Thiamin, Riboflavin, and

NiacinBy: Kaitlin Deason and

Confidential Group Members

Objectives:

Brief history and fun facts of thiamin, riboflavin, niacin

Overview of absorption, digestion, and transportation

Overview of RDAs, sources, deficiencies, toxicities, and assessment tests

Overview of metabolism

ThiaminVitamin B1

History 1880s Dr Takaki: relationship

between “the nitrogenous substances …in the food” and the disease beriberi (I can’t –I can’t) in Japanese Navy

1890 Diet to prevent beriberi was written into law

1886 Dr. Christian named beriberi as polyneuritis gallinarum

“anti-polyneuritis factor” could be extracted from rice hulls with water and ethanol

History con’t. 1911 Dr. Funk crystallized

an amine substance from rice bran

1926 Dr. Jansen and Dr. Donath crystallized vitamin B1 from rice bran named antineuritic vitamin; however, they missed the sulfur atom and their formula was incorrect

1936 Williams published the correct formula

Thiamine as reflection of amine nature of vitamin

Thiamin: Absorption Transport Storage

Water soluble vitamin Absorption in the

jejunum Passive diffusion if

thiamin intake is high Active diffusion

Sodium Dependent if thiamin intake is low

Ethanol ingestion interferes with active transport of thiamin

In the blood bound to albumin

Storage: 30 mg ~90 % within

blood cells Small amount in

the liver, skeletal muscles, brain, heart, kidney

Thiamin: Main Active FormsThiamin Di- or Pyrophosphate (TDP/TPP)

Thiamin: Main Active Forms Thiamine triphosphate (TTP)

Thiamine di-phosphate + ATP Thiamine triphosphate (TTP)+ ADP

Metabolism Thiamin: Energy Transformation TDP in Enzyme Systems

Oxidative Decarboxylation of- Pyruvate- -ketoglutarate- Three branched chain amino acids:

isoleucine, leucine and valine

Physiological & Biochemical functions

Noncoenzyme: Membrane and nerve conduction

Coenzyme: Energy transformation Synthesis of pentoses and NADPH

Recommended Daily AmountsRDA

Men: 1.2 mg/day

Women: 1.1 mg/day

Pregnant: 1.4 mg/day

Lactating : 1.5 mg/day

Sources of thiamin

Excellent sources: Pork and Sunflower

seeds Good sources:

Enriched and fortified or whole grains: (bread, ready-to-eat cereals)

Funny fact

If you can’t get enough of sushi you might want to think twice. Raw fish contains thiaminase – an enzyme that deactivates thiamin. Cooking fish makes the enzyme inactive.

Thiamin Deficiency: Groups at Risk

Biological half-life of thiamin in the body is about 15 days, deficiency symptoms can be seen in people on a thiamin deficiency diet as little as 18 days.

Groups with a greater risk: individuals with kidney diseases on dialysis Malabsortion syndrome or genetic metabolic disorders Pregnant women with more then one fetus Seniors Chronic dieters Elite athletes Alcoholics

Thiamin Deficiency

Beriberi –true deficiency is not common in USA

Dry beriberi from low thiamin intake in older adults

Wet beriberi with cardiovascular system involvement

Acute beriberi in infants

Failure to oxidize -keto acids from leucine, isoleucine and valine causes accumulation in blood the branched –chain acids

Findings are characteristic of Maple Syrup Urine Disease (MSUD)

Thiamin Deficiency Symptoms

Associated with alcoholism Wernicke-Korsakoff Syndrome:

muscle wasting and encephlopathy

Mental confusion Speech difficulties Nystagmus Diarrhea Edema Fatigue Weight loss Burning pain in the extremities Ataxia Coma Heart failure

Toxicity Symptoms

Oral intake of 500 mg/day for 1 month Headache Convulsion Cardiac arrhythmia Anaphylactic shock No tolerable upper intake level

Assessment Thiamin

Measurement of erythrocyte transketolase activity ( an increase in transcetolase activity >25% indicates thiamin deficiency

Measurement of urinary thiamin excretion Clinical response to administered thiamin

(symptoms improve after the person is given thiamin supplements)

Thiamin: Disease implications

Benfotiamin- lipid-soluble thiamin derivative can activate pentose phosphate transketolase to prevent experimental retinopathy

Hammer, H-P, Du, X., Edelstein, D (2003) Benfotiamine Blocks Three Major Pathways of Hyperglycemic Damage and prevents Experimental Diabetic Retinopathy. Nature Medicine, 9,3,294-299

Case study: 5-week girl was hospitalized due heart failure. The infant was diagnosed with dilated cardiomyopathy. Parents refused the heart transplantation and treatment with thiamine hydrochloride was started. 48 hours later the patient condition was improved, suggestion that her condition was due to defect of thiamin intake.

Conclusion: All patient with early dilated cardiomyopathy should have their thiamin plasma measured.

Rocco, M.D., Patrini, C., Rimini, A. (1997) A 6-month-old Girl with Cardiomiopathy Who Nearly Died. Lancet, 349, 616

RiboflavinVitamin B2

Description of Riboflavin

Water Souble Vitamin Riboflavin = Flavin + Ribitol Flavin means yellow in

Latin Ribitol is a alcohol sugar Yellow fluorescent

characteristic of Riboflavin comes from Flavin

Greatest concentrations of B2 found in liver, kidneys, and heart

Ribitol

Flavin

http://themedicalbiochemistrypage.org/images/riboflavin.jpg

History of Riboflavin

1933 Riboflavin was discovered by Kuhn, Szent, Wagner

In the US, originally known as vitamin G Riboflavin’s unique fluorescent orange-

yellow color help researchers identify B2

http://sandwalk.blogspot.com/2007/09/nobel-laureate-richard-kuhn.html

Main Coenzymes

FMN - Flavin Mononucleotide FAD - Flavin Adenine Dinucleotide Most commonly found in foods In the intestinal lumen the coenzymes are

converted into riboflavin

FAD FMN Riboflavin

FAD pyrophosphatase FMN phosphatase

Physiological and Biochemical Functions of Riboflavin

Main Function - Electron Hydrogen Transfer Reactions Oxidative Decarboxylation of pyruvate Succinate Dehydrogenase Fatty Acid Oxidation Sphinganaine Oxidase Xathine Oxidase Aldehyde Oxidase Pyridoxine phosphate oxidase Active form of folate Synthesis of niacin from tryptophan Choline Catabolism Thioredoxin reductase Monoamine oxidase Oxidized form of glutathoine

Metabolism of Riboflavin

Riboflavin most commonly found bonded to protein in foods. Prior to absorption, riboflavin must be freed of the protein Divalent metals such as Copper, Zinc, Iron inhibit the

absorption of riboflavin Alcohol – impairs Riboflavin digestion and absorption ~ 95% Riboflavin is absorbed from foods up to 25 mg ~7% of FAD is covalently bound to AAs; Histidine or Cysteine,

can’t function in the body and remains bound Excreted in the urine

Absorption of Riboflavin

Mucosal cells: Riboflavin FMN

Serosal surface: FMN is dephophorylated to Riboflavin B2 is transported to the liver

Converted to FMN or other coenzyme by flavokinase

FAD is most predominant flavoenzyme in tissues

Flavokinase

ATP ADP

Transportation of Riboflavin

Systemic plasma Most flavins are found as riboflavin

Riboflavin, FMN, and FAD are transported in the plasma by a variety of proteins Albumin, fibrinogen, and globulins

Albumin is the primary transport protein Free riboflavin uses carrier mediated

process to traverse most cell membranes In the brain riboflavin uses a high affinity

transport system for B2 and FAD

Deficiency of Riboflavin

Ariboflavinosis Cheilosis – lesions on outside of lips Angular Stomatitis – Corners of mouth Glossitis – Inflammation of tongue Hyperemia – Redness or bleeding in oral cavity Edema – swollen mouth/ oral cavity Seborrheic Dermatitis – inflammatory skin condition Anemia Nueropathy- peripheral nerve dysfuction

Populations with greatest risk of deficiency

Congential heart disease

Some Cancers

Excess alcohol intake

Thyroid disease

Diabetes Mellitus, trauma, stress

Women who take oral contraceptives

Sources of riboflavin

Excellent Sources – animal origin products Beef Liver, Sausage, Steak, Mushrooms, Ricota Cheese,

Nonfat Milk, Oysters

Significant Sources – Eggs, meat, legumes

Fairly Good Sources – Green Vegetables

Minor Sources – Fruit and Cereal grains

Forms of Riboflavin in Foods

FMN and FAD Most common

Free or protein bound milk, eggs, enriched breads and cereals

Phosphorous bound

RDA of Riboflavin

Men – 1.3 mg/day

Women – 1.1 mg/day Pregnant – 1.4 mg/day Lactating – 1.6 mg/day

Toxicity Levels of Riboflavin

Level has yet to be determined Fun Fact - 400 mg of Riboflavin – is an effective

treatment dose for migraine headaches without any side effects

Assessment of Riboflavin

Erythrocyte glutathione reductase Good measurement because requires FAD for a coenzyme If reaction is limited than Riboflavin intake is low

Riboflavin disease Implications

Riboflavin increase lowers homocysteine reducing the risk of coronary atherosclerosis Riboflavin and folate work together to reduce plasma

tHcy (total homocysteine)Moat, S., Pauline A. L., Ashfield-Watt, Powers, H. J., Newcombe R.G, and McDowell, I. (2003). Effect of Riboflavin Status on

the Homocysteine-lowering Effect of Folate in Relation to the MTHFR (C677T) Genotype. Clinical Chemistry. 2003;49:295-302

Riboflavin can increase the amount of antioxidants in a breast cancer patient, increasing DNA repair Supplemented with 100 mg co-enzyme Q10, 10 mg

riboflavin and 50 mg niacin (CoRN), one dosage per day along with 10 mg tamoxifen twice per day.

Premkumar, V. G., Yuvaraj, S., Shanthi P., and Sachdanandam, P . (2008). Co-enzyme Q10, riboflavin and niacin supplementation on alteration of DNA repair enzyme and DNA methylation in breast cancer patients undergoing

tamoxifen therapy. British Journal of Nutrition 100: 1179-1182

NiacinVitamin B3

History of Niacin Niacin was discovered because of its deficiency pellagra Documentation of pellagra dates back to the 1760’s in

Spain and Italy Joseph Goldberger was the first to come up with a

scientific reason to explain pellagra He discovered that pellagra could be cured by milk and

concluded that it was not an infectious disease Continuing the work of Joseph Goldberger, Conrad

Elvehjem was able to isolate and identify niacin. Fun fact: Originally, referred to as only nicotinamide, it

was renamed to niacin because it was thought that nicotinamide too closely resembled nicotine and the didn’t want people getting confused and thinking they were harming themselves or that cigarettes contained vitamins.

Niacin is the general term to classify both nicotinic acid and nicotinamide

Suave, A. A. (2007). NAD+ and Vitamin B3: From metabolism to therapies. The Journal of Pharmacology and Experimental Therapeutics, 324(3), 883-893.

Absorption

Most absorption of niacin occurs in the small intestine.

Absorption/transportation occurs in one of two ways:

1. Passive diffusion- this happens when it is at high concentrations (ex. Pharmacological doses)

2. Facilitated diffusion- This is a sodium dependent reaction that occurs when niacin is in lower concentrations

Transportation Niacin is transported through the blood

stream and then is able to move across cell membranes by simple diffusion The exception is when nicotinic acid is being

transported into the kidney tubules or the RBC’s. This requires a carrier.

However, this is not very often because in the blood plasma, niacin is most commonly in the form of nicotinamide

Niacin is used by all tissues so it is transported throughout the body

Importance of Niacin

Nicotinamide is the primary precursor for NAD and NADP

Approximately 200 enzymes require NAD or NADP

NADNADH: main role is to transport electrons through the ETC, but also acts as a co-enzyme for: Glycolysis β-oxidation of fatty acids Oxidative decarboxylation of pyruvate Oxidation of acetyl CoA via Krebs cycle Oxidation of ethanol

Importance of Niacin cont.

NADPNADPH: main role is as a reducing agent in the hexosemonophosphate shunt but also also acts as a co-enzyme for: Fatty acid synthesis Cholesterol and steroid synthesis Oxidation of glutamate Synthesis of deoxyribonucleotides Regeneration of glutathionine, vit. C, and

thioredoxin Folate metabolism

Mechanism of action

NAD+ and NADP act as electron acceptors (and donors)

Boyer, R. (2002). Concepts in biochemistry. Canada: John Wiley and Sons. Fig. 16.7

Synthesis of Niacin Our body can synthesize NAD from the

amino acid tryptophan in the liver. This requires other vitamins and minerals. Despite this, we still require niacin from

dietary sources.

WHY? This only happens when we have adequate

amounts of tryptophan, AND it only occurs at a rate of 60:1. This ends up being about 3% of tryptophan being used to synthesize NAD

RDA for Niacin The RDA is expressed in niacin equivalents

(NE) For men: 16 mg (NE)/day For Women: 14 mg (NE)/day During pregnancy and lactation this increases

to 18 mg (NE) and 17mg (NE)/day

To determine NE we assume the 60:1 mg tryptophan to niacin ratio

Approximately 1% of each gram of protein is tryptophan

Sources of Niacin

http://www.nlm.nih.gov/medlineplus/mobileimages/ency/fullsize/18104_xlfs.png

• Foods high in protein such as, fish*, chicken*, beef, and pork

• Enriched/fortified breads and cereals

• Legumes• Small amounts from dairy products and green vegetables

*Excellent sources are chicken breast and canned tuna

Calculating NE Determine RDA for protein.

0.8g/kg body wt. So, for someone who weighs 61 kg they need 49g of protein

Anything above this (leftover protein) will be used to convert to niacin. So lets say this person eats 79g protein

Divide leftover protein by 100 to determine grams of tryptophan and then x1000 to get mg

Finally divide by 60 to determine niacin mg synthesized

79g-49g= 30g ; 30g÷100=0.3 g tryptophan ; 0.3x1000= 300mg tryptophan ; 300mg tryptophan÷60= 5mg niacin

Pellagra: niacin deficiency

Fred, H. L., & Van Dijk, H .A. (2007). Images of memorable cases: 50 years at the bedside. Houston: Long Tail Press/Rice University Press.

Characterized by the 4 D’s:

1.Diarrhea2.Dermatitis3.Dementia

4.Death

Pellagra cont.

Niacin can be covalently bound to proteins (niacinogen) or carbohydrates (niacytin)

The covalent bond is not sensitive to HCl in the stomach and therefore niacin is not released for absorption

Niacin is not absorbed and deficiency occurs Niacinogen and niacytin are most common

in corn which was a major source of food during the depression

Now we know how to solve the problem

Niacin deficiency Besides pellagra, deficiency or diminished

niacin status can also occur

Populations at risk: Those taking certain medications (Ex.

Antituberculosis drug isoniazid) Malabsorptive disorders- chronic diarrhea,

inflammatory bowel disease, some cancers… Those with Hartnup disease- impairs

tryptophan absorption decreasing synthesis to niacin

Alcoholics

Niacin toxicity Nicotinic acid is used as a treatment for

high hypercholesterolemia. High doses (4g/day) have been shown to increase HDL and lower LDL. The mechanism of action is unknown.

Side effects occur when consuming >1g niacin (usually in form of nicotinic acid for benefits)

Niacin toxicity con’t. Side effects include:

Niacin flush- redness, burning, itching, and tingling of the skin.

Gastrointestinal problems Hepatic toxicity Hyperuricemia- Niacin competes with uric acid

for excretion which causes a build-up and possibly gout

Elevated blood glucose (glucose intolerance)

Tolerable Upper Intake Level: 35mg/day for adults

Assessment of niacin Measurement of urinary metabolites of the vitamin:

<0.8 mg/day N’ methyl nicotinamide= deficiency <0.5 mg N’ methyl nicotinamide/1 g creatinine= poor

niacin status 0.5-1.59 mg N’ methyl nicotinamide/1 g creatinine=

marginal status >1.69 mg N’ methyl nicotinamide/1 g creatinine=

adequate status Sometimes other ratios of urinary excretion are used to

assess status

Measurement of ratio of erythrocyte concentrations of NAD to NADP and just NAD has been used to assess status.

Niacin disease Implications

Cardiovascular disease Niacin has been shown to increase HDL while

at the same time decreasing LDL and total TG. One review even stated that niacin, “is

considered the most efficacious agent currently available for therapeutic elevation of subnormal HDL-C concentrations, and typically produces a 15 to 35% increment as a function of dose” (Chapman, Redfern, McGovern, & Giral, 2010)

Athersclerosis Niacin helps slow the progression of

atherosclerosis by slowing the thickening of arteries

Niacin disease implications con’t.

Alzheimer’s Disease Niacin is though to have a protective effect

against niacin although more research is needed to determine mechanism of action and significance.

Cancers Niacin is plays a role in DNA repair and

therefore supplementation may improve cancer outcomes by helping prevent tumor growth.

MetabolismThiamin, Riboflavin, Niacin

Important in reactions:

Glycolysis (fig. 4.14) β-oxidation of fatty acids

(fig 6.24) Oxidative decarboxylation

of pyruvate (fig 9.12) Oxidation of acetyl CoA via

Krebs cycle (fig. 4.15) Oxidation of ethanol (fig.

4.23) Fatty acid synthesis (fig.

6.30) Cholesterol and steroid

synthesis (see ch. 6) Oxidation of glutamate

(fig. 7.23) Choline Catabolism (see pg. 305) Thioredoxin reductase (see ch. 12)

Synthesis of deoxyribonucleotides

Regeneration of glutathionine, vit. C, and thioredoxin (pg. 285, 269, & 460)

Sphinganaine Oxidase Xathine Oxidase (fig. 7.18) Aldehyde Oxidase (fig. 10.4) Pyridoxine phosphate oxidase (fig.

9.39) Active form of folate(fig. 9.31) Synthesis of niacin from

tryptophan (fig. 9.18) Monoamine oxidase

Oxidative Decarboxylation of Pyruvate: Vitamins

B1, B2, & B3

Oxidative decarboxylation of pyruvate

In order for the formation of Acetyl CoA, thiamin diphosphate must first be present.

Pyruvate dehydrogenase combines thiamin diphosphate with pyruvate in order to form Acetyl CoA.

NAD and FAD are also required as reducing agents are oxidized to NADH and FADH2

Krebs Cycle and vitamin B1, B2, & B3

Krebs Cycle and vitamin B1, B2, & B3

NAD and FAD act as electron acceptors in the Krebs Cycle. They are oxidized to NADH and FADH2

NADH and FADH2 then move to the ETC where they donate the hydrogen necessary to ultimately start ATP synthase and produce ATP

Thiamin is also required for the oxidative decarboxylation of α-ketoglutarate to succinyl CoA

Hexosemonophosphate Shunt

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Hexosemonophosphate Shunt: thiamin and niacin only

Important in the formation of NADPH and is most active in tissues with a high need of NADPH for fatty acid synthesis.

Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase both require NADP as a cosubstrate.

Transketolase requires thiamin in order to work.

Fatty acid synthesis: niacin only

http://ull.chemistry.uakron.edu/Pathways/FA_synthesis/index.html

QUESTIONS?

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References Boyer, R. (2002). Concepts in biochemistry. Canada: John Wiley and Sons.

Chapman, M.J., Redfern, J.S., McGovern, M.E., Giral, P. (2010) Niacin and fibrates in atherogenic dyslipidemia: Pharmacotherapy to reduce cardiovascular risk. Pharmacology and Therapeutics, 126, 314-345.

Gropper, S.S., Smith, J.L., & Groff, J.L. (2005 ,2009). Advanced nutrition and human metabolism. Belmont, Ca: Thomson Wadsworth.

http://www.vitaminsworld.org/vitamins/vitamin-b2.html

Morris, M.C., Evans, D.A., Bienias, J.L., Scherr, P.A., Tangney, C.C., Herbert, L.E., Bennett, D.A., Wilson, R.S., Aggarwal, N. (2004). Dietary niacin and the risk of incident Alzheimer’s disease and of cognitive decline. Journal of Neurology, Neurosurgery, and Psychiatry, 75, 1093-1099.

Premkumar, V.G., Yuvaraj, s., Satish, S., Shanthi, P., Sachanandam, P. (2008). Anti-angiogenic potential of CoenzymeQ10, riboflavin and niacin in breast cancer patients undergoing tamoxifen therapy. Vascular Pharmacology, 48, 191-201.

Suave, A. A. (2007). NAD+ and Vitamin B3: From metabolism to therapies. The Journal of Pharmacology and Experimental Therapeutics, 324(3), 883-893.

Wrenger, C., Knöckel, J, Walter, R. D. & Müller, J. B.(2008). Vitamin B1 and B6 in the malaria parasite: requisite or dispensable? Braz J Med Biol Res, 42: 82-88.