Vitamin B12: Plant Sources, Requirements, And Assay - AmJClinNutr 1988

Vitamin B-i 2: plant sources, requirements, and assay

852 Am J C/in Nuir l988;48:852-8. Printed in USA. © 1988 American Society for Clinical Nutrition

Victor Herbert, MD, JD

ABSTRACF Vitamin B-l2 is of singular interest in any discussion of vegetarian diets be-cause this vitamin is not found in plant foods as are other vitamins. Many ofthe papers in theliterature give values of vitamin B-12 in food that are false because as much as 80% of theactivity by this method is due to inactive analogues of vitamin B-l2. Am J Clin Nuirl988;48:852-8

KEY WORDS Vitamin B-l2, vitamin B-l2 analogues, corrinoids, cyanocobalamin,

vegans

Introduction

Vitamin B-l2 is of singular interest in any discussionof vegetarian diets because this vitamin is not found inplant foods as are other vitamins. Confusion about whatsources may yield vitamin B-b2 to strict vegetarians hasarisen because the standard US Pharmacopeia (USP) as-say for vitamin B-l2 does not assay only vitamin B-12(1). In the USP method the content of vitamin B-l2 ofany given food is determined by making a water extractofthat food and feeding the extract to a bacterium (Lac-tobacillus leichmannii). The quantity ofvitamin B-l2 isdetermined by the amount of bacterial growth. Theproblem is that what is active vitamin B-b2 for bacteriais not necessarily active vitamin B-12 for humans (1-6).Many of the papers in the literature give values of vita-

mm B-l2 in food that are false because as much as 80%ofthe activity by this method is due to inactive analoguesof vitamin B-l2. In this paper we review the origins ofvitamin B-l2 and its analogues, the effect of vitamin B-12 structure on absorption, assay methods for the vita-mm, and issues relating to the requirements for thevitamin.

Origins ofvitamin B-12

There is no active vitamin B-12 in anything that growsout ofthe ground; storage vitamin B-l2 is found only inanimal products where it is ubiquitous and where it isultimately derived from bacteria (1, 2, 4). All the vitaminB-b2 in plants is there fortuitously in bacteria contami-nating the food. That contamination is usually on theoutside of the plant but occasionally is internal. For cx-ample, in certain pulses in India in the nodules and onthe root some bacteria ofthe rhizobium species grow andproduce small amounts ofvitamin B-l2. They also pro-duce analogues ofvitamin B-b2 (1, 4).

The more frequent source of vitamin B-l2 in associa-

tion with plant food is external contamination with bac-teria, often of fecal origin. In one of the less appetizingbut more brilliant experiments in the field of vitamin B-12 metabolism in the 50s, Sheila Callender (7) in En-gland delineated that human colon bacteria make largeamounts of vitamin B-b2. Although the bacterial vita-mm B-l2 is not absorbed through the colon, it is activefor humans. Callender studied vegan volunteers who hadvitamin B-l2 deficiency disease characterized by classicmegalobbastic anemia. She collected 24-h stools, madewater extracts ofthem, and fed the extract to the patients,thereby curing their vitamin B-l2 deficiency. This exper-iment demonstrated clearly that 1) colon bacteria of veg-ans make enough vitamin B-l2 to cure vitamin B-12 de-ficiency, 2) the vitamin B-b2 is not absorbed through thecolon wall, and 3) if given by mouth, it is absorbed pri-marily in the small bowel. Vitamin B-12 is one of thosefew nutrients absorbed primarily from the lower half ofthe small bowel (3, 4, 6).

Structure of vitamin B-12 and analogues

The structure of the vitamin B-12 molecule is shownin Figure 1. This molecule (cobalamin) consists of fourbasic parts, the core of which is almost identical to theheme ofhemogbobin, suggesting ontogenic developmentfrom the same precursor. This core structure (corrin)differs from heme in only two things: the attached metalin corrin is cobalt (it is iron in heme) and one ofthe alphamethene bridges(there are four in heme) is missing; thereare only three alpha methene bridges in the corrin nu-cleus. The corrin nucleus is the central structure of allthe corrinoids.

Corrinoids are cobalt-containing cyclic structures in

� From the Mt Sinai School ofMedicine, New York, New York.2 Reprints not available.

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(Factor Viol

VITAMIN B-12: SOURCES AND REQUIREMENTS 853

FIG 1. The structural formula ofvitamin B-l2. From reference 1.

the human body, in foods of animal origin (meat, poul-try, eggs, fish, milk, and milk products), and in the bacte-ria, which make not only vitamin B-b2 but also variousanalogues thereof (1-6). Vitamin B-l2 and all its ana-bogues are corrinoids. The human uses as vitamin B-b2only those corrin nuclei to which are added the threeother basic parts ofthe cobalamin molecule: aminopro-panol, sugar, and a nucleotide (5). To use it as a vitamin,the human cell must see it as depicted in Figure 1, ie, asa cobalamin with no alterations except in the R adduct.Cobalamins remain vitamin active for humans with avariety of R adducts, which are named cobalamin withwhatever is attached to cobalt as a prefix. Thus, we havehydroxocobalamin (Fig 1), aquocobalamin, 5’-deoxy-adenosylcobalamin, methylcobalamin, and cyanocobal-amin among the naturally occurring human-active andpotentially human-active forms ofvitamin B-12 in van-ous foods.

One can sequentially remove parts of the vitamin B-12 molecule, add side chains, or alter it in other ways. Insuch cases it ceases to be a cobalamin and thus is not avitamin for humans. However, it may remain a corn-noid vitamin for one or more ofa wide variety of simplerlife forms, such as algae and bacteria, which need onlythe corrin nucleus for vitamin activity in contrast to hu-mans who need the entire cobalamin structure for vita-mm B-12 activity. As noted, in addition to cobalamin,bacteria synthesize a number of vitamin B-12 analoguesthat are noncobalamin comnoids and therefore not vita-mm active for humans.

Cyanocobalamin is stable but not vitamin active

Cyanocobalamin is the form in most pharmaceuticalpreparations because adding cyanide stabilizes the mole-cube. This was accidentally learned when vitamin B-l2was first isolated in the eluate from charcoal columns (4).The fortuitous reason that the beautiful red crystallinecobalamin structure came out of the charcoal columnsintact, but not out of other columns, was because the

charcoal columns contained cyanide, which exchangedwith the naturally present adduct groups attached to thecobalt. The cyanide stabilized the vitamin B-12 mole-cube, which otherwise is so unstable that exposure to lightalone can destroy it.

Cyanocobalamin is not vitamin active for humans un-til the cyanide is removed within the body. This fact isdramatically illustrated in the rare infant born with a de-fect in the ability to enzymatically remove cyanide fromvarious substances. Such infants are unable to use cyano-cobalamin as vitamin B-12 because they cannot removethe cyanide from it (8). In fact, such infants, when theyhave a vitamin B-l2 deficiency, are made worse by givingthem cyanocobalamin because for them it acts as an anti-metabolite. This was demonstrated by Rosenblatt andhis group at Yale Medical School (8) in studies of chib-dren with genetic defects in vitamin B-l2 metabolism.

Differential radioassay

How does one, then, differentiate the nonvitamin ana-bogues from the true B-12, which is vitamin active forhumans, because it cannot be done by microbiologic as-say? This is done by differential radioassay (1, 3, 6). Themixture of vitamin B-l2 and vitamin B-l2 analogues isassayed for the total content ofcorrinoids (ie, total B-12)by using a binder that attaches primarily to the cornnnucleus. Such a binder is ubiquitous in human and ani-mal tissues; it is a heterogenous glycoprotein called R (forrapid mobility on electrophoresis) binder. It is also calledtranscobalamin 1+111 (TC 1+111), haptocorrin, or coba-bophilin (4, 6). R binder attaches only to the corrin nu-cleus and thereby measures the total number of corrinnuclei (true B-l2 plus noncobalamin analogues). Thenthe vitamin B-12 active for humans (ie, cobalamin) isassayed by using a substance which attaches to both endsofthe cobalamin molecule, the corrin end and the nude-otide end. The substance that accomplishes this is intrin-sic factor (IF), first discovered by William Castle at Ha-yard and the molecule that makes it possible to absorbfree vitamin B-b2. This gastric parietal-cell secretion is aglycoprotein that attaches specifically to vitamin B-b2with a high affinity coefficient but does not attach to ana-bogues of vitamin B-l2 (9). When we use IF as a binder,we essentially measure only the cobalamins in the mix-ture.

By subtracting the value for cobalamin determined us-ing pure IF(true B-l2) from the value for total corrinoidsdetermined by using R binder (total B-l2), we determinethe amount of analogues by difference, hence the termdifferential radioassay of analogues.

(noncobalamin) analogues = total B-l2

-trueB-l2 (1)

Fecal contamination as a vitamin B-12 source in vegans

When we apply differential radioassay to human stool,we find that an enormous amount of vitamin B-l2 in

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854 HERBERT

TABLE 1

Cobalamin and analogue levels in 6-d 200 C-dehydrated feces collections from six men

Patient

Feces 8-12 (pgJ24 h)*

Folate (�g/24 h):L caseiL. /eichmanniit

Radioassay

Corrimoids Cobalamins Analogues

1 32.9 52.9 1.7 51.2 94.5

2 37.36 70.3 1.1 69.2 61.3

3 31.4 103.3 1.6 101.7 150.7

4 12.5 29.8 2.1 27.7 230.75 72.9 1 14.8 12.8 102.0 576.7

6 28.1 46.3 0.6 45.7 56.8i±SEM(�g/24h) 35.6 ±20.0 69.5 ±13.6 3.31 ±1.08 66.25±12.5 195.11±80.8j:� SEM (ILg/g) 1.83 ± 0.52 3.63 ± 0.33 0.159 ± 0.068 3.47 ± 0.32 10.53 ± 3.66

* The dehydration process destroys approximately one-third each of cobalamim and analogues as compared with fresh (refrigerated) feces.

Therefore, actual content is 150% ofthe values in the above table. Reprinted from reference 1.

t Not alkali boiled. Boiling in alkali destroys -�-85% ofstool growth activity for L leichmannii(ie, -.‘85% is 8-12 by L /eichmannii assay).

human stool is from analogues (Table 1). In this process24-h stools are collected over 6 d and dehydrated downto a few ounces of powder. These results represent two-thirds ofthe vitamin B-12 and analogue content becauseapproximately one-third ofeach is destroyed in the dehy-dration procedure. Correcting for this loss, we find thatnormal, 24-h human stool output contains -� 100 �g oftotal B-12 (vitamin B-12 plus analogues) of which only,-,.,5 �g is cobalamin (vitamin B-l2-active for humans)and 95% are various analogues (1).

From Callender’s work we know that a water extractof stool will correct human vitamin B-b2 deficiency.Therefore, although about 19 out of 20 B-l2 moleculesin the stool are not active vitamin B-12, these analoguesdo not block the absorption of that one vitamin B-l2molecule when gastric intrinsic factor secretion is nor-mal. However, some analogues do compete with vitaminB-l2 for absorption and may block residual vitamin B-12 absorption when it is already impaired (3, 10).

The fact that stool vitamin B-l2 can be important inhuman vitamin B-l2 economy was delineated by JamesHalsted (1 1) working with Iranian vegans who did notget vitamin B-b2 deficiency. It was difficult to under-stand why these people, who were strict vegetarians (veg-ans) for religious reasons, did not get vitamin B-l2 defi-ciency. Halsted went to Iran and found that they grewtheir vegetables in night soi/(human manure). The vege-tables were eaten without being carefully washed and theamount of retained vitamin B-l2 from the manure-richsoil was adequate to prevent vitamin 8-12 deficiency.Thus, strict vegetarians who do not practice thoroughhand washing or vegetable cleaning may be untroubledby vitamin B-l2 deficiency.

Limitations of the standard Schilling test

As we get older we gradually develop, on a geneticallydetermined basis, gastric atrophy. About 1 person in 100has vitamin B-l2 deficiency through gastric atrophy by

age 60. We have calculated that everybody in the UnitedStates will develop vitamin B-12 deficiency by age 127;it will be difficult to prove us wrong!

The sequence ofevents in developing vitamin B-l2 de-ficiency is indicated in Figure 2 (12). Long before gastricIF is lost we lose our gastric acid and gastric digestiveenzyme secretion and the ability to absorb vitamin B-12from foods. This is because vitamin B-l2 is peptidebound in milk and all other foods. To be absorbed, thevitamin must first be cleaved from its peptide bonds.This cleavage is brought about by gastric acid and diges-tive enzymes.

Negative vitamin B-l2 balance characterized by theinability to absorb vitamin 8-12 from food can be diag-nosed by a food Schilling test (ie, vitamin B-l2 in an om-elet is not absorbed). Doscherholmen (3, 13) showed thatthe inability to absorb vitamin B-b2 from food can occurin a 1-3 y period during which crystalline vitamin B-l2is still normally absorbed (ie, the standard Schilhing testgives normal results). This occurs because there is stillsubstantial IF secretion but the gastric acid and enzymesecretion has been lost. It only takes -‘�-20% ofnormal IFsecretion for normal absorption of0.5-l.5 �g vitamin B-12(3,6).

B-12 produced by intestinal bacteria

What is the role of intestinal bacteria above the colonin vitamin B-l2 absorption? We have seen that the 5 igof vitamin B-l2 made by colon bacteria per 24 h is oflittle, ifany, value to individuals unless they ingest someof their own feces because vitamin B-12 is not absorbedacross the colon mucosa. If one takes gastric aspiratesfrom humans and looks for quantities ofviable bacteria,one finds that as the gastric pH becomes closer to neutralthe quantities of bacteria gradually increase (1). In thenormal, healthy, acid-secreting stomach, there are veryfew bacteria. As we grow older and our gastric acid secre-tion decreases, gradually more bacteria grow in our

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Stage:

Uv� B,2

HOIOTC II

RBC+WBC B,2

VITAMIN B-l2: SOURCES AND REQUIREMENTS 855

<2%

1<150 1dU Suppression Normal Normal Normal Normal Normal

<1%

<100

Abnormal

Yb

<10%

<10%

<100

Mwoovalocytlc

EI#{149}vatsd

Low

Elsvatsd

Yb

Friquint

L -I/----

9-

08-

� +<: � � � � � � � I

C�strcGastric pH oChiOrhydro

FIG 3. Increasing stomach bacteria with decreasing gastric pH.From reference 14.

5

Duodenum Je�unum � Itsum

Lscloboc.II. S*’�co�’ �cwrs (,w,obw�.�o (Mswbscwb Dw*1*o bhdsbicIs,w

FIG 4. Flora ofthe small intestine. From reference 14.

Excess Positive B,2 Negative B,2 B,2 B,2 -Deficient B,2 -DeficiencyBalance Anemia�uz�nE�es4 _Norm� Balance

H�IoTCII(p�n�) >50 >40 >30 � � � ‘�#{176} <12 <12

TCII%sat. >5% >5% >5% l_<5s I _______Haloh�(pg/M) >500 >300 >150 >150

<1%

<100

Abnormal

Hyp.rs.gmentation No No No No No Yb

TBnI��t%$at. >50% >40 >15% >15% >15% <15%

H� % s�. >50% >40 >20% >20% >20% <20%

RBCFolate(ng/mI) >160 >160 >160 >160 >160 <140

Er�ihrocyt.s Normal Normal Normal Normal Normal Normal

MCV Normal Normal Normal Normal Normal Normal

H.mo�ob� Normal Normal Normal Normal Normal Normal

TC II Normal Normal Normal Normal Normal Elevatsd

M.*ll4malonaI.+* No No No No No 1

MyalIn Damage No No No No No

FIG 2. Sequential stages ofvitamin B-12 status. sCyanocobalamin excesses (injected or intranasal) produce tram-

sient rise in B-l2 analogues on B-l2 delivery protein (TC II); the significances of rises is unknown. tTBBC, total

B-l2 binding capacity. tIn serum and urine. From reference 12.

stomachs and upper small bowel(Fig 3)(1). This is a veryinteresting phenomenon and we need to explore whetherthese bacteria release any unbound vitamin B-b 2.

The average American omnivore or vegetarian who isnot a vegan get vitamin B-l2 from food in which the vita-mm B-b2 is peptide bound. There is adequate vitaminB-12 in milk or milk products for the needs ofany personwith normal gastric, pancreatic, and intestinal secretionsand functions. However, as noted, a negative vitamin B-12 balance may result when those secretions are de-creased. Thus the potential contribution of gastric andsmall intestine bacteria to overall vitamin B-b2 nutritureis of interest.

There is normal distribution of viable bacteria in thesmall intestine and the quantity ofbacteria increases pro-gressiveby (Fig 4) (1) down the small intestine to the cc-

cum where we have the highest colony count before thecolon. Of particular importance may be bacteroides,which are present in the upper halfofthe small intestineand which make both vitamin B-l2 and analogues. Al-bert, Mathan, and Baker(1 5) found that Lactobaciii, thestreptococci, the bacteroides, and other enteral bacteriain the small intestine made primarily vitamin B-12.However, their studies used microbiologic assays withorganisms that grow on some noncobalamin comnoids.It is thus uncertain how much ofthose bacterial productswere cobalamin rather than noncobalamin comnoids.

Enterohepatic circulation of vitamin B-i 2

The enterohepatic circulation of vitamin B-12 is ofcrucial importance in human vitamin B-12 economyparticularly for vegetarians (4, 6). The reason is that any-where from 1 to 10 �ig of vitamin B-l2 is secreted in the

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FIG 5. The ileal vitamin B-12-intrinsic factor(IF) receptors.

856 HERBERT

bile each day. Nobody needs > 1 jzg vitamin B-b2/d. Wenormally reabsorb much of the vitamin B-b2 in bile se-cretions. In addition, enterohepatic circulation has theeffect of removing unwanted analogues from the body,returning vitamin B-12 relatively free of analogues (3,16, 17).

The vegetarian often may be getting more vitamin B-12 by reabsorption from bile than from external foods.This would be true for those who eat very little animalprotein. The reabsorption of bile vitamin B-b2 explainswhy it takes ��-20 y to run out of vitamin B-12 and getvitamin B-b2 deficiency disease after one stops consum-ing dietary B-b2 but only 3 y to run out and get vitaminB-l2 deficiency disease if one stops absorbing the vita-mm (3, 4, 6).

The mechanism of vitamin B-i 2 absorption

In the average omnivorous American diet there are 5-1 5 �tg of vitamin B-12 (2). The food vitamin B-b2 has tobe removed from its peptide bonds in the food by prote-ases and acids in the stomach. When removed from food,it does not immediately attach to IF but rather to theubiquitous R binder, which has a higher affinity for corri-noids (including cobabamin) than does IF. Because we allregularly swallow our own saliva, and saliva is loadedwith R binder, the vitamin B-b2 split from peptides inour food attaches to R binder and not to IF. Similarly,the vitamin B-b2 secreted in bile (along with analogues)is attached to R binder. Vitamin B-12 cannot be ab-

sorbed or reabsorbed as long as it is attached to R binder.The pancreas secretes proteases which, at the slightly

alkaline pH of the upper intestine, selectively digest theR binder, releasing its vitamin B-12, which then for thefirst time is taken up by the dilute-alkali-resistant IF notin the acidic stomach but in the mildly alkaline uppersmall bowel. The vitamin B-12-IF complex then passes

down into the ileum, where it attaches to specific recep-tors for the vitamin B-b2-IF complex (Fig 5) and is thenabsorbed (9).

Pancreatic secretion not only digests the R binder andreleases the food vitamin B-b2 but also digests the Rbinder that comes out in the bile with vitamin B-l2 at-tached to it, thereby allowing that vitamin B-12 also tomigrate to IF and then be absorbed across the ileum (16).Thus, a healthy pancreas is ofcrucial importance in theabsorption ofvitamin B-b2 (17).

The ileal receptor is not just for IF but, as we showed25 y ago, is a key-and-lock receptor for the complex ofvitamin B-l2 and IF. That is a very important distinctionbecause the receptor is for the complex and there can besome absorption ofvitamin B-l2 (an incomplete key) inthe absence of IF and there is evidence that does in factoccur. This direct vitamin B-l2 absorption can beblocked by analogues and is an area of active researchright now (3). Shaw delineated that the main site of ab-sorption of analogues is in the ileum just as is the mainsite ofabsorption ofvitamin B-12 itself(18).

There are two separate mechanisms for vitamin B-12absorption: the IF-dependent physiologic mechanismand the mass-action pharmacologic mechanism whereby1% of any quantity of free vitamin B-b2 is absorbed bydiffusion across the ileum (Table 2) (1, 4, 6).

Needed dietary intake of vitamin B-i2

How much vitamin B-12 do we need? No more than1 �g daily (2). Figure 6 is the laboratory data ofan mdi-vidual, aged 54 y, with relatively early pernicious anemia(PA), the form of vitamin B-b2 deficiency disease that isdue to inadequate or absent secretion of gastric IF. Thisindividual, when given just 1 �g cyanocobabamin/d byinjection, had a beautiful hematobogic response with a

TABLE 2

Absorption of2 vs 30 �g oral cyanocobalamin without vs withintrinsic factor’

Subject

Vitamin B-l2 in 48-h urinet

After2��goral

cyanocobalamin

B-l2 B-l2+IF

After 30 �igoral

cyamocobalamin

B-12 B-l2+IF

I234

5

0.02 0.36

0.01 0. 170.01 0.35

0.02 0.14

0.01 0. 1 1

0.24 0.180.44 0.45

0.48 0.600.16 0.24

0. 12 0.27

Average 0.02 0.23 0.29 0.35

C When 2 or 30 �g ofB-l2 is fed to patients with pernicious anemia,

there is -� 1% urinary excretion in a Schilling test (suggesting ‘-3%diffusion absorption?) Reprinted from reference 1.

t Flushed into urine in 48 h by injection of 1 mg nonradioactivecyanocobalamin at 0 time and again at 24 h after the oral dose of B-12.IF, hog intrinsic factor concentrate.

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VITAMIN 3-12: SOURCES AND REQUIREMENTS 857

PLASMA IRON (yiq/l00 ml)

PLASMA FOLATE (n�uq/mI)

1401 8 42

47 22 21 7. 5.3

24 26 Se ase 571 348

�

LASMA�T.

�

�.!_#s,s!.�)

�24

W.M. ��54;78kq

PERNICIOUS

ANEMIA

t!��oMl.AMlN (I�4 dolly, l.rn.)I

-IS -10 -5 0 5 0 15 20 25

I TIME (Days)-Word D..t-..4-.--DIst Dsvoid Of Fruit Juics, FrssliVsg.,LIvsr

FIG 6. Excellent hematologic response ofpatient with vitamin B-12

deficiency (pernicious anemia) to I pg/vitamin B-l2/d. (To convert�gFe/l00 mL to �imol/L, multiply by 0.1791. To convert mg folate/mL to nmol/L, multiply by 2.266. To convert pg vit B-12 to pmol/L,multiply by 0.7378.)

sharp increase in very young red cells and reticulocytesand a rise to normal in red cell count, hemoglobin, andhematocrit. Characteristically, as happens when onetreats vitamin B-12 deficiency, the serum Fe plummets,in this case from 180 to 8 over the first 10 d, as the plasmaFe is drawn into the formation of new hemoglobin forthe new red cells.

Nobody needs more than 1 zg/d because 1 &g wouldactually treat and return to normal people with no storesof vitamin B-12 (2). The average omnivorous human, ifone does sequential studies each decade, has a progres-siveby rising liver level of vitamin B-b2 throughout life,indicating the average omnivore is eating much more vi-tamin B-12 than needed and is in continuous positivebalance and progressively storing the excess. There isnothing about continuous positive balance that is inher-ently desirable (12).

What is the minimal daily amount we need? Less than1 �g. In studies carried out at Harvard with then researchfellow Louis Sullivan, we showed that one can treat vita-mm B-12 deficiency with as little as 0. 1 �tg/d (2). At thisbevel stores are not rapidly replenished and the responseis submaximal but it only takes 0. 1 �g to produce a re-sponse.

The minimum daily requirement (MDR) for vitaminB-12 to sustain normality is probably in the range of�.-0. 1 �tg, 0.2-0.25 �tg/d absorbed from food is probablyadequate for anybody (2). There are no objective pub-lished data that larger amounts ofvitamin B-b2 have anyadded value for greater health or longer life. The currentCanadian Recommended Dietary Allowances (RDA)and the recent Recommended Dietary Intakes (RDI)

for vitamin B-12 are lower than previous recommenda-tions (2).

Sources of vitamin B-i 2

Fermented products, such as soy products bike tem-peh, do not contain substantial amounts ofB-b2 (1). Theamounts given on the labels cannot be trusted becausethey were obtained by the US Pharmacopeia (USP) assaymethod, which sellers ofproducts containing vitamin B-12 are required to use.

The label-stated content of vitamin B-l2 is in fact thecontent of all comnoids in which L leichmannii growsand not just cobalamin. It should say corrinoids ratherthan vitamin B-12. The Food and Drug Administration(FDA) was petitioned several years ago to require vita-mm B-b2 assay for true vitamin B-12 and analogues andperhaps the assay will eventually be changed.

We studied several types oftempeh, including OriginalSoy Tempeh, a Rhizobus oligosporus culture with a labelclaim of 160% of the US RDA for vitamin B-12 per 4oz. Using the differential radioassay we found there waspractically no vitamin B-l2 in it (1).

We also studied most of the spirulinas sold in healthfood stores as sources ofvitamin B-12; there is practicallyno vitamin B-l2 in them. The so-called vitamin 8-12 isalmost exclusively analogues of vitamin B-b2 and wehave extracted the two largest peaks of analogues andthey actually block vitamin B-12 metabolism. We sus-pect that people taking spirulina as a source of vitaminB-12 may get vitamin B-12 deficiency quicker becausethe analogues in the product block human mammaliancell metabolism in culture and we suspect they will alsodo this in the living human. Remember that the labelclaim of vitamin B-12 is actually a claim of comnoidcontent, not vitamin B-12 content.

The vegan diet, if it is a diet exclusively of productsthat grow out ofthe ground, which are then well washed,contains no vitamin B-12 except trace amounts in somerhibozium-bacteria-containing root nodules. Carefulstudies from England (19) on several hundred vegansshowed that they all eventually get vitamin B-l2 defi-ciency disease with anemia and pancytopenia, low whitecounts, low red counts, low platelet counts, and slowedDNA synthesis (19). Vegans all eventually have slowedDNA synthesis, which is corrected by vitamin B-12. Myadvice to the vegan parents of a vegan child is that youhave to provide a supply of vitamin B-l2. Yeast grownon vitamin B-b2-enriched medium is only the answerwhen some of the vitamin B-12-enriching medium ismixed in with the yeast that is eaten because the yeastitselfdoes not contain active vitamin B-b2; it contains alot ofanalogues but not active vitamin B-12. Differentialradioassay show that all the vitamin B-b2 is accountedfor by vitamin B-12-enriched medium rather than bythe yeast itself. Vegans must get a source for vitamin B-12. It can be 1 �tg/d of vitamin B-12 in a tablet or insomething else but it has to be cobabamin.

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858 HERBERT

Bindra et al (20) note that vegetarians who boil theirmilk before drinking may destroy much ofthe milk vita-mm B-l2 and place themselves at risk; they also sug-gested that the high dietary fiber levels of Punjabi dietsmay increase fecal excretion ofvitamin B-b2 (20).

Vitamin B-i2 as snake oil

Just before this conference, Time magazine inquiredabout the latest California health food fad: sniffing vita-mm B-l2 gel up the nose. Like most health food fads,it was created by entrepreneurs with heavy advertisingbudgets and light consciences. The heavily promoted fadmoved east to just about every health food store acrossthe United States. Given a deceptive name suggesting en-ergy, it is falsely represented as giving an energy boost,which, ofcourse, is biochemically impossible because vi-tamin B-12 neither supplies nor releases energy except inthe vitamin B-12--deficient individual. Vitamin 8-12 isinvolved in intermediary metabolism as a catalyst muchbike a traffic cop at an intersection.

Like many other pharmaceutical agents, vitamins areabsorbed better through the nasal mucosa than throughthe alimentary mucosa. However, the increased percent-age absorption of vitamin B-l2 through the nasal routeis of no value whatsoever to the person with a normalserum vitamin B-b2 level. All that vitamin B-l2-normalpeople get from wasting their money on vitamin B-12nasal gels is expensive urine. In addition, the gel may beallergenic for some. Nasal gel vitamin B-l2 gets no morevitamin 8-12 into the bloodstream than a 10-fold greateroral dose and may not be as reliable as injected vitaminB-l2 (2 1). The FDA has been petitioned to stop the saleofthe gel as a food supplement.

What is the RDA for vitamin 8-12? The suppressed(3) 1980-85 RDA appears in the April 1987 issue of theAmerican Journal of Clinical Nutrition (2) as the RDI(for which read “ 1985 RDA”) for vitamin B-l2. RDI isthe international term used by the World Health Organi-zation (WHO) and many other countries. RDA is a termused primarily in the United States. We (the 1980-85RDA Committee) reduced the RDA for vitamin B-l2 to2 �ig for adults (which is still more than anybody needs)because there was no scientific basis for higher amounts(2). As delineated above, nobody needs that much, thereis no evidence that it has any value whatsoever for hu-mans and, as we discover more and more about excessesofany nutrient, we discover hams we did not know cx-isted (12, 21, 22). It will probably turn out eventually thattoo much vitamin B-b2, like too much of anything, isharmful(l2).

References

1. Herbert V, Drivas 0, Mamusselis C, Mackler B, Eng J, Schwartz E.

Are colon bacteria a major source of cobalamim analogues in hu-man tissues? 24-hour human stool contains only about 5 �sg of co-

balamin but about 100 pg of apparent analogue (and 200 �g of

folate). Trans Assoc Am Phys l984;97: 161-71.2. Herbert V. Recommended dietary intakes (RDI) ofvitamin B-l2

in humans. Am J Qin Nutr l987;45:671-8.3. Herbert V. The 1986 Herman Award Lecture. Nutrition science as

a continually unfolding story: the folate and vitamin B-l2 para-

digm. Am J Oin Nutr 1987;46:387-402.4. Herbert V, Colmam, N. Folic acid and vitamin B-12. In: Shils M,

Young V, eds. Modern nutrition in health and disease. 7th ed. Phil-

adeiphia: Lea & Febiger, 1988:388-416.5. Herbert V. Vitamin B-l2. In: Olson RE, ed. Present knowledge in

nutrition. 5th ed. Washington, DC: Nutrition Foundation, 1984:

347-64.6. Herbert V. Biology ofdisease: megaloblastic anemias. Lab Invest

1985;52:3-19.

7. Callender ST, Spray, OH. Latent pernicious anemia. Br J Haema-

tol l962;8:230-40.8. CooperB, Rosenblatt D. Inherited defectsofvitamim B-l2 metabo-

lism. Ann Rev Nuts l987;7:29l-320.9. Cannel R, Rosenberg AH, Lau K-S, StreiffRR, Herbert V. Vita-

mm B-l2 uptake by human small bowel homogenate and its en-hancement by intrinsic factor. Gastroenterology l969;56:548-55.

10. Kanazawa 5, Kondo H, Terada H, Okuda K. R-binder blocks co-balamin analogue binding to intrinsic factor-cobalamin receptor.Fed Proc l984;43:1859(abstr).

1 1. Halsted JA, Carroll J, Rubert S. Serum and tissue concentration ofvitamin B,2 in certain pathologic states. N Engl J Med l959;260:575-80.

12. Herbert V. Stating nutrient status from too little to too much byappropriate laboratory tests. In: Livingston GE, Vanderveen JE,IaconoJM, eds. Nutrition statusassessment ofthe individual. New

York: American Health Foundation (in press).

13. Doscherholmen A, McMahom J, Ripley D. Inhibitory effect of eggson vitamin B-l2 absorption: description of a simple ovalbumim“Co-vitamin B-l2 absorption test. Br J Haematol 1976;33:261-72.

14. Drasar, BS, Hill MJ. Human intestinal flora. New York: AcademicPress, 1974.

15. Albert Mi, Matham VI, Baker Si. Vitamin B-l2 synthesis by hu-

man small intestinal bacteria. Nature l980;283:781-2.16. Kamazawa S, Herbert V. Mechanism ofenterohepatic circulation

ofvitamin B-l2; movement ofvitamim B-l2 from bile R-binder tointrinsic factor due to the action ofpancreatic trypsin. Trans Assoc

Am Physicians 1983;96:336-44.17. Hei-zlich B, Herbert V. The role ofthe pancreas in cobalamin (vita-

mm B-l2) absorption. Am J Gastroenterol l984;79:489-93.

18. Shaw 5, Meyers S, Colman N, Jayatilleke E, Herbert V. The ileumis the major site ofabsorptiom ofvitamin B-l2 analogues. Fed Procl987;46:(abstr).

19. Chanarim I, Malkowska V, O’Hea A-M, Rinsler MG, Price AB.Megaloblastic anemia in a vegetarian Indian community. Lancetl985;2:l 168-72.

20. Bindra OS, Gibson R, Berry M. Vitamin B-l2 and folate status of

East Indian immigrants living in Canada. Nutr Res(in press).21. Herbert V, Stopler T, Huebscher T. Nasal vitamin B-12 gel may

not be a reliable alternative to injectable vitamin B-l2; both bind

preferentially to TC II and produce analogue incrementjust on TCII. Blood l987;70(suppl):45A(abstr).

22. Herbert V. The inhibition of some cancers and the promotion ofothers by folic acid, vitamin B-l2, and theirantagonists. In: Butter-worth CE Jr, Hutchinsom ML, eds. Nutritional Factors in the In-

ductiom and Maintenance of Malignancy. New York: AcademicPress, 1983:273-87.

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