering a sulfur atomfrom cysteine to the thiazole component (3a).

6.6.6. Mapping and Cloning of ThiaminBiosynthetic Genes from Other Species

Thi genes complementing thiA32 (hydroxy-ethylthiazole (3a) requirement), thiC34 (hy-droxymethylpyrimidine (2a) requirement) andto some extent thiB33 mutations (coupling de-fect) of E. coli mutants have been mapped onthe second symbiotic megaplasmid of Rhizo-bium meliloti [592].

Three classes of B. subtilis thimutants can bedistinguished according to the dependence onhydroxymethylpyrimidine (2a) (thiA mutants),hydroxyethylthiazole (3a) (thiBmutants) or thi-amin (thiCmutants) for growth [593]. (Note thatthe nomenclature of thimutants inB. subtilis andE. coli is not consistent.) Whereas in E. coli thegenes involved in biosynthesis of the two thi-amin precursor components and the couplingenzyme are tightly clustered, the thiA, -B, and-C mutations of B. subtilismapped at 70◦, 102◦,and 331◦ of the bacterial chromosome, respec-tively [594]. During the course of the Bacillussubtilis genome sequencing project a 97 kb re-gion from 325◦ to 333◦ including the thiC locushas been cloned and sequenced [595].Schizosaccharomyces pombe thi2 mutants

required hydroxyethylthiazole (3a) for growth.The thi3 and thi4 mutants could be comple-mentedwith hydroxymethylpyrimidine (2a) andthiamin (1a), respectively [596]. The thi3 gene,which is identical to the nmt1 gene [597], hasbeen isolated and sequenced. The thi2 genehas been cloned [598], but the sequence hasnot been published so far. The thiamin biosyn-theticnmt2geneofSchizosaccharomyces pombehas been cloned and sequenced [599]. The thi4gene [600], which should be involved in phos-phorylation of one of the two thiamin precursorcomponents or in the coupling step, has some

76 Vitamins

weak homology to the E. coli thiE gene, whichis required for biosynthesis of the thiazole com-ponent (3a). There was no further sequence ho-mology observed between the thi genes ofE. coliand Schizosaccharomyces pombe in accordancewith the different biochemical routes leading tothiamin pyrophosphate (12b) in yeast and bac-teria. Recently the DNA sequence of the thi80gene encoding thiamin pyrophosphate kinasefrom yeast has been reported [582].

6.6.7. Regulation of Thiamin Biosynthesis inProkaryotes

Thiamin biosynthesis is tightly controlledby feedback and repression mechanisms. InS. typhimurium, thiamin, which has no effect onPRPP aminotransferase, interfered with biosyn-thesis of pyrimidine (2a) and thiazole com-ponent (3a) at 40 ng/mg dry cell weight, andwith the coupling reaction at 100 ng/mg drycell weight [601,602]. In addition, hydroxy-ethylthiazole (3a) inhibited its own synthesis at1 ng/mg dry cell weight, whereas hydroxymeth-ylpyrimidine (2a) did not interfere with its ownsynthesis.

The hydroxymethyl- and phosphomethylpy-rimidine kinases, hydroxyethylthiazole kinase,and the coupling enzyme thiaminphosphate syn-thase (see Section 6.6.4) are repressed in E. coliby intracellular thiamin [584,603,604]. MutantE. coli strains resistant towards the thiamin an-timetabolite pyrithiamin exhibited similar ac-tivities of the coupling enzyme and hydroxy-ethylthiazole kinase, irrespective of the intracel-lular thiamin concentration. Themutation in oneof these strains, designated PT-R1, mapped at90′ of the E. coli genome and is probably veryclose to the E. coli thi locus.

6.6.8. Regulation of Thiamin Biosynthesis inYeasts

In S. cerevisiae two positive regulatory genes ofthiamin biosynthesis have been described andcloned [605–607]. These genes were designatedthi2 ( pho6) and thi3 similar to the structuralthi genes of S. pombe. Mutations in S. cerevisiaethi2 and thi3 resulted in thiamin auxotrophy

and affected synthesis of thiamin monophos-phate (12a) from the pyrimidine (2a) and thi-azole component (3a). In addition, thi3mutantshad a markedly impaired thiamin transport sys-tem.

As in S. typhimurium and S. cerevisiae,biosynthesis of thiamin is negatively regulated inS. pombeby thiamin [596]. Expression of the thi-amin biosynthetic genes thi2 and thi3 involved inbiosynthesis of the thiazole (3a) and pyrimidine(2a) components in S. pombe, respectively (seeSection 6.6.6), is strongly repressed by thiamin[598]. In addition, the thi2 gene is repressed byhydroxyethylthiazole (3a), but not by hydroxy-methylpyrimidine (2a), similar to the situationin S. typhimurium. Several mutations designatedtnr1, 2, and 3 have been mapped in the genomeof S. pombe, resulting in de-repressed expres-sion of genes involved in thiamin biosynthesis[608]. Intracellular levels of thiamin (1a), butnot of thiamin monophosphate (12a) or thiaminpyrophosphate (12b), in tnr3 were 10- to 20-fold higher (ca. 10 ng/mg dried cells) than intnr1, 2 or in the wildtype. In addition to mu-tations in the tnr genes that might encode nega-tive regulators of thiaminbiosynthesis, a positiveregulatory gene designated thi1 has also beenidentified in S. pombe. Thi1mutants are thiaminauxotroph. They can be complemented with hy-droxyethylthiazole (3a), but not with hydroxy-methylpyrimidine (2a), indicating that thesemu-tants can not produce the thiazole component(3a). Accordingly, no thi2 mRNA encoding astructural gene of thiazole biosynthesis could bedetected in S. pombe thi1 mutant strains.

6.6.9. Thiamin-OverexpressingMicroorganisms

E. coli PT-R1 mutants which are deregulatedfor thiamin biosynthesis (Section 6.6.7) ac-cumulated ca. 100 ng thiamin/mg dry cellularweight [584], which corresponded to a three-to fourfold increase of thiamin levels comparedto wildtype E. coli strains. Similar levels ofthiamin pyrophosphate (12b) were reached inS. typhimurium 1.5 h after derepression of thi-amin biosynthesis caused by adenosine prein-cubation [602]. An S. typhimurium thi mutantdefective in thiazole biosynthesis accumulatedhydroxymethylpyrimidine (2a) in the medium.

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Certain Neurospora crassa mutants con-taining a defect in biosynthesis of the thiazolecomponent (3a) secreted up to 0.2mg/L of sub-stances closely related to the pyrimidine com-ponent (2a) into the culture broth. The chemicalnature of one of themwas identified as being thediamine (2e) [609,610]. A broad search amongmany yeast species has been reported [611]. TheSaccharomyces cerevisiae strain 1271 secretedup to 200mg/L thiamin into the culture broth.

6.7. Chemical Synthesis

Several syntheses of thiamin (1) have been pub-lished, and basically two general methods haveevolved over the years [612–616].

6.7.1. Condensation of the Pyrimidine andThiazole Rings

The first approach, developed after the origi-nal method reported by Williams et al. [514],consists of separate syntheses of 4-amino-5-bromomethyl-2-methylpyrimidine hydrobro-mide (2f) [2908-71-6] and of the thiazolemoiety(3a) or its acetate (3b) [656-53-1]. Condensationof the two intermediate heterocycles gives thi-amin bromide hydrobromide [4234-86-0] (1d)which is converted to thiamin chloride hydro-chloride (1b) by treatment with silver chloridein methanol or with an ion-exchange resin.

The first industrial synthesis of vitaminB1,developed by Merck-Rahway (United States),followed this synthetic route.

6.7.2. Construction of the Thiazole Ring ona Preformed Pyrimidine Portion

The second general method, based on theprocedure of Todd and Bergell [516,517]and Andersag and Westphal [518], con-structs the thiazole ring on a preformed py-rimidine intermediate. Thiamin manufacturersgenerally follow this approach. Some use mal-onitrile [109-77-3] as the starting material forconstruction of the 4-amino-5-aminomethyl-2-methylpyrimidine ring (2e) [95-02-3] (alsocalled Grewe diamine). Others start with acry-lonitrile but all proceed via the key Grewe di-amine intermediate (2e) through to thiothiamin[299-35-4] (16) (see page 82).

For the preparation of malonitrile, β-ami-nopropionitrile (17a), obtained by addition ofammonia to acrylonitrile, is subjected to oxida-tive dehydrogenation in the gas phase at hightemperature and in the presence of molecularoxygen and a metallic catalyst [617].

Pyrimidine Moiety. A convenient synthe-sis of the pyrimidine moiety starts from ac-etamidine hydrochloride [124-42-5] or thecorresponding imino ether and a suitablysubstituted C4 unit carrying the requiredfunctional groups, such as methoxymethy-lenemalononitrile [672-25-3] (18a), amino-methylenemalononitrile [672-81-1] (18b), 2-dimethoxymethyl-3-methoxypropionitrile (19),or α-amino-β-formylaminopropionitrile (20)(aniline, o-chloroaniline or N-methylaniline arethe most frequently used α-amino groups).

Subsequent conversion of the resulting in-termediates – 5-cyanopyrimidine [698-29-3](21a), N-[(4-amino-2-methyl-5-pyrimidinyl)-methyl]-acetamide [23676-63-3] (2g), N-[(4-amino-2-methyl-5-pyrimidinyl)-methyl]-form-

78 Vitamins

amide (2h) – provides the desired Grewe di-amine (2e) [613,618–623].

The Takeda andUbe in Japan have jointly de-veloped a new process to prepare the key inter-mediate Grewe diamine (2e) [624,625]. Acry-lonitrile and carbon monoxide are used as ba-sic raw materials. Nitrite-catalyzed oxidation ofacrylonitrile in the presence of an alcohol af-fords 3,3-alkoxypropanenitrile (22). This sameintermediate can also be obtained, probablymore expensively, by formylation of acetoni-trile, followed by acid-catalyzed acetalization[626,627]. Alternatively, 22 can be generated byelectrooxidation of β-aminopropionitrile (17b)[628].

Carbonylation of dialkoxypropanenitrile (22)followed by acid-catalyzed acetalization andsubsequent thermal elimination of alcohol yields2-(dialkoxymethyl)-3-alkoxy-2-propenenitrile(23). On reaction with acetamidine, 23 gives inhigh yield the acetal (21b), which after hydro-lysis to the 2-methyl-4-amino-5-pyrimidinecar-boxaldehyde [73-68-7] (21c) and reductive am-ination provides the desired Grewe diamine (2e)[629–631].

In a different approach [632], 5-cyano-1,6-dihydropyrimidine (25), prepared byThorpe –Ziegler cyclization of N ′-cyano-N-(2-cyanoethyl)-acetamidine (24), is converteddirectly to the desired Grewe diamine (2e) by

dehydrogenation in the presence ofRaney cobaltand ammonia.

Thiamin Synthesis. Having obtained 4-ami-no-5-aminomethyl-2-methylpyrimidine (2e),three chemical steps are still required to ob-tain thiamin (1a): extension of the amino-methyl side chain at the 5-position, cycliza-tion to the thiazole ring, and conversion to thi-amin. Commonly used synthons for extendingthe side chain are 3-chloro-5-hydroxypentan-2-one [13045-13-1] (26a), the 3-mercaptoketone[15678-01-0] (26b) or their corresponding ac-etates [13051-49-5] (26c) and [55289-66-2](26d) [633,634]. Besides the multistep prepara-tion of chloroketone (26c), a catalytic procedurehas been developed in which a mixture of α-chloroacetylacetone (27) and ethylene oxide isreacted at 80 ◦C in an autoclave in the presenceof nickel(II) acetylacetonate as catalyst [635].

Condensation of 4-amino-5-aminomethyl-2-methylpyrimidine (2e) with carbon disulfideand chloropentanone (26a) provides in verygood yield the dithiocarbamate (28), which isthen converted by acidic treatment to the 4-thiazoline-2-thione derivative (16) [636–639].Oxidative desulfurization with hydrogen perox-ide followed by anion-exchange resin completesthe synthesis of thiamin hydrochloride (1a).

Vitamins 79

Similarly, thiamin hydrochloride (1b) canbe prepared by condensation of N-[(4-amino-2-methyl-5-pyrimidinyl)methyl]methanethio-amide (2i) [31375-20-9], or N-formyl Grewediamine (2h) with ester (26c) or (26d), respec-tively [633,634,640]. Reaction of Grewe di-amine (2e) with α-mercaptoketone (26b) andformaldehyde provides dihydrothiamin (10)which can then be converted to thiamin (1a)[641,642].

A more convergent synthesis of thiamin(1b) has been developed by Hoffmann-LaRoche [643,644]. Condensation of 3,4-di-hydro-7-methylpyrimido[4,5-d]pyrimidine (29)[31375-19-6] with the mercaptopentanone(26d) in formic acid followed by treatment withhydrochloric acid gave thiamin in high yield. 29is obtained by heating Grewe diamine (2e) indimethylformamide dimethyl acetal [645–647]or alternatively, in an excess of triethyl orthofor-matewith a catalytic amount of p-toluenesulfon-ic acid.

6.8. Commercial Forms

The commercially available forms of thiamin arethe chloride hydrochloride (known as thiaminhydrochloride) (1b) and the mononitrate (1c).World production of thiamin in 1993 was esti-mated at ca. 4200 t; the principal producers areHoffmann-La Roche, Takeda Chemical Indus-tries, and Chinese State Companies. The pricefor thiamin in 1993 on the U.S. market was ca.$ 30 – 35/kg.

The discovery in 1951 by Fujiwara et al.[648] that treatment of thiamin with an ex-tract of garlic or other Allium species convertsit to a product that is physiologically very ac-tive opened the door to derivatives of thiaminthat act as “prodrugs.” The mixed thiamin allyldisulfide, TAD, (30a) (allithiamin [554-44-9])was the first representative of a variety of thi-amin alkyl sulfides. The most important com-mercial forms, almost exclusively marketed in

Japan (they are not yet approved in the UnitedStates) are TAD, thiamin propyl disulfide,TPD, (30b) (prosulthiamin [59-58-5]), thiamintetrahydrofurfuryl disulfide (30c) (fursulthiamin[804-30-8]), O-benzoylthiamin disulfide (31)(bisbenthiamin [2667-89-2]), and dibenzoylthi-amin [299-88-7] (32). These forms aremore sta-ble, less water soluble and more lipid soluble,being thus more rapidly absorbed and better re-tained in the body than the more common thi-amin hydrochloride. In vivo they are convertedback to thiamin [649].

6.9. Derivatives, Analogues, andAntimetabolites

Thiamin forms esters at the hydroxyethyl sidechain with various acids, the most important be-ing themono- (12a), di- (12b), and triphosphates(12c). The pyrophosphate (12b) acts as a coen-zyme (cocarboxylase) in the decarboxylation ofα-keto acids in association with carbohydratemetabolism. The palmitate (33) is as effectiveas thiamin and is used for supplementation ofvarious feed and food products due to its stabil-ity and good lipid solubility [650].

In Japan, derivatives of thiamin with sugars,especially with saccharide, have also been pre-pared as food additives. They are readily hydro-

80 Vitamins

lyzed to physiologically active thiamin and, un-like thiamin, have no unpleasant smell.

Other derivatives are obtained from the thiolform of thiamin (see Section 6.8).

Several analogues have been synthesized bystructural modification of the pyrimidine or thia-zole ring, but none exceeds the biological activ-ity of thiamin. The 2-ethyl analogue (34) showsactivity comparable to that of thiamin, whereasthe 2-butyl (35), 2-methoxy (36), and 2-methyl-thio (37) analogues are potent antagonists [651–653].

Replacement of the 4-amino group by hydro-gen or an alkylamino group generally results inloss of activity [652,654]. Oxythiamin (5) (seesection 6.4), the 4-hydroxy (or 4-oxo) analogueis a known antagonist.

From analogues 38 and 39 it was concludedthat the nitrogen atom in the 1-position is moreimportant for coenzyme activity than that at the3-position [655,656].

Compounds 40 and 41 [657], the 2-methylsubstituted analogue 42 [658], oxothiamin

(43) and thiothiamin (16) are completelyinactive [659–661], whereas the homothi-aminglycol (44) is an antagonist [662], demon-strating that hydrogen at the 2-position and the3-hydroxyethyl group at the 5-position in the thi-azole ring are essential for biochemical activity.

Replacement of the thiazole ring by other het-erocycles, as in imidazolthiamin (45) [652,663],pyrithiamin (46) [659,664,665] or Amprolium(47) [666] provides potent thiamin antagonists.The latter is a good inhibitor of thiamin diphos-phate dependent enzymes. In small doses, it af-fects thiamin transport across bacterial cell wallsand is therefore an effective coccidiostat. It isused commercially for the treatment of poultry[667,668].

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6.10. Biochemical and PhysiologicalFunctions

6.10.1. Metabolic Functions in the Organism

Thiamin serves a number of essential metabolicfunctions and its deficiency is associated withimbalances in carbohydrate status, with conse-quent deleterious effects on nerve functions. Inliving systems, the only known biologically ac-tive form of thiamin is the diphosphate ester(12b) – otherwise known as thiamin pyrophos-phate, TPP, or cocarboxylase –which is formedby the reaction between thiamin and ATP inliver cells [669]. As a cofactor of enzymes in in-termediate metabolism, TPP participates in thedecarboxylation of α-keto acids (pyruvate andα-ketoglutarate dehydrogenase complex) andin the reversible α-ketol transfer reactions cat-alyzed by transketolase in the pentose phosphatecycle [670,671].

Oxidative Decarboxylation. Pyruvate is animportant intermediate product of glycolysis,a largely anaerobic process. Under these con-ditions, pyruvate is decarboxylated to acetyl-coenzymeA (acetyl-CoA), a universal productof the metabolism of carbohydrates, fats, andamino acids. The decarboxylation reactions areinitiated by addition of pyruvic acid to the 2-position of the thiazole ylid (9) of TPP to formthe adduct (48a). The adduct after the splitting-off of carbon dioxide forms the so-called ac-tive aldehyde (49) from which the subsequentbiochemical reactions are derived, leading ulti-mately to acetyl-coenzymeA [672] (see bottomof page).

To support this mechanism, the protonatedform of the adduct between thiamin and acetal-dehyde – 2-(1-hydroxyethyl)thiamin (50) – hasbeen synthesized [673] as well as the thiamin –pyruvate adduct and its pyrophosphate (48b)[674,675].

In the citric acid cycle, TPP similarly takespart in the oxidative decarboxylation of α-ketoglutaric acid to succinyl coenzymeA. Anal-ogously, the oxidative decarboxylation of thethreebranched-chain α-ketoacids derived fromthe deamination of leucine, isoleucine, and va-line is catalyzed by TPP among a multienzymecomplex.

Transketolase Reactions. In the transketo-lase reactions (direct glucose oxidation), TPPadds to appropriate α-ketosugars to break thecarbon – carbon bond between C2 and C3 to

82 Vitamins

form an active intermediate which is then trans-ferred to a suitable acceptor. This metabolicpathway is important for the production of pen-toses for RNA and DNA synthesis.

Other Functions. Thiamin may be an activesubstance in the nervous system. It has been pos-tulated that thiamin, probably as thiamin triphos-phate (12c), could play an essential role in thestimulation of peripheral nerves [676–678].

This could be a distinct function of thiamin,independent from its role as a coenzyme inthe metabolism of carbohydrates. This remains,however, to be established.

6.10.2. Thiamin Requirements andDeficiency

The maintenance requirements for thiamin de-pend on carbohydrate intake. An average figureof 20 – 30µg per kilogram body weight is givenfor humans. This corresponds to a recommendeddaily allowance ranging from 0.3 to 1.5mg/d,depending on age and sex; this recommendationdiffers from country to country. Factors such aspregnancy, lactation, fever, alcoholism, liver dis-ease, stress, or dietary practices can substantiallyraise the minimum requirement.

Beriberi is the most prominent pathologicalcondition resulting from a thiamin deficiency.Symptoms of the disease can vary, depending onindividual, diet, duration and severity of the de-ficiency. The disorders affect the central nervoussystem, but are also accompanied by cardiovas-cular damage.

In most cases, parenteral administration ofthiamin in large doses is sufficient to restore thenormal supply status of thiamin. For better ab-sorption from the digestive organs, derivativesof the thiol form of thiamin are used in Japan(see Section 6.8).

6.11. Analytical Methods

For the determination of thiamin in foods,feeds, pharmaceutical preparations and othersubstances, physicochemical [679,680] and mi-crobiological [680] methods are employed.

Since thiamin occurs principally as phos-phate esters and a significant part is therefore

protein bound, extraction procedures have beendesigned to free thiamin before analytical meth-ods are employed. For some more complex bi-ological materials, further treatment or purifi-cation may be necessary to remove compoundswhich might interfere with the analyses.

The fluorometric thiochrome method is atpresent the most widely used method for mea-suring free thiamin. It is based on the oxida-tion of thiamin (1a) to thiochrome (8) by an al-kaline solution of potassium ferricyanide (seeSection 6.4). Mercury(II) chloride or cyanogenbromide can also be used as oxidant. Thethiochrome is extracted into isobutanol and thefluorescence of the extract at an emission wave-length of 436 nm compared with that of a stan-dard thiochrome solution.

Thiamin can also be determined spectropho-tometrically by measuring its UV absorption at266 nm, but only in cases where no other ma-terials absorbing at this wavelength are presentin significant amounts. A less sensitive and lessspecific method is a colorimetric test based onthe formation of a diazotized aromatic aminefrom thiamin. Methods based on high perfor-mance liquid chromatography offer the advan-tages of specificity, simplicity, ease of use, andreduced time of analysis [681].

Although earlier bioassays were performedon animals, modern methods make use of mi-croorganisms, a large variety ofwhich have beenshown to require thiamin for their growth andreproduction (see Section 6.6). The main disad-vantage of usingmicroorganisms is their relativelack of specificity, as some respond either to thethiazolemoiety or to the pyrimidine precursor orto intact thiamin. Assays based on Lactobacil-lus viridescens and Lactobacillus fermenti arepresently the most widely used, partly becauseboth are thiamin specific.

Microbiological assays are simple, inexpen-sive and quite sensitive (detection limit of 5 –50 ng thiamin), but their main drawback is thelonger period of time to obtain the results.

7. Riboflavin

7.1. Introduction

According to IUPAC rules [691], riboflavin[83-88-5] (1) is the valid designation for 7,8-

Vitamins 83

dimethyl-10-(d-1′-ribityl)isoalloxazine, alsoknown as vitaminB2 or lactoflavin.

Until about 1970, this compound wasgenerally called 6,7-dimethyl-9-(d-1′-ribityl)-isoalloxazine in the literature because of a dif-ferent numbering of isoalloxazine.

Riboflavin occurs in nature as the free vita-min, as the 5′-phosphate (2) or flavin mononu-cleotide [130-40-5] (FMN), and as the 5′-adenosine diphosphate (3) or flavin adenine din-ucleotide [146-14-5] (FAD).

7.2. History

A water-soluble growth factor present in milkwasmentioned for the first time in 1913 [692]. In1917, this heat-stable growth factor was isolatedfrom rice bran and separated as a homogeneousfraction from another, heat-sensitive compoundhaving vitamin character. The second compoundwas able to cure a certain type of polyneuritis[693]. Thus, vitaminB, whichwas considered tobe homogeneous until then, was separated intothe vitamins B1 and B2.

In 1932,Warburg and Christian describeda yellow enzyme which they obtained fromaqueous extracts of yeast [694]. In the follow-ing year, they separated this enzyme into a pro-tein and a dye component and showed that nei-ther component alone is enzymatically active[695]. The isolation of the protein-free dye inthe crystalline state from chicken egg white wasachieved in 1933 [696].

It was shown later that the flavins iso-lated from various sources, which were calledovoflavin, lactoflavin, uroflavin, hepatoflavin,vitaminB2, and vitaminG, depending on thesource, were all identical. The name riboflavingained acceptance after 1937when it was recog-nized that this vitamin is a derivative of ribose.

The structure of riboflavin was established in1933 – 1935 by the groups of Kuhn and Kar-rer. The key reactions were:

1) Formation of lumiflavin (4) on exposure tolight under alkaline conditions. The identifi-cation of one of the cleavage products as atetrahydroxybutyl group [697].

2) Alkaline degradation of lumiflavin to4,5-dimethyl-2-(methylamino)aniline [698],which allowed the identification of lumi-flavin as 7,8,10-trimethylisoalloxazine.

3) Total synthesis of riboflavin, whereby thesynthesis of different stereoisomeric flavin-10 derivatives led to the establishment of theconfiguration of the tetrahydroxybutyl sidechain of riboflavin [699–701].

7.3. Physical and Chemical Properties

Riboflavin (1), Mr 376.36, crystallizes as yel-low, bitter-tasting needles. The melting pointwith decomposition is between 271 and 293 ◦C,

84 Vitamins

depending on the heating rate. The solubility in100mLwater is 5mg at 7 ◦Cand 13mg at 27 ◦C.It can be improved by the addition of, e.g., boricacid, urea, nicotinamide [702], or xanthine com-pounds [703]. At 27 ◦C, 4.5mg of riboflavin dis-solves in 100ml of ethanol. Riboflavin is poorlysoluble in pentanol, cyclohexanol, benzyl alco-hol, and phenol. It is insoluble in acetone, ether,chloroform, and in aliphatic or aromatic hydro-carbons. It is freely soluble in dilute aqueousalkali solutions, but decomposes rapidly. Neu-tral aqueous solutions of riboflavin have an in-tense yellow color (λmax at 445, 372, 265, and220 nm) and exhibit a green fluorescence (max-ima at 530 and 565 nm).

The specific rotation [α]25D is +59◦ (c = 0.5in 37% HCl). Light decomposes riboflavin inneutral or weakly acidic solutions to give lu-michrome [1086-80-2].

Irradiation of alkaline solutions with visiblelight produces lumiflavin [1088-56-8] (4).

In acid solution, riboflavin is resistant to ox-idizing agents (e.g., H2O2, HNO3, or halogens)up to ca. 100 ◦C. This property is used in the pu-rification of crude products during production.Sodiumdithionite (Na2S2O4) reduces riboflavinwith the uptake of two hydrogen atoms to givethe colorless leucoriboflavin, which can readilybe reoxidized to riboflavin by shaking with air.This reaction can be used in the analytical deter-mination of riboflavin (see Section 7.9.).

Riboflavin binds heavy metals (e.g., Fe, Mo,Cu, Ag, Cd, Ni, Zn, Co), forming deeply coloredcomplexes (metal chelates) [704], for example:

Flavin mononucleotide (2) crystallizes as themonosodium salt with two molecules of waterof crystallization. Its solubility (pH 4.5) in wa-ter, 5 g/100mL, is much higher than that of ri-boflavin. It is poorly soluble or insoluble in eth-anol, ether, chloroform, and benzene. Owing toits high solubility in water, it is used instead ofriboflavin in the production of solutions of thevitamin.

Like riboflavin, FMN is instable in alkalinesolution. In an acidic medium, this phosphateester is hydrolyzed to riboflavin. FMN is moststable at pH 6.

7.4. Occurrence

Riboflavin is one of the vitamins that occur ubiq-uitously. It is contained in all plant and animalcells. Certain bacteria, yeasts, fungi as well asthe organs liver, heart, and kidneys are especiallyrich in riboflavin. Appreciable amounts are alsofound in egg white, meat, and milk.

The concentration of riboflavin in foods (inµg/g) is as follows [683]:

White bread 20 – 100Rye bread 73 – 250Cows milk 20 – 300Cheese 330 – 565Hen’s egg 275 – 350Pork 90 – 350Beef 40 – 350Pork liver 2900 – 4400Spinach 57 – 340Potatoes 7.5 – 200

The concentration of riboflavin in human tis-sues (in µg/g) [682] is:

Kidney 20.0Liver 16.0Heart 8.3Adrenal gland 8.2Stomach 5.2Ovary 4.3Ileum 4.2Spleen 3.6

Vitamins 85

Brain 2.5Mammary gland 2.4Muscle 2.0 – 2.3Colon 2.1Testes 2.0Lung 1.9Skin 1.2Spermaduct 1.0

In the culturemedia of various species of bac-teria and fungi (e.g.,Ashbya gossypii,Eremothe-cium ashbyii, Bacillus subtilis,Candida flareri),riboflavin is present in concentrations far above10 g/L (see Section 7.6.3).

7.5. Biosynthesis [684]

Riboflavin is synthesized by many plants andmicroorganisms. In the animal kingdom, manyherbivores which do not form this vitamin intheir tissues can cover their riboflavin require-ment with the help of bacteria in their digestivetract.

According to Bacher [684,705–710], thebiosynthesis of riboflavin starting with guano-sine triphosphate (GTP) involves the follow-ing steps (Scheme 21): opening of the imida-zole ring of GTP (5), catalyzed by the enzymeGTP cyclohydrolase II, produces the pyrimidinederivative (6). Conversion of 6 to 5-amino-6-ribitylamino-2,4(1H ,3H )-pyrimidinedione (10)proceeds via 8 in fungi and via 7 in bacte-ria, respectively. The condensation of 10 withthe C4 building block l-3,4-dihydroxy-2-buta-none-4-phosphate (12) leads to 6,7-dimethyl-8-ribityllumazine (13). In Bacillus subtilis, thisstep is catalyzed by the β-subunit of riboflavinsynthetase. The α-subunit of riboflavin syn-thetase catalyzes the dismutationof the lumazine(13) to give riboflavin (1) and the pyrimi-dinedione (10).

7.6. Production

7.6.1. Syntheses

The structural elucidation of riboflavin (1)was completed with the first syntheses con-ducted by Karrer [711–713] and Kuhn[714–716]. Karrer et al. reacted 2-(ethoxycar-bonylamino)-4,5-dimethylaniline (14) with d-ribose, hydrogenated the resulting Schiff base

to give the corresponding N-ribitylamine (15),and obtained N-(2-amino-4,5-dimethylphenyl)-d-1′-ribitylamine (16) after alkaline hydrolysis.Reaction with alloxan [2244-11-3] (17) in hy-drochloric acid medium gave riboflavin (1) in15% yield, based on d-ribose. Kuhn et al. di-rectly hydrogenated the Schiff base formed fromnitroxylidine (18) and d-ribose to compound 16(Scheme 22).

An improved yield of 16 was attained byintroducing the amine group at the stage ofN-(3,4-dimethylphenyl)-d-1′-ribitylamine (19),which is obtainable from 3,4-dimethylani-line and d-ribose. This was achieved bycoupling 19 with benzenediazonium chlorideto give N-(2-phenylazo-4,5-dimethylphenyl)-d-1′-ribitylamine (20), followed by reductivecleavage of the azo double bond with sodiumdithionite [717]. Cyclocondensation of 16 withalloxan (17) produced riboflavin (1) in a yield of38%, based on d-ribose.

Substantial progress in the synthesis of ri-boflavin was made by Tishler [718] who foundthat in a weakly acidic medium, the azo com-pound (20) can be directly reacted with barbi-turic acid [67-52-7] (21) to give riboflavin (1),with elimination of aniline. Thus, the reductionof 20 to16wasno longer required, and instead ofthe relatively expensive alloxan (17), the morereadily available barbituric acid (21) was usedin the synthesis. The total yield of riboflavin (1)was increased to 46%, based on d-ribose.

86 Vitamins

Scheme 21. Biosynthesis of riboflavin

Subsequently, major efforts were made to re-place d-ribose, which is not widely available, byother sugars. For example, ribamide (22), madefrom d-ribonolactone and 3,4-xylidine, was di-rectly [719,720] or, after acetylation via theimide chloride (23), catalytically hydrogenatedto give 19 [721]. d-Arabinose, which is read-ily available, was reacted with 3,4-xylidine toyield arabinoside (24). Amadori rearrangement

with benzoic acid gave the keto compound (25),which could then be catalytically hydrogenatedto 19 in alkaline solution [722,723]. As a resultof moderate total yields, none of these synthe-ses has achieved importance in the productionof flavins.

In the 1970s, a novel synthesis of flavin wasdescribed by Yoneda et al. They condensed 19with 6-chlorouracil (26) to give 6-(N-3,4-di-

Vitamins 87

Scheme 22. Synthesis of riboflavin

methylphenyl)-N-d-1′-ribitylamino)uracil (27).Nitrosation with nitric acid and reduction of theresulting N-oxide (28) with sodium dithioniteyielded riboflavin (1) in a total yield of ca. 70%[724].

Riboflavin (1)was also obtained in goodyieldby autoxidation of the amine (29), which wasmade from 19 and 6-chloro-5-nitrouracil (30),followed by catalytic hydrogenation of the nitrocompound (31) [725]. Nevertheless, these syn-theses have not yet attained economic impor-tance, mainly because of the multistep produc-tion of the uracils (26) and (30) from barbituricacid (21).

7.6.2. Industrial Chemical Production

Commercial syntheses of riboflavin require eco-nomic pathways for the production of d-ribose(32). Three processes, all startingwithd-glucose[492-62-6] (33) should be mentioned here.

Oxidation of d-glucose with oxygen in aque-ous potassium hydroxide solution [726] yieldspotassium arabonate (34) in ca. 90% yield. 34 isconverted to crystalline calcium arabonate (35).Heating 35 in aqueous solution to ca. 140 ◦Cleads to an equilibriummixture of epimeric salts,containing ca. 30% calcium d-ribonate (36).

88 Vitamins

After separation of high-molecular coloredside products by adsorption on activated carbon,35 is isolated by crystallization [727], themotherliquor is acidified with sulfuric acid, and thepoorly soluble calcium sulfate is separated. Thed-ribonolactone [5336-08-3] (37) in the motherliquor is purified by crystallization from n-buta-nol and then reduced with sodium amalgam inaqueous solution in the presence of boric acid[728]. The sodium hydroxide solution formedis neutralized with sulfuric acid, sodium sulfatebeing precipitated with methanol and separated.A d-ribose solution is obtained which containslow amounts of starting material and ribitol andis directly further processed. The total yield ofd-ribose (32) is 40%, based on 33.

In a more recent chemical process, d-ribose(32) is made from d-glucose (33) in threesteps with a total yield of ca. 56% [729].33 is fermentatively oxidized to sodium d-gluconate [527-07-1] (38), which is, in turn, ox-idatively degraded to d-arabinose [28697-53-2](39) with sodium hypochlorite. After separationof sodiumchloride, 39 is crystallized frommeth-anol by electrodialysis. Epimerizationof 39withmolybdic acid in aqueous solution gives an equi-librium mixture containing ca. 25% d-ribose

(32), from which the starting material can beseparated by crystallization and recycled [730].The resulting d-ribosemother liquor can be usedwithout further purification for the synthesis ofriboflavin (1). In an advantageous process vari-ant, molybdic acid is fixed to a basic ion ex-changer and the process is conducted continu-ously [731].d-Ribose (32) is obtained directly from d-

glucose (33) by fermentation with microor-ganisms of the genus Bacillus which have notransketolase activity and do not form spores[732]. Especially productive are genetically en-gineered strains which overexpress the glu-conate operon [733]. After separation of thebiomass, decoloration, desalting, and evapora-tion of the filtrate, 32 is obtained by crystalliza-tion from water – ethanol mixture in a yield ofca. 45%.

In the commercial synthesis of ri-boflavin, the three step reaction sequence,32 → 19 → 20 → 1, conceived by Karrer andTishler, is used. The total yield has been in-creased to >60% by optimizing the originalreaction conditions.

The readily available compound 3,4-xylidineis reacted with d-ribose (32) in methanol. Theresulting riboside is catalytically hydrogenatedto give N-(3,4-dimethylphenyl)-d-1′-ribamine(19) without isolation, and the product is pu-rified by crystallization.

Coupling of 19 with phenyl diazonium saltin a buffered aqueous solution yields crys-talline N-(2-phenylazo-4,5-dimethylphenyl)-d-1′-ribamine (20). Dry 20 is converted to ri-boflavin (1) by cyclocondensation with barbitu-ric acid in a weakly acidic medium. Mixtures ofdioxane and acetic acid or di-α-substituted car-boxylic acids have proved suitable as solvents[734]. Crude 1 can be purified by reprecipita-tion from hydrochloric acid or dilute sodium hy-droxide solution, possibly with the addition ofhydrogen peroxide [735,736].

7.6.3. Industrial Production byFermentation

Riboflavin (1) can be produced microbiologi-cally. At present, the microorganisms Bacillussubtilis, the ascomycetesEremotheciumashbyii,Ashbya gossypii, and the yeasts Candida flareri

Vitamins 89

and Saccharomyces cerevisiae are used [737–740]. The nutrientmedia employed aremolassesor plant oils as carbon source, inorganic salts,amino acids, animal or plant peptones and pro-teins, as well as vitamin additives. In a sterileaerobic submerged process, yields much higherthan 10 g of riboflavin per liter of culture brothare obtained in a few days with good aerationand stirring at temperatures below 30 ◦C.

After separation of the biomass, evapora-tion and drying of the concentrate, an enrichedproduct with a vitaminB2 content of up to80% is obtained. The majority of the riboflavinproduced by fermentation is used in animalfeeds. The companies ADM (United States) andBASF (Germany) produce riboflavin microbio-logically.

7.6.4. Syntheses of Flavin Mononucleotide(FMN)

Flavin mononucleotide, FMN, (2) was first pro-duced by Kuhn by reacting riboflavin (1) withphosphorus oxychloride [741]. Of the numer-ous phosphorylation reagents tested [742], par-tially hydrolyzed phosphorus oxychloride hasbeen used most often [743]. A complex mix-ture of various riboflavin phosphates is alwaysproduced in this process. These can be separatedby RP-HPLC and identified with the aid of au-thentic preparations [744].

In a commercial process employed since1954 by Hoffmann-La Roche [745,746], afterphosphorylation, repeated crystallization is usedto separate 2 as the diethanolamine salt fromisomeric phosphates and unreacted 1. Resaltingwith sodiumacetate and crystallization fromwa-ter – ethanol yields FMN (2) in yields of>80%.

In amore recent process, unreacted riboflavin(1) is separated on polar adsorption resins madeby the copolymerization of acrylic acid deriva-tiveswith divinylbenzene [747]. Subsequent finepurification can be achieved by evaporative crys-tallization or by chromatography on RP silicagel with water –methanol. Products purified bycrystallization contain ca. 75% FMN (2) andca. 10%4′-FMN.Chromatographically purifiedpreparations exhibit a ca. 90% content of activeingredient but still contain ca. 5% of 4′-FMN.

7.6.5. Syntheses of Flavin AdenineDinucleotide (FAD)

The synthesis of flavine adenine dinucleotideFAD (3) comprises the condensation of FMN(2) with adenosine 5′-phosphate (AMP). Themain problem is preventing formation of sym-metrical phosphate esters; this is achieved by us-ing activated AMP. The compounds morpholid[748], imidazolid [749], and products of reac-tion of AMP with α-pyridone [750] or withdiphenylphosphoryl chloride [751] are used asactivated derivatives of AMP. FAD yields of upto 70% are attained, depending on the methodemployed.

FAD can also be isolated from the myceliumof cultures of Eremothecium ashbyii [752].

FAD can also be produced from riboflavinand AMP by fermentation with genetically en-gineered E. coli strains that overexpress the en-zymes flavokinase and FAD synthetase [753,754] (Scheme 23).

Table 26. Flavoproteins and their functions

Enzyme Coenzyme Function

Acyl-CoA dehydrogenase FAD first dehydrogenationstep in the β-oxidation offatty acids

Electron transferringflavoprotein (ETF)

FAD transfer of hydrogen fromacyl-CoA dehydrogenaseto ubiquinone

Xanthine oxidase FAD oxidation ofhypoxanthine to xanthineand xanthine to uric acidin purine degradation

Succinate dehydrogenase FAD dehydrogenation ofsuccinate to fumarate inthe citric acid cycle

Dihydroliponamidedehydrogenase

FAD transfer of hydrogen fromdihydroliponamide toNAD in the 2-oxo acidoxidase system

NADH cytochrome creductase

FMN transfer of hydrogen fromNADH to ubiquinone inthe respiratory chain

Monoamino oxidase FAD oxidation of monoaminesand diamines

Aldehyde oxidase FAD oxidation of aldehydes tocarboxylic acids

Glutathione reductase FAD reduction of oxidized toreduced glutathione

7.7. Importance for the Organism

In the human and animal organism, riboflavinacts as the coenzyme of a series of oxi-dation and reduction enzymes classified as

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Scheme 23. Fermentative production of FAD

flavoproteins. More than 100 flavoproteins areknown and more than 40 are found in the hu-man organism. Examples are succinate dehy-drogenase, lactate oxidase, glutathione reduc-tase, and NADH/NADH2 cytochrome c reduc-tase (see Table 26).

Riboflavin is specifically bound to these en-zymes as flavinmononucleotide (FMN) or flavinadenine dinucleotide (FAD) via the 8-methylgroup of isoalloxazine.

In energy producing metabolism, flavopro-teins act as intermediate hydrogen carriers inthe oxidation of substrates and are reoxidized byenzymes with a higher oxidation potential (e.g.,cytochrome c). In this process, the hydrogen orelectron acceptor and donor are the coenzymesFAD or FMN [685,755]. Since flavoproteins areinvolved in intermediary metabolism in diverseways, riboflavin deficiency in the diet has an ef-fect on many processes and causes deficiencysymptoms.

Riboflavin is resorbed exclusively in the freeform,mainly in the small intestine. After resorp-tion, riboflavin is rephosphorylated to FMN byriboflavin kinase in the mucosa cells. FMN isthen converted to FAD, primarily in the liver.

The elimination of riboflavin proceeds pre-dominantly via the kidneys (urine).

7.8. Requirements, DeficiencySymptoms, and TherapeuticApplication [686]

To prevent clinical deficiency symptoms inadults, 0.8 – 0.9mg of riboflavin per day are gen-erally sufficient.However, this amount cannot beconsidered adequate in the sense of an optimalsupply.

The German Society for Nutrition (1991)recommends the following amounts: women1.5mg, men 1.7mg, pregnant women 1.8mg,nursing mothers 2.3mg.

In humans, the most characteristic symptomsof a diet deficient in riboflavin (ariboflavinosis)are rhagades and fissures at the corners of themouth and lips (cheilosis), atrophic glossitis,magenta colored tongue, seborrheic dermatitisin the facial area, and dystrophy of the fingernails. Of the diverse eye problems, conjunctivi-tis, corneal vascularization with the feeling of aforeign body and photophobia are noteworthy

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[687]. Neurological symptoms such as pares-thesia in the legs, a burning sensation in thefeet, ataxia, and tremor have been observed. Oncontrolled administration of only 0.5mg of ri-boflavin per day, deficiency symptoms occur af-ter 4 – 10 months [756].

Although clinical B2 deficiency symptomsare now practically unknown in the industrialcountries, the supply of this vitamin in all groupsof society cannot be regarded as satisfactory.The 1991 national study on consumption andthe nutritional report of 1992 show that the ri-boflavin intake of a large percentage of younggirls and women in Germany is much less thanthe amounts recommended by the German So-ciety for Nutrition.

Proven applications of vitaminB2 are in theprophylaxis and treatment of clinical riboflavindeficiencies. These can have various causes:

1) Inadeqate and false nutrition2) Increased requirement (e.g., during preg-

nancy and lactation)3) Malabsorption4) The result of phototherapy in newborn hyper-

bilirubinemia

In prophylaxis, vitaminB2 is generally ad-ministered in daily doses of 1 – 5mg, usually inthe form of multivitamin or vitaminB complextablets. In the treatment of an established ari-boflavinosis, daily doses of up to 25mg are ad-ministered.

In the food industry, riboflavin is used on alarge scale together with other vitamins in thefortification of various foods, such as flour, babyfoods, cereals, and multivitamin juices.

Riboflavin is of great importance in animalfeeds, especially as an additive to poultry and pigfeed. In these animals, a lack of riboflavin causesgrowth retardation, poor utilization of feed, anddiarrhea. For laying hens, which have a greatlyincreased riboflavin requirement because of thehigh production of eggs, the addition of 5mg perkilogram of mixed feed is recommended. Thetypical manifestation of riboflavin deficiency inthe chicken is toes bent inwards.

7.9. Analysis [688,689]

Microbiological, enzymatic, and physicochem-ical methods are used for the analytical identi-fication of flavins. The growth tests conducted

earlier with experimental animals on deficiencydiets no longer play a role because they are timeconsuming and inaccurate.

Microbiological methods are especially suit-able for the determination of low concentra-tions in biological media and in pharmaceuticalpreparations. They have the advantage of highspecificity and relatively high accuracy. They arebased on the effect of riboflavin on the growth ofcertain microorganisms, e.g., the unicellular or-ganismTetrahymenapyriformisor the lactic acidbacterium Lactobacillus casei. In the widelyused assay with Lactobacillus casei [690], ei-ther the turbidity produced or the lactic acid lib-erated is used as the measure of the riboflavinconcentration. If FMN or FAD is present, priorrelease of riboflavin is necessary.

The enzymatic method is based on the in-crease in the activity of glutathione reductase onaddition of the cofactor FAD. This method pro-vides an indication of the riboflavin status via thedetermination of the degree of saturation in theblood. It is a suitable method for the diagnosisof riboflavin deficiencies [757].

Physicochemical methods include the photo-metric measurement of the yellow color of ri-boflavin and the measurement of the character-istic yellow-green fluorescence. In both cases,riboflavin is determined either directly or as lu-miflavin (4), which is formed on irradiation. Po-larography is also employed for determination.

In the direct photometric method, the absorp-tion ismeasured at 445 nm, themaximum exhib-ited by the yellow color of riboflavin. Since thespecific extinction (ε1 %

1 cm = 323) is relatively low,this method is not very sensitive. In addition, itrequires the absence of other yellow substances.Therefore, this method is restricted to the mea-surement of high-percentage products withoutinterfering components [688].

The fluorometric method is more sensitive.It involves the measurement of the fluorescencewith a maximum at 530 nmwhich appears on ir-radiation with UV light. Its intensity is constantin the pH range 3 – 5 and depends only on theconcentration of riboflavin. In some cases, inter-ference by accompanying substanceswith a sim-ilar fluorescence can be eliminated by quench-ing the fluorescence of riboflavinwithNa2S2O4.This method is suited to the determination of ri-

92 Vitamins

boflavin in pharmaceutical preparations and inbiological media [688].

In the lumiflavin method, riboflavin is con-verted to lumiflavin (4) by irradiation. This com-pound can be extracted by shaking with tri-chloromethane and measured either photomet-rically at 450 nm or fluorometrically at 513 nm.

The specificity and the sensitivity, especiallyof thefluorometric lumiflavinmethod, are higherthan those of the direct methods. For this reason,this method is used more frequently [688].

Riboflavin can also be determined by using astraightforward polarographic method [758].

High-pressure liquid chromatography(HPLC) is the most important method for theanalysis of riboflavin, FMN, and FAD. HPLCis the fastest and most sensitive method, espe-cially for the determination of these componentsin complex mixtures, such as foods, and in theanalysis of fermentation samples [759–761].

7.10. Economic Aspects

Thepresentworldwide requirement of riboflavinis ca. 2400 t/a. Of this, ca. 70% is used as feedadditive, 20% for pharmaceutical purposes, and10% in foods.

The world market price has remained fairlyconstant over the past ten years, at ca. 45¤/kgfor feed quality and ca. 54¤/kg for pharmaceu-tical quality.

Producers of riboflavin are Hoffmann-LaRoche (Switzerland), BASF (Germany), ADM(United States), and Takeda (Japan). There arealso local producers in the Commonwealth ofIndependent States and in China.

7.11. Tolerance

Riboflavin causes no pharmacological side ef-fects in animals or human beings and can beregarded as completely nontoxic. The adminis-tration of oral doses of up to 10 g/kg to rats and2 g/kg to dogs produced no acute toxicity. In-traperitoneal administration to rats gave anLD50value of 560mg/kg.

8. Vitamin B6

8.1. Introduction

VitaminB6 is a water-soluble vitamin. The termvitaminB6 refers to a group of six compoundsthat are chemically closely related and, underphysiological conditions, biochemically (enzy-matically) interconvertible. These compounds(1) have in common the structural element 2-methyl-3-hydroxypyridine and bear two othersubstituents in positions 4 and 5 (Table 27).

Table 27. The vitamins B6

R1 R2 Name

CH2OH OH pyridoxine (2)CH2OH OPO3H2 pyridoxine

5′-phosphate (3)CHO OH pyridoxal (4)CHO OPO3H2 pyridoxal

5′-phosphate (5)CH2NH2 OH pyridoxamine (6)CH2NH2 OPO3H2 pyridoxamine

5′-phosphate (7)

In 1973, the IUPAC– IUB Commission onBiological Nomenclature [762] proposed achange in the numbering of the pyridine ring.Accordingly, the common structural unit (1)should be called 5-hydroxy-6-methylpyridine.Until now, however, this proposal has beenlargely ignored in the literature (especially withregard to the 5′-phosphates which should nowbe named 3′-phosphates).

The commercial form of vitaminB6 is pyri-doxine hydrochloride [58-56-0] (8), which ismore stable than the other compounds in thisseries.

8.2. History

In 1926, a syndrome similar to pellagra (pella-gra is characterized by skin changes and distur-bances of the nervous system, digestion, growth,fertility, blood count, etc.) was induced in rats on

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a diet deficient in “vitaminB complex” [763].This syndrome was called rat acrodynia. From1934 to 1936, Gyorgy et al. [764] showed thatvitamins B1 and B2, which were both known atthat time, could not cure this rat dermatitis. Theyshowed that another vitamin, designated antider-matitis vitamin, adermin, or vitaminB6, must beresponsible for the cure. Based onGyorgy’s ex-act animal model tests, the isolation (from yeastand rice bran) and structural elucidation of pyri-doxine hydrochloride (8) were achieved simul-taneously by five groups in 1938 [765].

Pyridoxine hydrochloride (8) was synthe-sized in 1939 by two teams working indepen-dently [766]. Between 1942 and 1945, Snellet al. showed by using microbiological experi-ments that vitaminB6 is not a homogeneous sub-stance, but a group of six interconvertible com-pounds 2–7 [767].

Although 8 was isolated as early as 1932[768], its vitamin function was not recognizedat that time.

8.3. Physical Properties

A compilation of the physical properties of 2–8 is given in [769–772]. The properties of thecommercial product pyridoxine hydrochloride(8), including its spectra, are described in de-tail in [770,771]. All vitaminB6 compounds aresensitive to light and, as pyridine bases, formsalts with acids. They decompose when heatedfor longer periods in neutral or basic aqueoussolutions.

Pyridoxine [65-23-6] (2), (CA: 3,4-pyr-idinedimethanol, 5-hydroxy-6-methyl), 2-methyl-3-hydroxy-4,5-bis(hydroxymethyl)pyr-idine, C8H11 NO3, Mr 169.18. Colorless nee-dles (from acetone),mp 159 – 160 ◦C, soluble inwater, methanol, ethanol, and acetone; thermo-labile as solid and in solution.

Pyridoxine hydrochloride [58-56-0] (8),(CA: 3,4-pyridine-dimethanol, 5-hydroxy-6-methyl, hydrochloride), C8H12ClNO3, Mr205.64.

Colorless platelets or rods from ethanol –acetone, mp 205 – 212 ◦C (decomp.). Solublein water (22 g/100mL at room temperature),ethanol (1.1 g/100mL), and propylene glycol.Poorly soluble in methanol and acetone, and in-soluble in ether and chloroform. Aqueous so-lutions are stable at room temperature (120 ◦Cfor at least 30min). The pH of a 10% aqueoussolution is 3.2.

Pyridoxine 5′-phosphate [447-05-2] (3),[CA: 3,4-pyridine-dimethanol, 5-hydroxy-6-methyl, α-3-(dihydrogen phosphate) ],C8H12NO6P, Mr 249.16.

White needles fromconcentrated aqueous so-lutions, mp 212 – 213 ◦C (decomp.), soluble inwater, and insoluble in ethanol.

Pyridoxal [66-72-8] (4), (CA: 4-pyridine-carboxaldehyde, 5-hydroxy-3-(hydroxymeth-yl)-6-methyl), 3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridinecarboxaldehyde, C8H9NO3,Mr 167.16.

The aldehyde form is in equilibrium with thecyclic hemiacetal form. Readily decomposingcompound, independent of pH.

94 Vitamins

Pyridoxal hydrochloride [65-22-5] (9),(CA: 4-pyridinecarboxaldehyde, 5-hydroxy-3-(hydroxymethyl)-6-methyl, hydrochloride),C8H10ClNO3, Mr 203.63.

Almost colorless rhombic crystals, mp177 –181 ◦C (decomp.), soluble in water(50 g/100mL) and 96% ethanol (1.7 g/100mL),insoluble in acetone, ether, and chloroform. Insolution, 9 is less stable than 8.

Pyridoxal 5′-phosphate [54-47-7] (5),(CA: 4-pyridinecarboxaldehyde, 5-hy-droxy-6-methyl-3-[(phosphonooxy)-methyl]),C8H10NO6P, Mr 247.14.

The monohydrate of 5 [41468-25-1],C8H12NO7P, Mr 265.15, forms yellowish nee-dles, mp 139 – 142 ◦C (decomp.), solubility inwater 0.5%, less soluble in ethanol, and insolu-ble in acetone and ether.

Pyridoxamine [85-87-0] (6) (CA: 3-pyr-idinemethanol, 4-(aminomethyl)-5-hydroxy-6-methyl), 3-hydroxy-4-aminomethyl-5-hydroxy-methyl-2-methylpyridine, C8H12N2O2, Mr168.20. Crystals, mp 193 ◦C, soluble in waterand ethanol.

Pyridoxamine dihydrochloride [524-36-7](10), C8H14Cl2N2O2,Mr 241.12. Colorless hy-groscopic platelets,mp 228 ◦C (decomposition),soluble in water (50 g/100ml) and in 96% etha-

nol (0.65 g/100mL), insoluble in ether and chlo-roform.

Pyridoxamine 5′-phosphate [529-96-4] (7)[CA: 3-pyridinemethanol, 4-(aminomethyl)-5-hydroxy-6-methyl, α-(dihydrogenphosphate)],C8H13N2O5P, Mr 248.18.

Thedihydrate of7[84878-64-8],C8H17N2O7P,Mr 284.2, forms rhombic platelets; mp of thehydrochloride 240 ◦C (decomp.).

8.4. Chemical Reactions [770,772,773]

Many of the reactions and syntheses in vita-minB6 chemistry date from the years follow-ing the discovery and isolation of pyridoxine.These reactions often aided the structural eluci-dation and proof of structure of compounds 2 to8. Since the commercial form of vitaminB6 ispyridoxine hydrochloride (8), some characteris-tic reactions of pyridoxine (2) are described here(see Scheme 24).

Pyridoxine (2) contains three reactive hy-droxyl groups. The OH group in position 3 ofthe pyridine ring reacts with iron(III) ions togive the red or orange color typical of aro-matic hydroxyl groups. This OH group can beesterified and etherified. Together with the 4-hydroxymethyl group, six-membered cyclic ke-tals or acetals (10) can be formed with ke-tones and aldehydes. The two hydroxymethylgroups in positions 4 and 5 undergo reactionstypical of groups of this type, e.g., ester for-mation. Under acid catalysis, reactions with ke-tones and aldehydes produce, apart from 10,seven-membered cyclic ketals or acetals (11).Of the two CH2OH groups, the more reactiveis the one in position 4. For example, oxidation

Vitamins 95

of 2 with MnO2 gives pyridoxal (4), which canbe further oxidized by O2 under catalysis withcyanide to give pyridoxine 4-carboxylic acid(12) [82-82-6]. Although acidic aqueous solu-tions of 2 are quite stable even at 100 ◦C, it reactswith inorganic acid halides or hydrohalic acidsunder drastic conditions.WithHCl, for instance,depending on the reaction conditions, the com-pounds 13 [5196-20-3], 14 [102694-13-3], and15 [13983-22-7] are formed. Acetylation cangive the 4′,5′-diacetate 16 [26280-83-1] or the3,4′,5′-triacetate 17 [10030-93-0] depending onthe reaction conditions. Heating 2 with aqueousammonia produces pyridoxamine (6).

8.5. Occurrence

VitaminB6 occurs in plants mainly as pyri-doxine. Grains, potatoes, green vegetables, andlegumes are especially rich in vitaminB6.A con-siderable part of the vitamin occurs in plantsas 5′-O-(β-glucopyranosyl)pyridoxine. Animalexperiments show that this glucosylated form isapparently well absorbed but appears to be notentirely bioavailable. The main forms observedin the animal kingdom are pyridoxal and pyri-doxamine. Accordingly, these forms are foundin animal foods, such as meat, fish, and eggs.The concentration of vitaminB6 in some foodsis given in Table 28 [774–777].

Table 28. VitaminB6 content of some foods [774–777]

Food VitaminB6, Pyridoxinemg/100 g β-glucoside %

Potatoes 0.14 – 0.39 42Spinach 0.208 – 0.22 50Carrots 0.17 – 0.70 51Cabbage 0.14 – 0.27 46Bananas 0.26 – 0.313 3Legumes 0.381 – 0.57 42Meat (chickenbeef, pork) 0.30 – 0.70 0Fish 0.316 – 0.45 0Eggs 0.19 – 0.25

8.6. Biosynthesis

Pyridoxine is synthesized in bacteria, fungi,and plants. Experiments with radioactively la-belled or deuterated compounds, such as glu-cose and glycerol, contributed to the elucidation

of the biosynthesis of vitaminB6. Hill et al.found later that d-1-deoxyxylulose is incorpo-rated into the pyridoxine ring and supplies thecarbon atoms C 2, 2′, 3, 4, and 4′ [778,779].According toDempsey, 4-hydroxythreonine is acentral intermediate in the biosynthesis of pyri-doxine [780]. Lam and Winkler reported that4-hydroxythreonine is made from erythrose 4-phosphate in four enzymatic steps and subse-quently condensed with d-1-deoxyxylulose togive pyridoxine [781]. The enzymes that cat-alyze these reactions are as yet not completelyknown. In E. coli, the corresponding genes arespread over the entire chromosome and orga-nized in complex operons. At present, biotech-nological productionbygeneticallymanipulatedmicroorganisms or the industrial isolation fromnatural sources is not yet of interest.

8.7. Production of Vitamin B6Compounds

8.7.1. Pyridoxine [770,772,782]

Pyridoxine hydrochloride (8) is the form of vi-taminB6 that is produced on a large scale. Itis mainly used in pharmaceutical preparationsand as an additive in food and feed. The otherB6 compounds, pyridoxal (4), pyridoxamine (6),and the accompanying 5′-phosphates (3), (5),and (7) can be made from 8.

The five synthetic pathways known for 2 and8 are outlined here in the order of their discovery.At first, 8 was produced entirely by the methodsdescribed in Section 8.7.1.2. Today, themethodsdescribed in Section 8.7.1.4. are exclusively em-ployed. Intensive work on these synthetic path-ways began in 1939 and 1957 and led to manypatents because of the commercial interest inB6.Since the expiry of all the basic patents on theseprocesses, the number of newpatents in this fieldhas clearly decreased in the past years.

8.7.1.1. Oxidative Degradation of BicyclicHeterocyclic Compounds [773]

In 1939, the teams of Kuhn [766] and Ichiba[783] published the first syntheses of 2 start-ing with suitably substituted isoquinoline or

96 Vitamins

Scheme 24. Reactions of pyridoxine (2)

quinoline derivatives [784], e.g., 4-methoxy-3-methylisoquinoline [103988-78-9] (18).

The synthesis starts with the oxidative degra-dation of the carbon ring to give the dicarboxyl-ic acid (19) [7442-22-0], which is subsequentlyconverted in several stages to 2. The commer-cialization of syntheses of this type was pre-vented by the poor availability of the startingmaterials, the multiple stages involved, and thestill unsolved problem of finding an inexpensivereduction of such carboxyl functions to alcohols.

8.7.1.2. Condensation Reactions withAliphatic Precursors [770,773,782]

In 1939, Harris and Folkers [785] also pub-lished a synthesis of 2 which was subsequently

developed into the first commercial process. Thereaction of ethoxyacetylacetone [20754-01-2](20) with cyanoacetamide [107-91-5] (21) givesthe pyridone (22) [17718-67-1].

Following the same synthetic principle, thereaction of 2-amino-4-oxo-5-ethoxypent-2-eneand malodinitrile provides improved commer-cial

access to 22. This method was described bySchnider as early as 1941 [786].

The conversion of 22 to 2 occurs in manystages and involves relatively heavy losses. Inthe following 20 – 25 years, a number of syn-thetic pathways for 2, which use many differentaliphatic building blocks, were described. A sur-vey is given in [773].

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8.7.1.3. From Furan Compounds

Synthesis of 3-hydroxypyridine from 2-ami-nomethyl-2,5-dimethoxydihydrofuran [787]was published in 1950 and used in themid-1950s to synthesize 2 [788]. In thekey step, N-[1-[3,4-bis[acetyloxymethyl]-2-furanyl]ethyl]acetamide [84878-62-6] (23) isconverted to its 2,5-dimethoxy derivative (24)[84878-63-7] by anodic oxidation in methanol.Compound 24 is then converted to pyridoxine(2) by alkaline saponification and acidification.

Even after the availability of the starting com-pounds was improved, the cost of the startingmaterials, the multiple stages, and the modesttotal yield of the synthesis hindered its commer-cialization [789,790].

8.7.1.4. Diels – Alder Syntheses withOxazoles [770,772]

This synthetic principle described by Kon-dratyeva in 1957 [791] is now used exclu-sively for the industrial production of pyridoxine(2). She found a new synthesis for pyridines viathe Diels –Alder reaction of oxazoles (25) withmaleic acid (26) (or derivatives). Under the reac-tion conditions, the Diels –Alder adduct (27) isaromatized to the cinchomeronic acid derivative(28) by the elimination of water.

An important discoverywas that if good leav-ing groups Z are incorporated into position 5 of4-methyloxazole (29), after reaction with thedienophile (30), the bridging oxygen atom ofthe bicyclic Diels –Alder adduct remains in thesubsequent product and forms an OH group inposition 3 of pyridine (31) [792].

For good results, these oxazoles should ex-hibit low steric hindrance and high thermal sta-bility.

Diene Components. From the extensivepatent literature, it appears that the compoundsthat are industrially applied aremainly 5-ethoxy-4-methyloxazole (29, Z =OEt) and 5-cyano-4-methyloxazole (29, Z =CN).

The starting material for the production of5-ethoxy-4-methyloxazole [5006-20-2] (34) isd,l-alanine ethyl ester (32) [17344-99-9], its N-formyl derivative (33) [4289-99-0] being dehy-drated to 34 by P2O5 [793].

Attempts were made to find alternatives tothe formerly expensive d, l-alanine and theoxazole (34), which is patented. One synthe-sis started with inexpensive maleic anhydride(35) and produced d, l-aspartic acid [617-45-8]by NH3 addition to the double bond. The di-ethyl ester of aspartic acid (36) [43101-48-0]was then used to produce 5-ethoxy-4-oxalaceticacid [10500-75-1] (37) via a known reactionsequence (N-formylation and dehydration) andsaponification [794]. Under Diels –Alder reac-tion conditions, compound 37 is decarboxylatedto 34.

98 Vitamins

Another precursor of 34 is 5-ethoxy-4-methyl-oxazole-2-carboxylic acid [23429-05-2](40), which is also decarboxylated to 34 in theDiels –Alder reaction. Here, propionic acid (38)is halogenated and converted to d,l-alaninewithammonia. Esterification to give 32 and reac-tion with ethyl oxalate gives the intermediate 39[23460-73-3] which on cyclocondensation andsaponification produces 40 [795].

The starting material for 4-methyl-5-cyanooxazole [1003-52-7] (41) is diketene,which is converted to ethyl acetoacetate withalcohol. Chlorination produces chloroethyl ace-toacetate (42), which is reacted with form-amide (43) to give 4-methyl-5-oxazolecar-boxylic acid ethyl ester [20485-39-6] (44),which is dehydrated to the nitrile (41) viathe 4-methyl-5-oxazole carboxamide (45)[4866-00-6]. This dehydration can be conductedwith P2O5/quinoline [796], acetic anhydride[797], in a catalytic gas-phase reaction [798], orunder very mild condition with cyanuric chlo-ride [799].

Dienophiles. Derivatives of maleic acid orfumaric acid (e.g., their esters or nitriles) reactas dienophiles with the oxazoles used as dienesin the Diels –Alder reaction (e.g., 34 or 41),giving very good results. However, the above-mentioned expensive reduction steps of the re-sulting pyridoxine dicarboxylic acid (deriva-tives) have prevented industrial application.

With regard to the oxidation state, 2-butene-1,4-diol would be the ideal dienophile if theoxazole diene 34 or 41 is used. However, thefirst-named compound reacts very poorly. Itsopenchain derivatives, e.g., the ether or es-ter, react better but are still fairly unreactive.(Nevertheless, (Z )-2-butene-1,4-diol-diacetate[25260-60-0] appears to have been used in aproduction process by a Japanese company).In contrast, the cyclic seven-membered acetalsand ketals of (Z )-2-butene-1,4-diol [6117-80-2](45) [800] – e.g., the isopropyldioxepin (47)[5417-35-6] obtainable from isobutyraldehyde(46) [78-84-2] – have proved to be sufficientlyreactive and stable in an inert-gas atmosphere.The compound n-propyldioxepin (48), derivedfrom n-butyraldehyde, is also a suitable reagent.

Diels – Alder Reaction. The reaction of theoxazole (34) or (41) with the dioxepin (47)or (48) requires high temperatures (ca. 115 –180 ◦C) and longer periods of time (ca. 10 – 20 hor more). The primary Diels –Alder adductsformed have three chiral carbon centers, result-ing inmixtures of eight stereoisomers in the formof four diastereomeric pairs. Excess or unreactedstarting material can be separated and recycled.

The use of 5-cyano-4-methyloxazole (41)and isopropyldioxepin (47) results in a Diels –Alder adduct that is not stable under the reactionconditions and decomposes with the eliminationof hydrogen cyanide and aromatization to give49 [1622-67-9] in > 90% yield [800].

5-Ethoxy-4-methyloxazole (34) reacts withthe dienophile (47) under somewhat milder con-ditions than 41. In this case, the Diels –Alderadduct is much more stable and can be iso-lated (yield > 95%). Under mildly acidic con-

Vitamins 99

ditions, aromatization occurs with the elimina-tion of ethanol to give the bicyclic acetal (49)[772]. Heating 49with hydrochloric acid resultsin almost quantitative conversion to isobutyr-aldehyde (46), which is separated and reused,and pyridoxine hydrochloride (8).

The above reactions of the oxazoles (34)and (41) with the dienophile (47) are “in-verse”Diels –Alder reactions inwhich electron-deficient dienes react with an electron-richdienophile. The variant corresponding tothe “normal” Diels –Alder reaction has alsobeen published [801]. Here, 4-methyloxazole[693-93-6] (50) reacts with 2,5-dihydro-3-(methylsulfonyl)furan [41409-84-1] (51).

In this combination, the dienophile and notthe oxazole carries a good leaving group in theform of a sulfonyl group. This group makes theDiels –Alder reaction possible and is also re-quired for the aromatization of the Diels –Alderadduct. However, this synthesis has problemsteps which make industrial application doubt-ful.

AnotherDiels –Alder reaction for the synthe-sis of pyridoxine uses 1,2,4-triazine derivativesas dienes instead of oxazoles [802]. However,the elaborate synthesis of the triazines makesthis synthetic principle appear totally uninterest-ing for an industrial process. Since all the basicpatents for the various oxazole routes to 2 haveexpired and the price of d,l-alanine has fallenconsiderably in the past years, it appears thatthe combination 34+47 is used by Takeda andDaiichi, 41+47 by Roche, and 40+48 by at leastone of the Chinese producers for the industrialproduction of pyridoxine hydrochloride (8).

8.7.1.5. Cobalt-Catalyzed[2+2+2]-Cycloaddition Reactions ofAcetylenes and Acetonitrile

Independently, two groups have developeda new pathway to pyridoxine using cobalt-catalyzed [2+2+2]-cycloaddition reactions of

protected α,ω-diyne ethers and acetonitrile[803].

However, the yields of 8 obtained were lessthan 10%. For instance, the problem of the con-version of the (CH3)3Si group at position 3 ofpyridoxine to an aromatic OH group could notbe satisfactorily solved.

8.7.2. Pyridoxine 5′-Phosphate

The OH groups in positions 3 and 4 ofpyridoxine (2) can be protected with ace-tone/hydrochloric acid, forming the ketal (52).Phosphorylation of 52 at 60 ◦C with H3PO4 –P2O5, followed by gentle acid hydrolysis, yields3 [804]. Pyridoxine 5′-phosphate (3) can alsobe obtained by the reduction of pyridoxal 5′-phosphate (5) with NaBH4 [805].

Phosphorylation of 52 is also achieved withmetaphosphoric acid (HPO3)3. The resultingmixture of oligophosphates can be hydrolyzedto 3 [806]. Compound (3) can also be producedby treating pyridoxamine 5′-phosphate (7) withHNO2 [807].

8.7.3. Pyridoxal [808]

Oxidation of pyridoxine (2) with permanganatein the presence of hydroxylamine produces pyri-doxal oxime (53), which can be converted to

100 Vitamins

pyridoxal (4) by treatment with nitrite and hy-drochloric acid [809].

Direct oxidation of 2 to 4 can be conductedwith MnO2 –H2SO4 [806,810]. Isolation of 4from solution can be advantageously achievedvia its bisulfite addition product [811]. A totalsynthesis of 4 from aliphatic building blocks hasbeen described [809].

8.7.4. Pyridoxal 5′-Phosphate

Pyridoxal 5′-phosphate (5) (“codecarboxylase”)[772] canbemade frompyridoxine 5′-phosphate(3) [803, a (Baddiley; Targa)] or from pyri-doxamine 5′-phosphate (7) [803, a (Wilson)][804,805] by oxidation with MnO2 or withpyruvic acid in the presence of Cu, Fe, Ni, orCo salts [812]. Direct phosphorylation of pyri-doxal (4) is described in [813]. Derivatives ofpyridoxal in which the aldehyde group is pro-tected by readily removable groups can be phos-phorylated and 5 released by mild hydrolysis.The following protective groups have been used:N-dimethylglycylhydrazone [806,814], Schiffbases of aromatic amines [815] and alkene-diamines [816], oxazolidines [817], and thia-zolidines [818]. Starting with pyridoxine 5′-phosphate (3) or pyridoxamine 5′-phosphate (7),pyridoxal 5′-phosphate (5) was made by enzy-matic oxidationwith immobilized pyridoxamine5′-phosphate oxidase [819]. Starting with pyri-doxine 5′-phosphate (3), the synthesis of 5 usingimmobilized bacterial cells has been described[820].

8.7.5. Pyridoxamine [772,808]

Pyridoxamine (6) can be produced from pyri-doxal oxime (53) by catalytic hydrogenationwith PtO2 in acetic acid [821], Pd/C [822], orby reduction with zinc dust [823]. Pyridoxam-ine (6) is obtained from pyridoxine (2) by re-action with NH3 in CH3OH [824]. It is also

produced by the reductive amination of pyri-doxal (4) [825]. Pyridoxamine (6) is also synthe-sized in a Diels –Alder reaction of 5-ethoxy-4-methyloxazole (34) with γ-hydroxycrotonitrile,followed by reduction of the nitrile group in po-sition 4 of the pyridine ring to an aminomethylgroup [826].

8.7.6. Pyridoxamine 5′-Phosphate

Pyridoxamine 5′-phosphate (7) is obtained frompyridoxal 5′-phosphate (5) by reaction with glu-tamic acid in an autoclave [827]. Starting withpyridoxamine (6), 7 is made by treatment withPOCl3 in aqueous solution [808]. However, bet-ter results are obtainedwhen 6 is phosphorylatedby heating with metaphosphoric acid, followedby degradation of the resulting 5′-triphosphateby mild acid hydrolysis [828] or by the action ofH3PO4/P2O5 at 60 ◦C on 6 [807,829].

8.8. Metabolism and Importance for theOrganism

The three nonphosphorylated B6 vitamins areabsorbed in the region of the upper small in-testine (proximal jejunum) by a nonsaturableprocess (passive diffusion) [830]. They arephosphorylated in the liver by a pyridoxal ki-nase and ATP, bound to albumin, and releasedinto the blood [831]. As a result of the bind-ing to albumin, pyridoxal 5′-phosphate is pro-tected from hydrolysis in circulation and isable to reach the tissues. The two phosphoforms, pyridoxine 5′-phosphate and pyridoxam-ine 5′-phosphate, are converted to pyridoxal 5′-phosphate (PLP) by a FMN-dependent oxidase[832]. After dephosphorylation by a membrane-bound alkaline phosphatase, pyridoxal can en-ter the cell through the cell wall. Pyridoxal isirreversibly converted to 4-pyridoxic acid byan NAD-dependent dehydrogenase or an FAD-dependent aldehyde oxidase. This acid is ex-creted in the urine as the main metabolite of B6metabolism [833]. The enzymatic interconver-sion of the vitamins B6 are shown in Scheme 25.

Pyridoxal 5′-phosphate acts as a coenzymefor many enzymes which, above all, regulateamino acid metabolism. The coenzyme is boundto an ε-amino group of a lysine residue of

Vitamins 101

Scheme 25. Metabolic interconversion of the vitamins B6

the apoenzyme through its formyl group. Thus,the active enzyme complex is formed via theresulting Schiff base. In this way, reactionssuch as transamination, nonoxidative deami-nation, decarboxylation, and hydrodesulfuriza-tion are catalyzed. The majority of pyridoxal5′-phosphate (75 – 80%) is found in glycogenphosphorylases, which are involved in gluco-neogenesis in plants and animals. However, thereaction mechanism is still unknown. Further-more, pyridoxal 5′-phosphate acts in microbialracemases which catalyze the conversion of l-amino acids to d-amino acids. In addition, itappears to have a regulatory effect on steroidhormone receptors. Under physiological condi-tions, pyridoxal 5′-phosphate binds reversibly tosteroid receptors and can modulate or inhibit theinteractionbetween the steroid receptor complexand the specific DNA sequence [834,835]. Thephysiological importance of the interaction bet-ween PLP and steroid receptors is not yet under-stood.

8.9. Deficiency Symptoms andApplications

A food-related vitaminB6 deficiency manifest-ing itself in clinical symptoms is largely un-known in humans. Since vitaminB6 plays an es-sential role in numerous biochemical processes,a lack of this vitamin is easily detectable fromthe resulting metabolic disturbances. Deficien-cies are produced by an unbalanced diet, inter-ference with medicines, excessive loss due tofunctional kidney disorders, intestinal diseases(diarrhoea, disturbances of absorption), hered-itary disorders (cystathioninuria, homocystein-uria), or neurological diseases.

Patients with liver cirrhosis (e.g., alcoholics)often have a low vitaminB6 level in the blood[836,837]. In 15 – 20% of women, estrogen-containing contraceptives cause an impairmentof tryptophan metabolism which can be normal-ized by 2 – 25mg of vitaminB6 per day [838]. Inthe treatment of tuberculosis with isoniazid, sideeffects appear (neuritis, convulsions) which canbe prevented by 25 – 50mg of vitaminB6 perday [839,840]. Treatment of the rare Wilson’s

102 Vitamins

disease with d-penicillamine causes impairedtryptophan metabolism which responds well to100mg of vitaminB6 per day [841]. The verylow plasma values found in patients with kid-ney diseases (chronic kidney failure, hemodial-ysis) or after kidney transplantation can be nor-malized by administration of 5 – 50mg of vi-taminB6 per day. Some genetic diseases resultin B6 malfunctions. Included in this categoryare cases of infantile convulsions caused by thepoor binding of vitaminB6 to the apoenzymeglutamate decarboxylase; primary cystathionin-uria, which is marked by a greatly reduced affin-ity of the enzyme cystathionase for vitaminB6;or homocysteinuria, which is caused by lowamounts of cystathionineβ-synthase. In general,intake of 5 – 50mg of vitaminB6 per day re-sults in improvements. High doses ormegadosesof vitaminB6 are often applied in the treatmentof rheumatic diseases, carpal tunnel syndrome,autism, schizophrenia, learning difficulties, andmental retardation [842,843].

8.10. Analysis

Numerous methods have been developed forthe determination of vitaminB6. Preparationsenriched with pyridoxine can be determinedby using UV spectra or color reactions (e.g.,the reaction between pyridoxine and diethyl-p-phenyldiamine). The analysis of vitaminB6 infoods, animal organs, and body fluids is compli-cated. Reasons for this are the different chemi-cal forms (pyridoxine, pyridoxal, pyridoxamine,and their phosphate esters), sensitivity to heatand light, and the covalent bindingof these formsto proteins. Microbiological methods are oftenused for the determination of vitaminB6. Theyeast strain Saccharomyces uvarum is generallyused for this purpose [844,845]. Frequently, allthe vitaminB6 forms can be determined by usingcombinations with other microorganisms, e.g.,Lactobacillus casei, Streptococcus faecium. Thedetection limit is 0.5 – 1.0 ng. However, thesemethods are very time consuming. The microor-ganisms can mutate or suffer growth inhibitionunder the influence of the extracts. Pyridoxal 5′-phosphate is enzymatically determined in theblood, serum, or in erythrocytes by the decar-boxylation of amino acids [846,847]. Pyridoxal5′-phosphate can be indirectly determined in

erythrocytes by measuring the enzyme activitiesof erythrocyte glutamate oxaloacetate transam-inase (EGOT) and erythrocyte glutamate pyru-vate transaminase (EGPT) [847–849]. Gas chro-matography and HPLC have also been success-fully used [850,851].

8.11. Economic Aspects

World production of pyridoxine hydrochloride(8) is estimated at 2550 t/a for 1993. Themain producers are Roche (Germany), Takeda(Japan), and Daiichi (Japan). In the past years,the production capacity in several plants hasbeen drastically increased, especially in China,and the estimated world capacity of > 4100 t/afor 1993 greatly exceeds demand. From 1992,the market has been increasingly supplied byChinese companies (especially by New Asiatic,Shanghai). The list price for pyridoxine hydro-chloride given in [852] is $ 50/kg. However, theactual retail prices on the world market fluctuateand are considerably lower than the list price (asof October 1994).

The historically highest retail price for 8 was7600 $/kg in the year of its introduction and thelowest price was $ 30/kg in 1967.

Trade names of vitaminB6 and its pro-ducers are, e.g., Benadon (Roche), Hexo-bion (Merck), Beelith (Beach Pharmaceuti-cals, USA), VitaminB6 Richard (Lab. M.Richard, France), VitaminB6 Streuli (Streuli,Switzerland), B6-Vicotrat (Heyl, Germany),Pyragamma (Worwag, Germany), and Vita-minB6 Jenapharm (Jenapharm, Germany). Allthese products contain pyridoxine hydrochlorideas the active agent in doses of 20 to 300mg, andin one case 600mg.

8.12. Requirements and Tolerance

The requirement for vitaminB6 is influenced byvarious factors, especially by the daily intakeof protein. The requirement increases with in-creasing protein absorption. In theUnitedStates,the average daily intake of vitaminB6 in 1985was 1.87mg for men, 1.16mg for women, and1.22mg for children of 1 – 5 years [853]. Vari-ous methods are used to estimate the require-ment, e.g., direct determination of the different

Vitamins 103

forms in the blood and/or urine (excretion of4-pyridoxic acid), various load assays (e.g., de-termination of the tryptophan metabolites xan-thurenic and kynurenic acid in the urine afteroral tryptophan loadingwith 2 – 5 g), or themea-surement of vitaminB6 dependent enzymes (in-direct functional determination). Since 1989, therecommended daily allowance (RDA) for vita-minB6 in theUnitedStates has been set at 2.0mgfor men, 1.6mg for women, 2.1mg for preg-nant and nursing women, 1.0mg for children ofage 1 – 3, 1.1mg for 4 – 6 year old children, and1.4mg for children at the age of 7 – 10 [777,853].

Pyridoxine and related compounds havingvitaminB6 activity show only low acute tox-icity [854,855]. Teratogenic and carcinogeniceffects of high pyridoxine doses are unknown[856,857]. After ingestion of excessively highdoses (2 – 6 g of pyridoxine per day) over longerperiods of time, peripheral neurological disor-ders have been observed in humans [858]. Adaily intake of up to 250mg of pyridoxine overlonger periods is harmless [859,860]. The limitat which toxic effects can appear is ca. 500mg/dor more [861].

9. Vitamin B12 (Cobalamins)

9.1. Introduction

Vitamin B12 [68-19-9] belongs to the groupof cobalamins, which also includes adeno-sylcobalamin [13870-90-1], methylcobal-amin [13422-55-4] and hydroxocobalamin[13422-51-0]. The cobalamins and other com-pounds similar to B12 are corrinoids (Fig. 12).The corrinoids have a tetrapyrrole ring systemwhich, compared to the porphyrins, lacks themethine bridge between rings A and D; centralatom is cobalt.

The cobalt atom is coordinated by two lig-ands perpendicular to the plane of the tetrapyr-role ring. One of these ligands is a nucleotidelinked to a propionyl group of the tetrapyrrolering by an aminopropanol group.

All corrinoids that contain 5,6-dimethylbenz-imidazole [582-60-5] as the nucleotide ligandare known as cobalamins. Depending on the an-ionic ligand, a distinction is made between vi-tamin B12 (cyanide as ligand), coenzyme B12

(deoxyadenosyl group as ligand), etc. The cobal-amins are presented in Figure 13.

Deoxyadenosylcobalamin and methylcobal-amin are physiologically active as coenzymes.Another compound found in the bodies of mam-mals is hydroxocobalamin, a precursor of thecoenzymes. Cyanocobalamin is a form madefrom the naturally occurring cobalamins. It isreconverted to the coenzyme forms in the body.

Other B12 derivatives are found in bac-teria. They have in common that the rib-azole group (5,6-dimethylbenzimidaole) is re-placed by imidazoles or purines. Important re-presentatives are pseudovitamin B12 (Co α-[α-(5,6-dimethylbenzimidazoyl)]cobamide) ofmethanogenic bacteria. In addition, other cor-rinoids are found in acetogenic bacteria,clostridia, and sulfate-reducing and sulfur-metabolizing bacteria [862].

In 1925, Whipple and Robscheit-Robbinspostulated for the first time that vitaminB12 is ananimal protein factor. In 1926,Minot andMur-phy showed that raw liver cured pernicious ane-mia. Subsequent work showed that this diseaseis caused by vitamin B12 deficiency. In 1948,vitamin B12 was isolated from liver samplesby Rickes and Smith. The structure was elu-cidated by Hodgkin et al. in 1955. In 1972, thetotal chemical synthesis, with more than 70 syn-thetic steps, was achieved by Woodward andEschenmoser.

Neither animals nor plants can synthesize vi-tamin B12. According to present knowledge, thebiosynthesis of corrins is restricted to microor-ganisms.

In humans, vitamin B12 is absorbed in twoways: by diffusion at higher vitaminB12 concen-trations, and via the intrinsic factor, a mucopro-teid that is formed in the mucous membrane ofthe stomach and specifically binds cobalamins.[The name “intrinsic factor” comes from the factthat two factors can be responsible for vitaminB12 deficiency: lack of B12 (extrinsic factor) andlack of binding protein (intrinsic factor).] Themucoprotein complex reaches the ileum, whereresorption is mediated by a transport system.Evenwithout intrinsic factor, vitaminB12 can beabsorbed by diffusion throughout the small in-testine. However, the doses needed to cover thedaily requirement in this way are 10 to 100 timeslarger than the amounts available in foods. In theliver, vitamin B12 is converted to coenzyme B12

104 Vitamins

Figure 12. Classification of corrinoids

Figure 13. Structure of cobalaminsR = CN− : cyanocobalamin = vitamin B12R = 5′-desoxyadenosyl group: adenosylcobalamin = coenzyme B12R = methylgroup: methylcobalamin = mecobalaminR = hydroxyl group: hydroxocobalamin = aquocobalamin = vitamin B12 a

in a reaction dependent on adenosyl triphosphate(ATP) and flavin adenine dinucleotide (FAD),and stored. VitaminB12 is excretedwith the bile.

With a balanced diet, humans ingest 10 –30µg of vitamin B12 or other cobalamins perday, of which ca. 5µg is resorbed. This amounthas also been stipulated by the German Societyfor nutrition and by the U.S. Food and NutritionBoard as the daily requirement for humans.

Cobalamins are predominantly ingested withanimal products. In experiments with pigs, feed-

ing exclusively with plant proteins led to stop-page of growth due to B12 deficiency.

In the large intestine of humans, the intestinalflora produce ca. 50µg of cobalamins per day,which are, however, not resorbed.

In humans vitaminB12 deficiency causes per-nicious anemia,which is characterized by a largereduction in the erythrocyte count due to a distur-bance in the maturation of red blood cells. Theanemia is accompanied by neurological symp-toms. In most cases, pernicious anemia is due

Vitamins 105

to a lack of intrinsic factor, resulting in reducedB12 absorption.

In the treatment of pernicious anemia, vita-min B12, hydroxocobalamin, or adenosylcobal-amin is administered in a dosage of ca. 100µg.In the case of cyanide poisoning, high doses ofhydroxocobalamin are given to bind the cyanide.

Vitamin B12 is used as a component of dietfoods, children’s foods, vitamin mixtures, cere-als, beverages, vitamin preparations, and as afeed additive [863].

In pig breeding, vitamin B12 is used in a con-centration of 10 – 30mg/t of feed. The GermanFeed Law permits the addition of 10 – 20µg ofvitamin B12 per kilogram of feed for young pigs,hens, turkeys, and ducks. Ruminants cover theirrequirements via the formation of B12 by rumenbateria.

9.2. Properties of Vitamin B12

Cyanocobalamin forms dark red, odorless, taste-less crystals (needles and prisms). The crystalsare hygroscopic and can absorb up to 12% ofmoisture. All the corrinoids are soluble in wa-ter (ca. 1%), poorly soluble in lower alcoholsand phenols, and insoluble in acetone, chloro-form, ether, and other organic solvents. Aque-ous solutions are optically active. The followingoptical rotations are measured in dilute aqueoussolutions of cyanocobalamin: [α]20656:− 59± 9◦,[α]20644: −110± 10◦.

Aqueous solutions of cyanocobalamin arestable. Decomposition occurs only at tempera-tures above 120 ◦C at pH values below 3 orabove 8. Solutions of adenosyl-, methyl-, andhydroxocobalamin are, however, sensitive tolight. The decomposition temperature and solu-bility of some B12 derivatives (solids) are listedin Table 29.

Table 29. Decomposition temperature and solubility of someimportant derivatives of vitamin B12 [864]

Name Decompositiontemperatures, ◦C

Solubility inwater, g/L

Vitamin B12 210 14.1Adenosylcobalamin 150 15.0Methylcobalamin 210 13.4Hydroxocobalamin 200 107.0

According to IUPAC– IUB nomenclature,the Co complexes are designated as Co

α-agylkonyl-(Co β-ligandyl) cobamides. Themeaning of α and β is the same as in the steroidseries. Thus, cyanocobalamin is [(Co α-(5,6-di-methylbenzimidazoyl)]-Co β-cyano-cobamide,abbreviated:

Apart from cyanide, other ions can com-plex cobalt, in the following order of stability:CN− SO2−

3 >OH− halides.In alkaline solution, the cobalamins can be

reduced by thiols, giving reduced vitamin B12r,a Co(II) cobalamin. Further reduction gives riseto Co(I) cobalamin (vitamin B12s).

Vitamin B12s can be converted by methyl io-dide to methylcobalamin with the formation ofan organometallic bond:

Reaction with 5′-tosyl-2′3′-isopropylideneadenosine gives adenosylcobalamin.

Most of the known biochemical reac-tions involving adenosyl- or methylcobalaminhave been found to occur in microorgan-isms. These include ribonucleotide reductase(DNA synthesis), propanediol dehydratase (iso-propanol degradation), glutamate mutase (glu-tamate degradation), ethanolamine ammoniumlyase (ethanolamine degradation), lysine mu-tase (lysine degradation), ornithine mutase (or-nithine degradation), and glycerol dehydratase(glycerol degradation). In human metabolism,methyl-cobalamin is required by the enzymemethionine synthase (E.C. 2.1.1.13), and adeno-sylcobalamin is requiredbymethylmalonylCoAmutase (E.C. 5.4.9.9.2). It is probable thatleucine mutase (E.C. 5.4.3.7) is also dependenton adenosylcobalamin.

106 Vitamins

9.3. Analysis

A large number of methods for the detection ofvitamin B12 have been described [865]:

1) Microbiological growth tests (detection limitof 10−11 mol/L≈ 10 ng/L) with Lactobacil-lus leichmanii [American Type Culture Col-lection (ATCC) 4797 and 7830], which re-acts very specifically to cobalamin. Growthtests with Escherichia coli (ATCC 9637) arealso possible. These are either turbidimet-ric assays in test tubes or growth tests onagar plates. The plate tests are mainly usedin screening for a quick assessment of theproductivity of mutants.

2) Chromatographic processes (detection limitof 10−6 mol/L≈ 1mg/L) with HPLC meth-ods (XAD-2 columns or reversed phase RP-18 columns and photometric detection).

3) Radioimmunological methods (RIA); com-mercial test sets are available for serial as-says.

4) UV–VIS spectroscopic methods (detec-tion limit of 10−5 mol/L≈ 10mg/L). Thismethod is only suitable for pure substances.

Aqueous solutions of cyanocobalamin haveabsorption maxima at (Fig. 14): λ= 278 nm(± 1 nm), ε1 %

1 cm = 93; λ= 360 nm (± 1 nm),ε1 %1 cm = 168; λ= 550 nm (± 1 nm), ε1 %

1 cm = 52.

Figure 14. UV-VIS spectrum of cyanocobalamin

The absorption maxima of the other corri-noids are similar to those of cyanocobalamin.

Owing to the very low concentrations of vi-tamin B12 in foods and feeds, its detection byspectroscopic methods is possible only after pu-rification or isolation. Microbiological methodsallow very specific B12 detection without pre-treatment of the samples. These tests are, how-

ever, less accurate and require a microbiologicallaboratory.

Before analysis, each of the corrinoids in cor-rinoid mixtures must be isolated and determinedseparately. If the total amount of corrinoids issufficient, the corrinoids are converted to thecyano form and determined as vitamin B12. Ifcoenzyme B12 or methylcobalamin is isolated,it is necessary to work under red light becausethese B12 variants are sensitive to light.

9.4. Biosynthesis

The biosynthesis of vitamin B12 is one ofthe most complex metabolic pathways known.Many of the steps have been studied [866–868].Themetabolism has, however, not yet been fullyelucidated, and various groups are working onthe biochemistry and genetics of B12 biosyn-thesis. The synthesis of B12 in the bacteriaEscherichia coli, Propionibacterium shermanii,Pseudomonas denitrificans, and Salmonella ty-phimurium has been thoroughly studied.

As in the case of hemes and chlorophylls, theprecursor of the corrinoids is uroporphyrinogenIII [1976-85-8] (uro’gen III). Uro’gen III is con-verted to precorrin 8× by a series of methylation

Vitamins 107

steps and elimination of the methine bridge bet-ween rings A and D.

Cobalt insertion gives rise to cobyrinicacid, insertion of a molecule of threonine[72-19-5] produces cobinamide [14709-02-5],and linkage of the “nucleotide loop” with 5,6-dimethylbenzimidazole from riboflavin (vita-minB2) [83-88-5] and adenylation yields coen-zyme B12.

The biosynthesis of vitamin B12 is controlledby ca. 30 genes. In the bacterium Salmonella ty-phimurium, most of these genes are organizedin a functional unit, the cob operon. The genesof Pseudomonas denitrificans are similarly or-ganized.

9.5. Production

Vitamin B12 is produced exclusively by fermen-tation. With more than 70 steps, chemical syn-thesis is not economical.

In the past, vitamin B12 was isolated fromfermentation residues obtained from the pro-duction of antibiotics, e.g., from streptomycinproduction with Streptomyces griseus, fromneomycin fermentation with Streptomyces fra-diae, or from chlorotetracycline production withStreptomyces aureofaciens [869]. In comparisonwith fermentation processes, however, this pro-cess is no longer economical.

Clarification sludge fromanaerobicwastewa-ter treatment is rich in vitaminB12 andpseudovi-tamin B12, but extraction is not profitable.

Although more than 100 processes for thefermentative production of vitamin B12 are de-scribed in the literature and in patents, only fiveto ten are used in practice worldwide.

The most important processes are fermenta-tion with Pseudomonas denitrificans and withbacteria of the genus Propionibacterium. Fer-mentations with other species have also beendescribed [870].

Bacteria which produce B12 or B12 deriva-tives are listed in the following:

Propionibacteria Propionibacterium shermaniiPropionibacterium freudenreichii

Pseudomonada Pseudomonas denitrificansPseudomonas methanolignicaPseudomonas aeroginosa

Methanogenic bacteria Methanosarcina barkeriMethanobacterium thermo-autotrophicum

Acetogenic bacteria Acetobacterium woodiiAcetobacterium spec.

Other species Arthrobacter hyalinusMicromonaspora purpureaStreptomyces olivaceusBacillus megateriumNocardia methanophilusNocardia gardeneriKlebsiella spec.Eubacterium spec.Rhodopseudomonas spec.Butyribacterium metholo-trophicum

All fermentations produce a mixture ofmethyl-, hydroxo-, and adenosylcobalamin. Themethanogenic bacteria produce pseudovitaminB12 or Factor III. Isolation of the individualcorrinoids is tedious. For this reason, work-upis usually carried out after the corrinoids havebeen converted to the cyano form by addition ofcyanide.

9.5.1. Fermentation

Vitamin B12 is produced in fermenters whichare filled with a production medium in whichthe bacteria grow and produce. The productionmedium is suited to the needs of the strains used.The fermenter is inoculated from a preculturewhich, in turn, comes from a working cell bank[871].

The bacterial strains used to produce vitaminB12 are improved in a strain-development pro-

108 Vitamins

gram. In this way, the productivity (amount ofB12 per liter of fermentation medium per unittime) of the strains can be greatly improved.Spella et al. [872] have described in a programwhich was used to improve the productivity ofPseudomonas denitrificans in eight steps from4mg/L of B12 to 150mg/L of B12. In this pro-gram, the strains were treated with an agent thatcauses mutations (e.g., UV radiation or nitro-somethylurea [684-93-5]). Among the mutants,strains with improved characteristics were se-lected (mutation – selection process). Improvedcharacteristics can include not only higher pro-ductivity, but also improved growth rate, im-proved nutrient utilization, higher tolerance toend products (e.g., propionic acid) and start-ing materials (e.g., dimethylbenzimidazole), orimproved genetic stability. The search for im-proved mutants is very tedious (1 improvedstrain per 105 – 106 strains).

The number of hits can be increased if it ispossible to specifically search for characteris-tics which are connected with improved pro-duction. For instance, it is known that porphyrinsynthesis reduces B12 production because bothcompounds have a common precursor in theirbiosynthesis. Mutants with reduced porphyrinsynthesis can be recognized, e.g., by their re-duced catalase activity. In the screening pro-cess, strains with reduced catalase activity aresearched for first. These strains are then testedfor B12 productivity.

It is also possible to search for improvedstrains by using antimetabolites (e.g., ethionine)which permit the selection of strainswith alteredenzyme activities.

Propionibacterium Process. The propioni-bacterium process is the oldest described fer-mentation process. Propionibacterium sher-manii (ATCC 6707) and Propionibacteriumfreudenreichii (ATCC 13 673) are the preferredspecies. The carbon and energy sources are car-bohydrates that are fermented to propionic acidwithout oxygen. The acid produced (ca. 10% ofthe fermentation volume) must be neutralized(pH 7) during fermentation. An example of aproduction medium follows:

Glucose 120 gCorn steep liquor 80mLGlycine 5 g

NH4NO3 3 gMgSO4 · 5H2O 0.5 gNa2MoO4 10µgCalcium pantothenate 5mgCoCl2 · 6H2O 10mgDimethylbenzimidazole 20 gTap water 1 L

Propionibacteria are anaerobic organismswhich grow and produce B12 very poorly inthe presence of oxygen. However, the synthe-sis of 5,6-dimethylbenzimidazole, a componentof B12, is dependent on oxygen. Furthermore,the biosynthesis of B12 is inhibited by the pres-ence of B12 (feedback inhibition). These prob-lems are avoidedbyusing a two-stage process. Inan anaerobic fermentation that takes ca. 3 d, thebacteria grow and produce the vitamin B12 pre-cursor cobamide (B12 without dimethylbenzim-idazole). Subsequently, the culture ismoderatelyaerated for 1 – 3 d to promote the biosynthesis ofdimethylbenzimidazole and to complete the for-mation of B12. As a rule, dimethylbenzimidaz-ole is added in the second stage of fermentation,increasing the yields and reducing the fermenta-tion time. In this way, B12 yields of > 150mg/Lcan be achieved.

PseudomonasProcess. Miller andRosen-blum [873] have isolated mutants of Pseu-domonas denitrificans characterized by goodproductivities. Taxonomically, P. denitrificansno longer exists, having been assigned to thespecies Pseudonomas fluorescence and Alcali-genes xylosoxidans [874].Pseudomonas denitrificans converts glucose

to carbon dioxide, consuming oxygen in the pro-cess. In contrast to the Propionibacterium pro-cess, growth occurs with parallel synthesis ofB12. The fermentation is aerated and conductedat ca. pH 7 and 30 ◦C. After a fermentation pe-riod of 1 – 2 d, a yield of> 150mg/L is achieved.The culture is well aerated during the logarith-mic growth phase. However, the oxygen con-centrations in the medium must not be high be-cause otherwise a decrease in growth and pro-duction occurs. Dimethylbenzimidazole as B12precursor is added to the fermentation medium.The growth of Pseudomonas denitrificans is im-proved by glutamate, and the synthesis of B12 isstimulated by betaine, choline, dimethylbenzim-idazole, and cobalt. Sugar beet molasses is of-ten used in industrial fermentations because of

Vitamins 109

its high betaine content. An example of a B12production medium is present in the following:

Sugar beet molasses 105 gSucrose 15 gBetaine 3 gAmmonium sulfate 2.5 gMagnesium sulfate 0.2 gZinc sulfate 0.08 g5,6-Dimethylbenzimidazole 0.025 gTap water 1 L

9.5.2. Work-Up

Vitamin B12 and its derivatives are present in-side the cell. Vitamin B12 is either isolated andpurified or the dried cell mass is used as a con-centrate in animal feeds (Fig. 15).

Figure 15. Work-up of vitamin B12

Hydroxocobalamin is obtained if the naturalcobalamins are converted to the chloro, sulfate,

or nitrate form during extraction and then sub-jected to alkaline ion exchange [e.g., on Am-berlite IRA400 (OH−)] [875]. Adenosyl- andmethylcobalamin are isolated directly from thefermentation broth.

To produce a concentrate, the cells are sepa-rated from the medium with separators or cen-trifuges, and the cellmass is dried. Alternatively,the entire contents of the fermenter can be evap-orated or concentrated and spray dried. The con-centrates produced in this manner contain ca. 1 gof corrinoids per kilogram of dry matter.

For use in the feed industry, the centrifugedcell mass is heated as an aqueous suspensionto lyse the cells. Depending on the productionorganism, other processes can also be used forlysis. The dissolved corrinoids are convertedto cyanocobalamin by the addition of KCN,usually in the presence of sodium nitrite andheat. The vitamin B12 solution is then clarifiedby filtra-tion, treatment with zinc hydroxide, orsimilar processes. Precipitation of this aqueoussolution produces a product of ca. 80% puritywhich is a suitable animal feed additive. Theprecipitation can be performed by concentrationand addition of auxiliaries such as tannic acid orcresol.

For further purification to give B12 of foodand pharmaceutical quality, the clarified vita-min B12 solution can be extracted with organicsolvents (cresol, carbon tetrachloride), and thenwith water after the addition of butanol, andagain with organic solvents. Adsorption pro-cesses, e.g., with ion exchangers, aluminum ox-ide, or activated carbon, canbeused alternativelyor in addition. Pure vitamin B12 is obtained bycrystallization after the addition of organic sol-vents. A detailed description is given in [871].

9.5.3. Patents and Scientific Survey

More than 1000 patents and articles on the fer-mentative production of vitamin B12 have beenpublished.

A general protection by patent for the Pro-pionibacterium and Pseudomonas processes nolonger exists. However, a number of specialprocess and preparation variants have beenpatented. Furthermore, other production organ-isms and substrates have been decribed. A sur-vey is presented in Table 30.

110 Vitamins

Table 30. Survey of the patent and literature on the fermentative production of vitamin B12

Company/year Organism Productivity Remarks

Asahi Chemical (J) 1988 fermentation with cell recyclingDaicel Chemicals (J)1987 – 91

Acetobacterium, Methanosarcina 4.1mg/L in 124 h, 23mgB12/g DM∗

fermentation with methanol mutants

Dainippon (J) 1978 – 79 Pseudonocardia, Nocardia 1.85mg/L fermentation with methanolGedeon Richter (HUN) bisdato

Propionibacterium shermanii 26mg/L in 10 d different fermentation andpreparation patents

Kobe Steel (J) 1992 Methanobacteriumthermoautotrophilum

fermentation with methane

Kureha Chemicals (J) 1983 Protami-nobacterium+Rhodospirillum

cell fusion

Kyowa Hakko (J) 1975 Klebsiella methanol mediumMitsubishi Gas Chemicals(J) 1987

Eubacterium/Butyribacterium 37 – 39mg/L in 40 h methanol medium, partly mixedculture

Nippon Oil (J) 1976 – 1995 Arthrobacter, Propionibacteriumshermanii

43mg/L in 7 d fermentation with glucose,isopropanol, patents on work-up

Nippon Point (J) 1988 isolation of B12

Nippon Zeon (J) 1980 Candida tropicalis 0.2mg/L in 80 h ethanol mediumRhone-Poulenc,Rhone-Poulenc Rohrer (F)1979 – 1985

Pseudomonas denitrificans studies on metabolic elucidation,patents on cloning of metabolicpathways of pseudomonas

Shimizu Construction (J)1990 – 1993

Methanogenic 1.4mg/L in 4 d

Shiseido (J) 1979 – 1987 reinfarction of B12

∗DM=dry matter.

9.6. Specifications and Legal Aspects

A USP cyanocobalamin reference standardis available (USP 207) with the followingUV–VIS spectroscopic properties: maxima at278± 1 nm, 361± 1 nm, and 550± 2 nm. Thequotient of the absorptions at 361 and 278 nmmust be 1.70 – 1.90.

The toxicity of vitamin B12 is extremely low.In animal experiments, vitamin B12 in doses ofup to 1 g/kg of body weight showed no toxiceffects. In humans, allergic reactions have beenobserved in a few cases. A connection betweenmethylcobalamin and tumor development hasalso been reported. However, these effects arestill disputed [876]. There is no limitation to thedosage of B12; excess B12 is excreted.

9.7. Economic Aspects

With reference to the pure substance, the to-tal sales of vitamin B12 amount to more than10 t/a and the market volume is ca. ¤ 77×106.The feed sector accounts for ca. 55% of thesales, and the food/pharmaceutical sector forca. 45%. Apart from cyanocobalamin, hydroxo-and adenosylcobalamin are traded, especially onthe pharmaceutical market.

NorthAmerica andWesternEurope representthe largest market potentials. The Asian coun-tries including Japan are gaining importance.The remaining market volume is almost equallydistributed among Brazil, the other countries inLatinAmerica, andEastern Europe. TheAfricanand Western Asian markets are of less impor-tance.

More than 80%of the total annual productionof vitamin B12 comes from France. The com-pany Rhone-Poulenc occupies a dominant po-sition in both feed and pharmaceutical sectors.Roussel Uclaf, another French manufacturer, isactive exclusively in the pharmaceutical sector.

Other producers are: Gedeon Richter, Hun-gary, with an estimated share of themarket of ca.10%; Nippon Petrochemicals, Japan; Merind,India; and smaller producers in Eastern Europe.

10. Vitamin C (l-Ascorbic Acid)

10.1. Introduction

Ascorbic acid [50-81-7] (1) (or l-ascorbic acid,l-xylo-ascorbic acid, l-threo-hex-2-enoic acidγ-lactone) is the official IUPAC designation forvitaminC [893]. Ascorbic acid is the naturallyoccurring antiscorbutic vitamin. VitaminC defi-

Vitamins 111

ciency causes scurvy, one of the oldest diseasesknown to humankind.

In 1928 vitaminC was isolated for the firsttime by Szent-Gyorgyi [894] and five yearslater its chemical structure was determined byHaworth and Hirst [895]. At the same timeindustrial production of ascorbic acid was de-veloped, and has since reached a volume of ca.60 000 t/a. The most significant characteristic ofl-ascorbic acid is its oxidation to dehydro-l-ascorbic acid [490-83-5] (2), withwhich it formsa reversible redox system. This reducing prop-erty, together with its nutritional qualities andlow toxicity, is the main reason for the numer-ous applications of vitaminC in the food andpharmaceutical industries.

Of the other three diastereomers of ascor-bic acid only d-arabo-ascorbic acid [89-65-6](3) (isoascorbic acid, erythorbic acid, d-erythro-hex-2-enoic acid γ-lactone) shows antiscorbu-tic activity (ca. 5% of that of vitaminC). Insome countries it is used as an antioxidant in thefood industry, as a cheaper substitute for ascor-bic acid.

10.2. History [882]

Although a disease remarkably similar to scurvywas known by the ancient Egyptians, it was notuntil 1753 that James Lind described the pre-vention of scurvy by dietary means. He revealedscurvy to be a dietary deficiency arising from alack of fresh fruits or vegetables. Nevertheless,scurvy incidents still occurred in times whenfruits and vegetables were unavailable.

A breakthrough in scurvy research was thediscovery by Holst and Frolich [896] in 1907that guinea pigs are susceptible to scurvy. Thus,they had an animal to test various diets for theirantiscorbutic activity, and they conclusively con-firmed that scurvy was produced by a defectivediet.

In 1928 Szent-Gyorgyi accomplished theisolation of pure vitaminC from the adrenal cor-

tex of cattle, orange juice, cabbage water, andlater from paprika. He demonstrated its relation-ship to antiscorbutic factor and named it hex-uronic acid [894]. In 1933 the structure was elu-cidated by Haworth and Hirst [895] and con-firmed by a synthetic route to d-ascorbic acidand l-ascorbic acid (Reichstein, Haworth[897]). In the same year Haworth and Szent-Gyorgyi suggested the name be changed to l-ascorbic acid to reflect its antiscorbutic proper-ties [898]. Both scientists were awarded the No-bel Prize in 1937 for their work on vitaminC.

10.3. Physical and Chemical Properties[883,884]

A summary of physical properties of l-ascorbicacid is given in the following [899]:

Appearance white, odorless, acidic tasteFormula C6H8O6

Mr 176.13Crystalline form monoclinic, usually plates, sometimes

needlesmp 190 – 192 ◦C (decomp.)Density 1.65 g/cm3

Optical rotation [α]25D in H2O: +20.5◦ to +21◦ (c = 1)

[α]20D in EtOH: +52◦ (c = 0.5) [900][α]20D in MeOH: +49.5◦ (c = 0.5) [900]

pH 3 (5 g/L); 2 (50 g/L)pK1 4.17 (3-OH)pK2 11.57 (2-OH)Redox potential first stage: E1

0 =+0.127V (pH 5)Solubility, g/mL 0.33 (H2O)

0.02 (absolute EtOH)0.01 (glycerol USP)0.05 (propylene glycol)insoluble in ether, benzene, oils, fats

UV λmax = 245 nm (acid solution)λmax = 265 nm (neutral solution)

Spectral data UV [901,902]; IR [901,903]; 1HNMR[901,904]; 13CNMR [905,906]; MS [907]

l-Ascorbic acid exists in at least five tau-tomeric forms [885], of which tautomer 1 re-presents crystalline l-ascorbic acid [908] andalso predominates in aqueous solution at pH 2[909]. The 2-OH and 3-OH groups are acidic.With bases ascorbic acid forms monobasicsalts; the dibasic salts are unstable. In sodiumascorbate (C6H7O6Na) and calcium ascorbate[(C6H7O6)2Ca · 2H2O] the metal atom is asso-ciated with the O atom in the 3-position [908].The negative charge of the ascorbate monoan-ion is delocalized in a π-bonding system, which

112 Vitamins

explains the stability of its lactone ring towardalkali:

When boiled in methanolic potassium hy-droxide l-ascorbic acid isomerizes to l-isoascorbic acid [910].l-Ascorbic acid (1) is a strong reducing agent

due to its enediol structure. Two-step oxidationof 1 proceeds via intermediate radical anionsemidehydroascorbic acid (4) to dehydroascor-bic acid (2) [911]. Semidehydroascorbic acid is astrong acid (pK = 0.45) and a radical scavenger;it reacts with itself, yielding 1 and 1a [912], orwith other free radicals.

The autoxidation of ascorbic acid to dehy-droascorbic acid (2) is catalyzed by transitionmetals (e.g., Cu, Fe). In the presence of oxygenthey form a ternary intermediate complex (e.g.,5)which dissociates to dehydroascorbic acid (2),hydrogen peroxide, and the metal ion [913].

The autoxidative process accelerates abovepH 7 and can be retarded by complexing themetal ions with chelating agents such as EDTA,metaphosphoric acid, citric acid, or oxalic acid.Treatment of ascorbic acid with oxygen inmeth-anol containing activated carbon gives a syrupymethanol adduct, which on refluxing in methylethyl ketone forms dehydroascorbic acid (2) in64% yield as a crystalline dimer [914]. 2 isstable for several days at 4 ◦C in aqueous so-lution at pH 2.5 – 5.5 and can be quantitativelyreduced back to ascorbic acid by, e.g., hydrogensulfide, dithiothreitol, or cysteine [915]. Dehy-

droascorbic acid (2) still exhibits vitaminC ac-tivity, which is lost on irreversible opening ofthe lactone ring to give 2,3-diketogulonic acid[3445-22-5] (6).

Ascorbic acid readily undergoes degradationin aqueous solution under aerobic and anaero-bic conditions (Fig. 16). The decomposition de-pends on pH, temperature, light, concentration,and heavy metal catalysts. The loss of ascorbicacid is often ten times higher in the presence ofoxygen than the loss under anaerobic conditions.Final oxidation products are l-threonic acid (7)andoxalic acid.Anaerobic degradation proceedsto furfural (8) and CO2 [883,916].

In the absence of moisture and in darknesssolid ascorbic acid is stable for a long time. Pro-tection against oxidative degradation in aque-ous applications is achieved by coating solidascorbic acid with fats and polymers or bychemical substitution of the enediol hydroxylgroups (l-ascorbate 2-sulfate [917], l-ascorbate2-phosphate [918], 2-O-methyl ascorbic acid[919]).

10.4. Analysis [886,887]

Methods for determination of ascorbic acid arenumerous. Thefirst testwas the biological assay:preventionor cure of scurvy in guinea pigs [920].Despite being time consuming it is still used tomeasure the antiscorbutic activity of products.Chemical methods are mainly based on the re-ducing character or derivatization of ascorbicacid. They provide less expensive and faster as-says, but do not distinguish between the differ-ent stereoisomers, and often other reducing sub-stances interfere. Therefore, the selection of ananalytical procedure for ascorbic acid in biolog-ical fluids, foods, and pharmaceutical prepara-tions should be made carefully. Increased speci-ficity can be achieved by using commerciallyavailable ascorbate oxidase. Ascorbic acid is se-lectively oxidized by this plant enzyme. HPLCallows rapid separation and detection of ascorbicacid and isoascorbic acid. High analytical speci-ficity can be achieved by using a HPLC methodwith a post-column oxidation/reduction systemin conjunction with UV, electrochemical (EC),or fluorometric measurement of ascorbic acid.HPLC and electrochemical methods are fully

Vitamins 113

Figure 16. Degradation of ascorbic acid

Table 31. Selected analytical procedures for ascorbic acid

Method, reagents Notes Ref.

Iodine standard method in pharmacopeias, wide analytical range, not specific [921]Chloramine T KI/starch, mixtures with sulfite, cysteine, glutathione, pharmaceutical preparations [922]2,6-Dichlorphenol-indophenol

standard redox reagent, automated procedures, poor specificity, dye instability [923]

MTT thiazolyl blue, ascorbic acid oxidase, marketed in kit form [924]2,4,6-Tris-(2-pyridyl)-s-triazine

colored iron complex, clinical method, determination of AA/DHA [925]

2,4-Dinitro-phenylhydrazine

classical colorimetric method, interfering substances, clinical samples [926]

Diazotized4-methoxy-2-nitroaniline

pharmaceutical stability studies, automated serum and urinary analysis, blankcorrection

[927]

o-Phenylenediamine official method, automatable, food analysis [928]Ascorbic acid oxidase enzyme isolation, increase of specificity, food and clinical analysis [929]Electrochemical methods specific oxidation at an electrode, automated procedure, 0.4 ng detection limit [930]GLC trimethylsilyl derivatives of ascorbic acid, sugars and related substances [931]TLC qualitative analysis of AA and degradation products [932]HPLC detection by UV/refractive index, IAA/AA/diketo-gulonic/gluconic acid [933]HPLC, derivatization withDNPH

electrochemical detection, biological fluid analysis, IAA/AA, 2 ng detection limit [934]

HPLC, reversed phase, ionpair

fluorescence detection, biological fluid analysis, AA/DHA/IAA, 10 ng detection limit [935]

114 Vitamins

automatable and therefore suitable for routineassays or process control in production.

A selection of analyticalmethods is presentedin Table 31.

10.5. Occurrence and Sources ofVitamin C

Ascorbic acid is considered to be an ubiquitousconstituent of greenplants.Ascorbic acid has notbeen encountered in yeast andprokaryotes, otherthan cyanobacteria, and it is not clear whether itoccurs in colorless forms of plants [888]. Dur-ing winter potatoes and cabbage were probablythe most important sources of vitaminC for theWestern population. Today, fresh fruits and veg-etables are available all year. Storage and cook-ing partly destroy ascorbic acid [936].

The content of vitaminC varies in the dif-ferent plant tissues. High concentrations are en-countered in tropical fruits and in the leaves ofgladiolus. The latter source was used industri-ally before the first chemical synthesis had beenworked out. Some plants contain ascorbic acidin bound form: ascorbigen (Brassica species)[937] or elaeocarpusin [938].

In the animal kingdom ascorbic acid is alsowidespread, but in lower concentrations. Thehighest contents are found in the endocrinic or-gans (cow: hypophysis 126, adrenal gland 100 –160mg/100 g). Some species of animals, e.g.,primates (including humans), guinea pig, flyingmammals, some birds, and certain fishes and in-sects, cannot produce ascorbic acid.

The vitaminC content of some foods (inmg/100 g) follows [939]:

Fish 0 – 3 Asparagus 15 – 30Liver, kidney 10 – 40 Beans 10 – 30Meat (beef,pork)

0 – 2 Broccoli 70 – 160

Milk (human) 3 – 6 Brussels sprouts 90 – 150Milk (cow) 1 – 2 Cabbage 30 – 60Acerola 1300 Carrots 5 – 7Apples 10 – 30 Cauliflower 60 – 80Bananas 5 – 10 Oat, rye, wheat 0Black currant 150 – 230 Parsley 170Cherries 10 Peas 10 – 30Guava 300 Peppers 125 – 200Hawthornberries

160 – 800 Potatoes 10 – 30

Lemons 50 – 80 Rice 0Oranges 40 – 60 Spinach 50 – 90Rose hips 1000 Tomatoes 10 – 30Strawberries 40 – 90 Turnips 15 – 40

10.6. Biosynthesis [888]

Tracer experiments with radiolabeled sugarshave revealed different biosynthetic pathwaysfor ascorbic acid in animals and plants.

Animals convert d-glucose via glucuronicacid to vitaminC (Fig. 17). The biosynthesisincludes an inversion of the carbon chain se-quence, whereby C-1 and C-6 of d-glucose be-come C-6 and C-1, respectively, of ascorbicacid [940]. In scurvy-prone animals such as theguinea pig, the final step, which is catalyzed byl-gulonolactone oxidase, is missing. Adminis-tration of this enzyme enables guinea pigs to liveon an ascorbic acid deficient diet [941]. Humansalso lack l-gulonolactone oxidase. The ability toproduce ascorbic acid was probably lost in theprogress of evolution [942]. l-Gulonolactoneoxidase resides in the liver of mammals andin the kidneys of other vertebrates (amphibians,reptiles, etc.) [943].

Biosynthesis in plants follows a noninversionpathway. d-Glucose [50-99-7] (9) is oxidized atC-2 to d-glucosone [1854-25-7] (10), epimer-ized at C-5 and finally oxidized at C-1. A specu-lative scheme of ascorbic acid synthesis in plantsbased on results of tracer studies is presentedin Figure 18 [888]. Since plants also convert l-galactono-1,4-lactone to ascorbic acid very ef-ficiently, an inversion-type pathway was postu-lated, too [944].

Metabolic degradation in plants leads to ox-alic and tartaric acid [945], and in animals toCO2, oxalic acid, C5 aldonic acids, and undeter-mined products [946].

Biosynthesis is inhibited by deficiencies ofvitamins such as vitaminA, E, or biotin, andis stimulated by xenobiotics and drugs (3,4-benzpyrene, barbiturates, etc.) [947].

10.7. Manufacture of Vitamin C

The first synthesis of l-ascorbic acid [895,948]was accomplished before complete elucidationof its chemical structure. The starting material,l-xylosone (11), was treated with potassiumcyanide, forming l-xylonitrile (12). After lac-tonization and enolization l-ascorbic acid (1)

Vitamins 115

Figure 17. Biosynthesis of ascorbic acid in animals

Figure 18. Biosynthesis of ascorbic acid in plants

was obtained in moderate yields (ca. 40% froml-xylosone). However, this synthesis never at-tained economical importance, because of thelack of an easy and inexpensive access to l-xylosone.

About a year after publication of the firstsynthesis Reichstein and Grussner describedtheir route to l-ascorbic acid from the readilyavailableC6 sugar d-glucose [949]. Thismethodremains the most important synthetic pathway

for commercial manufacture. Today, ascorbicacid is produced by slight modifications of thescheme first devised in 1934.

10.7.1. Reichstein Synthesis [684]

The basic principle of the Reichstein synthesisis a sequence of easy, high-yield steps withoutcleavage or formation of a carbon – carbon bond.The essence of this pathway is reduction of C-1of d-glucose and oxidation at positions 5 and6. The chirality at C-2 and C-3 is preserved togive the l-threo-configuration at C-4 and C-5 ofl-ascorbic acid.

The classical Reichstein –Grussner synthe-sis starts with reduction of d-glucose (9) to d-sorbitol (13) by hydrogenation over a nickelcatalyst. The microbiological oxidation of d-sorbitol [50-70-4] (13) to l-sorbose [87-79-6](14) is carried out with Acetobacter xylinum.On treatment of l-sorbose (14) with ace-tone at low temperature in the presence ofsulfuric acid, 2,3 : 4,6-di-O-isopropylidene-α-l-sorbofuranose [17682-70-1] (15) is formed.

116 Vitamins

The di-O-isopropylidenyl protection of the hy-droxy-groups at C-2, C-3 and C-4, C-6 al-lows high-yield oxidation to di-O-isopropyl-idene-2-ketogulonic acid [18467-77-1] (16),without over-oxidation or other side reactions.The oxidation is carried out with potassiumpermanganate in alkaline solution. Treatmentof 16 with hot water affords 2-keto-l-gulonicacid [526-98-7] (17), which is converted to l-ascorbic acid (1) by heating in water at 100 ◦C(20% yield) or by esterification and treatmentwith sodium methoxide in methanol followedby acidification with hydrogen chloride, yield-ing ca. 70% of 1. The overall yield of ascorbicacid from d-glucose is 15 – 18%.

10.7.2. Industrial Manufacture by theReichstein Route

The development of Reichstein’s classical pro-cedure into an industrial process is marked bygreat efforts to improve each reaction step. Asa result of many technical and chemical mod-ifications each step gives over 90% yield. Theoverall yield of ascorbic acid from d-glucose isnow ca. 60%.

d-Sorbitol. The catalytic hydrogenation ofd-glucose (9) to d-sorbitol (13) is accomplishedat high pressures and elevated temperatures in

the presence of a Raney nickel catalyst (see bot-tom of page).

The hydrogenation is carried out in batch orcontinuous operations and affords almost quan-titative yields with minimal formation of d-mannitol and l-iditol. After removal of the cata-lyst, the sorbitol solution is employed in fermen-tation without any further purification. There-fore, manufacturers of ascorbic acid make highdemands on the quality of d-glucose (9); crys-talline dextrose or purified starch hydrolysatesare used.

The cathodic and microbiological reductionof d-glucose (9) tod-sorbitol (13) have also beenreported [950].

l-Sorbose is obtained in the pyranose formby microbiological oxidation of sterile aqueoussolutions of d-sorbitol (13) in batch or con-tinuous operations in the presence of air. Themost frequently used strains belong to the Glu-conobacter oxydans family, which converts d-sorbitol (13) to l-sorbose (14) with more than90% efficiency. Large-scale fermentations haveto be carried out at pH 4 – 6 and at 30 – 35 ◦Cunder sterile conditions to avoid loss of productduring oxidation and work-up by filtration andcrystallization.

2,3 : 4,6-Di-O-isopropylidene-α-l-sorbofu-ranose. The protection of the 2,3- and 4,6-

Vitamins 117

hydroxyl groups by forming cyclic ketals isachieved in acetone with excess sulfuric acidas catalyst and dehydrating agent, generally atlow temperatures (e.g., 4 ◦C). In the final reac-tion mixture 2,3 : 4,6-di-O-isopropylidene-α-l-sorbofuranose (15) is obtained as main prod-uct. Byproducts are 2,3-O-isopropylidene-α-l-sorbofuranose and 1,2-O-isopropylidene-α-l-sorbopyranose. After neutralization, excessacetone is recovered by distillation. 2,3 : 4,6-di-O-isopropylidene-α-l-sorbofuranose (15) isextracted from the aqueous solution with aro-matic solvents (e.g., toluene). The remainingmonoisopropylidenesorboses are also recoveredfrom the aqueous solution and returned to theprocess.

2,3 : 4,6-Di-O-isopropylidene-2-keto-l-gulonic acid. Originally, the oxida-tion of 2,3 : 4,6-di-O-isopropylidene-α-l-sorbofuranose (15) to 2,3 : 4,6-di-O-isopropyl-idene-2-keto-l-gulonic acid (16) was performedat elevated temperatures in dilute sodium hy-droxidewithKMnO4, yielding ca. 90%of prod-uct. Much less expensive oxidation methods areapplied inmodern continuous processes: sodiumhypochlorite, electrochemical oxidation, or cat-alytic air oxidation.

Oxidation with hypochlorite in the presenceof catalytic amounts of nickel chloride or sulfateat 60 ◦C yields >93% of 16. The active oxidantis presumably nickel peroxide [951].

Electrochemical oxidation is performed withnickel or nickel oxide electrodes in alkalinesolution [952,953]. Hydrogen, evolved at thecathodes, can be used for hydrogenation of d-glucose. It can be advantageous to stop electro-chemical conversion to 16 at ca. 90% to avoida drop in selectivity at the end of electrolysis.Oxidation can be completed with sodium hypo-chlorite solution. At the end of the electrooxida-tion, selectivity can be improved by electrolyticcellswith high electrode surfaces, such as Swiss-roll cells [954] or cells with three-dimensionalelectrodes [955].

Alternatively, the oxidation has been per-formed by using air or oxygen and a metalcatalyst in alkaline solution. Good yields areachieved with palladium or platinum on car-bon [956,957]. The reaction product 2,3 : 4,6-di-O-isopropylidene-2-keto-l-gulonic acid (16)

is isolated by acidification and precipitation asthe monohydrate.

l-Ascorbic acid. The conversion of 2,3 : 4,6-di-O-isopropylidene-2-keto-l-gulonic acid (16)to l-ascorbic acid (1) is achieved by two differ-ent procedures:

1) Deprotection to give 2-keto-l-gulonic acid(17), followed by esterification with metha-nol and base-catalyzed cyclization

2) Acid-catalyzed cyclization to ascorbic aciddirectly from the protected or released 2-keto-l-gulonic acid.

The starting material for base-catalyzed re-actions ismethyl 2-keto-l-gulonate [3031-98-9](18), prepared by treatment of the acid 16 withacidic methanol. Finally, reaction of the methylester 18 with sodium hydrogencarbonate orsodium acetate affords sodium ascorbate in highyield [958,959].

The first acid-catalyzed route to ascorbic acidwas published only few years after discovery ofthe Reichstein process [960,961]. Today, the in-dustrial process is performed with an inert sol-vent (e.g., trichloromethane or toluene) in thepresence of hydrochloric acid. The advantageof this method is that ascorbic acid precipitatesfrom the mixture as it is formed, minimizingdecomposition during reaction. The crude prod-uct is obtained by filtration in high yield andhigh purity. After dissolution of crude ascorbicacid in water, impurities are removed by refin-ingwith activated carbon, decolorizing resins, orionexchange resins, followed by crystallization.

Environmental Aspects. The economicalefficiency of the Reichstein synthesis is stronglyinfluenced by environmental protection costs.

Wastewater is fed into a sewage plant. Thedischarged COD load is similar to the amountof ascorbic acid produced. Most constituents(sugar derivatives) are easily biodegradable. Ef-fluents containing ketalized sugar derivativeshave to be pretreated to hydrolyze nondegrad-able ketals. Loss of nickel catalyst in wastewa-ter is nearly quantitatively eliminated by filtra-tion and adsorption on ion-exchange resins. Theinorganic load consists of sodium chloride andsodium sulfate.

Contaminations in exhaust gases are mainlyremoved by absorption in water scrubbers.

118 Vitamins

Gases containing water-insoluble compoundssuch as chlorinated solvents are purified by ad-sorption on activated carbon and/or by low-tem-perature condensation.

Distillation residues are disposed of by in-cineration. Solid residues are mainly sewagesludge, spent carbon, and filter aids.

10.7.3. Other Methods for Production ofAscorbic Acid [879,885,889]

All syntheses of l-ascorbic acid (1) are partialsyntheses inwhich the chirality at C-4 andC-5 isderived fromnaturally occurring sugars. Startingmaterial is most frequently cheap d-glucose (9),and to some extent galactose and galacturonicacid obtained from natural sources. Synthesesinvolving the coupling of aC1 fragment and aC5fragment (e.g., l-arabinose [962]) or a C2 frag-ment and a C4 fragment are less important, butuseful for the preparation of radiolabeled deriva-tives and analogues of ascorbic acid (review in[879]). d-Glucose (9) is converted to l-ascorbicacid (1) either by inversion of the carbon chainsequence (C-2/C-3 of glucose become C-5/C-4of ascorbic acid) or by inversion of the configu-ration of C-5. A schematic overview of selectedpartial syntheses of l-ascorbic acid is given inFigure 19.

Many efforts have been made to reduce pro-duction costs by optimizing and shortening thevarious reaction routes. These attempts includethe combination of chemical and microbiologi-cal reaction sequences, direct chemical oxida-tions (avoiding the use of protecting groups),enzymatic catalysis, the use of genetically mod-ified microorganisms or plant tissues, and cofer-mentations. Main emphasis in the last decadewas on the development of the fermentativetransformation of d-glucose (9) to 2-keto-l-gulonic acid (17) via d-sorbitol (13), l-sorbose(14) or 2,5-diketogluconic acid [2595-33-7](28), and the direct conversion of d-glucose (9)to l-ascorbic acid (1) by mutagenized microal-gae. However, the Reichstein synthesis still pre-dominates in commercial production, althoughit is reported that a single fermentation step to2-keto-l-gulonic acid (17) has already reachedproduction scale [963].

Synthesis via l-Sorbose (Fig. 19B).Modifi-cation of ProtectingGroups.The replacement ofacetone as a protecting agent for the oxidationof l-sorbose (14) has been intensively studied inthepast [885].However, the useof other reagentssuch as formaldehyde, benzaldehyde, cyclohex-anone, etc. is not economic and little attentionis paid to further improvements of these meth-ods. Syntheses using partly protected l-sorbitol[964] or l-sorbose (e.g., methylsorboside [965])are also not viable alternatives.Chemical Oxidation. Direct oxidation of l-

sorbose (14) to 2-ketogulonic acid (17) has beentried by many authors. Best results are obtainedby catalytic oxidation with noble-metal cata-lysts, promoted by heavymetals such as bismuthor lead. The reaction however is not sufficientlyselective, the catalyst is rapidly deactivated, andpublished yields (up to 87%) are hardly repro-ducible [966].Microbiological Oxidation. Fermentative

production of 2-keto-l-gulonic acid (17) froml-sorbose (14) with microorganisms of the gen-eraPseudomonas, Bacillus, Gluconobacter, etc.is the subject of numerous patents. The biosyn-thesis of 2-keto-l-gulonic acid (17) in bacteriaproceeds via l-sorbosone [49865-02-3] (19).l-Idonic acid, reported earlier to be an inter-mediate, is probably a degradation product of2-keto-l-gulonic acid (17) [967].

Oxidation can be accomplished with singlemicroorganisms, mixed-culture fermentations,or by enzymatic catalysis. Continuous improve-ments to these methods over the last decades byusing bioengineering techniques has led to re-markable results. A Chinese patent claims a pro-cesswhich comprises themixed-culture fermen-tation of l-sorbose (14) to 2-keto-l-gulonic acid(17) in yields of 70 – 80% [968]. Even better re-sults are obtained by fermentation of l-sorbose(14) with Pseudogluconobacter saccharoketo-genes/Bacillus megaterium and supplementingthe culture medium with rare earth elements(yield 86%); isolation of the crystalline freeacid from the fermentation broth gave an over-all yield of ca. 70% [969]. Further improve-ment is achieved when d-sorbitol (13) is usedas starting material instead of l-sorbose (14).Mixed-culture fermentation of d-sorbitol (13)with strains of Gluconobacter suboxydans andGluconobacter oxydans results in yields of 75 –90% [970]. A purification method for 2-keto-l-

Vitamins 119

Figure 19. Synthetic pathways to l-ascorbic acid

gulonic acid (17) is given in [971]. Once scaledup, the microbiological oxidation of d-sorbitol(13) or l-sorbose (14) to 2-keto-l-gulonic acid(17) will enable a cheaper production route tol-ascorbic acid.

In a wider sense all these reaction sequencesvia l-sorbose represent modifications of the Re-

ichstein process, and the final step – conversionof 2-keto-l-gulonic acid (17) to ascorbic acid(1) – still has to be performed chemically.

Synthesis via d-Glucuronic Acids(Fig. 19C). Other routes from d-glucose (9) tol-ascorbic acid (1) start with oxidation at C-6,

120 Vitamins

leading to d-glucuronic acid derivatives. The C-1 aldehyde group is reduced in subsequent steps.Key intermediates of these syntheses are the ke-tolactone (22) and l-gulonolactone [1128-23-0](24).

In the synthesis of Bakke and Thean-der (9 → 21 → 22 → 25 → 1) l-ascorbic acidis obtained in 28% overall yield from d-glucose [972]. A modification of this methodhas been published by Kitahara, who synthe-sized ketolactone (22) from d-glucuronolactone[32449-92-6] (23) [973]. Due to low yields andhigh reagent costs these syntheses have not at-tained industrial significance.

A shorter production route to l-ascorbic acidproceeds via l-gulonolactone (24), which is ob-tained by hydrogenation of d-glucuronolactone(23) [974]. l-Gulonolactone (24) can be oxi-dized chemically [975] and microbiologically[976] to 2-ketogulonic acid (17) in good yield,or directly converted to l-ascorbic acid (1) withl-gulonolactone dehydrogenase [977]. To com-pete with the Reichstein process these synthesesrequire cheap access tod-glucuronolactone (23).However, 23 is obtained from protected glucose[978] or starch [979] only in low yields.

Synthesis via Gluconic Acids (Fig. 19A).In this pathway d-glucose is converted to l-ascorbic acid without carbon-chain inversion,and the correct configuration at C-5 is achievedby a oxidation/reduction sequence. Key inter-mediates of these pathways are 5-ketogluconicacid [5287-64-9] (26) and 2,5-diketogluconicacid (28).

Microbiological oxidation of d-glucose to 5-keto-d-gluconic acid (26) is carried out withstrains of Acetobacter suboxydans in almostquantitative yields. 26 is reduced by catalytichydrogenation to a mixture of d-gluconic andl-idonic acid [1114-17-6] (27) (77%). Fermen-tation of 27 with Pseudomonas strains yields 2-keto-l-gulonic acid (17) (90%) [885]. The lackof a selective reduction method for 26 seems tobe the reason that little attention has been paidto further development of this synthesis.

2,5-Diketogluconic acid (28) is efficientlyproduced from d-glucose (9) by several speciesof Acetobacter, Gluconobacter, or Erwinia[980]. Subsequent chemical (NaBH4) or mi-crobiological reduction converts 28 to 2-ketogulonic acid (17) [885]. An alternative

chemical route proceeds via the 5,5-dimethylketal of 28 and 5-keto-l-ascorbic acid (29) to1 [981]. 29 and its 5,5-dimethyl ketal exhibitantiscorbutic activity. The microbiological pro-cess → 17 has been optimized by introducinga two-stage fermentation method with mutantstrains of Erwinia sp. and Corynebacterium sp.(73% overall yield from glucose) [982]. Themutagenic treatment is necessary to repress thegene for 2-keto-d-gluconate formation, becausethe isolation of 17 from a mixture containing2-keto-d-gluconate is difficult [983]. Additionof nitrate to the culture medium has a stimula-tive effect on the production of 17 [984]. Fur-ther simplification of the process by combiningthe relevant traits ofErwinia sp. andCorynebac-terium sp. into a single organism was investi-gated by two different research groups, but theresults obtained still remain far from industrialapplication [985].

Synthesis via d-Galacturonic Acid(Fig. 19 E). This approach is similar to syn-thesis via d-glucuronic acid [6556-12-3] (20):inversion of carbon-chain sequence, similar re-action conditions, and difficult access to low-cost starting material. The differing chirality atC-4 of d-glucuronic acid (20) is lost in the finalreaction step (C-3 of l-ascorbic acid).d-Galacturonic acid [685-73-4] (31) is ob-

tained by enzymatic hydrolysis of pectic acid[885] or by chemical transformation of d-galactose [59-23-4] (30) which is available fromlactose-containing whey [986]. Catalytic reduc-tion of the aldehyde group affords l-galactonicacid [28278-08-2] (32), which is transformed tol-ascorbic acid (1) by fermentation with Can-dida sp. [987] or by chemical oxidation to 33and subsequent cyclization to 1 [885].

Synthesis by Microalgae (Fig. 19D). Directsynthesis of l-ascorbic acid (1) from d-glucose(9) can be achieved with mutagenized microal-gaeof the genusChlorella [988].Up tonowyieldand concentration are too low for industrial ap-plication, but the l-ascorbic acid (1) enrichedbiomass obtained may be useful as an aquacul-ture fish feed or feed additive.

Vitamins 121

10.8. Absorption and Metabolism

Ascorbic acid is readily absorbed intestinallyand to a lesser extent in the mouth and stomach.The absorption rate of low supplemental dosesis up to 80%; for example, 75% is absorbedat an oral intake of 1 g. There is an inverse re-lationship between supplement and absorptionrate because of limited intestinal absorption ca-pacity. The absorption rate is lowered to ca. 20%at an oral intake of 5 g [989]. Excess ascorbicacid is excreted unaltered in the urine.

VitaminC is themost abundantwater-solubleantioxidant in the body. The polarity that encour-ages its presence in aqueous fluids also hindersits passage across the hydrophobic cell mem-brane. This, and the high concentrations withinthe cell, makes it almost certain that the formof transport of ascorbic acid is active and facil-itated, i.e., requiring membrane carriers such assodium ions [990].

Although widely distributed in the body,ascorbic acid is concentrated in adrenal and pitu-itary glands, in the brain, eye lens, and in leuko-cytes [991]. A dosage of ca. 60mg of ascorbateper day is necessary to create and maintain abody pool of 1500mg, which protects againstscurvy for at least 30 d. However, the body poolcan be expanded to 2300 – 2800mg by 200mg/ddoses [992].

The main metabolites of ascorbate in the hu-man body are oxalate, dehydroascorbic acid,2,3-diketogulonic acid and ascorbic acid 2-sulfate. Large intakes of ascorbic acid do not in-duce higher levels of oxalate because humans arenot able to indefinitely increase the metabolismof ascorbate.

10.9. Medical Aspects of Vitamin C

The most significant chemical property of l-ascorbic acid is its oxidation to dehydro-l-ascorbic acid. This reversible oxidation is thebasis for many of its known physiological activ-ities.

A number of enzymes are stimulated byascorbic acid. Compared with other reducingagents vitaminC shows the greatest stimula-tion of dioxygenases that contain prostheticFe2+ andmonooxygenaseswith prostheticCu+.

Dioxygenases are responsible for several hy-droxylation reactions. In collagen synthesis,proline and lysine are hydroxylated on the grow-ing polypeptide chain.Many of the clinical signsof scurvy are explained by defects in collagensynthesis. Keeping iron in dioxygenase in itsrequired reduced form seems to be the role ofascorbic acid [992].

The requirement for ascorbic acid as a co-factor in hydroxylation reactions is also sug-gested in the biosynthesis of norepinephrinefrom dopamine, of carnitine, of homogentisicacid, of 5-hydroxytryptophan, and of 3,4-dihy-droxyphenylalanine.

Ascorbic acid is an essential cofactor forthe activation of peptidylglycine α-amidatingmonooxygenase. It provides reducing equiva-lents to the copper-containing enzyme whichis responsible for biosynthesis of neuropep-tides and endocrine peptides. Thus, ascorbic acidplays an important role in the proper functioningof the nervous and endocrine systems [993].

Much literature deals with the relationshipbetween vitaminC and the common cold.A highvitaminC level in the body does not decrease thenumber of incidences of the common cold.How-ever, it has consistently decreased the durationof cold episodes and the severity of symptoms.Ascorbic acid seems to decrease inflammatoryeffects by reduction of oxidizing compounds re-leased from activated leukocytes [994].

VitaminC appears to play many importantroles in protecting the body against cancer. Theability of ascorbic acid to inhibit formation ofcarcinogenic nitrosamines in the stomach is thebest documented cancer-protecting effect of vi-taminC [995]. It takes part in the protection ofDNA and lipid membranes from oxidative dam-age, an important factor in cancer incidence. Vi-taminC in connection with other antioxidantsmay also have beneficial effects on immune re-sponse, thereby reducing cancer risk by enhanc-ing tumor surveillance by the immune system[996].

Ascorbic acid serves as an intracellular andextracellular quencher of free radicals and ox-idants. Together with other antioxidants suchas carotenes, glutathiones or vitamin E, ascor-bic acid acts as a scavenger of oxygen species.In contrast to redox reactions, in which vita-minC donates two electrons, in the inactivationof highly reactive free radicals, ascorbic acid do-

122 Vitamins

nates only one electron. The reduction of toco-pheroxy radical to tocopherol, retaining the levelof vitamin E in tissues, follows this pathway. Vi-taminC reduces vitamin E radical not only in ho-mogeneous solution but also in liposomal mem-brane systems [997,998].

VitaminC influences the catabolism of cho-lesterol. Studies in humans show that for individ-uals with high total cholesterol concentrations asupplement of ascorbic acid may have a benefi-cial effect. These results suggest an inverse re-lationship between vitaminC level in the bodyand cardiovascular diseases [999].

Ascorbic acid possesses an essential physio-logical function in iron absorption. It enhancesthe absorption in a logarithmic fashion.Adosageof 25 – 50mg vitaminC per meal secures an op-timum promoting effect on absorption [1000].Despite some warnings, the ingestion of largequantities of ascorbic acid does not result incalcium oxalate stones, increased uric excre-tion, impaired vitaminB12 status, iron overload,systemic conditioning (dependence after pro-longed ingestion of high doses), or increasedmutagenic activity in healthy individuals [1001].High doses of ascorbic acid over long periods oftime do not lead to any side effects. In severalclinical studies daily intakes up to 10 g were ap-plied for several years without any adverse ef-fects [1002].

VitaminC is an essential nutrient for themaintenance of human health. The main appli-cation is the prevention and therapy of scurvy. l-Ascorbic acid supports wound healing and it en-hances the body’s immune response in prevent-ing and fighting infection. It has been suggestedthat l-ascorbic acid is helpful in preventing can-cer and cardiovascular diseases. A daily supple-ment to enhance iron-absorption from food isalso recommended in treating anemia [1003].

Some high risk groups, such as smokers, el-derly people, diabetics, and alcoholics, have alower vitaminC status than the rest of the pop-ulation. Pregnant and lactating women, womentaking oral contraceptives, sportsmen, sick andstressed people require a higher daily intake ofvitaminC. In these cases, supplementary vita-minC is beneficial.

The current recommended ascorbic acid in-takes of 60mg/d (United States, European Com-munity) [1004,1005] or 75mg/d (Germany)[1006] are based only on the prevention of

scurvy and on the urinary excretion threshold.The optimum amount of ascorbic acid for hu-man health is under discussion, and many ex-perts recommend a much higher daily intake.

10.10. Industrial Uses [890–892]

The chemical properties of l-ascorbic acid pro-vide a wide range of industrial applications.About one-third of total production is used forvitamin preparations in the pharmaceutical in-dustry. The rest is mainly applied as an additiveto food and feed to enhance product quality andstability.l-Ascorbic acid is an active component in

a variety of pharmaceutical products, includ-ing multivitamin preparations, tablets, syrups,elixirs, and effervescent tablets. Solid dosageforms are quite stable. In liquid formulationsascorbic acid is subject to oxidative decompo-sition and interaction with other ingredients.

The largest field of application is in the foodindustry, particularly in the processing of meat,fruits, and flour. The antioxidant and physiolog-ical vitamin activity of ascorbic acid is used topreserve foods, avoiding undesirable odors, fla-vors, and colors due to oxidative degradation, toenhance the nutrient value, or to improve pro-cessing:

1) Inhibition of nitrosamine formation in curedmeat products

2) Protection from enzymatic browning in pro-cessed fruits and vegetables

3) Nutritional restoration and fortification offoods and beverages

4) Improvement of flour and dough quality5) Retardation of oxidative rancidity in fats and

oils6) Increased clarity of wine and beer7) Synergistic activity in applicationswith other

antioxidants (e.g., tocopherols)

In the United States, ascorbic acid may notbe added to fresh meats, because it prevents dis-coloration and may thus give a false appearanceof freshness. Ascorbic acid, its salts, and ascor-byl palmitate are registered food additives in theEuropean Community and classified as gener-ally recognized as safe (GRAS) substances byFDA.

Vitamins 123

Ascorbic acid or the more stable 2-sulfate areused in fish farming to prevent scurvy and en-hance immune response.

The use of ascorbic acid in plant applica-tions (improvement of seed germination, ozoneprotection, harvesting aid, disease resistance,growth regulation) is limited by its economics.

Further use in the polymer, photographic, andcosmetic industries, etc. is less important.

10.11. Economic Aspects

Total demand for ascorbic acid in 1995 is esti-mated at 60 000 t/a. Since 1980 world produc-tion has increased by 50%. After a long con-solidation period at ca. 24DM/kg in Europe and$ 15 in overseas trade, the price of ascorbic acidbegan to fall in 1995 because of the appearanceof new suppliers, especially fromChina. The de-velopment of world market price is as follows:

1954 36¤/kg 1980 10¤/kg1957 20¤/kg 1982 12¤/kg1964 8¤/kg 1993 12.50¤/kg1974 9¤/kg 1995 10¤/kg

Principal manufacturers include: Hoffmann-La Roche; Dalry (United Kingdom), Belvidere(United States); Takeda Ind. Chem., Osaka(Japan), Wilmington (United States); Merck,Darmstadt (Germany); BASF, Grenaa (Den-mark); Pliva, Zagreb (Croatia); and several Chi-nese companies, including Northeast GeneralPharmaceutical Factory, Sunve Shanghai No. 2,Xing Xang Pharmaceutical Factory, JingshuPharma, and Jiang Xi.

The bulk of production is sold as ascorbicacid. However, specialities such as salts, es-ters (ascorbyl palmitate, sulfate), coated ascor-bic acid, and granulated acid for direct compres-sion are becoming increasingly important.

11. Pantothenic Acid

11.1. Introduction

The structure of pantothenic acid, [(R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-β-al-anine] (1), is characterized by a carboxyl group,a primary and a secondary hydroxyl group, anω-peptide bond, and a chiral center. Only the R

enantiomer occurs in nature; the S form has novitamin activity.

11.2. History

Pantothenic acid was discovered by Williamsin 1931 as a growth factor for microorganisms[1012]. In 1938, it was isolated in a purity ofca. 40% from sheep liver [1013]. It was thenidentified with the aid of two “active factors,”required for normal growth in rats [1015] and toprevent dermatitis in chickens [1014]. The namepantothenic acid results from its ubiquitous dis-tribution (Greek: from everywhere) [1016].

11.3. Physical and Chemical Properties

Pantothenic acid (1) is a hygroscopic oilwhich isinstable in acidic or alkaline solution. The com-mercial form is the calcium salt. The sodium saltand the corresponding alcohol, (R)-panthenol,are used in special pharmaceutical formulations.

Calcium and sodium (R)-pantothenate arewhite powders (mp ca. 200◦C, decomp., and160 – 165◦C, respectively). The sodium salt ishygroscopic.

(R)-Panthenol is a colorless, viscous oil.Other physical data are presented in Table 32.

11.4. Occurrence

Pantothenic acid (1) iswidely distributed in plantand animal tissues in varying concentrations.Liver, kidneys, egg yolk, muscle, brain, yeast,rice and wheat bran, green leafy vegetables, andlegumes are especially rich sources of this vita-min (Table 33).

124 Vitamins

Table 32. Physical data of pantothenic acid and some of its derivatives

Compound Empirical formula Mr [α]20D(c = 4,water)

Solubility in

water ethanol ether

(R)-Pantothenic acid C9H17NO5 219.2 + 37.5 readily soluble soluble slightly solubleCalcium (R)-pantothenate (C9H16NO5)2Ca 476.5 + 27.0 readily soluble poorly soluble insolubleSodium (R)-pantothenate C9H16NO5Na 241.2 + 27.5 readily soluble slightly soluble insoluble(R)-Panthenol C9H19NO4 205.3 + 30.5 readily soluble readily soluble poorly soluble

Table 33. Occurrence of pantothenic acid in foods according to theGerman Bundeslebensmittelschlussel (BLS), 1990 and Souci,Fachmann, Kraut (SFK) 1989 [1017]

Food Content, mg/100 g

BLS [1018] SFK

FruitApple 0.1 0.1Strawberries 0.3 0.3Oranges 0.25 0.24VegetablesBroccoli 1.3 1.29Cauliflower 0.9 1.01Corn 0.49 0.65Potatoes 0.35 0.4Tomatoes 0.32 0.31Milk productsSkimmed milk powder 3.4Cows’ milk 0.35 0.35Hens’ egg 1.6 1.6MeatCalf liver 8.0 7.9Pork liver 6.8 6.8Beef heart 2.6 2.78Pork 0.62 0.7Beef 0.5 0.6CerealsWheat bran 2.1Wheat (whole grain) 1.2 1.18Wheat flour 0.21 0.21Soymeal 1.4Rolled oats 1.1 1.09Oats (whole grain) 0.8Polished rice 0.25 0.63

Pantothenic acid mainly occurs in nature inthe bound form as a component of coenzymeA.It is not formed in the humanor animal organism.Only the conversion of pantothenic acid to themetabolically active molecule coenzymeA isbiosynthetically possible. In contrast, most mi-croorganisms are capable of synthesizing pan-tothenic acid from pantoic acid and β-alanine.

(R)-Panthenol does not occur in nature. It israpidly oxidized to pantothenic acid by warm-blooded animals [1019]. In feeding experimentson chickens, (R)-panthenol exhibits ca. 60% of

the vitamin activity of an equimolar amount ofcalcium (R)-pantothenate [1020].

11.5. Biosynthesis

The biosynthesis of pantothenic acid proceedsvia enzyme-catalyzed condensation of pantoicacid with β-alanine in the presence of adenosinetriphosphate. The intermediate enzyme complexof pantothenic acid synthetase, pantoic acid, andAMP has been thoroughly studied [1021].

The biosynthesis of pantoic acid (2) branchesoff from that of valine at the 2-oxo-3-methyl-butyric acid (3) stage [1022]. The introductionof a hydroxymethyl group by means of 5,10-methylenetetrahydrofolic acid [1023] results in4-hydroxy-3,3-dimethyl-2-oxobutyric acid (4),which is enzymatically reduced to 2 with for-mation of an asymmetric center.

At least four pathways are known for thebiosynthesis of β-alanine (5):

1) Hydrogenation and hydrolysis of uracil (6)via β-ureidopropionic acid (in Chlostridiumuracilicum) [1024]

2) Decarboxylation of l-aspartic acid (7)[1025]

Vitamins 125

3) Oxidative degradation of spermine or sper-midine (8) (in Pseudomonas aeruginosa)[1026]

4) Transamination ofmalonylaldehydic acid (9)with amino acids, especially with glutamicacid inmammals andmicroorganisms [1027]

11.6. Production

The startingmaterials for the large-scale produc-tion of calcium (R)-pantothenate, the commer-cial form of pantothenic acid, are (R)-pantolac-tone and calcium β-alaninate.

Pantolactone. Racemic pantolactone (10)can be produced in a yield of ca. 90% fromisobutyraldehyde, formaldehyde, and hydrogencyanide without the isolation of intermediates[1028]. The intermediates are 3-hydroxy-2,2-di-methylpropanal (11) and 2,4-dihydroxy-3,3-di-methylbutyronitrile (12). Saponification of 12with strong acids directly produces the lactone(10), which can be isolated by extraction and/ordistillation.

Several processes have been developed forthe preparation of optically active (R)-pantolac-

tone (13): (1) resolution of racemates with re-cycling, (2) asymmetric hydrogenation, and (3)microbiological synthesis.

Diastereomeric salts or amides of pantoicacid can be separated by fractional crystalliza-tion. The separation can be achieved with alka-loids [1029] and with synthetic chiral amines[1030]. Amines such as dehydroabietylamine(from rosin) [1031] and (+)-3-aminoethylpinane(from (−)-α-pinene) [1032], which are easilyobtainable from inexpensive starting materials,are especially suited to industrial application.

Frequently, the enantiomers are separated byfractional crystallization after alternately seed-ing with the R or S form. Such processes areknown for pantolactone [1033], acetylpantolac-tone [1034], and salts of pantoic acid [1035].

Racemization and recycling of undesired (S )-pantolactone are achieved either by heating withaqueous NaOH for several hours at 150◦C togive sodium pantoate, or in the presence ofstrongly basic amines or alkoxides [1036].

Alternative processes for (R)-pantolactonebegin with the corresponding 2-oxolactone (16),which is obtainable by oxidation of 10 [1037] orof the 2-aminolactone 14 [1038], or by reactionof dimethylpyruvic acid (15) with formaldehyde[1039]. 2-Oxopantolactone can be enantioselec-tively hydrogenated to (R)-pantolactone (13)mi-crobiologically [1040] or catalytically with op-tically active rhodium complexes.

The biphosphines 17 and 18, obtainable from4-hydroxyproline, are the preferred chiral lig-ands [1041].

126 Vitamins

A large number of microbiological processesfor the production of (R)-pantolactone have beendescribed [1042]. For instance, stereoselectiveenzymatic oxidation of racemic pantolactone(10), followed by asymmetric reduction of theresulting 2-oxolactone, affords quantitative con-version to (R)-pantolactone [1043]. However,these processes have not yet gained economicimportance.

β-Alanine. Two processes are mainly usedfor the industrial synthesis of β-alanine (5).

The addition of ammonia to acrylonitrile(19) at 20 – 30 bar and ca. 125◦C yields β-ami-nopropionitrile (20), which is hydrolyzed withsodium hydroxide solution to give sodium β-alaninate [1044]. After acidification with sul-furic acid, evaporation, and extraction of theresidue with methanol, crystalline β-alanine isobtained [1045].

Startingwith acrylic acid (21), the addition ofammonia directly produces β-alanine with theavoidance of salt formation [1046].

Until now, microbiological processes [1047]have not been used on a large scale.

Aqueous solutions of β-alanine are obtainedby chromatography on weakly acidic cation

exchangers and, more recently, by electro-dialysis of aqueous alkaline solutions [1048].

β-Alanine is converted to the correspond-ing calcium salt (22) before further reactionwith (R)-pantolactone. After neutralization of 5

with Ca(OH)2 in water, filtration, and drying,a product is obtained which is free of water-insoluble components. Consequently, it fulfilsthe strict quality requirements of Pharm. Eur. IIand USPXXII.

Pantothenic Acid. Calcium (R)-pantothenate is obtained by the reaction ofcalcium β-alaninate with (R)-pantolactone inboiling methanol or ethanol. Depending on thecrystallization and drying conditions, three sol-ventless polymorphous forms, two solvates, andan amorphous product can be isolated [1049].Hygroscopic prisms with four moles of meth-anol and one mole of water are formed oncrystallization from 90% aqueous methanolat 0◦C. Drying of these prisms at 80◦C and30mbar produces X-ray amorphous calcium(R)-pantothenate while maintaining the crystalform.

Calcium (R)-pantothenate can be producedby seeded crystallization in 90% aqueous meth-anol by exploiting the higher solubility of theracemate compared with the solvates of theenantiomers [1050]. Racemization of the S formis achievedwith sodiummethoxide in anhydrousmethanol [1051].

Sodium (R)-pantothenate is isolated by react-ing sodium β-alaninate with (R)-pantolactone inmethanol or ethanol (1 h, 65◦C) [1052].On cool-ing, it crystallizes in the form of square platelets(mp 160 – 165◦C) and is highly hygroscopic.

Panthenol. (R)-Pantolactone reacts with 3-amino-1-propanol at 70◦C (2 h) to give (R)-panthenol [1053]. On distillation at 170◦C and10−3 bar, a colorless product is obtained whichmeets the requirements of Pharm. Eur. II andUSPXXII.

11.7. Metabolism and Importance forthe Organisms; Coenzyme A and itsPrecursors

Pantothenic acid exhibits its biological activityas a component of coenzymeA (23) [1054] andof some peptide coenzymes [1055] which, in theform of energy-rich thiol esters, participate inthe metabolism of carboxylic acids, especiallyof acetic acid.

Vitamins 127

Pantothenic acid (active form: acetyl coen-zymeA and derivatives) occupies a central po-sition in intermediary metabolism (Fig. 20). Itis involved in numerous biochemical reactionsand enzymatic processes and, via coenzymeA,is of fundamental importance in the entiremetabolism:

1) Reactive esters are formed from unreactivefatty acids

2) Degradation of carbohydrates, fats, and ami-no acids occurs mainly at the acetyl coen-zymeA stage

3) Acetyl coenzymeA provides active acetategroups for the biosynthesis of fatty acids,phosphatides, and sterols (cholesterol, bileacids, steroid hormones)

4) Acetyl groups bound to coenzymeA enterthe citric acid cycle (energy producing oxida-tive degradation of substances to CO2, H2O,and synthesis of ATP)

5) Acetyl coenzymeA transfers the acetylgroup to certain acceptors (e.g., formation ofthe transfer agent acetylcholine at the nerveends)

6) Amino sugars are acetylated in the syn-thesis of mucopolysaccharides (cartilage),hyaluronic acid (connective tissue), andother(mesenchymal) skeletal substances

7) Acetyl coenzymeA supplies the acetylgroups for acetylation reactions that are in-volved in the detoxification of foreign sub-stances (e.g., chemotherapeutic agents, sul-fonamides)

8) CoenzymeA participates in the synthesis ofthe porphyrin skeleton of hemoglobin bypro-viding succinic acid in an activated form

Further physiological functions in the organ-ism are attributed to pantothenic acid. It is im-portant for:

1) Formation and normal function of body tis-sues

2) Maintaining the resistance of mucous mem-branes to infections

3) The optimal course of metabolic processes,especially in the skin and epithelial tissue

4) Promotion of growth and pigmentation of thehair

The absorption of pantothenic acid in theintestine proceeds rapidly, absorption beingmainly by diffusion, less by active transport[1056]. It is distributed among practically all thecells in the entire body (tissue concentrationsbetween 2 and 45µg/g).

Especially high concentrations of coen-zymeA are found in the liver, kidneys, adrenals,and erythrocytes. The pantothenic acid presentin blood serum is bound to plasma proteins.In the human organism, pantothenic acid ishardly metabolized. Absorption and eliminationof this vitamin are almost in balance (ca. 70%of the pantothenic acid absorbed is excreted un-changed in the urine and ca. 30% in the feces)[1057]. Thus, the vitamin status can be deter-mined from either the plasma level or the rate ofexcretion in the urine.

The structural elucidation of coenzymeA in1954 [1054] was confirmed by its synthesis in1959 [1058]. The key product was the derivative(27) of adenosine diphosphate which was linkedto pantetheine 4′-phosphate (28). The R config-uration was unambiguously assigned in 1970 bychemical correlation of (−)-pantolactone with(S )-(+)-ethyl pinacolyl ether [1059].

In body cells, the biosynthesis of coenzymeA(23) from pantothenic acid (1) proceeds via fiveintermediate steps [1060].

By using high performance liquid chromato-graphy (HPLC), coenzymeA and its precursorscan now be separated and quantitatively deter-mined on a picomole scale [1061].

128 Vitamins

Figure 20. Intermediary metabolism

Pantetheine (25) was discovered as a growthpromoting substance in lactic acid bacteriaand was thus named Lactobacillus bulgar-iens factor (LBF) [1062]. Chemical synthesiswas first achieved by the reaction of methyl(R)-pantothenate with cysteamine (24) [1063].Later, higher-yield processes were developedwhich start with pantothenic acid nitrile (29) orcalcium pantothenate [1064]. Analogously, thecorresponding disulfide, pantethine (30), is ob-tainable from cystamine (31) and has interestingpharmaceutical properties [1064].

11.8. Deficiency Symptoms,Requirement, and Application

Spontaneous pronounced deficiency symptomsin humans are unlikely because of the ubiqui-tous occurrence of pantothenic acid. Clear de-ficiency symptoms appear in humans only afterapplication of the strong pantothenic acid antag-onist ω-methylpantothenic acid. A deficiency ofpantothenic acid can be manifested as follows:

1) Impaired adrenal function (insufficiencysymptoms), deficiency symptoms in the ner-vous system (peripheral perception disor-

Vitamins 129

ders, coordination problems, degeneration ofthe central/peripheral nerve tracts) [1065]

2) Damage to the skin (dermatitis) and to thehair and nails

3) Gastrointestinal disorders (nausea, vomiting,motility, disturbances) [1066]

4) Impaired reproduction5) Retardation of growth or loss of weight6) Degeneration of the liver cells (fat infiltra-

tion)7) Inhibition of immune resistance/antibody

formation (increased susceptibility to infec-tion, e.g., infections of the upper respiratorytract)

8) Burning feet syndrome in undernourishedpersons [1067]

A balanced mixed diet of 10 000 kJ containsca. 10mg of pantothenic acid. In Germany, theaverage daily intake of pantothenic acid is ca.5mg/d [1068]. The German Society for Nutri-tion (1991) set the daily requirement for adults at6mg. The Food and Drug Administration in theUnited States stipulates a daily dose of 4 – 7mgof pantothenic acid (1989). Symptoms due tolarge doses of pantothenic acid or panthenol arenot known. Extremely high doses are toleratedwithout side effects, so that pantothenic acid isdesignated as atoxic.

Apart from the application in acute (R)-pantothenic acid deficiency and in preventivetreatment against such a vitamin deficiency (e.g.,multivitamin preparations), favorable effects inthe local treatment ofwounds havebeen reportedin the literature.

Calcium (R)-pantothenate is preferably usedin solid drug forms. The hygroscopicity ofthe compound must be taken into account. Itis also suitable for direct tabletting. In liquidand semisolid forms – especially in combinationwith other vitamins – (R)-panthenol (provita-minB5) is preferred to calcium (R)-pantothenatefor reasons of stability. In the topical applicationof (R)-panthenol, a suitable form of this sub-stance (e.g., 5 g/100 g of ointment or solution) isapplied to or sprayed on the affected areas onceor several times a day.

Calcium (R)-pantothenate is added to var-ious foods to prevent pantothenic acid defi-ciency due to false nutrition or malnutrition orfor certain nutritional requirements (baby food;

athletes’ products; low-calorie, reduced-calorie,and vitamin-rich foods).

(R)-Pantothenic acid improves the pigmenta-tion and growth of hair and prevents skin dam-age. As a result of its higher stability and easierapplicability, (R)-panthenol is usually preferredto calcium (R)-pantothenate, e.g., in hair-careproducts, sunscreens, shaving lotions, and babycare products.

Pantothenic acid deficiency occurs more of-ten in working animals. The symptoms vary inindividual animal species. Apart from a poorgeneral condition, deficiency is characterized bylow utilization of fodder, retardation or even ar-rest of growth, diverse impairments of the skin,mucous membranes, nervous system, blood, di-gestive tract, reproductory system, and of theadrenals [1069]. In addition, severe patholog-ical changes can occur, including convulsionsand paralytic symptoms, especially in the pig[1070], and a higher sensitivity to stress. In thehen, the egg yield is reduced and the brooding re-sults are unsatisfactory [1071]. In severe cases,dermatitis, decolorization and loss of feathers,incrustations of the beak and around the eyes, aswell as degeneration of the myelinated nervesoccur [1072]. Pantothenic acid is also a vitaminfor insects [1073] and acts as a growth substancein plants [1074].

Native pantothenic acid in feedstuffs usu-ally does not cover requirements. Therefore, thecomposition of the total feed plays an importantrole.With modern feeding processes and perfor-mance demands, pigs and poultry are dependenton supplementation of the feed with pantothenicacid. In the case of ruminants with fully formedrumena, according to present knowledge, the ad-dition of pantothenic acid is not required if thetotal feed is suited to ruminants. The microbialsynthesis of pantothenic acid in these animalsis sufficient. However, in calves fed on the ba-sis of milk surrogates, the supply of pantothenicacid from the feedstuff is not adequate. There-fore, appropriate pantothenic acid additives arenecessary. Since mixed feed contains on aver-age 6 ppm of pantothenic acid, addition of ca.50% of the total requirement is recommendedas a safety margin [1076].

Recommended doses in milligrams per kilo-gram of solo feed are [1075]:

130 Vitamins

Laying hens 8Fattening turkeys 15Porkers 8Calves 20Breeding and show horses 60

11.9. Analysis

For calcium (R)-pantothenate, the followingmethods are stipulated in the pharmacopeias(Pharm. Eur. II, USXXII): complexometric de-termination of calcium, determination of nitro-gen by the Kjeldahl method, and measurementof optical rotation.

Furthermore, a 5% aqueous solution must beclearly water soluble and have a pH value in therange of 6.8 – 8.

As a result of their low sensitivity, chem-ical detection methods can be applied onlyto simple mixtures (e.g., multivitamin prepara-tions) [1077]. These analytical techniques in-clude chromatographic preliminary purification(ion exchanger) and hydrolytic cleavage of theactive agent, the pantolactone and β-alanineliberated being detected spectrophotometrically[1078], titrimetrically [1079], or colorimetri-cally (color reaction with ninhydrin or 1,2-naphthoquinone-4-sulfonic acid) [1080].

Pantolactone forms a purple-red hydroxam-ate with hydroxylamine in alkaline solution inthe presence of Fe(III) [1081] and a yellow-green complex with naphthalenediol in sulfuricacid [1079].

The enantiomeric purity of (R)-pantolactonecan be determined from its specific optical ro-tation and, more accurately, by gas chromato-graphy on columnswith a chiral stationary phase[1082].

As a component of pharmaceutical prepara-tions, (R)-panthenol is detected by thin layerchromatographic separation [1083] or directlyby ion-pair chromatograpy [1084]. Determina-tion of optical purity can be achieved by gaschromatography after derivatization [1085].

For the determination of pantothenic acidin complex substrates (e.g., in foods and feedsand in tissues and body fluids), microbiologicaltest systems give reliable results. The test or-ganism used is usually Lactobacillus plantarum(ATCC8014) [1086]. Pantothenic acid presentin the form of coenzymeA must first be en-

zymatically released. Combinations of alkalinephosphatase with liver enzymes that cleave pan-tetheine to pantothenic acid and cysteamine aregenerally used [1087,1077].

(R)-Panthenol can be determined microbi-ologically by its growth-inhibiting effect onLeuconostoc mesenteroides (ATCC8042 P-60)[1088].More recently, immunoassays have beenused for the determination of pantothenic acidin blood and tissues. Both the RIA method(radioimmunoassay) [1089] and the ELISAmethod (enzyme linked immunosorbent assay)[1090] yield results comparable to microbiolog-ical tests.

The curative chick growth test [1091] is a bio-logical determinationmethod for free and boundpantothenic acid and for (R)-panthenol.

11.10. Economic Aspects

Pantothenic acid is supplied almost exclusivelyin the form of the calcium salt. The worldmarket volume in 1993 was ca. 6000 t/a. Themost important producers are: Hoffmann-LaRoche (Scotland), Daiichi (Japan), and BASF(Germany) for calcium (R)-pantothenate; Alps(Japan), Terapia (Rumania) and Polfa (Poland)product is also supplied by other smaller pro-ducers for the feed sector [1076]. World marketprices in 1993 were: calcium (R)-pantothenateca. ¤ 18/kg, calcium (R,S )-pantothenate ca.¤ 7 – 8/kg.

About 80% of the calcium (R)-pantothenateis used in animal feeds and the rest in the foodand pharmaceutical sectors.

World annual production of (R)-panthenol isca. 1000 t with a price of ca. DM43 – 45/kgin 1993. The most important producers of (R)-panthenol are Hoffmann-La Roche, Daiichi, andBASF. This product has found diverse applica-tions in the pharmaceutical and cosmetic sec-tors, especially in the treatment of wounds andfor skin- and hair-care products [1092,1093].

12. Biotin

12.1. Introduction

Biotin [58-85-5], 2,3,3a,4,6,6a-hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic

Vitamins 131

acid, has three asymmetric centers and can oc-cur in eight stereoisomeric forms.However, onlythe isomer with the configuration (3aS,4S,6aR),d-(+)-biotin (1), has full biological activity asvitaminH. Some important derivatives of biotinare presented in Formulas 1 – 11 above.

12.2. History

Biotin was first isolated in 1935. In addition tomesoinositol (Bios I) [87-89-8] and pantothenicacid (Bios IIA) [79-83-4], a factor Bios IIB wasseparated from a mixture of substances [1100]knownas growth factorBios formicroorganisms[1101]. Bios IIB was named biotin. A year latercrystalline biotin methyl ester [608-16-2] wasisolated from dried egg yolk [1102]. It was ver-ified [1103] shortly afterwards that biotin wasidentical to both vitaminH [1104], a protectivefactor against egg white injury caused by one-sided consumption of large amounts of raw eggs[1105], and coenzymeR [1106], a factor neces-sary for the growth and respiration of the bacteriaof the root tubercles of leguminous plants.

In 1942, the structure of biotin was estab-lished by degradation reactions [1107,1108].This was followed by the first, albeit stereo-chemically nonspecific, total synthesis of biotin[1109]. The first stereospecific synthesis of pureenantiomers and diastereoisomers of biotin wasachieved in 1949 [1110–1112].

12.3. Physical and Chemical Properties

d-(+)-Biotin (1) C10H16N2O3S, Mr 244.31, isreadily soluble in dilute alkalis, hot water, and

95% ethanol, slightly soluble in cold water anddilute acids, and almost insoluble in organic sol-vents. It crystallizes fromwater as long thin nee-dles, mp 230 – 232 ◦C (decomp.), [α]22D +92.0◦(c = 1, 0.4% NaOH) [1113]. Crystalline biotinhas an orthorhombic structure [1114]. The thio-phane ring has the same conformation in so-lution and in the solid state. The sulfur liesabove the plane of the ring and the valeric acidside chain occupies a quasi-equatorial position[1115]. Crystalline biotin is stable in air, ther-mally stable, and resistant to sunlight. It is, how-ever, instable when exposed to UV light, oxidiz-ing agents, and strong acids and strong bases.

With avidin [1405-69-2], biotin forms a mo-lecular compound, in the ratio of 4 : 1, havingone of themost stable noncovalent bonds known[1116,1117]. Avidin, a glycoprotein containedin raw eggwhite, has amolar mass of ca. 70 000.This molecular compound can be cleaved nei-ther enzymatically (peptidases) nor by acids, butonly by the denaturation of avidin by radiation orby heating at 100◦C for a longer period of time.Biotin forms similarly strong complexes withthe related proteins streptavidin [9013-20-1] andstravidin [97276-57-8] which are formed in thefungi Streptomyces and Saccharomyces [1118].

12.4. Occurrence

Biotin occurs in varying amounts, either in thefree state or bound to proteins, in most food-stuffs. The richest natural sources of biotin areyeasts [1119], lower fungi [1120], and microor-ganisms [1121]. Microorganisms excrete suchlarge amounts of biotin into the surrounding

132 Vitamins

medium that most animals can cover their bi-otin requirement through microbiological syn-thesis within the intestinal tract. In humans, thefecal excretion of biotin exceeds external intake,indicating endogenous biosynthesis by intesti-nal flora. In plants, biosynthesis of biotin is lo-calized in the leaves; therefore, cultivated greencells of Lavendula vera have an especially highbiotin content [1122]. Since biotin can also beresorbed from the soil through the roots, it is dis-tributed throughout the entire plant. Some typesof phytoplankton are capable of biosynthesiz-ing biotin as well, e.g., Skeletonema costatum,Stephanopyxis turris or Gonyaulax polyedra.

Fresh cow’s milk, with a biotin content of2.0 – 3.5µg/mL, is one of the most importantexogenous natural sources for the biotin re-quirement of humans [1123]. Depending on thestage of lactation, breastmilk also contains vary-ing amounts of biotin (0.06 – 0.29µg/mL in thecolostrum stage and 0.81 – 1.30µg/mL duringthe nursing period) [1124]. In contrast to meat,beef liver (up to 100µg/100 g) and kidney havean especially high biotin content. Among hu-man foodstuffs, other fairly good sources of bi-otin are eggs, some vegetables (e.g., soya with60µg/100 g or lentils), cereals (e.g., rolled oatswith 20µg/100 g), and nuts [1123]. In smalleramounts (0.1 – 5.0µg/100 g), biotin is a ubiqui-tous component of all other foodstuffs. Remark-ably, the biotin content of cheese increases four-fold during maturation due to bacterial synthe-sis.

In some foodstuffs, such as meat or plantshoots, biotin is present in the bound form asbiocytin (ε-N-biotinyllysine) [576-19-2]. Sincethe body can resorb only free biotin, this amide isquantitatively hydrolyzed to biotin and l-lysine[56-87-1] in the gastrointestinal tract by the en-zyme biotinidase [1125,1126]. However, hu-mans utilize only ca. 50% of ingested biotin; therest is excreted in the feces. Accordingly, sedi-mentation sludgewith 50 – 70µg of biotin/100 gof solids represents a further, albeit not directlyexploitable, source of biotin.

12.5. Biosynthesis

The biosynthesis of biotin in individualmicroor-ganisms is now well studied and described indetail in the literature [1127,1128]. Although

there is no uniform precursor, biosynthesis usu-ally starts with pimelic acid [111-16-0] andacetyl coenzymeA, which are converted bypimelyl CoA synthetase to pimelyl coenzymeA,which undergoes decarboxylation with l-ala-nine [56-41-7] to give 7-oxo-8-aminopelargonicacid [4707-58-8]. This reaction is catalyzedby the pyridoxal phosphate dependent en-zyme 7-oxo-8-aminopelargonic acid synthetase.Subsequently, 7,8-diaminopelargonic acid ami-notransferase transfers the amino group fromS-adenosylmethionine [29908-03-0] to give7,8-diaminopelargonic acid [21738-21-6]. Thiscompound then undergoes ring closure with de-thiobiotin synthetase and bicarbonate, ATP, andMg2+ to give dethiobiotin [533-48-2]. Finally,sulfur is incorporated into biotin with the aid ofthiobiotin synthetase (Scheme 26) [1132].

In this unusual reaction, the sulfur presum-ably comes from l-cysteine [56-89-3] [1129]and is incorporated at the prochiral C-4 of de-thiobiotin with retention of configuration, elim-inating the pro-4S hydrogen [1130]. Accord-ingly, cystine, pimelic acid, and dethiobiotinpromote biosynthesis in biotin culture media.However, biotin itself causes negative feedbackinhibition of biosynthesis, probably by repress-ing formation of most of the enzymes requiredfor synthesis [1131].

Other precursors include glutaric acid[110-94-1] and pelargonic acid [112-05-0], thelatter being the precursor for biosynthesis in astrain of Pseudomonas [1133].

For conversion to the biologically active bi-otin enzyme, biotin is subsequently activated byadenosine triphosphate with the formation of bi-otin adenosinylate [4130-20-5]. This substancereacts with the ε-amino group of an l-lysineresidue of apoenzymes to give the active cofac-tor biocytin.

12.6. Function as Prosthetic Group

Enzymes which contain biotin as cosubstrate orcoenzyme are classified as carboxylases (car-boxylation via hydrogencarbonate), transcar-boxylases (transfer of carboxyl function), anddecarboxylases (Na transport with decarbox-ylation), depending on their metabolic function.However, only carboxylases play a role in higherorganisms [1134,1135].All three classes have in

Vitamins 133

Scheme 26. Biosynthesis of biotin (enzymes and E. coli genes)

common a two-step mechanism. The carboxylgroup is first transferred to the biotin enzyme,and then reacts further in a substrate-specificmanner. In this process, the carboxylases mustbe activated with ATP, while the trans- anddecarboxylases are independent of ATP [1136]:

E-Biotin +ATP+HCO−3 �E-Biotin-CO−

2 +ADP+P

E-Biotin-CO−2 +R-H�E-Biotin +R-CO−

2

∑: R-H+ATP+HCO−

3 �RCO−2 +ADP+P

In addition, metal cations such as Mg2+, K+ orMn2+ are required for enzyme activity. How-ever, they play a more structural than partici-pative role. As expected, avidin inhibits all thebiotin-dependent steps of the carboxylases.

Some of the important biotin-dependent car-boxylases are listed in Table 34. A series of rev-

iew articles are available for detailed infor-mation on biotin enzymes [1134,1135].

Table 34. Biotin dependent enzymes (carboxylases)

Enzyme Catalyzed reaction Role inmetabolism

Acetyl CoAcarboxylase

acetyl CoA→malonylCoA

fatty acidbiosynthesis

Pyruvate carboxylase pyruvate→ oxalacetate gluconeogenesis,lipogenesis

Propionyl CoAcarboxylase

propionylCoA→methylmalonylCoA

propionatemetabolism

3-MethylcrotonylCoA carbo- xylase

3-methylcrotonylCoA→ 3-methylglutaconylCoA

leucine catabolism

Geranyl CoAcarboxylase

GeranylCoA→ carboxygeranylCoA

catabolism ofisoprenoidcompounds

Urea carboxylase urea→N-carboxyurea metabolization ofurea in yeasts to autilizable form

134 Vitamins

12.7. Isolation and Production

Isolation from Natural Sources. Biotin wasfirst isolated from the natural sources egg yolk[1102], ox liver [1137], and milk [1138]. Theextracts are subjected to an elaborate purifica-tion procedure, and crude biotin is finally precip-itated with phosphotungstic acid [1343-93-7].After conversion to the methyl ester, purifica-tion is carried out by high-vacuum sublimationor molecular distillation. However, the amountsisolated are out of all proportion to the effort re-quired. Genetically modified strains of E. coliand Bacillus sphaericus have proved to be aricher source of natural biotin [1128]. Concen-trations of up to 230mg/L have already beenobtained in fermenter charges [1139].

However, all the biotin used in the animal feedor pharmaceutical industry is still produced syn-thetically and corresponds to the natural productin all respects.

Industrial Synthesis [1099]. The first non-stereospecific total synthesis of biotin startedwith l-cysteine and involved the formation ofthe thiophane ring, followed by cyclization ofthe imidazolone segment. It produced all eightisomers of biotin [1109]. Following this proofof structure, Goldberg and Sternbach wereable in their synthesis to reduce the numberof isomers to one separable pair of diastere-omers (3aS,4S,6aR-(+)- and 3aR,4S,6aS-(−)-biotin) [1110–1112]. Their process providedthe foundation for the modified synthesis ofGerecke [1140], which is the basis for almostall industrial syntheses of biotin. This improvedprocess has the advantage of the economicallymore favorable separation and recycling of thebiologically inactive l-(−) isomer to an earlierstage in the synthesis (Scheme 27).

More recent efforts to optimize this synthesisinclude shifting the isomeric ratio to favor (+)-biotin by asymmetric induction with opticallyactive amines [1141] or alcohols [1142], recy-cling and isomerization of (−)-biotin [1143],or a simpler introduction of the exocyclic va-leric acid group by means of the Wittig reaction[1144] or by using ortho esters [1145].

The recent literature contains numerous ef-forts to find new methods for the synthesis ofracemic and diastereoisomerically pure biotin(Table 35).

In the latter case, efforts are made to trans-fer asymmetric centers from natural chiral sub-strates, such as sugars or amino acids, to thetarget molecule. Until now, however, none ofthese syntheses can compete with the modifiedGerecke process in industrial application.

In the past years, rapid progress has beenmade in the biosynthetic preparation of biotinvia genetically modified bacterial strains (seeabove). To seriously compete with the stereo-specific total chemical synthesis, biotin concen-trations of at least 1 g/L of fermentation solutionmust be attained. Therefore, the current biosyn-theses which give concentrations of not morethan 230mg/L are still not feasible.

12.8. Metabolism and Importance forthe Organism

Biotin ingested with food is present in the bodyeither in the free state or protein-bound in theform of biocytin. The latter is cleaved into biotinand ε-lysine by the enzyme biotinidase, whichis found in most mammals [1168]. The uptakeof free biotin in the intestinal tract is not yet wellunderstood. However, it appears that an active,carrier-mediated transport mechanism plays arole at low biotin concentrations, and a passivediffusion mechanism at high biotin concentra-tions [1169,1170]. The intracellular accumula-tion of biotin in various bacterial strains alsoproceeds in an energy-dependent protein carrierprocess [1171].

In the plasma, 80% of the biotin present isprotein bound [1172]. Transport probably oc-curs via biotinidase and is not linked to any en-ergy source [1173]. Biotin cannot be degraded inmammalian metabolism. The only reaction thathas been observed is oxidation to biotin sulfox-ide [3376-83-8] and tetrabisnorbiotin in the mi-tochondria after activation of the carboxyl group[1174]. Bothmetabolites aswell as biotin are ex-creted in the urine and feces. The total excretionof biotin usually exceeds the intake [1175].

Various bacterial strains and molds are ca-pable of metabolizing biotin completely. Thus,strains of Pseudomonas use biotin as the solesource of C, N, and S. Degradation proceeds viastepwise β-oxidative decomposition of the sidechain and the thiophane ring. Finally, the imida-zolidine ring is metabolized to urea [1128].

Vitamins 135

Scheme 27. Industrial preparation of (+)-biotin from fumaric acidR∗= oxyalkyl, aminoalkyl; Bn = benzyl

Table 35. Alternative syntheses for (±)- or (+)-biotin

Starting material Product Isomer separation Steps Refs.

l-Cysteine (+)-biotin + 13 [1146]l-Cysteine (+)-biotin − 12/14 [1147–1149]l-Cystine (+)-biotin − 11 [1150]d-Glucose (+)-biotin − 23/18 [1151,1152]d-Glucos-amine [3416-24-8] (+)-biotin − 10 [1153]d-Mannose (+)-biotin − 16 [1154]d-Arabinose (+)-biotin − 11/ – [1155,1156]3-Amino-2-carbmethoxythiophene[22288-78-4]

(±)-biotin − 9 [1157]

Chromene (±)-biotin − 12 [1158]Cycloheptene (±)-biotin − 14/10 [1159,1160]6-Methoxy-2H-pyran [4454-05-1] (±)-biotin − 10 [1161]Fumaric acid (+)-biotin + 11 [1162]Pimelic acid (±)-biotin − 11 [1163]ω-Ketohexanoic acid [928-81-4] (±)-biotin − 9 [1164]ω-Ketohexanoic acid (+)-biotin + 12 [1165]ω-Ketoheptanoic acid [35923-65-0] (+)-biotin + 9 [1166]1,4-Butenediol (±)-biotin − 14 [1167]

136 Vitamins

As a coenzyme (see above) for a series of im-portant metabolic functions (e.g., CO2 fixationand transfer) biotin is of essential importancefor bothmicro- andmacroorganisms. Therefore,several metabolic functions are simultaneouslydisturbed in biotin deficiency.

12.9. Deficiency Symptoms,Requirement, and Application

Biotin deficiency can be produced in humansand animals by diets low in this vitamin, con-sumption of large amounts of raw egg white, orby the administration of sulfonamides,which actas antivitamins by competitive enzyme inhibi-tion [1175]. Manifestations of biotin deficiencyin mammals include alopecia, pruritus, dermati-tis, central nervous disorders, formation of crustsand rhagades, and hypercholesterinemia [1176].

12.9.1. Symptoms and Therapy in Humans

Biotin deficiency can be produced in humansby a one-sided diet of raw eggs [1177], alco-hol abuse [1178], side effects of some drugs(e.g., sulfonamides or antiepileptics) [1179], in-born genetic defects (absence of biotin enzymes)[1180], or by incomplete parenteral nutrition[1181]. In babies, biotin deficiency can alsooccur after an excessively long nursing period(>4months) due to the sinking biotin levels inbreast milk [1182].

In 1942, biotin avitaminosis [1183] was firstdescribed by Sydensticker. He fed four adulttest persons with a one-sided diet rich in eggwhite. The first typical symptoms, such as de-presion,muscular pain, and irritability, appearedafter 3 – 4 weeks. Longer term biotin deficiencyleads to desquamative, maculosquamous, andseborrheic dermatitis; sleepiness; local paraes-thesiae, anorexia; alopecia; ataxia; and glossitis.The normal blood values with regard to hemat-ocrit, cholesterol, and bile pigments are altered;the excretion of biotin in the urine decreasesfrom 30 – 50 to 3 – 7µg/d.

Is is also possible that sudden infant deathsyndrome (SIDS) is also due to vitamin defi-ciency [1184], as suggested by the low hepaticbiotin level found in the dead babies.

All the symptoms, which are biochemicallycaused by a disturbance in glucogenesis andpotassium deficiency in the muscles, can be re-versed in a very short time by oral or parenteraladministration of ca. 150µg/d of biotin. Bioki-netic studies on the turnover of biotin and val-idated deficiency experiments are not yet avail-able. Hence, all the known biotin requirementvalues are estimates. The recommendationmadeby the DGE (German Society for Nutrition) is30 – 100µg of biotin per day for adults.

12.9.2. Symptoms and Therapy in Animals

Symptoms of biotin avitaminosis in higher or-ganisms were first observed in rats given a dietrich in egg white [1104]. After 2 – 4 weeks, therats suffered from egg white injury, which ex-hibits the typical biotin deficiency symptoms.It starts with progressive dermatitis, especiallyof the genitals, spreading to the neck and muz-zle. In the further course of the syndrome, thedermatitis becomes pronounced and resemblesLeiner’s disease. This is followed by alopecia,pruritus, edema, and a characteristic spastic gait(kangaroo posture). A low fat content in the dietincreases the symptoms,whereas administrationof ascorbic acid (vitaminC) slows down the syn-drome [1185].

Before the outer signs become significant,there is a marked decrease in the activity of thebiotin-dependent enzymes pyruvate and acetylCoA carboxylase. The enzymes are transformedinto vitamin-free apoenzymes, which, however,can be reactivated by addition of biotin inan ATP-dependent process [1186]. Apart frompotassium deficiency in the striated neuromus-cles, a further pathological symptom is the in-crease in the C16 fatty acid content and the cho-lesterol level in the tissue [1187].

Aswith other higher organisms, all the symp-toms can be completely reversed within 2 – 4weeks by the external supply of 3 – 8µg of biotinper 100 g of animal food.

Most of these avitaminosis symptoms, suchas dermatitis, loss of hair and feathers, depig-mentation, encrustations of the eye lids, growthinhibition, and weight loss, are also found inother animals, including fish, hens, pigs, dogs,andmonkeys [1175,1188]. In the case of chicks,marginal deficiency symptoms also include per-

Vitamins 137

osis as well as fatty liver and kidney syndrome.The latter results in a very high mortality of theyoung animals due to disturbed hepatic glucoge-nesis via pyruvate carboxylase. The symptomsare similar to those of SIDS (see above) [1189].

12.10. Biotin Analogs

Of the 8 possible stereoisomers of biotin, onlythe d-(+) form (1) has biological activity. Its nat-urally occurring form, having the same activity,is biocytin, biotin ε-lysine (2). Biotin can be lib-erated from this compound by acid hydrolysisor enzymatically with biotinidase [1190].

Some analogs also show biological effects,but only in the configuration corresponding tothe d-(+) form. Dethiobiotin (3), which canreadily be made by reduction of biotin, canfully substitute for biotin as a growth factorin some microbial substrates, such as E. colior Bacillus sphaericus [1191]. Therefore, it iscertain that dethiobiotin is a precursor of bi-otin [1130]. After incorporation into acetyl CoAcarboxylase, selenobiotin [57956-29-3] (4) ex-hibits coenzymatic properties comparable tothose of biotin [1192]. Selenobiotin can bemadesynthetically or isolated microbially from Phy-comyces blakesleanus. Like biotin sulfoxide (5),it can totally replace biotin in lower organ-isms. In the case of higher organisms, oxybiotin[14474-91-0] (6) and biotinol [53906-36-8] (7)are the only analogs capable of replacing biotinin the diet without the appearance of deficiencysymptoms. Vitamin deficiency symptoms in ratsare also modified by these analogs, but to a lim-ited extent.

Biotin derivatives that are modified in theside chain, such as norbiotin [669-72-7] (8),homobiotin [1784-22-1] (9) or α-dehydrobiotin[10118-85-1] (10) are generally active an-tagonists for microorganisms such as Lacto-bacillus casei or Saccharomyces cerevisiae. α-Dehydrobiotin is one of the strongest antago-nists known until now. When incubated withapocarboxylases, it irreversibly prevents thebinding of biotin to the enzyme [1193]. Bi-otin sulfone [40720-05-6] (11) has a minorresorption-inhibiting effect. The most effectivebiotin antagonists are the glycoprotein avidinfound in raw egg white and the proteins stra-

vidin and streptavidin produced by microorgan-isms [1194].

12.11. Analysis and Standardization[1098]

For the determination of biotin concentrations inserum, bioassays using biotin-deficient animalswere mainly employed in the past. The disad-vantages of this method are the low accuracyand high time consumption [1195].

Highly specificbiotin determinations are pos-sible with microbiological and radioisotope di-lution assays. Microbiological determination isbased on the measurement of the increase inthe amount of the corresponding substrates (seeTable 36) after administration of a sample ofunknown biotin concentration compared withseveral standard solutions. The biotin containedin the sample must be converted to a bioavail-able form by treatment with acid or papain[9001-73-4], and unsaturated fatty acids mustbe removed because they interfere with the as-say. After inoculation and incubation, the deter-mination of the biotin concentration (detectionlimit 0.1 ng) is carried out by turbidimetry ortitrimetry. The disadvantage of this process isthat diverse biotin analogs are also included, de-pending on the microbe [1196].

More recent methods, including the radioiso-tope assay, make use of biotin – avidin technol-ogy. Identical samples of radioactively labelledavidin are treated in parallel with the unknownsample and standard biotin solutions. Subse-quently, the samples are fixed via immobilizedbiotin. The concentration – amounts as low as0.1 ng can be determined [1199] – is directly ob-tained from the count rate [1200]. Apart from la-belling bymeans of doping, fluorescence [1201]and chemiluminescence measurements [1202]have also been used for detection.

In general, chemicophysical methods are em-ployed for the purification and definition of pu-rity. The purification is conveniently conductedby ion exchange [1203,1204], paper, thin-layer,or liquid chromatography [1098]. Detection iscarried out refractometrically or by reactionwith color reagents. The sensitivity of the lat-ter methods, which detect either visually or byUV/fluorescence measurements, varies between0.3 and 50 ng [1205]. Finally, purity testing can

138 Vitamins

Table 36. Microorganisms for biotin bioassays and their activity towards biotin analogs∗

Microorganism Biotin Biotinmethylester

Biotinsulfoxide

Biotinsulfone

Oxybiotin Norbiotin Dethiobiotin Biocytin 7-Oxo-8-ami-nopelargonicacid

7,8-Diamino-pelargonicacid

Saccharomycescerevisiae∗∗

+ + + inhibitor +/− inhibitor + + + +

Lactobacilluscasei

+ − − inhibitor +/− inhibitor − +

Lactobacilluslantarum∗∗∗

+ − + inhibitor +/− inhibitor − − − −

Neurosporacrassa

+ inhibitor inhibitor

Ochromonasdanica

+ inhibitor inhibitor + −

Allescheria boydii + +/− inhibitor +/− inhibitor +/− +Streptococcusfaecalis

+ inhibitor − inhibitor

∗+ shows activity similar to pure biotin; +/− shows activity, but less than pure biotin; − shows no activity. ∗∗Detection of“total biotin” [1197]. ∗∗∗Detection of “true biotin” [1198].

be completed by using NMR, LC/GC–MS, andpolarography.

There is no international standard for biotin.

12.12. Uses and Economic Aspects

The annual production of 25 t of biotin is largelyused in the animal feed, pharmaceutical, andfood industries. To a minor extent, it is also usedtogether with avidin for analytical purposes inimmunoassays, as an additive in nutrient mediafor microbial substrates, and in the cosmetic in-dustry.

In the large-scale husbandry of domestic ani-mals, the use of biotin as a feed additive is basedon the prevention of the readily occurring bi-otin deficiency symptoms caused by the lack ofthis coenzyme, which is essential in a series ofimportant metabolic functions. In pharmacy, bi-otin is used – in the form of multivitamin tablets,dragees, capsules, or syrup – exclusively to pre-vent vitamin deficiency symptoms, which canoccur, e.g., after operations, during pregnancyand lactation, and in growth phases.

In immunology, diagnostics, and biochemi-cal research, the use of biotin – avidin technol-ogy [1096,1206] has undergone rapid develop-ment in the last few years. The method is basedon two principles. First, biotin can easily befunctionalized via its carboxyl group and, sec-ondly, the bicyclic unit with which binding toavidin occurs is spatially far removed due toa spacer of four methylene groups. Avidin can

also be activated with suitable substrates. Thus,two otherwise chemically inert substrates can bejoined together by means of the biotin – avidincomplex. In tumor therapy, for instance, antitu-mor antibodies are biotinylated and the tumorcells are treated with these immunoglobulins.Subsequently, the damaged cells are incubatedwith an avidin – cytostatic agent complex. Theresulting formation of the avidin – biotin com-plex results in the drug being brought directly tothe cancer cell without interfering with healthycells [1207,1208].

The application of biotin – avidin technologyin numerous immunoassays [1209], in the sepa-ration and purification of immunoglobulins fromsera [1210] (see Fig. 21), and in the detection ofDNA or RNA fragments in hybridization exper-iments [1211] is based on the same principles.

These uses and pharmaceutical and foodtechnological purposes, however, require only5 – 10% of the annual production of 25 t of bi-otin (100%). The majority is employed in theanimal feed industry.

The main producers of biotin are Hoffmann-La Roche (Switzerland), Tanabe (Japan), Sum-itomo (Japan), Merck (Germany), Il Sung (Ko-rea), Lonza (United States), and, for feed-qualityonly, BASF (Germany).

The world market price of 1 g of 100% pured-(+)-biotin is ca. 5¤(1995).

Vitamins 139

Figure 21. Isolation of antibody/antigen complex by means of biotin – avidin technologyA)Binding of biotinylated protein (antibody) to immobilized avidin;B)Addition of specific antigen;C)Binding of immobilizedantibody – antigen; D) Elution of biotinylated antibody – antigen complex via displacement of avidin with free biotin

12.13. Tolerance and EnvironmentalProtection

Even after the administration of large amountsof biotin, no acute toxicity in humans or animalshas been observed [1212]. Thus, doses of up to10mg of biotin per 100 g of bodyweight showedno side effects in rats. A six-month therapeutictreatment of humans with 10mg/d resulted inno harmful effects on body functions. Injectionsof 1mg of biotin/kg of body weight in pregnantrats, however, caused resorption of the fetus andplacenta and disturbances of estrogen formation[1213].

Biotin is considered environmentally harm-less and is subject to no special regulations withregard to storage and transport. No explosionlimits exist for biotin. Water, carbon dioxide,

foam, or powder can be used as extinguishingagents in the case of fire. As a result of its acidiccarboxyl function, classification as aweaklywa-ter endangering substance is being considered.Normal personal and hygienicmeasures (protec-tive glasses, washing of hands) should be takenwhen working with biotin. Ecological problemsare not to be expected if biotin is properly han-dled and used [1214].

13. Folic Acid

13.1. Introduction

Folates are a group of biologically active com-pounds which are structurally derived from

140 Vitamins

pteroylglutamic acid or folic acid (1). The chem-ical structure can be divided into the three sub-structures 6-methylpterin, p-aminobenzoic acidand l-glutamic acid. The Chemical Abstractsname is N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-l-glutamicacid.

Table 37. H4folate cofactors

∗ [1215].

Enzymatic reduction of folic acid leads to7,8-dihydrofolic acid (H2folate), a key sub-stance in biosynthesis. Further reduction, cat-alyzed by the enzyme dihydrofolic acid reduc-

tase, provides (6S )-5,6,7,8-tetrahydrofolic acid(H4folate) (2). This is the parent compound ofthe folates 3–8 possessing at N-5 and/or N-10 anadditional C1 substituent (Table 37). These sub-stances and the corresponding polyglutamatesare essential coenzymes for the transfer of C1units of different oxidation levels. In cofactor 8with a methenyl group (−CH+−) bridging po-sitions 5 and 10, a positive charge is associatedwith the substructure [N(5) - CH -N(10)]+. Allnatural folates have the same absolute configu-ration at position 6 as (6S )-tetrahydrofolic acid(2).

In contrast, chemical reduction of folic acidgives (6RS )-5,6,7,8-tetrahydrofolic acid, a dia-stereomeric mixture of (6S,αS )- and (6R,αS )-H4folate. Nomenclature and symbols for folicacid and related compounds are subject of rec-ommendations published by the IUPAC– IUBJoint Commission on Biochemical Nomencla-ture (1986) [1215].

In living cells folates are predominantlystored as polyglutamates in which the secondand subsequent molecules of l-glutamate areeach linked by amide bonds to the γ-carboxylof the preceding glutamate unit.

13.2. Historical Notes

In the 1930s, a dietary factor in yeast extractsand crude liver was found to cure megaloblas-tic anemia in pregnant women [1216]. Experi-mentally, this disorder could also be induced inmonkeys and cured by antianemic factor (vita-minM). Two bioassay procedures, the microbi-ological growth assay and the antianemic factorassay in chickens (vitaminBc), were developedand used to follow the isolation and purificationof folates [1217–1219]. A close relationship bet-ween the yeast growth factor and the antianemicfactor was soon established, but it became evi-dent that the yeast growth factor and vitaminBcwere different entities. A crystalline preparationof a highmolecularmass derivative of folatewas

Vitamins 141

isolated [1220] and later shown to be a peptide[1221]. Theunequivocal elucidation of the struc-ture of pteroylglutamic acid (1) was achieved in1946 by Angier et al. [1222]. The discovery offolates and developments during the followingdecade are reviewed in [1223].

13.3. Properties

This chapter describes some properties of se-lected folates [1224–1227]. UV data are givenas λmax (ε) in nanometers.

Folic acid (1), pteroylglutamic acid, PteGlu[59-30-3], C19H19N7O6, Mr 441.41; yellowthin platelets, no mp, chars above 250◦C; UV(pH 13): 256 nm (30 000), 282 nm (26 000),365 nm (9800); [α]27D =+19.9◦ (0.1N NaOH).Contains ca. 8% water which is lost in vacuumat 90◦C. Soluble in aqueous alkali metal hy-droxides and carbonates and in aqueous mineralacids, less soluble in acetic acid and pyridine,slightly soluble in hot water, very slightly solu-ble in cold water, insoluble in acetone and chlo-roform. Stable in crystalline form in the darkat room temperature; unstable in alkaline andacidic solution.

Dihydrofolic acid, 7,8-dihydropteroylglutamicacid, H2folate [4033-27-6], C19H21N7O6,Mr 443.42; UV (pH 7.2): 282 nm (28 600).Preparation from folic acid by catalytic hy-drogenation or sodium dithionite reduction inpresence of ascorbic acid [1228]. Highly airsensitive compound.

Tetrahydrofolic acid (2), 5,6,7,8-tetrahydropteroylglutamic acid, H4folate[135-16-0], C19H23N7O6, Mr 445.44; UV(pH 7.85): 296 nm (28 000). Synthesized fromfolic acid by catalytic hydrogenation in acidicsolution or by reductionwith sodiumcyanoboro-hydride [1229]. Very sensitive to oxygen.(6RS )-H4folate has [α]27D = +14.9◦ (0.1NNaOH). Natural (6S )-H4folate [71963-69-4]has [α]27D =−16.9◦ (0.1N NaOH).

5-Methyltetrahydrofolic acid (3), 5-CH3-H4-folate [134-35-0],C20H25N7O6,Mr 459.46;UV(pH 7): 290 nm(32 000). Synthesis fromH4-folate by condensation with formaldehyde and

reduction of the resulting 5,10-CH2-H4-folatewith sodium borohydride [1230]. 3 is more sta-ble towards oxygen than H4folate.

5,10-Methylenetetrahydrofolic acid (4),5,10-CH2-H4folate [3432-99-3], C20H23N7O6,Mr 457.45; UV (pH 7.2): 294 nm (32 000).Preparation from H4folate and formaldehyde.Very sensitive to hydrolysis. Synthesis of thenatural (6R)-stereoisomer by enzymatic reduc-tion of H2folate and treatment with formalde-hyde [1231]. Coenzyme in thymidylate biosyn-thesis.

10-Formyltetrahydrofolic acid (6), 10-HCO-H4-folate [2800-34-2], C20H23N7O7,Mr 473.45; UV (pH 7.5): 260 nm (17 000). Theunstable compound 6 is prepared by hydro-genation of 10-HCO-folate or by hydrolysisof 5,10-CH+-H4folate [1232]. Coenzyme inpurine synthesis.

5-Formyltetrahydrofolic acid (5), (6RS )-5-HCO-H4folate, folinic acid, leucovorin[58-05-9], C20H23N7O7, Mr 473.45; UV(pH 13): 282 nm (32 600); [α]25D =+16.0◦ (H2O,Ca salt). Most stable of the H4 folate cofac-tors. Preparation by hydrolysis of 5,10-CH+-H4folate in neutral or alkaline solution or bydirect formylation of H4folate [1233]. (6S)-5-HCO-H4folate, citrovorum factor, l-leucovorin[68538-85-2]; [α]D =−13.3◦ (H2O). Enzy-matic synthesis of the natural (6S )-stereoisomerfrom H2folate [1234,1235]. Preferential crys-tallization of the Ca salt of natural (6S )-5-HCO-H4folate from (6RS )-5-HCO-H4folate in water[1236]. 5-HCO-H4folate is used in medicineas an antidote to folic acid antagonists such asmethotrexate.

5-Formiminotetrahydrofolic acid (7), 5-NHCH-H4folate [2311-81-1], C20H24N8O6,Mr 472.46; UV (pH 7): 285 nm (35 400). Fasthydrolysis in aqueous solution.

5,10-Methenyltetrahydrofolic acid (8),5,10-CH+-H4folate, anhydroleucovorin[65981-89-7], C20H22N7O

+6 (cation); UV

(pH 1): 352 nm (23 900). Preparation by de-hydration of 5-HCO-H4folate in acidic medium[1235].

142 Vitamins

13.4. Content in Food andBioavailability

Folic acid is synthesized de novo bymicroorgan-isms and plants; mammals require it in their diet.Folates are present in all food products of animaland plant origin, especially in green-leaved veg-etables. Liver has a notably high content. The fo-late content (in µg/100 g) of some foods is givenin the following [1237–1241]:

Lettuce 106 – 200Spinach 78 – 194Asparagus 50 – 195Cabbage 30 – 79Broccoli 71 – 189Tomatoes 6 – 73.4Beans 68 – 100Apples 3 – 6Oranges 24 – 37Bananas 22 – 36Avocados 30 – 36Beef 5 – 18Beef liver 141 – 330Kidney 63Chicken 5 – 28Eggs 65 – 140Milk 5 – 20.5Cheese 20 – 66Fish 4.5 – 15

Most sources list the total folate content[1237–1240], but occasionally a distinction ismade between mono- and polyglutamates. Totake into account the lower bioavailability ofpolyglutamates, a folate equivalent can be calcu-lated [1241]. The wide range of folate contentsmost likely reflects different methods used fordetermination.

Foodprocessing, storage, and cooking reducethe content of folates considerably [1242–1244].Different forms of folate vary in stability. Inparticular, oxidation results in inactive cleavageproducts. Folates can be stabilized for longer pe-riods in the presence of reducing agents such asascorbate.

In humans, folic acid is almost completely ab-sorbed under fasting conditions. The absorptionof folylpolyglutamates is 50 – 80% of that ofmonoglutamates as judged by urinary excretion[1245,1246]. This percentage may be loweredin the presence of many foods.

13.5. Biosynthesis

Folic acid is synthesized in microorganismsand plants from the precursors guanosine 5′-triphosphate (GTP), p-aminobenzoic acid, andl-glutamic acid. The enzymatic steps involvedin folate biosynthesis in enteric bacteria havebeen extensively reviewed by Brown andWilliamson [1247,1248]. The pathway isshown in Figure 22A. GTP is converted byGTP cyclohydrolase I (EC 3.5.4.16) to 7,8-di-hydroneopterin triphosphate. This transforma-tion includes the hydrolysis of the annelatedimidazole ring, elimination of the C-8 atom asformate, Amadori rearrangement, and ring clo-sure to the pyrazine ring involving the riboseC-2′ atom. The pyrophosphate group is sub-sequently hydrolyzed chemically [1249] or bya nonspecific pyrophosphatase so far only de-scribed in E. coli [1250]. The residual phos-phate is removed by an as yet uncharacterizedphosphomonoesterase. Hydroxymethyl-7,8-di-hydropterin pyrophosphate is then obtained bya retro-aldol reaction with removal of a hy-droxyacetaldehydemoiety and subsequent addi-tion of a pyrophosphate group. Condensation ofp-aminobenzoic acid with hydroxymethyl-7,8-dihydropterin py-rophosphate is carried out bydihydropteroate synthase (E.C. 2.5.1.15) lead-ing to 7,8-dihydropteroate. l-Glutamate is thenattached to this intermediate by dihydrofolatesynthetase. In some microorganisms, this en-zyme also has folylpolyglutamate synthetase ac-tivity [1251].

Organization of genes encoding for the fo-late biosynthetic enzymes varies greatly amongbacteria. In Bacillus subtilis and Streptococ-cus pneumoniae folate operons were shown tocarry six and four biosynthetic genes, respec-tively [1252,1253], while in E. coli biosyntheticgenes are spread along the chromosome. InB. subtilis, genes for folate biosynthesis werefound at three different locations on the chro-mosome (Figure 22B). They have been clonedand sequenced [1252,1254,1255]. First, themtroperon maps at position 204◦ and encodes GTPcyclohydrolase I and tryptophan RNA-bindingattenuation protein (TRAP) [1254]. Second, thefolC gene encoding dihydrofolate synthetasemaps at position 258◦ and is not linked to theother genes of folate biosynthesis [1255]. Third,a folic acid biosynthetic operon mapping at po-

Vitamins 143

Figure 22. Biosynthesis of folic acidA) Biochemical pathway; B) Genes encoding enzymes of folate biosynthesis in Bacillus subtilis

sition 10◦ encodes for the three subunits of p-aminobenzoate synthase, dihydroneopterin al-dolase, hydroxymethyl pyrophosphokinase, anddihydropteroate synthase [1252].

Industrial production of folic acid by genet-ically engineered microorganisms or extractionfrom natural sources is not yet economically vi-able.

13.6. Chemical Synthesis

Folic acid (1) was first synthesized by condensa-tion of 2,5,6-triamino-4(3H )-pyrimidinone (9),p-aminobenzoyl-l-glutamic acid (10), and 2,3-dibromopropanal (11) [1222,1256].

144 Vitamins

Later variants of this one-pot process re-placed the C3 component 11 by variousother compounds, such as 1,1,3-tribromo-2-propanone [1257], 2,2,3-tribromopropanal[1257], 2,2,3-trichloropropanal (12) and 1,1,3-trichloro-2-propanone (13) [1258]. Addition ofsodiumbisulfite to the reactionmedium substan-tially increased the yield of folic acid [1258].

Syntheses of folic acid by stepwise con-densation of 9, 10, and a C3 component havealso been reported. For example, 2-amino-6-(hy-droxymethyl)pteridinone (14), prepared by con-densation of 9with 1,3-dihydroxy-2-propanone,was reacted with 10 to give folic acid (1)[1259]. In a different two-step synthesis, 2-hy-droxymalondialdehydewas condensedwith twoequivalents of 10. Subsequent reaction of theresulting diimine with 9 provided folic acid[1260].

Condensation of triaminopyrimidinone (9)with unsymmetrical C3 compounds invariablyleads to regioisomeric byproducts substituted inposition 7.

6-Formylpterin (15b) is another viable in-termediate for the synthesis of folic acid. Itcan be prepared from triaminopyrimidinone (9)and 2-bromo-3,3-diethoxypropanal with H2O2as oxidizing agent, followed by acetylation andhydrolysis [1261]. A synthesis of 15a from thepyrazine derivative 16 has been developed as a

route giving a single regioisomer [1262]. Finally,condensation of 15b with the diester of 10, re-duction of the Schiff base with sodium borohy-dride and hydrolysis leads to folic acid [1263].

The various schemes to synthesize folic acidare extensively discussed in [1225].

13.7. Metabolism and BiochemicalFunctions

Prior to absorption by the jejunum, dietaryfolylpolyglutamates are hydrolyzed tomonoglu-tamates by folylpolyglutamate hydrolases (con-jugases). Deconjugation occurs at the surfaceof the absorptive cells and within lysosomes inenterocytes [1264–1267]. Intestinal absorptionand transport are optimal in a slightly acidicenvironment at pH 6 [1268]. A single proteinseems to be responsible for the transport ofthe monoglutamates, as shown by competitiveinhibition [1269]. Some conversion of folates,mostly to 5-CH3-H4folate, can occur during thepassage across the intestinal mucosa and beforerelease into the portal circulation. At pharma-cological doses (≥1mg), significant amounts offolate appear unchanged in portal blood. Theyare converted to methylated forms in the liver[1270,1271]. Apart fromH4folate and 10-HCO-H4folate, mainly 5-CH3-H4folate is observedin blood, bound to plasma proteins such as al-bumin, α-macroglobulin, and transferrin (low-affinity binders). High-affinity folate-bindingproteins are also known, but their functionsare not yet well understood [1272,1273]. Theenyzme folylpoly-γ-glutamate synthetase con-verts folic acid to folylpolyglutamates and islocated in the cytosol as well as in mitochon-dria [1274,1275]. The liver exhibits the high-est activity of this enzyme; appreciable amountsare also observed in most other mammalian tis-sues [1276]. Folylpolyglutamates seem to be thestorage forms, but they can also increase thespecificity of some folate-dependent enzymes.The enzyme dihydrofolate reductase transformsthe different forms of folate to the active coen-zymes, the tetrahydrofolylglutamates, wherebyNADPH functions as hydrogen donor. Tetrahy-drofolates are involved in a variety of bio-chemical processes, such as methylation reac-tions, amino acid metabolism and biosynthesisof purines and pyrimidines [1239,1277]. They

Vitamins 145

acquire C1 units at N-5 and/orN-10 and functionas coenzymes in the transfer and utilization ofC1 fragments of different oxidation states, suchas methanol, formaldehyde, and formate.

In humans, plasma levels of 7 – 17µg/L arefound. The concentration of folate in erythro-cytes is ca. 200 – 500µg/L. The total folate poolin adult humans is estimated to be ca. 5 – 10mg,half of which is stored in the liver. The daily ex-cretion of folate in urine is ca. 40µg and in feces200µg [1278,1279].

13.8. Nutritional Requirements andMedical Use

Recommended Daily Intake (RDI). Ap-proaches that have been used to estimate folaterequirements in humans are the determinationof folate amounts needed to normalize the fo-late status after depletion, the evaluation of theaverage daily folate intake, the measurements ofurinary metabolites, and kinetic studies. Basedon several studies, the official recommendationin the United States was lowered in 1989 and isset at 3µg/kg body weight per day, resulting inca. 200µg folate for adult males and 180µg foradult females [1280]. Since pregnancy and lac-tation are often associated with poor folate sta-tus, 400µg/d and 260 – 280µg/d, respectively,is recommended. The recommended intake forinfants from birth until 1 year of age is set at3.6µg/kg bodyweight per day (25 – 35µg). Thisamount is also recommended for children bet-ween 1 and 10 years of age (50 – 150µg). Aspointed out by Bailey [1240], the present U.S.value [1280] may not be sufficient to maintainnormal folate levels and will most likely have tobe reevaluated.

Medical Use. Folate deficiency leads to im-paired amino acid metabolism, protein synthe-sis, and cell division. The effects are most no-ticeable in growing tissue.Amajor clinical prob-lem associated with folate deficiency is macro-cytic anemia in which the bone marrow pro-duces giant, immature red blood cells. An in-creased incidence of megaloblastic anemia hasbeen reported during pregnancy and followingchild birth. Folate antagonists (e.g., methotrex-ate and certain antiepileptics) are used in therapyfor various diseases, but their administration can

lead to a functional folate deficiency. In casesof infections (e.g., protozoal infections in AIDSpatients) or certain types of cancer (e.g., chori-ocarcinoma, cancer of the head and neck, child-hood acute lymphocytic leukemia) high dosesof 5-HCO-H4folate (5 – 30mg) may be given as“rescue dose” [1281–1283]. Precancerous con-ditions such as bronchial squamous metaplasiaimproved in some smokers after taking 10mg/d[1284].

Homocystinemia is a heterogeneous group ofinherited diseases of folate, vitaminB12, and ho-mocysteine metabolism and is associated withsevere arteriosclerosis and thromboembolismas well as mental retardation. Of the inheritedfolate disorders, deficiency of methylenetetra-hydrofolate reductase [1285] reduces or elimi-nates the generation of 5-CH3-H4folate, whichis needed as cosubstrate to convert homocysteineto methionine by methionine synthase. In menwith homocysteinemia, daily administration for6weeks of 1mg folic acid, 10mg vitaminB6,and 0.4mg vitaminB12 normalized serum ho-mocysteine levels [1286]. In psoriasis patientsreceivingmethotrexate, increased plasmahomo-cysteine levels could be lowered with folic acid[1287,1288]. In healthy men and women, a sig-nificant inverse correlation between plasma lev-els of folate and vitaminB12 and total plasmahomocysteine was observed [1289,1290].

Neural tube defects (NTDs), including spinabifida and anencephaly, are polygenic condi-tions. However, epidemiological studies haveshown an increased incidence of NTDs associ-ated with environmental factors. Several stud-ies have provided evidence that multivitaminor folic acid (0.8 – 4mg/day) supplementationprevents the majority of these NTDs [1291–1294]. Health authorities in the United Statesand United Kingdom advise women of child-bearing age to take daily folate supplements of0.4mg [1295,1296].

Toxicity. Daily oral supplementation with5 –10mg of folic acid is not toxic for nor-mal non-pregnant humans [1297]. Problems thatmight occur are themasking of vitaminB12 defi-ciency resulting in irreversible damage to spinalcord (subacute combined degeneration of thecord), and recurrence of epilepsy in epilepticstreated with drugs with antifolate activity. Ahypervitaminosis has not yet been reported. The

146 Vitamins

acute toxicity (LD50) is 500 and 600mg/kg bodyweight for rats and mice, respectively [1298].

Animal Nutrition. In animal nutrition, folicacid supplementation is required to obtain opti-mal performances of farm animals [1299]. Ac-cordingly, doses of 0.2 – 0.5mg/kg feed (drymatter) and 0.3mg/kg for chickens and pigsare required, respectively. Under commercialproduction conditions, however, increased folicacid supplementation is recommended [1300].Thus, the recommended amounts are 0.5 –1.0mg/kg feed for chickens and 0.5 – 2.0mg/kgfor swine.

13.9. Analysis

Analytical procedures, such as physicochemi-cal, chromatographic, radioisotopic, and micro-biological methods, are available to determinefolate concentrations. However, analysis is stillvery difficult, because of the many naturally oc-curring derivatives and their rapid conversion bynumerous enzymes, their lability, and the lowamounts in biological samples. Methods for de-termining the C1 unit, the reductive state of thecofactor, or the chain length of folylpolyglut-amates are also important. For general discus-sions and detailed procedures see [1301–1303].Pure folic acid, folate in enriched preparationsor various monoglutamates can be measuredby spectrophotometry, polarography, or HPLC[1304–1307]. Several microorganisms such asLactobacillus casei, Streptococcus faecalis, orPediococcus cerevisiae do not synthesize folateand can, therefore, be used to measure folatefrom natural sources [1308]. Since folylpoly-glutamates with more than 3 glutamate residuesare less active or inactive, hydrolysis of poly-glutamate chains by γ-glutamyl hydrolase is re-quired to obtain maximal growth of the microor-ganisms. Differential analysis of folates can beobtained by using P. cerevisiae, as this microor-ganism does not respond to nonreduced folate or5-CH3-H4folate. To determine folateactive sub-stances in erythrocytes and plasma, a number ofradioisotope kits based on competitive proteinbinding are commercially available. Various fo-lates, however, bind with different affinities tothe corresponding folate binding proteins, re-sulting in determination of preferential folates.

In addition, the results obtained with differenttest kits fluctuate considerably. Interpretation ofthe results with regard to the folate status may,therefore, pose problems.

13.10. Economic Aspects

The present world market for folic acid is ca.400 t/a, commercially available as pure crys-talline material (water content 8%) or as pre-mix. The major part (ca. 80%) is used for feedenrichment in animal nutrition. The pharmaceu-tical industry offers folic acid for therapeuticand prophylactic use, either as a monoprepara-tion or in combination with iron and/or othervitamins. Folic acid is also increasingly usedfor food enrichment (flour, cornflakes). The ma-jor producers of folic acid are Hoffmann-LaRoche; the Japanese companies Takeda, Sum-ica Fine Chemicals (previously Yodogawa Phar-maceuticals), and Kongo; the Chinese compa-nies Changzhou Pharma and Changshu Hua-gang Pharma; as well as some smaller compa-nies in China, India, and Russia. The sales pricein 1995 varied between ¤ 36 and ¤ 77/kg.

14. Niacin (Nicotinic Acid,Nicotinamide)

14.1. Introduction

Niacin is one of the vitamins of the B complex.In accordance with the rules on nomenclaturethe Institute of Nutrition [1309] suggested thatniacin be used as the generic name for both nico-tinic acid and nicotinamide.

Other commonly used names include pyri-dine 3-carboxylic acid and vitamin PP for nico-tinic acid, and pyridine 3-carboxylic acid amide,pyridine 3-carboxamide, vitamin PP, and niaci-namide for nicotinamide.

Nicotinic acid was first synthesized in 1867by oxidative degradation of nicotine. It had beenchemically identified long before its importance

Vitamins 147

as an essential nutrient (pellagra-preventing vi-tamin) was recognized [1310].

In the early 1900s itwasdiscovered that pella-gra, a disease with symptoms affecting the skin,gastrointestinal tract, and central nervous sys-tem, was caused by nicotinic acid deficiency.Experiments in 1917 with yeast, known forits high natural nicotinic acid content of up to500mg/kg, showed a curative effect on pellagra[1310,1311].

In 1935 nicotinamide was shown to be amoiety of the coenzymes NAD (nicotinamideadenine dinucleotide) and NADP (nicotinamideadenine dinucleotide phosphate). These coen-zymes are indispensable for many biochemicalreactions in all living cells [1310].

Niacin is a naturally occurring substance.Nicotinic acid is the formpresent in food of plantorigin, whereas nicotinamide occurs in animalproducts. Not all of the natural niacin is fullybioavailable for humans and animals. Nicotinicacid in cereals and in oil seeds is present in abound complex. Average contents of niacin arein the range of 10 – 200mg/kg of product.

The symptoms of niacin deficiency are usu-ally described as a disease of the “three Ds”:dermatitis, diarrhea, and dementia. However, thethree Ds represent the last stage of the disease.The early stages include lassitude, impaired di-gestion, and inflammation of the mucous mem-branes of the mouth, stomach, or intestine.

In developed countries, pellagra symptomsare found in connection with chronic alcoholismand one-sided nutrition. Animals exhibit skinproblems, diarrhea, weight loss and decreasedfeed intake.

Niacin is a component ofmultivitamin prepa-rations, breakfast cereals, and soft drinks. Flouris enriched with niacin. For domestic animalsniacin is an important part of compound feed.More than 60% of the niacin produced is re-quired by poultry, swine, ruminants, fish, andpets.

14.2. Physical and Chemical Properties

Physical properties of nicotinic acid and nicoti-namide are summarized in Table 38. The acidand the amide differ substantially in meltingpoint and solubility. Most producers guaranteestability of 2 – 3 years. However, nicotinamide

is a hygroscopic substance and tends to formlumps. It is therefore recommended that storage,in a closed package and under cool, dry condi-tions, should not exceed six months. Aqueoussolutions are stable, and sterilization is possiblewithout special precautions.

Table 38. Physical properties of niacin

Nicotinic acid Nicotinamide

IUPAC name pyridine 3-carboxylicacid

pyridine3-carboxamide

CAS no. [59-67-6] [98-92-0]Formula C6H5NO2 C6H6N2OMolecular mass 123.11 122.13Appearance white crystals white crystalsMelting point 234 – 237 ◦C

(>238 ◦C decomp.)128 – 131 ◦C

Solubility in water 1.3 g/100mL (15 ◦C) 38.3 g/100mL(20 ◦C)

9.8 g/100mL(100 ◦C)

14.3. Biochemical Functions

Niacin is absorbed almost completely by sim-ple diffusion across the intestinal mucosa. Theabsorbed niacin is taken up by the tissues, andis thought to be incorporated directly into coen-zyme forms.

Niacin serves as the precursor of two es-sential coenzymes: nicotinamide adenine dinu-cleotide (NAD) and nicotinamide adenine dinu-cleotide phosphate (NADP) [1310,1312,1313].Both coenzymes catalyze the metabolic transferof hydrogen – one of the basic functions in themetabolism of proteins, fats, and carbohydrates.This function is required for both the synthesisand degradation of amino acids, fatty acids, andcarbohydrates.

Another important task of the niacin coen-zymes is their repeated intervention in the citricacid cycle. The citric acid or Krebs cycle com-prises many steps in which activated acetate isrepeatedly oxidized.

Compounds originating from the degradationof fatty acids, carbohydrates and amino acids arefirst broken down to pyruvate oxalacetate andthen enter the cycle with the active acetate. Thevarious oxidative steps of the cycle yield a con-siderable amount of energy, which is stored inadenosine triphosphate (ATP). This energy canbe released by conversion of ATP to adenosine

148 Vitamins

diphosphate (ADP), which is then ready to pickup more energy. Niacin thus plays an essentialpart in the metabolic production and utilizationof energy.

14.4. Production

Industrial production of nicotinic acid andnicotinamide are based on the raw materials5-ethyl-2-methylpyridine and 3-methylpyridine(3-picoline).

3-Methylpyridine is obtained by catalyticgas-phase reaction of acetaldehyde, formal-dehyde, and ammonia (Figure 23). However,the principal product is pyridine, while 3-methylpyridine is obtained as a byproduct in30 – 50% yield. 5-Ethyl-2-methylpyridine isproduced by reaction of paraldehyde with am-monia (→Pyridine and Pyridine Derivatives,Chap. 2.2.6.).

Nicotinic acid is obtained directly by ni-tric acid oxidation of 5-ethyl-2-methylpyridine(Fig. 23) or by total hydrolysis of 3-cyanopyr-idine. Nicotinamide is available either fromthe amidation of nicotinic acid or from partialhydrolysis of 3-cyanopyridine. The latter is ob-tained by ammoxidation of 3-methylpyridine.

Oxidation of 5-Ethyl-2-methylpyridine.The world’s largest plant for the production ofniacin is located in Visp, Switzerland, and is op-erated by Lonza.

The principles of the Lonza process are sum-marized in the following and in the flow diagramof Figure 24 [1314]:

In an aqueous solution 5-ethyl-2-methylpyri-dine is oxidized to nicotinic acid in a single-tubereactor by using an excess of nitric acid. Thecontinuous process takes place at 230 – 270 ◦C

and 6 – 8MPa. Pyridine-2,5-dicarboxylic acid isformed as an intermediate, but is not stable un-der the reaction conditions, undergoing decar-boxylation to nicotinic acid nitrate. Neutraliza-tion of the nitrate with 5-ethyl-2-methylpyridineyields nicotinic acid. Water and carbon dioxideformed in the reaction are separated from theproduct. The nitrogen oxide formed is oxidizedwith air to nitrogen dioxide and then absorbedin water to yield nitric acid, which is recycledinto the process.

Ammoxidation of 3-Methylpyridine andHydrolysis of 3-Cyanopyridine (→Pyridineand Pyridine Derivatives, Chap. 3.7.).

In a multitubular reactor 3-methylpyridine,air, ammonia, and hydrogen react at ca. 350 ◦Cand moderate pressure to give 3-cyanopyridine[1315]. Heterogenous catalysts containing ox-ides of antimony, vanadium and titanium [1316]or antimony, vanadium and uranium [1317] arehighly effective and commonly used. For in-stance, with an antimony – vanadium – titaniumcatalyst, a reactor temperature of 360 ◦C, anda molar feed ratio of 3-methylpyridine : am-monia: air : water of 1 : 6 : 30 : 6 yields 96%of 3-cyanopyridine [1318]. 3-Cyanopyridine isconverted to nicotinamide by alkaline hydroly-sis. This reaction has the advantage that saponi-fication to the amide is fast compared to totalhydrolysis to nicotinic acid. The hydrolysis tothe amide is normally carried out with catalyticamounts of bases, mainly sodium hydroxide,at 130 – 150 ◦C [1319]. Other catalysts are alsomentioned. A copper – chromium oxide catalystgives good yields of nicotinamide [1320].

Conversion of 2-Methylglutaronitrile. 2-Methylglutaronitrile, a byproduct of adiponi-trile production, is converted to 2-methyl-1,5-diaminopentane. Cyclic hydrogenation gives 3-methylpiperidine. Dehydrogenation yields 3-methylpyridine [1321], which is then ammox-idated and partly hydrolyzed to nicotinamide.

Vitamins 149

14.5. Quality Specifications

Nicotinic acid and nicotinamide are available invarious forms. Pharmaceutical grade meets therequirements of current pharmacopoeias, andfood grade, which is usually equal to pharma-ceutical grade in quality, meets the standards ofUSP and FCC (Food Chemical Codex). Theseproducts have an assay of >99.8% of the activesubstance. The technical and feed grades of theleading producers have a purity of min. 99.5%(nicotinic acid) and min. 99% (nicotinamide).Lonza offers a DC quality (DC= direct com-pression), which is preferably used in the tablet-ting of vitamin or multivitamin tablets withoutauxiliary substances, thus improving the processin terms of energy consumption and productiontime.

There are only minor differences in productquality caused by variations in production pro-cesses or in the raw material used.

14.6. Analysis

Assays of the pure substances are most readilydetermined by titration. Nicotinic acid is deter-minedby titrationwith sodiumhydroxide [1322]or UV spectroscopy [1323]. Nicotinamide isdetermined by titration with perchloric acid inacetic acid [1324] or UV spectroscopy [1325].For the pharmaceutical grade, the official stan-dards of purity of the pharmacopoeias, such asUSP [1323,1325] or European Pharmacopoeia[1324,1326] apply.

To ensure a vitamin supply oriented tomeeting requirements in human and animalnutrition, foods and feeds are enriched withniacin or mixtures of vitamins. Microbiological[1327], spectrophotometric [1328], and chro-matographic [1329] procedures are described

for the quantitative determination of nicotinicacid or nicotinamide in foods and feeds. A mostsuitable method is the AOAC spectrophotomet-ricmethod [1328].Microbiologicalmethods andthe above-mentioned photometric method havethe disadvantage that only the total amount ofnicotinic acid and nicotinamide can be detected.With HPLC it is possible to determine the singlecompounds [1330,1331].

14.7. Uses

Pellagra in humans was formerly widespread inmany parts of theworldwhere cornwas themainsource of nutrition. Subclinical symptoms of de-ficiencies still appear under conditions of mal-nutrition or one-sided nutrition. Recommendeddietary allowances for humans are listed in Ta-ble 39 [1332].

Table 39. Recommended dietary allowances for niacin

Age, a Weight, kg Niacin, mg/d

Infants 0 – 0.5 6 50.5 – 1 9 6

Children 1 – 3 13 94 – 6 20 127 – 10 28 13

Men 11 – 14 45 1715 – 18 66 2019 – 24 72 1925 – 50 79 19>51 77 15

Women 11 – 14 46 1515 – 18 55 1519 – 24 58 1525 – 50 63 15>51 65 13

Pregnantwomen

17

Nursingmothers

20

Enrichment of cereal products with B vita-mins, especially with nicotinic acid or nicoti-namide, became feasible in the early 1940swhen the vitamin became commercially avail-able, and is now standard practice [1333]. Ni-acin is present in breakfast cereals, multivitamindrinks, and multivitamin tablets [1334].

Niacin is also essential to most animals[1335]. The requirements can be covered by var-ious sources. Biosynthesis from tryptophan isnot of importance since the conversion rate isvery low and most natural feedstuffs are rela-tively poor in this amino acid. Nicotinic acid

150 Vitamins

Figure 23. Industrial production of nicotinic acid and nicotinamide

Figure 24. Flow diagram of the oxidation of 5-ethyl-2-methylpyridine to nicotinic acid (Lonza process)a) Reactor feed preparation; b) Piston pump; c) Steam-heated reactor; d) Separator; e) Air compressors; f) Absorption columnsfor nitrogen oxides; g) Concentration column; h) Condensor; i) Crystallization; j) Centrifugation; k) Dissolver; l) Neutraliza-tion; m) Crystallization; n) Tray dryer

is synthesized by the microflora in the large in-testineofmonogastrics and in the rumen of rumi-nants. In monogastrics, biosynthesis is insignif-icant because it occurs in the large intestine,located after the main niacin-absorption sitesof the duodenum and small intestines. Most of

the nicotinic acid synthesized by the microflorais therefore excreted in the feces. Synthesis ofnicotinic acid in the rumen is suboptimal whenthe animals are under stress for high produc-tion or when the rumen microflora has been dis-turbed. The nicotinic acid production by the ru-

Vitamins 151

men microflora may be affected by antiobioticsand other feed additives.

Niacin deficiency reduces the activity ofthe coenzymes NAD and NADP, resulting inmetabolic disorders and hence in reducedweightgain, decreased feed intake, reduced feed effi-ciency, health problems (e.g., ketosis), and im-paired reproduction.

To achieve peak performance and to main-tain good health all domestic animals and petsneed a dietary source of niacin to meet their re-quirements. Niacin levels should therefore behigher than the NRC (National Research Coun-cil) recommendations [1336], because it is gen-erally accepted that the NRC recommendationsare based on ideal dietary and management con-ditions, since no safety margin is included orlimited bioavailability considered. Recommen-dations for niacin addition to animal feed (inmg/kg feed) are summarized in the following[1335]:

PoultryChicks 50Broilers 60Hens (laying) 40Hens (breeding) 50Turkeys (starting) 90Turkeys (growing and fattening) 80Turkeys (breeding) 80Ducks and geese (starting) 50PigsPiglets (early-weaned) 40Piglets (starting) 35Pigs (growing) 35Pigs (fattening) 30Sows (breeding) 35RuminantsCalves (0 – 3months) 60Cattle (fattening) 1000mg per animal

per dayDairy cows 3000 – 6000mg

per animal per daySheep and goats 1000mg per animal

per dayHorsesFoals and yearlings 40Working horses 90Race horses 90PetsDogs 25Cats 70FishCyprinids 100Salmonids 200Shrimps 200OthersRabbits 50Mink 50Foxes 50

Nicotinic acid andnicotinamide used in dosesin excess of nutritional requirements generate anumber of readily recognizable pharmacologi-cal responses, some of which are of therapeuticsignificance. In view of the relationship of nico-tinic acid to nicotinamide it is remarkable thatfew if any of the considerable pharmacologicalactivities of nicotinic acid are shared by nicoti-namide.

Some of the observed effects of pharmaco-logical doses of nicotinic acid are as follows:

1) Effects on lipid metabolism: Plasma cho-lesterol and triglycerides concentrations fellsignificantly with 1 – 3 g/d doses of nicotinicacid [1310,1337,1338]. Different productswith nicotinic acid or esters of nicotinic acidare commercially available.

2) Vasodilation: The flush reaction caused bythe increased blood flow rate appears veryquickly [1310,1313]. This effect is also usedin medicine [1310,1313] (e.g., in treatmentof smokers’ legs).

3) Activation of fibrinolysis [1310].

Pharmacological doses of nicotinamide havebeen shown to improve islet-cell regenerationand appear to have a protective effect on resid-ual insulin secretion in Type 1 diabetic patients.Thus, it is conceivable that nicotinamide mayhave a place in preventing diabetes in predis-posed subjects [1339].

Niacin is used in electroplating baths. Zincelectroplating baths contain niacin in a quater-nized form obtained by the reaction of niacinwith benzyl chloride, resulting in uniform andbrilliant surfaces.

14.8. Economic Aspects

In 1995, ca. 22 000 t of nicotinic acid and nicoti-namide were produced worldwide. 16 000 t orca. 75% of total niacin production was used inanimal nutrition and 6000 t as food additive. Themost important markets are the United Stateswith a market share of 35 – 40%, followed byEurope with 30%, and Japan and South EastAsia with 20%.

Table 40 shows the development of the niacinmarket over the last 25 years. Producers of nico-tinic acid and nicotinamide and their capacitiesare presented in Table 41.

152 Vitamins

Table 40. Niacin market 1970 – 1995 in t

1970 1980 1995

Nicotinic acid 3000 9 000 14 000Nicotinamide 1000 3 000 8 000Total 4000 12 000 22 000

Table 41. Producers and their capacities of nicotinic acid andnicotinamide in 1995

Producer Location Nicotinicacid, t/a

Nicotinamide,t/a

Lonza Visp, Switzerland 13 000 1500China∗∗ 3000

Vitachem∗ Antwerpen, Belgium 1 000 3500Indianapolis, USA 3500

Nepera Harriman, USA 3000Yuki Gosei Japan 200 1000Others 1 000 500

∗ Joint Venture of Degussa and Reilly Tar.∗∗ Joint Venture of Lonza/GPF Guangzhou, start-up plannedfor 1998.

Demand for niacin is rising by ca. 2 – 3%/ain the industrialized countries, while it is soar-ing by as much as 10%/a in the developing andnewly industrialized countries.

14.9. Toxicology

Nicotinic acid and nicotinamide are essential forhuman and animal health. The daily requirementto avoid deficiencies in humans is in the rangeof 15 – 30mg. For the treatment of hyperlipopro-teinemia and hypercholesterinemia daily dosesup to 6000mg of nicotinic acid are used [1337].

Nicotinic acid and nicotinamide are acutelynontoxic (oral LD50 greater than 5000 and3000mg/kg in rats, respectively), not irritant tothe skin but moderately irritant to the eye of rab-bits [1340].

Nicotinic acid, up to 1000mg kg−1 d−1, didnot show either cumulative toxicity in a 28 d oralstudy or teratogenicity in rats [1340]. No muta-genicity was found in a battery of mutagenic-ity tests [1340], and no carcinogenicity was ob-served in mice treated for a lifetime with nico-tinic acid in drinking water [1341].

Rare cases of skin flushing may occur in hu-mans, but this effect is reversible after termina-tion of exposure to nicotinic acid.

Nicotinic acid is practically nontoxic tofishes, daphnia, algae, and bacteria and is readilybiodegradable [1340].

Based on the available information nicotinicacid and nicotinamide do not represent a hazardto humans or the environment.

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Vitamins 153

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52. T. Moore, Biochem. J. 23 (1929) 803. H.Euler, V. Demole, P. Karrer, O. Walker, Helv.Chim. Acta 13 (1930) 1078.

53. P. Karrer, W. E. Bachmann, Helv. Chim. Acta12 (1929) 28. P. Karrer, A. Helfenstein, Helv.Chim. Acta 12 (1929) 1142. P. Karrer, R.Morf, Helv. Chim. Acta 14 (1931) 1033; 13(1930) 1084. R. Kuhn, H. Roth, Ber. Dtsch.Chem. Ges. 66 (1933) 1274. P. Karrer, A.Ruegger, A. Geiger, Helv. Chim. Acta 21(1938) 1171. R. Kuhn, H. Brockmann, Ber.Dtsch. Chem. Ges. 67 (1934) 1408.

54. P. Karrer, R. Morf. K. Schopp, Helv. Chim.Acta 14 (1931) 1036, 1431. P. Karrer, A.Helfenstein, H. Wehrli, A. Wettstein, Helv.Chim. Acta 13 (1930) 1094.

55. K. R. Farrar, J. C. Hamlet, H. B. Henbest,E. R. H. Jones, J. Chem. Soc. 1952, 2657.

56. P. Karrer, O. Solmessen, Helv. Chim. Acta 20(1937) 682. P. Karrer, O. Solmessen, W.Gugelmann, Helv. Chim. Acta 20 (1937)

154 Vitamins

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57. O. Wiss, H. Thommen, Wiss. Veroff. Dtsch.Ges. Ernahr. 9 (1963) 179.

58. G. Wald, R. Hubbardin, P. D. Boyer, H.Lardy, K. Myrback (eds.): The Enzymes, 2nded., Academic Press, New York 1960, vol. 3,p. 369.

59. M. Rosenberger, A. Lovey, J. F. Grippo,Nature (London) 355 (1992) 359 – 361. A.Wyss, L. J. Sturzenbecker, J. F. Grippo, P.LeMotte, Nucleic Acids Res. 21 (1993)1231 – 1237.

60. F. Kienzle, Pure Appl. Chem. 47 (1976) 183.61. O. Isler, Experientia 33 (1977) 555.62. H. Matsumoto, R. S. H. Liu, C. J. Simmons,

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63. O. Isler, G. Brubacher: Vitamine I,Fettlosliche Vitamine, Thieme Verlag,Stuttgart 1982.

64. W. E. Oberhansli, H. P. Wagner, O. Isler, ActaCrystallogr. Sect. B Struct. Crystallogr. Cryst.Chem. 30 (1974) 161.

65. T. Hamanaka, T. Mitsui, T. Ashida, M.Kakudo, Acta Crystallogr. Sect. B Struct.Crystallogr. Cryst. Chem. 28 (1972) 241.

66. C. H. Stam, Acta Crystallogr. Sect. B Struct.Crystallogr. Cryst. Chem. 28 (1972) 2936.

67. O.A. Roels in W.H. Sebrell, Jr., R. S. Harris(eds.): The Vitamins, vol. I, Academic Press,New York 1967, p. 182. L. Zechmeister:Cis-trans-Isomeric Carotenoid, Vitamin Aand Aryl Polyenes, Springer Verlag, Wien,1962. J. P. Sweeney, A. C. Marsh, J. Nutr.103 (1973) 20. J. C. Bauernfeind, C. R.Adams, W. L. Marusich, in C. Bauernfeind(ed.): Carotenoids as Colorants and VitaminA Precursors, Academic Press, New York1981, p. 577.

68. P. Karrer, E. Jucker, Helv. Chim. Acta 29(1946) 229.

69. S. Ball, T.W. Goodwin, R. A. Morton,Biochem. J. 42 (1948) 516.

70. B. Frank, M. Dust, A. Stange, P. P. Hoppe,Naturwissenschaften 69 (1982) 401.

71. F. B. Jungalwala, H. R. Cama, Biochem. J. 95(1965) 19.

72. Eastman Kodak, US 2 839 585, 1958 (O.D.Hawks).

73. Hoffmann-La Roche, DE-OS 2 061 507, 1969(R. Marbet).

74. H. B. Henbest, E. R. H. Jones, T. C. Owen, J.Chem. Soc. 1957, 4909.

75. G. Wald, P. K. Brown, R. Hubbard, W.Oroshnik, Proc. Natl. Acad. Sci. USA 41(1955) 438. P. K. Brown, G. Wald, J. Biol.Chem. 222 (1956) 865.

76. F. H. Carr, E. H. Price, Biochem. J. 20 (1926)497.

77. P. Karrer, U. Solmssen, Helv. Chim. Acta 20(1937) 682, 1020.

78. R. Kuhn, H. Brockmann, Ber. Dtsch. Chem.Ges. 65 (1932) 894; Justus Liebigs Ann.Chem. 516 (1935) 95.

79. P. Karrer, E. Jucker, Helv. Chim. Acta 28(1945) 427, 471.

80. F. J. Petracek, L. Zechmeister, J. Am. Chem.Soc. 78 (1956) 1427. R. Entschel, P. Karrer,Helv. Chim. Acta. 41 (1958) 402.

81. H. Thommen in O. Isler (ed.): Carotenoids,Birkhauser Verlag, Basel 1971.

82. C. D. Robeson et al., J. Am. Chem. Soc. 77(1955) 4111.

83. S. R. Ames, W. J. Swanson, P. L. Harris, J.Am. Chem. Soc. 77 (1955) 4134, 4136. S. R.Ames, Ann. Rev. Biochem. 27 (1958) 375.D.W. Stainer, T. K. Murray, Can. J. Biochem.Physiol. 38 (1960) 1467. O. R. Braekan, G.Lambertsen, L. R. Njaa, F. Utne, Int. Z.Vitaminforsch. 30 (1960) 363. J.Bruggemann, J. Tiews, Int. Z. Vitaminforsch.30 (1960) 279.

84. BASF, US 3 838 029, 1974 (M. Fischer,W.W. Wiersdorf, A. Nurrenbach).Hoffmann-La Roche, US 4 026 778, 1977(1974) (M. Lalende, H. Stoller). W.H.Waddel, K. Chihara, J. Am. Chem. Soc. 103(1981) 7389. A.H. Levin, F. I. Carrol, J. Am.Chem. Soc. 103 (1981) 6527.

85. Hoffmann-La Roche, US 4 051 174, 1977 (H.Stoller, H. P. Wagner).

86. K.H. Bassler, E. Gruhn: Vitamin-Lexikon, G.Fischer Verlag, Stuttgart 1992, pp. 211 – 233.

87. W. Muller-Mulot, Fette, Seifen, Anstrichm.78 (1976) 18.

88. A. Winterstein, Angew. Chem. 72 (1960)902. H. Thommen, O. Wiss, Z.Ernahrungswiss. Suppl. 3 (1963) 18.

89. T.W. Goodwin: The Biochemistry of theCarotenoids, 2nd ed., vol. I: Plants,Chapman and Hall, London 1980.

90. J. C. Bauernfeind, C. R. Adams, W. L.Marusich in J. C. Bauernfeind (ed.):Carotenoids as Colorants and Vitamin APrecursors, Academic Press, New York1981, p. 564. H. S. Huang, D. S. Goodman, J.Biol. Chem. 241 (1965) 2839. D. S.

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91. J. A. Olson, Am. J. Clin. Nutr. 22 (1969) 953.92. N.H. Fidge, T. Shiratori, J. Ganguly, D. S.

Goodman, J. Lipid Res. 9 (1968) 103. D. F.Moffa, F. J. Lotspeich, R. F. Krause, J. Biol.Chem. 245 (1970) 439.

93. D. S. Goodman et al., J. Clin. Invest. 45(1966) 1615.

94. H. B. Sewell, G. E. Mitchel, C. O. Little,B.W. Hayes, Int. Z. Vitaminforsch. 37 (1967)301. J. A. Boling et al., J. Nutr. 99 (1969)502. M. Kanai, A. Raz, D. S. Goodman, J.Clin. Invest. 47 (1968) 2025.

95. G.W. T. Gronendijk, P. A.A. Jansen, S. L.Bonting, F. J.M. Daemen, Methods Enzymol.67 (1980) 203.

96. K. C.D. Hickman, Ind. Eng. Chem. 29(1937) 968. K. C.D. Hickmann, Chem. Rev.34 (1944) 51.

97. H. J. Passino, Ind. Eng. Chem. 41 (1949) 280.98. R. Kuhn, C. J. O. R. Morris, Ber. Dtsch.

Chem. Ges. 70 (1937) 853. J. F. Arens, D.A.van Dorp, Recl. Trav. Chim. Pays-Bas 67(1948) 973.

99. H. Mayer, O. Isler: Carotenoids, Birkhauser,Basel 1971, p. 373. O. Isler, Pure Appl.Chem. 51 (1979) 447.

100. J. C. Bauernfeind: “Aspects of Human andNational Nutrition,” World Rev. Nutr. Diet.41 (1983) 110. W. Bollag, Lancet 1, 8329(1983) 860.

101. BASF, DE 951 212, 1956 (H. Pommer, G.Wittig). H. Pommer, Angew. Chem. 72(1960) 811.

102. G.W.H. Cheeseman et al., J. Chem. Soc.1949, 2031. J. Attenburrow et al., J. Chem.Soc. 1952, 1094.

103. BASF, DE 950 551, 1956 (H. Pommer); DE1 050 763, 1959 (H. Pommer, W. Sarnecki);DE 1 068 704, 1959 (H. Pommer, W.Sarnecki); DE 1 109 671, 1958 (H. Stilz).H. A.M. Jacobs, H.M. Berg, L. Brandsma,J. F. Arens, Recl. Trav. Chim. Pays-Bas 84(1965) 1113. Organon, NL 6 404 175, 1965(H.A.M. Jacobs).

104. Glidden, US 3 076 839 (R. L. Webb) 1963;US 3 062 875, 1962 (J. P. Bain); US3 278 623, 1964 (J.M. Deifer). H. Pauling,D.A. Andrews, N. C. Hindley, Helv. Chim.Acta 59 (1976) 1233.

105. O. Isler, R. Ruegg, A. Studer, R. Jurgens,Hoppe-Seyler’s Z. Physiol. Chem. 295(1953) 305.

106. W. Kimel et al., J. Org. Chem. 23 (1958) 153.107. G. Saucy, R. Marbet, Helv. Chim. Acta 50

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W. Hoffmann, Seifen, Ole, Fette, Wachse 101(1975) 89. W. Hoffmann, Chem. Ztg. 97(1973) 23.

109. Sumitomo Chem., US 2 951 853, 1960 (M.Matsui). M. Matsui et al., J. Vitaminol. 4(1958) 178.

110. H.O. Huisman, A. Smit, S. Vromen, L. G.M.Fischer, Recl. Trav. Chim. Pays-Bas 71(1952) 899. A. E. C., FR 1 234 824, 1960 (R.Courbron).

111. Eastman Kodak, US 2 676 990, 1954 (W. J.Humphlett, D.M. Burness); US 2 676 992,1954 (W. J. Humphlett); US 2 676 994, 1954(D.M. Burness, C. D. Robeson); US2 683 746, 1954 (C.H. Beuten, C. D.Robeson); US 2 688 747, 1954 (E. Marx).

112. Philips Gloeilampen-Fabrieken, DE1 041 950, 1958; NL 6 409 913, 1966 (H.O.Huisman, A. Smit). H. O. Huisman, A. Smit,H. P. von Leeuwen, J. H. van Rij, Recl. Trav.Chim. Pays-Bas 75 (1956) 977.

113. A. E. C., FR 1 243 824, 1960 (G. J.M.Nicolaux, E. A. Gray, J. Matet).

114. J. Cymerman, I.M. Heilbron, A.W. Johnson,E. R. H. Jones, J. Chem. Soc. 1944, 141.

115. O. Isler, W. Huber, A. Ronco, M. Kofler,Helv.Chim. Acta 30 (1947) 1911. O. Isler et al.,Helv. Chim. Acta 32 (1949) 489. C. v. Plantaet al., Helv. Chim. Acta 45 (1962) 517, 548.

116. O. Isler et al., Helv. Chim. Acta 30 (1947)1922. I.M. Heilbronn, A.W. Johnson,E. R. H. Jones, A. Spinks, J. Chem. Soc.1942, 727. S. Ishikawa, T. Matsuura, Chem.Zentralbl. 108 II (1937) 3452. N. Milas,Science (Washington D.C.) 103 (1946) 581.

117. O. Isler, R. Ruegg, P. Schudel, Chimia 15(1961) 212.

118. H. Pommer, Angew. Chem. 89 (1977) 437.W. Reif, H. Grassner, Chem. Ing. Tech. 45(1973) 646. H. Pommer, A. Nurrenbach, PureAppl. Chem. 43 (1975) 527.

119. G. Wittig, G. Geissler, Justus Liebigs Ann.Chem. 580 (1953) 44.

120. BASF, DE 943 648, 1956 (G. Wittig, H.Pommer); DE 1 003 730, 1957 (G. Wittig, H.Pommer).

121. BASF, DE 950 552, 1956 (G. Wittig, H.Pommer); DE 1 026 745, 1958 (G. Wittig, H.Pommer).

156 Vitamins

122. BASF, DE 1 046 046, 1958 (W. Sarnecki, H.Pommer).

123. U. Schollkopf, Angew. Chem. 71 (1959) 260.124. W. Oroshnik, G. Karmas, A.D. Mebane, J.

Am. Chem. Soc. 74 (1952) 295. W. Oroshnik,A.D. Mebane, J. Am. Chem. Soc. 71 (1949)2062. R. G. Gould, Jr., A. F. Thompson, Jr., J.Am. Chem. Soc. 57 (1935) 340.

125. L. A. Yanovskaya, Izv. Akad. Nauk SSSR Ser.Khim. 1960, 1435. Y. Ishikawa, Bull. Chem.Soc. Jpn. 37 (1964) 207.

126. BASF, DE 1 060 368, 1959 (H. Haeussler, M.Eucken); US 2 950 321, 1960 (W. Sarnecki,H. Pommer); DE 1 068 710, 1959 (W.Sarnecki, H. Pommer); NL 6 405 660, 1964(W. Sarnecki, H. Pommer); Hoffmann-LaRoche, US 3 932 485, 1976 (J. D. Surmatis).

127. H. Mayer, O. Isler in O. Isler (ed.):Carotenoids, Birkhauser, Basel 1971, p. 394.BASF DE-OS 2 004 675, 1970 (W.Himmele); DE-OS 1 941 632, 1969 (W.Himmele, A. Aquila). G. Eletti-Bianchi, F.Centini, L. Re, J. Org. Chem. 41 (1976) 1648.J. H. Babler, M. J. Coghlan, M. Fang, P. Fries,J. Org. Chem. 44 (1979) 1716. P. A. Wehrli,B. Schaer, Synthesis 1977, 649. Hoffmann-LaRoche, DE 2 621 224, 1976 (P. Fitton, H.Moffet). J. H. Babler, US 4 175 204, 1979.P. A. Wehrli, US 4 219 660, 1980. BASF EP268 931, 1987 (F. Merger, R. Fischer, H.Hasler). Eastman Kodak, WO 90/11989,1990 (J. R. Mannier, P. J. Muehlbauer); US5 120 877, 1992 (A. T. Fanning, H. F. Goss);US 5 138 077, 1992 (J. R. Mannier, P. J.Muehlbauer).

128. T. Onoda, Petrotech Tokyo 10 (1987) 810. Y.Tanabe, Hydrocarbon Process. 60 (1981)187. J. Haji, J. Yokotake, T. Yanraguchi, M.Sato, N. Murai, EP 289 725, 1988. A.V.Devekki, M. J. Yakushkin, Kinet. Katal. 28(1987) 1099. P. R. Stapp, US 4 044 041(1977).

129. BASF, DE 950 552, 1956 (G. Wittig, H.Pommer); DE 1 026 745, 1958 (H. Pommer,G. Wittig, W. Sarnecki).

130. BASF, US 3 838 029, 1974 (M. Fischer, A.Nurrenbach). Hoffmann-La Roche, US4 026 778, 1977 (M. Lalonde, H. Stoller).W.H. Waddel, K. Chihara, J. Am. Chem. Soc.103 (1981) 7389. A.H. Lewin, F. I. Carrol, J.Am. Chem. Soc. 103 (1981) 6527.

131. Hoffmann-La Roche, DE-OS 2 733 231, 1978(K. Schleich, H. Stoller).

132. J. Paust, W. Reif, H. Schumacher, JustusLiebigs Ann. Chem. 1976, 2194.

133. BASF, US 2 831 884, 1958 (H. Pommer, W.Arend). K. Sisido, K. Kondo, H. Nozaki, M.Tuda, Y. Udo, J. Am. Chem. Soc. 82 (1960)2286.

134. W. J. Conradi, C. F. Garbers, P. S. Steyn, J.Chem. Soc. C 1964, 594; G. Pattenden,B. C. L. Weedon, J. Chem. Soc. C 1968, 1984.

135. E. J. Corey, M. Chaykowsky, J. Org. Chem.28 (1960) 254. G.W. Fenton, C. K. Ingold, J.Chem. Soc. 1970, 705.

136. M. Julia, D. Arnould, Bull. Soc. Chim. Fr.1973, 743, 746.

137. P. Chabardes, J. P. Decor, J. Varagnat,Tetrahedron 33 (1977) 2799. A. Fischli, H.Mayer, W. Simon, H. J. Stoller, Helv. Chim.Acta 59 (1976) 397. G. C. Olson, H. Ch.Cheung, K.D. Morgan, Ch. Neukom, C.Saucy, J. Org. Chem. 41 (1976) 3287.

138. Rhone-Poulenc, DE-OS 2 202 689, 1972 (M.Julia).

139. Rhone-Poulenc, DE-OS 2 708 282, 1977 (J. P.Decor).

140. SCM-Corp., US 4 048 220, 1977 (C.G.Cardeus). G. Allmang, F. Grass, J.M.Grosselin, C. Mercier, J. Mol. Catal. 66(1991) L 27 –L 31. E. Facke, C. Mercier, N.Pagnier, B. Despeyroux, P. Pauster, J. Mol.Catal. 79 (1993) 117 – 131. C. G. Cardenas,US 4 001 307, 1977; US 4 048 220, 1977.

141. A. Fischli, H. Mayer, Helv. Chim. Acta 58(1975) 1492. Rhone-Poulenc, DE-OS2 305 267, 1973 (P. Chabardes, M. Julia);DE-OS 2 355 898, 1974 (P. Chabardes, M.Julia, A. Menet); DE-OS 2 708 210, 1977(J. P. Decor).

142. Rhone-Poulenc, EP 319 407, 1988. J.M.Grosselin, C. Mercier, Organometallics 10(1991) 2126.

143. Rhone-Poulenc, DE 2 708 282, 1976 (J. P.Decor); DE 2 708 304, 1977 (J. P. Decor); EP430 307, 1989 (J. P. Decor).

144. J. H. Babler, US 4 175 204, 1979. J. H. Babler,W. Buttner, Tetrahedron Lett. 1976, no. 4,239 – 242. Dunlop BP 978 892, 1974; US3 927 076, 1975 (J. Babler). J. E. Backvall, J.Am. Chem. Soc. 107 (1985) 3676 –3686.

145. Rhone-Poulenc, EP 544 588, 1993 (P.Duhamel, L. Duhamel).

146. Duphar International, EP 456 287, 1991 (G. J.Lagerweg, C. Bakker); EP 489 454, 1991 (C.Bakker). Kuraray, EP 187 259, 1985 (J.Otera, T. Mandai); EP 348 813, 1989 (S.Suzuki, T. Takigawa); EP 349 991, 1989 (T.Mori, S. Suzuki).

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147. J. Otera, H. Misawa, T. Onishi, S. Suzuki, Y.Fujita, J. Org. Chem. 51 (1986) 3834. F.Chemla, M. Julia, D. Ugnen, Bull. Soc. Chim.Fr. 1993, 130, 200. J. Otera et al., J. Am.Chem. Soc. 106 (1984) 3670. J. Otera et al.,Chem. Lett. 1985, 1883.

148. Kuraray, EP 282 913, 1988 (T. Onishi, S.Suzuki, T. Mori).

149. Kuraray, EP 412 551, 1990 (T. Mori, S.Suzuki, T. Onishi); EP 282 914, 1988 (S.Suzuki, T. Mori, T. Onishi); EP 234 496,1987 (J. Otera, S. Suzuki, T. Onishi).

150. L’Oreal, EP 292 350, 1988 (G. Solladie, S.Forestier). G. Solladie, A. Girardin, P. Metra,Tetrahedron Lett. 29 (1988) 209. G. Solladie,A. Girardin, Tetrahedron Lett. 29 (1988)213; J. Org. Chem. 54 (1989) 2620.

151. L. Duhamel, P. Duhamel, J. P. Lecouve, J.Chem. Res. Synop. 1986, 34 – 35.

152. L. Duhamel, P. Duhamel, J. P. Lecouve,Tetrahedron 43 (1987) 4339; 43 (1987) 4349.L. Duhamel, J. Guillemont, J.M. Poirier, P.Chabardes, Tetrahedron Lett. 32 (1991) 4499.L. Duhamel, P. Duhamel, Y. LeGallic,Tetrahedron Lett. 34 (1993) 319.Rhone-Poulenc, EP 237 438, 1987 (L.Duhamel, P. Duhamel); EP 242 247, 1987 (L.Duhamel, P. Duhamel).

153. E. Negishi, Z. Owozarczyk, Tetrahedron Lett.32 (1991) 6683. N. Okukado, D. E. VanHorn, W. E. Klima, E. Negishi, TetrahedronLett. 1978, 1027 – 1030. W. J. Scott, G. T.Crisp, J. K. Stille, J. Am. Chem. Soc. 106(1984) 4630. J. F. Normant, J. F. Alexakis, N.Jabri, Tetrahedron Lett. 22 (1981) 959, 3851;23 (1982) 1589. N. Miyaura, K. Yamada, A.Suzuki, Tetrahedron Lett. 30 (1979) 3437. N.Miyaura, K. Yamada, H. Suginome, A.Suzuki, J. Am. Chem. Soc. 107 (1985) 972.E. Negishi et al., J. Am. Chem. Soc. 109(1987) 2393. E. Negishi et al., J. Am. Chem.Soc. 100 (1978) 2254.

154. Hoffmann-La Roche, DE 855 399, 1952; DE858 059, 1952; DE 953 073, 1956 (O. Isler,M. Montavon); DE 953 074, 1956 (O. Isler,M. Montavon).

155. H. Pommer, Angew. Chem. 72 (1960) 911;89 (1977) 440.

156. BASF, DE 945 247, 1956 (H. Pommer, G.Wittig); DE 1 068 705, 1959 (H. Pommer, W.Sarnecki).

157. BASF, DE 1 068 709, 1959 (H. Pommer, W.Sarnecki); DE 1 158 505, 1963 (W. Sarnecki,A. Nurrenbach); DE 1 155 126, 1963 (H.Pommer, W. Sarnecki). U. Schwieter et al.,

Helv. Chim. Acta 49 (1966) 369. J. D.Surmatis, J. Gibas, R. Thommen, J. Org.Chem. 34 (1969) 3039. BASF, NL 6 909 081,1969 (W. Sarnecki, A. Nurrenbach).

158. Rhone-Poulenc, DE 2 224 606, 1972 (P.Chabarides, M. Julia).

159. Kuraray, EP 461 653, 1991 (T. Mori, H. Fujii,O. Onishi).

160. The Regents of the University of California,DE-OS 2 515 011, 1975 (J. E. McMurray).

161. H. J. Bestmann, L. Kisielowski, W. Distler,Angew. Chem. 88 (1976) 297. A. Nurrenbachet al., Justus Liebigs Ann. Chem. 1977, 1146.BASF, DE 2 505 869, 1976 (B. Schenk, J.Paust).

162. B. C. L. Weedon in O. Isler (ed.):Carotenoids, Birkhauser, Basel 1971, p. 270.

163. Hoffmann-La Roche, US 3 367 985, 1968(J. D. Sumatis).

164. Hoffmann-La Roche, DE 2 440 747, 1975(J. D. Sumatis).

165. K. Bernhard, H. Mayer, Pure & Appl. Chem.63 (1991) 35.

166. O. Isler et al., Helv. Chim. Acta 39 (1956)249. O. Isler, R. Ruegg, P. Schudel, Chimia15 (1961) 208. I. G. Farbenind., US2 165 962, 1939 (M. Muller-Conradi, K.Pieroh).

167. R. Ruegg et al., Helv. Chim. Acta 42 (1959)874. R. Ruegg et al., Helv. Chim. Acta 42(1959) 854. Hoffmann-La Roche DE1 112 730, 1962 (H. Lindlar, M. Montavon);DE 1 081 454, 1960;DE 1 090 658, 1961 (H.Lindlar, M. Montavon); DE 1 092 466, 1961(O. Isler, H. Lindlar, M. Montavon); DE1 088 951, 1961 (W. Guex, R. Ruegg, O.Isler).

168. H. Pommer, A. Nurrenbach, Pure & Appl.Chem. 43 (1975) 546. H. Pommer, Angew.Chem. 89 (1977) 440. BASF, DE 1 216 862,1966 (H. Pommer, A. Nurrenbach); DE1 211 616, 1966 (H. Pommer, A.Nurrenbach); DE 1 008 729, 1957 (H.Pommer, A. Nurrenbach).

169. O. Isler et al., Helv. Chim. Acta 42 (1959)864. Hoffmann-La Roche, DE 1 096 349,1961 (O. Isler, R. Ruegg); DE 1 197 085,1963 (V. Schwieter, R. Ruegg).

170. Hoffmann-La Roche, DE 1 247 299, 1968 (V.Schwieter, R. Ruegg).

171. B. C. L. Weedon in O. Isler (ed.):Carotenoids, Birkhauser, Basel 1971, p. 270.

172. J. H. Prystowsky, J. Biol. Chem. 256 (1981),4498 –4503. R. Blomhoff, Physiol. Rev. 71(1991) 951.

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173. D. R. Applung, F. Chytil, Endocrinology(Baltimore) 108 (1981) 2120 – 2133.

174. T. G. Ebrey, T. Yoshisawa, Photochem.Photobiol. 33 (1981) 417. K. Nakanishi, V.Balogh-Nair, M. Arnaboldi, J. Am. Chem.Soc. 102 (1980) 7945. W. Stockenius, R. H.Lozier, R. A. Bogomolni, Biochim. Biophys.Acta 505 (1979) 215.

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1314. Lonza, CH495 990, 1968 (A. Stocker, O.Marti, T. Pfammatter, G. Schreiner);CH 504 437, 1969 (A. Stocker, O. Marti, T.Pfammatter, G. Schreiner); CH 511 848, 1969(A. Stocker, O. Marti, T. Pfammatter, G.Schreiner); CH 527 193, 1969 (A. Stocker, O.Marti, T. Pfammatter, G. Schreiner);CH-A 548 394, 1969 (A. Stocker, O. Marti, T.Pfammatter, G. Schreiner).

1315. Yuki Gosei, JP 45 013 572, 1970 (M.Takeishi, K. Fuji, T. Matsui); Koei Chemical,JP 46 041 545, 1971 (M. Imazu, Y. Higata);Degussa, DE 1 948 715, 1969 (T. Lussling, H.Schaefer, W. Weigert); Degussa,DE 2039 497, 1970 (T. Lussling, H. Schaefer,W. Weigert); Lonza, CH582 151, 1973 (F.Moulin, K.-J. Boosen).

1316. Degussa, DE-OS 3 107 755, 1982 (H.Beschke, H. Friedrich, J. Heilos).

1317. Takeda, EP 0 037 123, 1981 (O. Isatsugu, F.Itsuo, M. Ichiro).

1318. Degussa, DE-OS 3 128 956, 1983 (H.Beschke, F. L. Dahm, H. Friedrich).

1319. Degussa, DE-OS 2 517 054, 1976 (H.Beschke, H. Friedrich, K.-P.Muller, G.Schreyer).

1320. Yuki Gosei, JP 74 127 976, 1973 (Y. Watabikiet al.)

1321. Lonza, WO94/22 824, 1994 (J. Heveling, E.Armbruster, W. Siegrist).

1322. DAB10 (1991), Suppl. 1993.1323. United States Pharmacopoeia, vol. 23, USP

Convention, Inc., Princeton, N.J., 1990,p. 1080.

1324. European Pharmacopoeia, 2nd ed.,Maisonneuve, Sainte Ruffine, France 1980,p. 47

1325. United States Pharmacopoeia, vol. 23, USPConvention, Inc., Princeton, N.J., 1990,p. 1082.

1326. European Pharmacopoeia, 2nd ed.,Maisonneuve, Sainte Ruffine, France 1986,p. 459.

1327. K. Helrich (ed.), Association of AnalyticalChemists : Official Methods of Analysis,15th ed., Arlington, Virginia 1990,Microbiology 960.46 and 985.43.

1328. K. Helrich (ed.), Association of AnalyticalChemists : Official Methods of Analysis,15th ed., Arlington, Virginia 1990,no. 961.14.

1329. R. T. Nuttall, B. Bush, Analyst (London) 96(1971) 875.

1330. G.W. Chase et al., J. Micronutr. Anal. 7(1990) 15 – 25.

1331. J. Fontaine, J. Horr, Agribiol. Res. 46 (1993)10 – 19.

1332. National Academy of Sciences:Recommended Dietary Allowances (RDA),10th ed., National Academy Press,Washington 1989, p. 285.

1333. C. L. Brooke, J. Agric. Food. Chem. 16(1968) 163 – 167.

1334. R. H. Anderson et al., Food Technol.(Chicago) 30 (1976) 110.

1335. Lonza, company brochure, Niacin in AnimalNutrition, Basel 1991, 20 p.

1336. Nutrient Requirements of Swine, 9th rev. ed.,National Academy Press, Washington, D.C.,1988. Nutrient Requirements of Poultry, 9threv. ed., National Academy Press,Washington, D.C., 1994.

1337. Bundesanzeiger, no. 76, April 21, 1990.1338. Lonza, company brochure, Niacin and

Cholesterol, Basel 1990, 14 p.1339. S. Sandler, J. Clin. Endocrinol. Metab. 77

(1993) no. 6, 1574 – 1576.1340. Lonza, unpublished data.1341. Anticancer Res. 2 (1982) 71 – 74.

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