Staging vitamin B12 (cobalamin) status in vegetarians13

Am J C/in Nuir 1994:59(suppl):1213S-22S. Printed in USA. © 1994 American Society for Clinical Nutrition 12135

Victor Herbert

ABSTRACT When one stops eating vitamin B-l2 (cobala-mins), one passes through four stages of negative cobalamin bal-ance: serum depletion [low holotranscobalamin II, ic, low vita-mm B- 12 on transcobalamin II (TCII)], cell depletion (decreasingholohaptocorrin and low red cell vitamin B-12 concentrations),biochemical deficiency (slowed DNA synthesis, elevated serumhomocysteine and methylmalonate concentrations), and, finally,clinical deficiency (anemia). Serum vitamin B- 12 is on two pro-teins: the circulating vitamin B-l2 delivery protein, TCII, and thecirculating vitamin B- I 2 storage protein, haptocomn. BecauseTCII is depleted of vitamin B-l2 within days after absorptionstops, the best screening test for early negative vitamin B- 12balance is a measurement of vitamin B- 12 on TCII (holoTCil).Ho1oTCII falls below the bottom of its normal range long beforetotal serum vitamin B- 12 (which is mainly vitamin B-12 on hap-tocomn) falls below the bottom of its normal range. Am JClin Nutr l994;59(suppl): 12 l3S-22S.

KEY WORDS Vitamin B-l2, serum holotranscobalamin II,transcobalamin II, cobalamin, vegans, serum total vitamin B-12,serum holohaptocomn, B- 12 haptocomn, haptocorrin

Introduction

In the 5 y since our report at the First International Congresson Vegetarian Nutrition ( 1 ), there has been increased understand-ing of vitamin B-12 status and its staging (2), increased under-standing of the role of genetics in determining individual predis-position to a deficiency of vitamin B-l2 (3, 4), and increasedunderstanding of selective vitamin B-l2 deficiency in one cellline rather than another (such as early vitamin B-12 deficiencyneural damage producing cognitive dysfunction before hemato-logic damage producing anemia) (5).

There arc six ways (etiologies) one can get a vitamin B-12deficiency (2, 6). They include three inadequacies-inadequateingestion, inadequate (defective) absorption, and inadequate util-ization (defects in vitamin B-l2 enzymes or other proteins)-and three excesses-increased requirement (ie, pregnancy, hy-perthyroidism), increased excretion (as in alcoholism), and in-creased destruction (as by megadoses of vitamin C) (7, 8). Inalcoholism, cobalamin deficiency occurs from a combination offour etiologies: inadequate ingestion, absorption, utilization, andincreased excretion (9). However, vitamin B- 1 2 deficiency in al-coholism is often masked by a holohaptocomn concentration thatis normal to elevated because of excess abnormal haptocomn

released from the damaged liver (9).Whereas most clinicians still talk in terms of either normal or

clinical vitamin B- 1 2 deficiency, nutrition professionals recog-

nize that, barring instant vitamin B-l2 deficiency produced by avitamin B-l2 antagonist or by nitrous oxide anesthesia (2, 10),the sequence from normality to deficiency (Fig 1) with respectto vitamin B-I 2 (or any nutrient) (I 1) passes through two stagesofdepletion (Stage I, low serum vitamin B-12; Stage II, low cell-store vitamin B-l2) followed by two stages of deficiency (StageIII, biochemical deficiency; and Stage IV, clinically manifest de-ficiency) (2). Additionally, one may have a deficiency of vitaminB- 1 2 in one cell line before it occurs in another, because stores

of vitamin B- I 2 are smaller and/or utilization is more rapid and!or resupply is slower in one cell line rather than another. Suchdeficiency may be early mild, or subtle. Negative balance is thedescriptor for the situation when, because of either inadequateingestion or defective absorption, the amounts of vitamin B- I 2absorbed daily decrease below the amount lost each day (Fig 1).

Inadequate ingestion of vitamin B-12

Dietary deficiency of vitamin B-I 2 results from a strict vegan(all plant food) diet because there is no vitamin B-l2 synthesizedby any plant, nor does any plant need, use, or store vitamin B-1 2 ( 1 ). The ultimate source of all vitamin B- I 2 is microbial syn-thesis; for any plant food to contain vitamin B-l2 the food mustbe contaminated with vitamin B-l2-synthesizing bacteria (1).Vegetarians who are not vegans (eg, lactoovovegetarians andlactovegetarians) ingest adequate amounts of vitamin B- I 2 inanimal (eg, egg, milk, and other) products (3). If these vegetar-ians develop vitamin B- 1 2 deficiency, it is for the same reasons

as do omnivorous humans. Whereas any structural or functionaldefect in the gastric, pancreatic, or small intestinal machinery forvitamin B-12 absorption can cause vitamin B-12 deficiency, themost frequent cause of omnivore vitamin B- 12 deficiency is ge-netically predetermined age-dependent loss of gastric secretoryfunction, ie, pernicious anemia (PA) (12).

Indicators of negative vitamin B-12 balance

Low serum total vitamin B- 1 2 concentrations indicate Stage IIto IV negative balance (Fig 1), and therefore are a relatively lateserum indicatorofnegative vitamin B-l2 status (13, 14), whereas

From the Mount Sinai and Bronx Veterans’ Affairs Medical Centers,New York City.

2 Supported in part by the V Herbert Research Fund at the Mount Sinai

School of Medicine, New York City.3 Address reprint requests to V Herbert, 130 West Kingsbridge Road,

Bronx, NY 10468.

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12145

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FIG 1 . Sequential stages of vitamin B- 12 status. Biochemical and hematological sequence of events as negativevitamin B-12 balance progresses. [©1990, 1993 Victor Herbert (modified 1993 to include homocysteine).l

low serum holotranscobalamin II (holoTCII) concentrations in-dicate Stage I negative balance, and are therefore the earliestserum indicator of negative vitamin B-l2 balance (15, 16). Totalserum vitamin B- I 2 is the sum of vitamin B- 12 on transcobala-mm II (TCII) and vitamin B-l2 on haptocomn (14). Total serumvitamin B- 12 concentrations are a relatively late indicator be-cause normally 80% of total serum vitamin B-l2 is on serumholohaptocomn, a late indicator, and only a20% normally is onthe early indicator, serum holoTCIl, which has a half-life of only6 mm (13, 17).

As cell stores decrease, the amount of vitamin B-12 on thecirculating storage protein, haptocomn, decreases pari passu. Ho-lohaptocomn (vitamin B-l 2 on haptocorrin) is a circulating storeof vitamin B-12 that is in equilibrium with body (particularlyliver) vitamin B-l2 stores, which fall slowly as negative vitaminB-12 balance progresses (13, 14). Additionally, vitamin B-l2-haptocomn has a half-life of 240 h ( 1 7). The only receptors forhaptocomn arc on vitamin B- 1 2 storage cells [liver cells andreticuloendothelial (RE) cellsj, whereas every cell that synthe-sizes DNA (including liver and RE cells) has cell-surface recep-tors for TCII, the ubiquitous vitamin B-l2 delivery protein (2,13). Ho1oTCII, made in the ileal enterocytes from intracellularlysynthesized TCII and absorbed vitamin B-12, is secreted into theserum with a subsequent half-life of only 6 mm, and thereforefalls rapidly when vitamin B- 12 absorption into the ileal brushborder enterocytes slows.

About one-third of the ‘ ‘vitamin B-l2’ ‘ in serum is in fact not

cobalamins (which are all forms of vitamin B-l2 that are activefor humans), but other corrinoids that are metabolically dead forhumans but active for bacteria. Thus, many microbiologic assays

may find normal “vitamin B-l2” concentrations in vitamin B-12-deficient people because the assay is reading as vitamin B-12 what is in fact noncobalamin comnoids. To avoid this prob-1cm, most laboratories use competitive inhibition radioassays thatmeasure only cobalamins and no other comnoids ( 1 . 2, 8). The

core of all corrinoids (cobalamins and noncobalamins) is corrin,which resembles the heme of hemoglobin, but has one less a-methene bridge and has cobalt instead of iron at the center. Theultimate source of all corrinoids is bacterial synthesis.

Vitamin B-12 may regulate the synthesis of proteins involvedin its own absorption, transport, and delivery (ie, intrinsic factor,haptocomn, transcobalamin, and their cell-surface receptor pro-teins) ( 18), just as iron regulates the proteins involved in its ownsynthesis (19). In his paper in the Addison book (20), Jacobsen(Vol 2: 105-17) describes how cell-surface receptors for TCII are

created, how they endocytose vitamin B- 12 and carry it from the

cell surface to internal organelles, and then turn around and goback to the cell surface for another vitamin B- 12 passenger. Healso discusses the up-regulation and down-regulation of thesecell-surface receptors (see also Fig I).

Mature red blood cells (RBCs) do not take up vitamin B-l2(21). The vitamin B-12 present in nucleated RBCs is largely cx-truded when the nucleus is extruded, and much of the remainingvitamin B-l2 is extruded when the reticulum of the reticulocyteis extruded. Much of the vitamin B- 12 in reticulocytes got there

via rcticulocytc cell-surface vitamin B-l2-TCII receptors (21).There arc no such receptors on mature RBCs (2 1 ). Total RBCvitamin B-l2 is largely that in the youngest RBCs. We thereforeagree (22) with Tisman et al (23) that measurements of RBCvitamin B- 1 2 may, like measurements of serum holoTCIl, prove

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STAGING VITAMIN B-12 STATUS 12155

to be more useful than total serum vitamin B-12 concentrationsin delineating early negative vitamin B-12 balance (Fig 1).

Inadequate absorption of vitamin B-12

The consequences of the much greater number of cell-surfacereceptor sites and much shorter half-life of the vitamin B-12-TCII complex (holoTCIl) as opposed to the vitamin B-12-hap-tocorrin complex (holohaptocorrin) can be seen when vitamin B-12 absorption across the ileum stops. Because of the very rapid

turnover of TCII, within 1 wk after absorption stops, vitamin B-12 on TCII will be below the normal range (13-15). However,

the amount of vitamin B-12 on haptocorrin will still be in thenormal range (13-16).

Because normal serum concentrations of total vitamin B-12range from 148 to 666 pmol/L (200 to 900 pg/mL), with a meanof 333 pmol/L (450 pg/mL), a week after absorption stops, themean serum total vitamin B-12 concentrations may drop from332 to 259 pmol/L (450 to 350 pg/mL). This total vitamin B-12

value is still within the normal range, but the normal 20% of totalvitamin B-12 that is on TCII may have dropped to 10% or less,below the normal range for holoTCIl. This indicates a negativebalance with reduced delivery of vitamin B-12 to all cells thatare synthesizing DNA, including those that have relatively lowvitamin B-12 stores, such as nervous system glial cells and bonemarrow hematopoietic cells. As Figure 1 shows, this negativebalance is undiagnosable by measuring total serum vitamin B-12concentration, which is now a90% vitamin B-12 on haptocorrinand remains within the normal range.

Iron from plant foods averages 3% absorbable; iron from an-imal foods averages 15% absorbable (3, 4, 19). Therefore, irondeficiency is twice as common in vegetarians as in omnivores (3,4, 19). Because some vegetarians, especially those consumingrestrictive diets, are at greater risk for deficiencies than omni-vores (24), all vegetarians should be tested for iron disorders (25).Prolonged iron deficiency damages the gastric mucosa and pro-motes atrophic gastritis and gastric atrophy, including loss ofgastric acid and IF secretion, and therefore diminished vitaminB-12 absorption (3, 4, 19). This would cause vitamin B-12 de-ficiency in twice as many vegetarians as omnivores (3,4, 19).

If daily vitamin B- 12 absorption continues to be less than dailyvitamin B-12 loss, negative balance will progress to Stage II,depletion of stores. At this point, many vegetarians stabilize foryears with depleted stores, because depleted stores trigger up-regulation of healthy machinery for absorption, making more ef-ficient the absorption of the trace amount of vitamin B-12 frombacterial contamination of the small intestine (26) and vitaminB-12 secreted in the bile (27). Eventually, however, continuingslight negative balance will deplete vitamin B-12 stores, afterwhich Stage III negative balance occurs (biochemical deficiency,ie, inadequate vitamin B-12 for normal vitamin B-12-dependent

biochemical reactions, sometimes called preclinical deficiency,see Fig 1).

Subnormal absorption was thought to be because of loss ofgastric intrinsic factor (IF) secretion; in the 1960s we directlyassayed for IF to prove it (28).

The role of haptocorrin relative to vitamin B-12 is similar tothat of ferritin relative to iron (ic, it is a circulating storage formof vitamin B-12 that is in equilibrium with body stores). TCII

serves much the same function for vitamin B-12 as does trans-ferrin for iron by being in equilibrium with vitamin B-12 that israpidly turned over in hematopoietic and other cells that are rap-idly synthesizing DNA.

Progression of biochemical deficiency produces clinical de-ficiency (ie, Stage IV negative balance). Biochemical defi-ciency is defined by biochemical laboratory tests; clinical de-ficiency is defined by clinical signs and/or symptoms. Becauseliver stores are greater than those in the blood-synthesizingbone marrow and in nervous tissue, liver cells remain in StageII negative balance (depletion) after bone marrow hematopoi-etic cells and nervous tissue glial cells (and sometimes alsomyelin-synthesizing cells) are already in Stage III, and per-haps even Stage IV.

Brain deprivation of vitamin B-12

Lack of delivery of vitamin B-12 to the glial cells of thebrain quickly wipes out their small vitamin B-12 stores, afterwhich they become vitamin B-12 deficient (13, 29, 30) withbuildup of homocysteine because vitamin B-12 is needed toconvert homocysteine to methionine (2, 6). Methylfolatetransfers a methyl group to vitamin B-12, which transfers it tohomocysteine, converting homocysteine to methionine (alsoknown as methylhomocysteine) (2, 6). Homocysteine, a nor-mal sulfur-containing amino acid at usual concentrations, is aneurotoxin and vasculotoxin when its concentration is dc-

vated (2, 31). Vitamin B-12 is also necessary to remove themethyl from methylfolate, a circulating storage form of folate,converting it to a metabolically active form, necessary for one-carbon transfers (2, 6).

We found decreased concentrations of hoIoTCII in 53% ofacquired immune deficiency syndrome (AIDS) patients (13).We found (13), and others confirmed (32), that some of thecognitive and hematopoietic dysfunction of such AIDS pa-tients is reversed by vitamin B-12 therapy. This allows higherazidothymidine (AZT) dosages (33). We are studying whetherAIDS patients with cognitive dysfunction that is due to vita-mm B-12 deficiency have elevated spinal fluid concentrationsof homocysteine as a partial explanation for such dysfunction,even though the serum concentration of homocysteine is stillnormal. Table 1 shows data from four AIDS patients. One hadnormal serum holoTCIl and no cognitive dysfunction. Thethree with cognitive dysfunction had almost no vitamin B-12on TCII. This means that no vitamin B-12 was being deliveredto the glial cells in their brains (13) and that homocysteinewas probably piling up in their brains. Note that, despite fac-tors that tend to give AIDS patients low serum homocysteineconcentrations (34), these three have borderline high serumhomocysteine.

Vitamin B-12 (or folate) deficiency, but not depletion, pro-duces elevated serum homocysteine concentrations. In theAddison book (20), Brattstrom (Vol 2:205-17) and Ueland sep-arately write of the accumulation of homocysteine in either adeficiency of vitamin B-12 or of folate (or an accumulation dueto other causes) being both neurotoxic and vasculotoxic, pro-moting heart attacks, thrombotic strokes, and peripheral vascularocclusions. This phenomenon may be much more widespreadthan previously appreciated (35). High serum homocysteine con-centrations may be reduced to normal by recommended dietary

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12165 HERBERT

TABLE 1Data on four patients with acquired immune deficiency syndrome (AIDS) who have normal total serum vitamin B- 12 concentrations

Average num ber of lobes’test

Beforevitamin B-12

Aftervitamin B-12

Schi lling

Free Foodtherapy therapy Vitamin B-12 Ho1oTCII2 vitamin B-12 vitamin B-12 Homocysteine

pmol/L pmol/L % tmo//L

Patient 1 2.61 2.64 358.9 77.7 25 15 20.9Patient 2 3.42 3.21 504.7 0 2 - 24.2Patient 33 3.32 3.04 275.3 1.5 3 - 26.4Patient 43 3.78 3.33 484.7 12.6 32 0 30.4

1 Granulocyte lobe average reference range = 3.2 ± 0.15.

2 Holotranscobalamin II.

3 Patients with cognitive dysfunction.

allowance (RDA) amounts of folic acid, vitamin B-12, or vitaminB-6, if the high concentration is related to a deficiency of one ofthose three vitamins (36). If the elevated concentration is not dueto such a deficiency, it still may often be reduced to normal bypharmacologic (well above RDA) doses of folic acid.

Hematopoietic effects of vitamin B-12 deficiency

When bone marrow stores of vitamin B-12 are gone, there isno longer enough vitamin B-12 for normal DNA synthesis, whichbecomes slow, resulting in large, oval red and white blood cells.When this deficiency progresses to Stage IV negative balance,overt clinical anemia appears (Fig 1). Thus, to wait for clinicalanemia to occur before looking for vitamin B-12 deficiency willresult in many patients with severe cognitive dysfunction that isdue to undiagnosed vitamin B-12 deficiency in the nervous sys-tem, which has not yet manifested clinically in the blood system.

Just as serum concentrations of total vitamin B-12 may bewithin the normal range when there is a vitamin B-12 deficiencyin brain glial cells, so may total serum vitamin B-12 still bewithin the normal range even when a biochemical deficiency ina number of cell lines has produced elevated serum methylma-lonate or homocysteine (29-35, 37). However, holoTCII willalways be low before there is a rise in methylmalonate or ho-mocysteine if that rise is due to a vitamin B-12 deficiency. A redherring is that serum homocysteine is elevated by renal diseasein the absence of vitamin B-12 or folate deficiency, and serum

homocysteine may be artificially low in AIDS patients despitevitamin B-12 or folate deficiency (34).

Inadequate utilization of vitamin B-12

The minimum daily dietary requirement to sustain normalityfor vitamin B-12 is only 0.1 .tg (38), so the recommended dietaryallowance (RDA) of 2 j. g (36, 39) is overgenerous (ic, allows asubstantial excess for storage). However, a healthy stomach, pan-creas, and ileum are all necessary for vitamin B-12 absorption.The digestion and absorption from food of vitamin B-12 requiresgastric acid, proteolysis, and gastric IF (2, 40-42). Gastric acidand proteolysis release vitamin B-12 from peptide bonds in food.The free vitamin B-12 does not attach to gastric IF, but ratherattaches to swallowed salivary vitamin B-12 binding protein,

which is a much more avid binder of vitamin B-12 than is IF(43). The salivary binding protein-vitamin B-12 complex passeswith free IF from the stomach into the upper small intestine,where the acid pH of the stomach is neutralized to slightly al-kaline. At mildly alkaline pH, pancreatic enzymes selectivelydigest the salivary binder, releasing the vitamin B-12, which thenattaches to IF, which is impervious to pancreatic enzyme diges-tion at mildly alkaline pH (40).

The vitamin B-12-IF complex then passes into the ileum,where the ileal cell-surface receptors for the complex are located.These receptors require free calcium to hold the vitamin B-12-IF complex against the ileal wall (40-42), as do reticulocytereceptors (21). Therefore, patients with pancreatic disease, whichreduces available free calcium, cannot absorb vitamin B-12.Their vitamin B-12 absorption is improved by giving them cal-cium and/or bicarbonate or pancreatic extract, each of which in-creases available free calcium (40-43).

The importance of free calcium for vitamin B-12 absorptionwas sharply etched in our study (44), which showed that the oralantidiabetic agent, metformin, produced vitamin B-12 malab-sorption by tying up free calcium.

Effects of excess vitamin C on vitamin B-12 status

Active vitamin B-12 can be destroyed by megadoses of vita-mm C, which convert vitamin B-12 to analogue forms that areworthless to humans (8). Vitamin C acts as an antioxidant pri-

manly at physiologic doses. At pharmacologic (or mega) doses,in the presence of iron, it is one of the most potent oxidantsknown and drives iron-catalyzed free radical generation (45),which can not only damage vitamin B-12 but can destroy IF (46).

Effects of genetics on vitamin B-12 status

Genetic factors affect an individual’s ability to digest, absorb,and utilize vitamin B-12 (2, 6). Genes determine when the stom-ach will stop producing gastric acid, and later, when it will stopproducing IF. If the family history includes a loss of gastric acidat an early age, such loss can be expected in offspring. With aloss ofgastric acid, vitamin B-12 will no longer be absorbed fromfood. Thus, genes determine when a predisposition to vitamin B-12 deficiency will be manifested. Total vegetarians (vegans), es-

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STAGING VITAMIN B-12 STATUS 12175

pecially those who have been so most of their lives, have lowvitamin B-12 stores and will more rapidly express a geneticallypredisposed vitamin B-12 deficiency.

Genetics also play a role in elevated concentrations of homo-cysteine, because of various genetically determined enzyme de-fects and because of the relationship of high homocysteine con-centrations to vitamin B-12 and folate status (2, 31).

Genes normally determine at what age gastric acid and gastricIF synthesis slow and stop. For genetic reasons, some peopleaged 45 y become totally unable to absorb vitamin B-12 fromfood, because to absorb vitamin B-12 from food, gastric acid andenzymes must split the vitamin from its peptide bonds. The ge-netically lucky have parents who do not lose their gastric acidand enzyme secretions until they are aged 100 y, and will prob-ably do the same.

Hematology and endocrinology are connected in part by a ge-netically predisposed autoimmunity, most strikingly seen in mul-tiple endocrine adenopathy syndrome, in which antibodies to hor-mones and to IF circulate in the body (47). In the same way thatthe presence of circulating antibody to the adrenal glands and thethyroid predicts adrenal and thyroid disease, the presence of cir-culating antibody to IF predicts PA (47).

Vitamin B-12 is unstable in its coenzymatically active forms(6). The American and British teams who successfully isolatedvitamin B-12 within weeks of each other in 1948 (2) were luckybecause, in the process of isolation, they passed their concen-trates through a charcoal column. The cyanide in the charcoalreplaced the unstable metabolically active adducts attached to thecobalt, resulting in the stable (but vitamin-inactive) molecule cy-anocobalamin, which, because of its stability, has been the pri-mary pharmaceutical form of the vitamin. As workers at Yalefirst noted [see chapters by Fenton (Vol 2:219-28) and Rosen-blatt (Vol 1 :219-28) in reference 20] infants are sometimes bornwith a rare genetic defect in the enzyme that removes cyanidefrom cyanocobalamin to render it vitamin-active (2, 6). In suchinfants, supplying cyanocobalamin is harmful because it attachesto cobalamin apoenzymes and produces a metabolically dead ho-

loenzyme, ie, a holoenzyme that acts as a monkey-wrench insteadof as a key in cobalamin-dependent biochemical reactions. Hu-mans have no circulating cyanocobalamin unless they are smok-ers (there is cyanide in tobacco smoke), eat cyanide-containingfoods (like bitter cassava or bitter apricot kernels), or take cya-nocobalamin parenterally, including via the nasal mucosa (48).

The 1993 Addison Symposium

In Padua and London, from May 20 to 27, 1993, I had the

honor of being the senior president at an international symposiumof the world’s leading researchers in the study of Addisonian PAand Addison’s disease, each of whom reviewed their most recentwork. These reviews are now a book, Advances in Thomas Ad-dison ‘s Diseases (also called ‘ ‘the Addison book’ ‘), published in1994 (20). In the Addison book, we reviewed the etiology ofcobalamin deficiency and the staging of vitamin B-12 status (Vol1:139-47). The book also includes a review by Green (Vol1:371-5) on his most recent work relating to the nerve damageof vitamin B-12 deficiency, and reviews by Metz, Allen, andLindenbaum. Goh et al (49) and Amin et al (50) have confirmedour status staging chart delineation (Fig 1) that low ho1oTCIIprecedes high homocysteine or methylmalonate as negative vita-mm B-12 balance develops.

Also in the Addison book (20), Linnell and Bhatt (Vol 1:371-5) write of abnormalities in cobalamin metabolism in a subgroupof patients with multiple sclerosis. Our laboratory has been study-ing two such families, each with two study subjects, with neu-rologist colleagues at the New York Hospital Cornell MedicalCenter. In these subjects, the evidence so far suggests that a com-mon genetic defect produces both disorders and that the vitaminB-12 deficiency leads to expression of an autoimmune multiplesclerosis. Vitamin B-12 therapy suppresses further expression.

Protecting enterohepatic circulation of vitamin B-12 invegetarians

The enterohepatic circulation of vitamin B-12 is very importantin vitamin B-12 economy and homeostasis (27). Nonvegetarians

normally eat 2-6 g of vitamin B-12/d and excrete from theirliver into the intestine via their bile 5-10 j. g of vitamin B-12/d.If they have no gastric, pancreatic, or small bowel dysfunctioninterfering with reabsorption, their bodies reabsorb 3-5 j tg of

bile vitamin B-12/d. Because of this, an efficient enterohepaticcirculation keeps the adult vegan, who eats very little vitamin B-12, from developing vitamin B-12 deficiency disease for 20-30 y(27) because even as body stores fall and daily bile vitamin B-12

output falls with body stores to as low as 1 g, the percentage ofbile vitamin B-12 reabsorbed rises to close to 100%, so that thewhole microgram is reabsorbed.

On the other hand, the infant of a macrobiotic mother, who has

almost no enterohepatic vitamin B-12 circulation because it gotalmost no vitamin B-12 stores from its mother and therefore has

little vitamin B-12 to put in its bile, rapidly develops vitamin B-12 deficiency (1, 2) (see also chapter by Ueland in reference 20).Unlike the vegetarian whose absorption machinery is normal, aperson whose absorption machinery is damaged by a defect ingastric secretion, by a defect in pancreatic secretion, or by a defectin the gut that produces intestinal malabsorption will develop vita-mm B-12 deficiency in 1-3 y because these absorption defectsblock not only absorption of food vitamin B-12, but reabsorptionof vitamin B-12 excreted into the intestinal tract in the bile (2, 6).

Stage I negative balance does not necessarily have to go on toStage II. It may stabilize as Stage I and then go back to normal,as, for example, when diminished vitamin B-12 absorption istransient rather than permanent. Similarly, as noted above, StageII lasts much longer in vegetarians than in people with absorptivedefects.

Whenever we find a serum ho1oTCII concentration below 29.6pmol/L (40 pg/mL) in our patient population at the Bronx Vet-erans Affairs or Mount Sinai Medical Centers, we find that thereis either chronic inadequate vitamin B-12 ingestion or a problemin the stomach, pancreas, or ileum. Sourial (20, Vol 2:241-5)reported on vegetarians who had relatively low total serum vita-mm B-12, but no biochemical or clinical problems. In our ownlaboratory we found such people have borderline low holoTCil,in the range between 29.6-44.4 pmollL (40-60 pg/mL), ratherthan concentrations below 40. These are people who became veg-etarians as teenagers or young adults, so they had significant liverstores of vitamin B-12 to protect them against vitamin B-12 de-ficiency for years because of their enterohepatic circulation ofvitamin B-12. The vitamin B-12 goes out in their bile, is reab-sorbed into their ileal cells, packaged therein onto TCII and theresultant holoTCil is exported via the bloodstream to all the cellsthat need it.

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Prediction and diagnosis of vitamin B-12 deficiency

The Schilling test measures absorbability, not stores

In the Schilling test, radioactive vitamin B-12 is fed to a patientto delineate whether there is malabsorption ofvitamin B-12. TheSchilling test is not a measure of vitamin B-12 stores, but a mea-sure of the absorbability of vitamin B-12 at the time the test isdone. If one misperceives the Schilling test as a test for vitaminB-12 deficiency, one often draws the wrong conclusion. Whencrystalline vitamin B-12 is used the test is a measure of whetherthe physiological machinery for vitamin B-12 absorption is nor-mal in the stomach, the pancreas, and the ileum, thereby allowingnormal vitamin B-12 absorption. If the Schilling test shows sub-normal absorption, repeating the test with the missing factoradded (eg, IF or pancreatic extract) delineates the reason for themalabsorption. If there is a defect in gastric acid or enzyme se-

cretion, which produces malabsorption only of food-bound vita-mm B-12, one can test for this defect via an animal protein-bound vitamin B-12 test (eg, egg, meat, or serum) rather than bya crystalline vitamin B-12 Schilling test.

Detection of circulating antibody

One can predict that an individual will get PA by finding cir-culating antibody to IF in his or her serum. Many people withautoimmune disease have circulating antibody to IF, but do notyet have PA. However, over a period of years they eventuallydevelop PA. Thus, the presence of circulating antibody to humanIF is diagnostic for incipient, latent, or full-blown autoimmunePA (47).

Genetic predisposition

As Bottazzo reviews in the Addison book (20, Vol 2:45-57),people of Finnish or Sardinian stock are genetically predisposedto PA. There is also genetic predisposition in other ethnic groups.Sullivan and I, studying the frequency of PA in Boston threedecades ago, found it almost as frequently among Italians in Bos-ton’s Little Italy as among Boston Irish. Often, if one looks forPA, one finds it. PA was allegedly rare in China, but our col-league Ran looked for it and she found it was not rare at all (51).

Similarly, when Johnson and Carmel (20, Vol 1:201-4) lookedin the 1970s for PA in African Americans in Los Angeles, as

Hift had previously done in the mid-1960s in Durban in SouthAfrica and Jack Metz first did the early 1960s in Johannesburgin South Africa, they found that African American females in thechild-bearing years have a genetic predisposition to get PA muchmore frequently than age-matched white females in the samegeographic area.

Screening population groups

If one is screening a population group solely for early diag-nosis of PA as an autoimmune disease, the ideal screening testwould look for antibody to IF in serum and/or gastric juice (47).However, to predict vitamin B-12 deficiency of any dietary ormalabsorptive cause, autoimmune or not, ie, to test for the earliest

stage of negative vitamin B-12 balance that is related to reducedabsorption, measurement of holoTCII is the appropriate screen-ing test(13-16), not only in cases ofAIDS (13) but also in casesof PA in elderly people (52, 53). Measurement of holoTCIl isnot only a surrogate Schilling Test (44) but is also much easierto perform because it requires only a sample of blood.

For three decades we recommended simultaneous determina-tion on a single blood sample of three laboratory tests to separatefour situations: normality, vitamin B-12 deficiency, folate defi-ciency, and deficiency of both. The three tests are measurementsof serum concentrations of vitamin B-12, folate, and RBC folate(12, 54). However, to diagnose vitamin B-12 depletion before

deficiency, we now know one must measure holoTCil, and mea-surements of RBC vitamin B-12 also may be useful (22, 23).

Disease prevention

With the increasing emphasis on disease prevention, measur-ing holoTCIl becomes particularly important because holoTCIlis a measurement of vitamin B-12 depletion (55) and the discov-cry of depletion allows for treatment before deficiency super-venes (Fig 1). Future study may show that the consideration oftotal serum vitamin B-12 concentration < 222 pmol/L (< 300pg/ml) as being subnormal (56) (rather than the concentration of

148 pmolIL or 200 pg/ml, which is generally accepted today)

may also be useful in the diagnosis of vitamin B-12 depletion,although this may lead to many more false positives and nega-tives than occur with diagnosing vitamin B-12 depletion basedon measurements of holoTCII < 44.4 pmol/L (< 60 pg/ml). De-ficiency is defined as not enough nutrient for normal biochemicalfunction. Damaged function comes only with deficiency.

Those who think only in terms of population screening forvitamin B-12 deficiency by measuring a marker of one or anotherdefect in a vitamin B-12-dependent biochemical pathway, suchas elevated methylmalonate or elevated homocysteine, will neverprevent vitamin B-12 deficiency. To prevent vitamin B-12 defi-ciency, one must screen for vitamin B-12 depletion, which pre-cedes deficiency. It is far wiser, and more economical, to treatvitamin B-12 depletion so that it never becomes deficiency, ratherthan waiting to treat the neuropsychiatric and/or hematologicand/or other damage of actual vitamin B-12 deficiency, espe-cially because some of that damage may prove to be irreversiblewith vitamin B-12 therapy (2, 6, 10).

If one is interested only in therapy and not interested in pre-venting vitamin B-12 deficiency disease, one uses only tests fordeficiency, such as measurements of methylmalonate and ho-mocysteine concentrations, and ignores the fact that in the two

most frequent (> 95% of all cases) causes of negative vitaminB-12 balance (inadequate ingestion and inadequate absorption),depletion (which is not disease) precedes deficiency (which isdisease) by months to years. It is possible to prevent disease bythe diagnosis of negative balance at either of the two stages ofdepletion before the negative balance reaches either of the two

stages of deficiency through the use of tests that diagnose deple-tion (eg, holoTCIl). It is important to remember that there can bevitamin B-12 deficiency in hematopoietic and neural cells, butnot in liver cells because liver cells not only have higher vitaminB-12 content but also have receptors for haptocorrin as well asfor TCII and therefore are not depleted ofvitamin B-12 as rapidlyas hematopoietic and neural cells (13).

If TCII does not pick up vitamin B-12 in the ileal enterocytes,it will have no vitamin B-12 to deliver via the bloodstream toDNA-synthesizing cell-surface TCII receptors. Cells with lowvitamin B-12 stores, such as blood and neural tissue cells, willthen quickly run out of vitamin B-12 and become deficient (13).Our studies two decades ago showed that when radioactive vita-mm B-12 is fed to vitamin B-12-deficient reticulocytes, theytake up less vitamin B-12 than do vitamin B-12-normal reticu-

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locytes, suggesting that TCII receptors are down-regulated byhigher concentrations of apoprotein TCII (apoTCIl) (21). Suchhigher concentrations of apoTCIl occur when there is not enoughvitamin B-12 to create hoIoTCII.

The diagnostic deoxyuridine suppression test

Our diagnostic deoxyuridine suppression test, which uses ci-ther whole blood (57) or bone marrow (12), is exceptionally use-ful for diagnosing early deficiency of vitamin B-12 or folate orboth. In the Addison book (20), Carmel and Wickramasinghewrite about how valuable this diagnostic test has been to theirresearch. Carmel also confirms our staging ofvitamin B-12 statusin which (Fig 1) the results of the deoxyuridine suppression testbecome abnormal before either methylmalonate or homocysteineconcentrations rise in early vitamin B-12 deficiency (29). In dis-cussing negative vitamin B-12 balance, Carmel uses the word‘ ‘subtle” as a synonym for the words ‘ ‘early” and “mild”(58-60).

In the Addison book (20), Jack Metz reviewed the work donein our laboratory in creating the diagnostic deoxyuridine sup-

pression test, and confirmed our methylfolate trap hypothesis bydemonstrating that in the deoxyuridine suppression test, meth-ylfolate is unable to function as folate, ic, it is trapped in “met-abolically dead’ ‘ status in vitamin B-12 deficiency. Our diag-nostic test is sometimes erroneously referred to as the ‘ ‘ Killman

experiment. ‘ ‘ Killman had shown that, in vitro, deoxyuridine

suppressed incorporation of thymidine into DNA differently innormal and megaloblastic bone marrow cells, but never deter-mined if this could be corrected in vitro. We converted Lhis bio-chemical finding into a diagnostic test by adding the missingvitamin(s) to bone marrow in additional test tubes and showingthat what was corrected in the additional test tubes (vitamin B-12 or folate or both) was also corrected in the patient.

Diagnosing vitamin B-12 deficiency

Vitamin B-12 deficiency as a clinical disease usually manifestsmost prominently in blood and/or neurologic damage, asLindenbaum and Allen discuss n their papers in the Addisonbook (20). There may be only mild (subtle) blood damage, man-ifested by granulocyte hypersegmentation (58-60), which, as wefirst showed (54), is always accompanied by an abnormal resulton the deoxyuridine suppression test. We suspect the neurologicdamage may prove to be associated with increased homocysteine(6) and/or cobalamin analogues (61) in the brain, although ho-mocysteine may not yet be clearly elevated in the spinal fluid orcirculating blood.

HL Menckcn, a leading American journalist, said, ‘ ‘ For everycomplicated problem there is a simple solution-and it iswrong. ‘ ‘ Such is the case in the vitamin B-12 field, where manyare looking for the golden grail-a magic single test that will be‘ ‘the’ ‘ diagnostic vitamin B-12 status test. They talk in terms ofa test that will be the gold standard. No test is a gold standardfor any other test. Each test is a gold standard only for itself (62).What is normal for one is not normal for another (59).

Elevated serum concentrations of methylmalonate or homo-cysteine diagnose biochemical damage that appears later than thebiochemical damage that produces an abnormal result on thedeoxyuridine suppression test. In every case where vitamin B-12deficiency produced elevated methylmalonate or elevated ho-mocysteine, the results of the deoxyuridine suppression test were

abnormal and were corrected to normal by the addition of vitaminB-12 (2, 29, 58, 60). Because hypersegmented granulocytes andmacroovalocytic red cells are the morphologic expression of thedamaged DNA synthesis that is biochemically expressed in thedeoxyuridine suppression test, hypersegmentation (and macroov-alocytes) also precede elevated concentrations of methylmalon-ate or homocysteine (2). For practical purposes in mass screeningfor deficiency, examining methylmalonate and homocysteineconcentrations is currently preferable to the deoxyuridine sup-pression test (but not to assessing hypersegmentation on a bloodsmear) solely because these measurements are currently less la-bor-intensive than the diagnostic test and can be carried outweeks after the blood sample is drawn. As we noted three dcc-ades ago, if one centrifuges a blood sample and looks at the top(reticulocyte and youngest) portion of the red cells under themicroscope, as one finds macroovalocytic red cells at almost thesame time as one finds hypersegmentation, within 7 wk of onsetof negative folate balance, whether the balance is negative dueto a loss of folate absorption or due to a vitamin B-12 deficiencythat makes folate unavailable (2).

The diagnostic deoxyuridine suppression test is a gold standardonly for itself. It diagnoses not only whether there is damagedDNA synthesis, but also whether the damage is due to deficiencyof vitamin B-12, of folate, or of both. This kind of diagnosisallow specific, rather than shotgun, therapy. Measurements ofmethylmalonate and homocysteine are also gold standards onlyfor themselves.

Measurement of holoTCIl as a surrogate Schilling test

We casually call a measurement of holoTCIl a surrogate Schil-ling test (44) but it is in fact both more and less than that. Becauseof the very short half-life of holoTCil, malabsorption of vitaminB-12 rapidly produces a decrease in the amount of holoTCII (44).The vast majority of apoTCil is made in enterocytes in the ileum.Ileal surface receptors for the vitamin B-12-IF complex take upthe complex and internalize it in mobile vesiclcs that haul it tolysosomes, which split the vitamin B-12 from the IF. The vitaminB-12 is transported to apoTCII, with the resultant ho1oTCII cx-ported into the bloodstream for delivery to every DNA-synthe-sizing cell in the body; all such cells have surface receptors forholoTCil (13).

A holoTCIl concentration < 44.4 pmol/L (< 60 pg/ml) is di-agnostic of reduced vitamin B-12 absorption that produced neg-ative vitamin B-12 balance at the time the blood sample wastaken and nothing else (13, 14). If one takes three serial samplesat weekly intervals and all samples show concentrations ofholoTCl! that are declining or are < 44.4 pmol/L (< 60 pg/mI),then one knows that subnormal vitamin B-12 absorption has con-tinued over that time, and one should treat the patient with vita-mm B-12 while continuing to determine whether the subnormalabsorption is reversible. This involves delineating whether the

etiology is dietary lack, gastric atrophy, pancreatic disease, sprue,metformin, or something else in our detailed tabular lexicon (2,10). A single holoTCII concentration that is < 29.6 pmolIL(< 40 pg/ml) diagnoses that negative vitamin B-12 balance hasbeen present long enough that vitamin B-12 therapy is indicated.Vitamin B-12 on serum TCII is in equilibrium with vitamin B-12 in hematopoietic and neural cells, analogous to iron on serum

transferrin (2).

The other circulating vitamin B-12 binding protein, haptocor-rin, is a marker of the size of stores of vitamin B-12 in liver cells

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and other cells (mainly macrophages) that have a significantnumber of surface receptors for haptocorrin (13). That equilib-rium is proportional to the number of cell-surface receptors forholohaptocorrin. The main body store for vitamin B-12 is in theliver; liver cells have many receptors for haptocorrin. It is roughlycorrect to state that vitamin B-12 on serum haptocorrin is in equi-librium with body stores of vitamin B-12 (13), which is analo-gous to iron on serum ferritin and body stores of iron (19). Ofcourse, ferritin stores more of its ligand, being capable of binding4500 atoms of iron per ferritin molecule of450 kDa (45), whereasone haptocorrin unit binds only one unit of vitamin B-12. Thevitamin B-12 in liver cells is in fact mainly not on haptocorrmnbut on excess (ie, well above need) intracellular quantities ofvitamin B-12 apoenzymes. The normal liver cell is programmedto make vitamin B-12 apoenzymes in 10-fold the quantity itneeds (2, 6).

Because liver cells have surface receptors for haptocorrin aswell as for TCII, but cell-surface receptors on hematopoietic cellsand glial cells (and possibly Schwann cells) are only for holo-TCII, hematopoietic and nervous system cells may be in StageII! or IV negative balance (ie, deficiency) while liver cells areonly in Stage II (ie, depletion with no deficiency). Similarly, be-cause of factors such as genetic (and acquired) variation thatproduce different receptor quantity and functionality in differentcell lines, as well as because of different internal processing ofholoTCil attached to cell-surface receptors that have been inter-nalized, some patients may have more neurologic damage thanhematopoietic damage and vice versa.

Folate slows vitamin B-12 deficiency blood damageprogression

Our studies in 1985-1989, 1964-1970, and 1977-1993, atMount Sinai, which has separate public and private wards thatreflect lower and higher economic status, all indicated that majormyclin synthesis damage that is due to vitamin B-12 deficiencywith only minor (ie, abnormal diagnostic deoxyuridine suppres-sion test and granulocyte hypersegmentation) hematopoieticdamage reflects better folate status because of higher economicstatus, which allows the purchase of fresh uncooked high-folatefruits and fruit juices in the colder months (September-April)(63). Folate per se does not protect myelin from vitamin B-12deficiency damage progression. We also found generally higherred cell folate in people with greater myelin damage that onlyvitamin B-12 deficiency produces, than in people with greaterhematopoictic damage, which either deficiency produces.

Detection of vitamin B-12 deficiency in elderly people

Serum holoTCIl should be measured every 5 y starting at age55 (55) because gradual loss of the ability to absorb vitamin B-12 occurs in everyone in a genetically determined, age-dependentpattern. Low concentrations of ho1oTCII are both a surrogateSchilling test (13, 44) and a measure of inadequate vitamin B-12delivery to cells that are synthesizing DNA (13). Low concen-trations of holoTCII occur before low concentrations of total se-rum vitamin B-12 or before deficiency (2) (Fig 1). MeasuringholoTCIl allows a monthly vitamin B-12 injection to be given toprevent deficiency from ever occurring. Health plans should un-derwrite these tests because abnormal results will trigger vitamin

B- 1 2 therapy to prevent early negative balance progressing toclinical harm, thereby saving billions of health care dollars (55).

Marcus et al (52) were the first to publish that negative vitaminB-12 balance was common in a large group of elderly people.They determined this by measuring holoTCIl. Subsequently,Pennypacker et al (64) screened 152 Veterans’ Affairs outpa-tients in Denver aged 65-99 y for Stage III-IV negative balanceby looking only for elevated serum methylmalonate and homo-cysteine and found negative balance in 14.5% of those studied.Based on earlier work by Marcus et al (52), it can be determinedthat at least an equal number of the subjects studied by Penny-packer et al (64) were probably in Stages I to II of negativevitamin B-12 balance and would have been found if the inves-tigators had assayed the same 152 sera for holoTCIl as didMarcus et al (52).

Supporting this probability is that by screening for low con-

centrations of holoTCII, we found negative vitamin B-12 balance(Stages I-IV) in 35% of 150 Veterans’ Affairs outpatients inBronx, NY, aged 65-95 y. Negative vitamin B-12 balance wasalso found in 37 of 100 upstate New York outpatient seniors,where the definition of negative balance was a serum vitamin B-12 concentration < 222 pmol/L (< 300 pg/ml) (56).Guzik et al (52) found negative vitamin B-12 balance as de-

termined by holoTCIl concentrations < 44.4 pmolfL (< 60 pg/ml) common among both well-fed (15%) and malnourished(36%) nursing home residents, but if one just looked at totalserum vitamin B-12, the negative balance was often concealedby elevated concentrations of total serum vitamin B-12 that weredue to elevated holohaptocorrin, perhaps secondary to liver dis-ease.

Circulating antibody to IF was widely used in the 1960s as ascreening test for vitamin B-12 malabsorption produced by gas-tric damage (47). This test has faded from use, but we recentlylooked for the circulating antibody and found it in the serum ofAIDS patients with vitamin B-12 malabsorption, despite theirnormal serum total cobalamin concentrations (65). We hope tosee circulating antibody to intrinsic factor being measured againfor screening in the elderly (63) and in nonelderly vegetarianswith chronic iron deficiency, which damages the gastric mucosa.However, chronic vitamin B-12 deficiency damages immunefunction (66), so circulating antibody may disappear as vitamin

B-12 deficiency progresses (47).Just as iron deficiency damages esophageal and gastric mucosa

and promotes esophageal cancer (67), so does vitamin B-12 de-ficiency (67, 68). Being a vegetarian gave no protection againstthe occurrence of esophageal cancer that was related to deficien-cies of vitamin B-12, vitamin E, or vitamin A and /3-carotene invegetarian Chinese people (67-69). El

References

1. Herbert V. Vitamin B-12: plant sources, requirements, and assay.Am J Clin Nutr 1988;48:852-8.

2. Herbert V, Das KS. Folic acid and vitamin B12. In: Sbus ME, OlsonJA, Shike M, eds. Modern nutrition in health and disease. 8th ed.Baltimore: Lea & Febiger, 1994:402-25.

3. Herbert V, Subak-Sharpe GJ, Hammock D, eds. The Mount SinaiSchool of Medicine complete book of nutrition. New York: StMartin’s Press, 1990.

4. Simopoulos A, Herbert V, Jacobson B. Genetic nutrition: designinga diet based on your family medical history. New York: Macmillan,1993.

5. Herbert V, Cohen L, Stopler T, Ness R, Handelsman L. Rediscov-ering selective nutrient deficiency in one cell line but not another:

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low serum holotranscobalamin II (holo-TCII) may identify negativevitamin B12 balance only in cells (cx: hematopoietic) with surfacereceptors solely for TCII, and not in cells (cx: liver) with receptorsalso for haptocorrin: in AIDS, there may be stage IV negative B12balance in hematopoietic and neuropsychiatric cells with liver cellsonly in stage I-Il. Clin Res 1990;38:23A(abstr).

6. Herbert V. Vitamin Bl2. In: Brown ML, ed. Present knowledge innutrition. Washington, DC: International Life Sciences Institute/Nu-trition Foundation, 1990:170-8.

7. Herbert V. Viewpoint: does mega-C do more good than harm, ormore harm than good? Nutr Today 1993;28(1):28-32.

8. Herbert V, Drivas G, Foscaldi R, et al. Multi-vitamin/mineral foodsupplements containing vitamin B12 may also contain analogues ofvitamin B12. N Engl J Med 1982;307:255-6.

9. Kanazawa S & Herbert V. Total corrmnoid, cobalamin (vitamin B12),and cobalamin analogue levels may be normal in serum despite co-balamin depletion in liver in patients with alcoholism. Lab Invest1985;53: 108-10.

10. Herbert V. Das KC. Anemias due to nuclear maturation defects(megaloblastic anemias). In: Hurst JW, ed. Medicine for the prac-ticing physician. 3rd ed. Boston: Butterworth-Heinemann, 1992:851-7.

1 1 . Herbert V. Staging nutrient status from too little to too much byappropriate laboratory tests. In: Livingston GE, ed. Nutritional statusassessment of the individual. Trumbull, C!’: Food & Nutrition PressInc. 1989: 147-67.

12. Herbert V. Megaloblastic anemias. Lab Invest 1984;52:3-19.13. Herbert V, Fong W, Gulle V, Stopler T. Low holotranscobalamin II

is the earliest serum marker for subnormal vitamin B12 (cobalamin)absorption in patients with AIDS. Am J Hematol 1990;34:132-9.

14. Herzlich B, Herbert V. Depletion of serum holotranscobalamin II:an early sign of negative vitamin B12 balance. L<a Invest1988;58:332-7.

15. Herbert V. Assay for vitamin B12 deficiency: vitamin B12 defi-ciency may be determined prior to the onset of anemia and/or nervedamage by determining a decrease in vitamin B12 carried by TCII.1987. United States Patent # 4,680,273.

16. Wickramasinge SN, Fida S. Correlations between holo-transcobala-mm II, holo-haptocorrmn, and total B12 in serum samples fromhealthy subjects and patients. J Clin Path 1993;46:537-9.

17. Allen TH. Cobalamin (vitamin B-12) absorption and malabsorption.Viewpoints Digest Dis 1982;l4:17-20.

18. Shevchuk 0, Huebscher T, Herbert V. Evidence that vitamin B12regulates synthesis of proteins involved in corrinoid metabolism.FASEB J l988;2(5):Al086(abstr).

19. Herbert V. Everyone should be tested for iron disorders. J Am DietAssoc 1992;92:1502-9.

20. Bhatt HR. James VHT, Besser GM, Bottazzo GF, Keen H, eds. Ad-vances in Thomas Addison’s diseases. Vols 1 and 2. Bristol, UnitedKingdom: Journal of Endocrinology Ltd, 1994.

21. Retief FP, Gottlieb CW, Herbert V. Delivery of Co57B12 to eryth-rocytes from alpha and beta globulin of normal, Bl2-deficient, andchronic myeloid leukemia serum. Blood 1967;29:837-51.

22. Das KC, Manusselis C, Herbert V. Determination of vitamin B12(cobalamin) in serum and erythrocytes by radioassay, and holo-transcobalamin II (holo-TC II) and holo-haptocorrin (holo-TCI andII) in serum by adsorbing holo-TC II on microfine silica. J NutrBiochem 1991;2:455-64.

23. Tisman G, Vu T, Amin J, et al. Measurement of red blood cellvitamin B12: a study of the correlation between intracellular Bl2content and concentrations of plasma holotranscobalamin II. Am JHemat 1993;43:226-9.

24. Craig WJ. Iron status of vegetarians. Am J Clin Nutr 1994;59(suppl): 1233S-7S.

25. Herbert V. Everyone should be tested for iron disorders. J Am DietAssoc l992;92: 1502-9.

26. Albert MJ, Mathan VI, Baker Si. Vitamin B12 synthesis by humansmall intestinal bacteria. Nature l980;283:781 -2.

27. Kanazawa 5, Herbert V. Mechanism of enterohepatic circulation ofvitamin B12: movement of vitamin B12 from bile R-binder to in-

trinsic factor due to the action of pancreatic trypsin. Trans AssocAm Physicians l983;96:336-44.

28. Sullivan LW, Herbert V, Castle WB. In vitro assay for human in-trinsic factor. J Clin Invest 1963;42:1443-58.

29. Herbert V. Cobalamin deficiency and neuropsychiatric disorders. N

Engl I Med 1988;319:1733(letter).30. Herbert V. B12 deficiency in AIDS. JAMA l988;260:2837(letter).31. Herbert V. Folate and neural tube defects. Nutr Today 1992;

27(6):30-3.32. Herbert V. Vitamin B12 deficiency neuropsychiatric damage in ac-

quired immunodeficiency syndrome. Arch Neurol l993;50:569(letter).

33. Herzlich BC, Ranginwala M, Nawabi I, Herbert V. Synergy of in-hibition of DNA synthesis in human bone marrow by azidothymi-dine plus deficiency of folate and/or vitamin B12? Am J Hematol1990;33: 177-83.

34. Jacobsen DW, Green R, Herbert V, Longworth DL, Rehm S.Decreased serum glutathione with normal cysteine and homo-cysteine levels in patients with AIDS. Clin Res 1990;38(2):556A(abstr).

35. Herbert V, Stopler-Kasdan T. Can genetic nutrition be used topredict and prevent heart attacks? The Mount Sinai heart at-tack prediction and prevention profile. Clin Res 1993;41:397A(abstr).

36. National Research Council. Recommended dietary allowances.10th ed. Washington, DC: National Academy Press, 1989.

37. Stabler SP, Allen RH, Savage DG, Lindenbaum J. Clinical spec-trum and diagnosis of cobalamin deficiency. Blood 1990;76:871-81.

38. Herbert V. Recommended dietary intakes (RDI)ofvitamin B12. AmJ Clin Nutr 1987;45:671-8.

39. Herbert V. The 1989 RDA is mainly the work ofthe 1980-85 (10th)RDA Committee, but with 9th RDA numbers for vitamins A and C.FASEB J 1990;4:A374(abstr).

40. Carmel R, Rosenberg AH, Lau KS, Streiff RR, Herbert V. VitaminB12 uptake by human small bowel homogenate and its enhancementby intrinsic factor. Gastroenterol 1969;56:548-55.

41. Herzlich B, Herbert V. The role of pancreas in cobalamin (vitaminB12) absorption. Am J Gastroenterol l984;79:489-93.

42. Herzlich B, Schiano T, Moussa, Zimbalist E, Panagopoulos G,Nawabi I. Decreased intrinsic factor secretion in AIDS: relation toparietal cell acid secretory capacity and vitamin B12 malabsorption.Am J Gastroenterol 1986;87: 1781-8.

43. Herzlich B, Herbert V. Rapid collection of human intrinsic factoruncontaminated with cobalophilin (R binder). Am J Gastroenterol1986;81:678-80.

44. Shaw 5, Jayatilleke E, Bauman W, Herbert V. Mechanism of Bl2malabsorption and depletion due to metformin discovered by usingserial serum holo-transcobalamin II (bob TCII) (Bl 12 on TCII) asa surrogate for serial Schilling tests. Blood 1993;82 (10, suppl1):432A(abstr).

45. Herbert V. Shaw S, Jayatilleke E, Stopler-Kasdan T. Most free-rad-ical injury is iron-related: it is promoted by iron, hemin, holoferritin,and vitamin C, and inhibited by desferrioxamine and apoferritin. In:Abraham NO, Shadduck RK, Levine AS, Takaku F, eds. Molecularbiology of hematopoiesis. Vol 3. Andover, UK: Intercept Press,1994.

46. Shaw 5, Herbert V, Colman N, Jayatilleke E. Effect of ethanol-generated free radicals on gastric intrinsic factor and glutathione.Alcohol 1990;7: 153-7.

47. Herbert V. Immunologic factors in pernicious anemia. Postgrad Med1967;42:298-303.

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48. Herbert V, Stopler T, Huebscher T. Nasal vitamin B12 gel may notbe a reliable alternative to injectable vitamin B12; both bind pref-erentially to TCII and produce analogue increment just on TCII.Blood 1987;70(suppl):45a(abstr).

49. Goh YT, Jacobsen DW, Green R, et al. Diagnosis of functional co-balamin deficiency: utility of transcobalamin Il-bound vitamin B12determination in conjunction with total serum homocysteinc andmethylmalonic acid. Blood 1991;78(suppl 1):100A(abstr).

50. Amin I, Vu T, Bateman R, et al. Measurement of red cell B12 andholoTCIl levels: the key to the evaluation of true B12 deficiency?Blood 1992;80:381A.

51. Herbert V. Megaloblastic anemia in China. Ann Intern Med1982;97: 139-40.

52. Marcus DL, Shaddick N, Crampz J, Gray M, Hcrnandez F,Freedman LM. Trancobalamin II levels in a hematologically normalelderly population. J Am Geriatr Soc 1987;35:635-8.

53. Guzik Hi, Tommasulo BC, Mandel FD, Kasdan TS, Herbert V.Prevalence of negative vitamin B 12 balance in well and malnour-ished frail elderly. J Am Geriatr Soc 1993;41(10):SA27(abstr).

54. Herbert V. The 1986 Herman Award Lecture: nutrition science as acontinually unfolding story: the folate and vitamin B12 paradigm.Am J Clin Nutr 1986;6:387-402.

55. Herbert V, Rudick A. Billions will be saved by assessing iron statusin all Americans, folate status in all fertile females, and vitamin B12status in all after age 55. FASEB I 1993;7:A412(abstr).

56. Yao Y. Low serum vitamin B12 in seniors. J Fam Pract1992;35:524-9.

57. Das KC, Asiz MA, Colman N, Manusselis C, Herbert V. Eliminatingfalse positive and false negative results in lymphocyte diagnostic dUsuppression test (Dx dUST) for folate deficiency by recognizing dif-ferent ‘ ‘normal’ ‘ individuals have different folate stores. Blood1991;78(Suppl 10):99a(abstr).

58. Herbert V. Don’t ignore low serum cobalamin (vitamin B12) levels.Arch Intern Med 1988;148:1705-7.

59. Herbert V, Memoli D, McAleer E, Colman N. What is normal? Van-ation from the individual’s norm for granulocyte ‘ ‘lobe average”

and holo-transcobalamin II (Holo-TC II) diagnoses vitamin B12 de-ficiency before variation from the laboratory norm. Clin Res1986;342:718A(abstn).

60. Carmel R. Reversal by cobalamin therapy of minimal defects in thedeoxyunidine suppression test in patients without anemia: furtherevidence for a subtle metabolic cobalamin deficiency. I Lab ClinMed 1992;119:240-4.

61. Herbert V, Memoli D, March R, et al. Vitamin B12 analogue levelstend to rise as cobalamin deficiency develops with cessation of then-apy in pernicious anemia. Blood 1986;68:46a(abstr).

62. Herbert V. Colman N, Palat D, et at. Is there a ‘ ‘gold standard’ ‘ forhuman serum vitamin B12 assay? J Lab Clin Med 1984;104:829-41.

63. Herbert V. Nutritional anemias in the elderly. In: Pninsley DM,Sandstead HH, eds. Nutrition and aging. New York: Alan R LissInc. 1990:203-27.

64. Pennypacker LC, Allen RH, Kelly JP, et al. High prevalence of co-balamin deficiency in elderly outpatients. I Am Geniatr Soc1992;40:1 197-204.

65. Herbert V, Shaw 5, Jayatilleke E, Lam P, Gulle V. Evidence inserum for food vitamin B12 (cobalamin) malabsorption in AIDS:high gastrin, low cobalamin on transcobalamin II, and circulatingantibody to intrinsic factor despite normal serum total cobalaminlevels. Clin Res 1990;38:361A(abstr).

66. Ran JY, Li XF, Rau HL, Wang LY, Herbert V. Reduced immuno-logic function in folate and/or B12 deficiency megaloblastic anemia(MA) and in vegetarians in China. Blood 1990;76(l0, suppl1):217a(abstr).

67. Herbert V. Nutrient deficiency and esophageal cancer in China. CNINutrition Week; 23 (October 29, 1993):6.

68. Ran JY, Dou P. Wang LY, Qin Y, Li XF, Herbert V. Correlation oflow serum folate and total B12 with high incidence of esophagealcarcinoma (EC) in Shanxi, China. Blood 1993;82(suppl 1):532a(abstr).

69. Herbert V. Diet and cancer prevention. Natl Council Against HealthFraud News 1992;15(3):3.

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