Megaloblastic anaemia, cobalamin, andfolateMegaloblastic anaemia, cobalamin, andfolate containing a varying amountofnative B12. Thenext stepis totakeoutanaliquotoftheB,2inthemixture:

J Clin Pathol 1987;40:978-984

Megaloblastic anaemia, cobalamin, and folateI CHANARIN

From the Medical Research Council Clinical Research Centre, Northwick Park Hospital, Harrow, Middlesex

SUMMARY Developments relating to cobalamin and folate are reviewed. Current work on therelations between these two coenzymes are discussed, particularly those that have emerged instudies using nitrous oxide, which inactivates cobalamin.

During the 1960s many ideas were developed aboutthe megaloblastic anaemias. Cobalamin assays on hu-man sera, initially at the Royal Postgraduate MedicalSchool, had been consolidated, and patients withmegaloblastic anaemia could be grouped into thosewith low B12 concentrations and those with normalconcentrations.' Isotopically labelled B,2 becameavailable and B,2 absorption tests became part ofclinical practice.2 A method for the assay of serumfolate was described3 and was quickly extended to themore useful red cell folate assay.4 By the end of 1963an assay for the intrinsic factor content of gastricjuice5 6and for the detection of antibodies in serum tointrinsic factor had been introduced as well as meth-ods for detecting gastric parietal cell antibodies.The use of urinary formiminoglutamic acid (Figlu)

as a test for folate deficiency reared its ugly head.7And, finally, two groups put forward a hypothesis toaccount for the strange interrelation between B12 andfolate that came to be known as the methylfolate traphypothesis.8 9

This review covers the developments of the pastquarter century and how the ideas of 25 years agohave stood the test of time.

Automated cel counters and mean corpuscular volume

The introduction into haematology laboratories ofaccurate and sophisticated blood counting machinesis the major technical development of the past 25years. It has highlighted the value of red cell size(mean corpuscular volume, MCV) in diagnosis andemphasised the raised MCV in many disorders thatare unrelated to impairment of B,2 and folate metab-olism. Normoblastic macrocytosis occurs in chronicalcoholism, hypothyroidism, normal pregnancy,healthy neonates, marrow failure, chronic haemolyticstates with raised reticulocyte values, many pre-leukaemic (myelodysplastic) disorders, and treatmentwith many cytotoxic drugs including, chlorambucil,mephalan, methotrexate, azathioprine etc. Finally,

there is a common macrocytosis occurring in pre-menopausal women whose only complaint is las-situde, which remains unexplained.Not all patients with megaloblastic anaemia will

have an increased MCV. Some who have thal-assaemia trait, anaemia of chronic disorders, or con-current iron deficiency may have a franklymegaloblastic marrow with a normal MCV."0 Whentreated with B,2 or folate, or both, the MCV falls tothat associated with the underlying disorder belowthe normal range of 80 to about 94 fl.

Finally, there is no agreement as to what consti-tutes the normal MCV: quoted ranges vary from 76 atthe lower end to 108 fl at the upper end. This is be-cause there is no absolute way to obtain this value andthe machine methods relate to the manner in whichthe settings are manipulated.

The serum cobalamin (B12) concentration

The early assays for B12 were all microbiological as-say procedures, and in essence these gave similar re-sults. The assays were tedious to perform and oftenassays were rejected because of loss of purity of theorganism, introduction of extraneous B,2 in the assaysystem, and poor growth of the assay organism. Forthese reasons the development of an alternativemethod based on dilution of isotopically labelled B,2by the native B,2 in a serum sample (saturation anal-ysis) was widely welcomed. These assays lent them-selves to the preparation of commercial kits that havebecome the mainstay of laboratory assays.

It soon became apparent that often a lot more B,2was being measured by the isotope methods than mi-crobiological methods." -'3 Thus the lower limit ofthe normal range could be 300 pg/ml with an isotopeassay but was about 170 pg/ml by microbiological as-say. A value of 250 pg/ml was low by isotope assaybut plumb normal when assayed microbiologically.When performing the isotope assay, a standard

amount of [57Co]B,2 is added to an extract of serum

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Megaloblastic anaemia, cobalamin, andfolate

containing a varying amount of native B12. The nextstep is to take out an aliquot of the B,2 in the mixture:this is done by adding a B,2 binding protein. Thegreater the amount of native B12 the greater the dilu-tion of [57Co]B12, and hence less [57Co]B,2 will at-tach to the binder (fig 1). The explanation of thediscrepancy between isotope and microbiological as-say lay in the nature of the B,2 binding protein usedin the assay. When purified gastric intrinsic factor wasused as the B12 binding protein the results were simi-lar to those obtained by microbiological assay. Whenother B12 binders, such as those present in serum or insaliva-collectively termed R-binders-were used theresult was always higher than that obtained with mi-crobiological assay. Kolhouse et al'4 suggested thatthe R-binders were picking up forms of B,2 in serumnot picked up by intrinsic factor. This suggestion wasshown to be correct by cross absorption studies."5 Byattaching intrinsic factor to polyacrylamide beadsand adding these to serum extracts, all B,2 analoguesdetectable both by microbiological assay and an iso-tope method with intrinsic factor were removed. Theserum extract, however, still had B,2 activity detectedby an isotope assay with an R-binder. It now seemsthat microbiological assays measure about half theB,2 present in serum. The same analogues are mea-sured by microbiological assay and an intrinsic factorisotope assay.16The identity of the B,2 analogues undetected by

microbiological assay remains unknown and its ori-

979

gin is equally conjectural. In practice, an isotope as-say for B12 using purified intrinsic factor is preferredbecause it gives a marginally better distinction be-tween normal and B12 deficient sera,"7 but claims thatlarge numbers of patients deficient in B12 were missedbecause R-binder assays were used, are exaggerated.Failure to detect low B,2 concentrations is morelikely to be due to a badly designed and badly per-formed assay than to the nature of the binder.

Finally, it is widely believed that a low serum B12concentration is synonymous with B12 deficiency.This is not the case.'0 B,2 deficiency is the common-est cause of low B,2 concentration, but there are othercauses. One third of patients with megaloblastic ana-emia due to folate deficiency have low B12 concen-trations, which return to normal within days oftreatment with folic acid.'8 Low B,2 concentrationsare common near term in normal pregnancy, in pa-tients with iron deficiency, after partial gastrectomyas well as in treated epileptics. Low B,2 concen-trations are often found in otherwise perfectly healthypeople. Except in vegetarians with nutritional B,2deficiency, all patients with clinically important lowB12 concentrations also have impaired B,2 absorp-tion. Where B,2 absorption is normal and the patientis taking a mixed diet, a low B,2 concentration is ofno clinical importance.

B, 2 ABSORPTIONTests for B12 absorption have become important and

57Co- B12 and intrinsic factor only

057CoB12 Intrinsic factor

solid phase'Free'' B12 Bound B12

57Co-B12 with test sample and intrinsic factor

0

0+

000

B12 in test sample

+ S=:Intrinsic factorsolid phase

00

'Free' 57CoB12increased

Bound 57CoB12reduced

Fig 1 When ['7Co]Bl2 is mixed with intrinsicfactor (in this example intrinsicfactor is attached to a solid matrix), someB12 attaches to intrinsicfactor and, when B,2 is present in excess, some remainsfree. When, in addition to ["7CoJB12, B12 isalso presentfrom the serum sample under test, the ["7CoJB12 is diluted and hence the B12 binding to intrinsicfactor is amixture of[57Co]B,2 and native B12 from serum. Thus the more native B,2 present thegreater the dilution of the [57CoJB12,and this is the basis on which the assay is performed.

57CoB12

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980reliable indicators of ileal function and of availabilityof gastric intrinsic factor. It is less widely appreciatedhow often misleading results are recorded because ofincomplete urine collection."9 Assessment of the re-sult by measuring the radioactivity of the labelled B12in plasma collected after eight to 12 hours is as re-liable an indicator of the result as a complete urinecollection; it should be mandatory to insure againstloss of urine, which occurs in between 25 to 50% of alltests, and in most tests carried out in the elderly, bycombining the standard Schilling test with mea-surement of plasma radioactivity.

In addition to the known permutations of the B12absorption test by giving B12 with intrinsic factor,performing the test after antibiotics etc, it has beensuggested that additional information can be ob-tained by "binding" the labelled B12 to protein. Somehave bound the labelled B12 to chick serum,20 othershave cooked the B12 with egg and served up an ali-quot of this material.21 The test thus requires di-gestive processes to release B12 from this unpromisingmaterial so that it can bind to intrinsic factor, whichin turn will enable B12 to be absorbed. Even normalsubjects are unable to achieve normal B12 absorptionwith this method, the mean urinary excretion with eggwhite being between 2-7 to 4 0% ofa 1-68 ig oral doseand 5 1 to 6-0 with egg yolk. By contrast, the meanresult in the standard Schilling test was 23-6%. Nor-mal has been defined as the B12 absorption of aque-ous B12 or absorption of food B12 as natural B12 inlamb.22 Not surprisingly, achlorhydric patients andpatients after gastric surgery fare even worse thancontrols with protein bound B12. This is presumablybecause they cannot activate pepsinogen to aid pro-teolytic digestion of the binding proteins. The test hasbeen interpreted, however, as detecting a lack of in-trinsic factor at a stage when the standard B12 ab-sorption test is still normal. There are no availabledata to suggest that this is likely; nor is there anyevidence that the test is of any clinical value.

Dietary B12 is present either as part of methioninesynthetase or as part of methylmalonyl CoA-mutase.Small amounts are linked to R-binders. Once B12 isreleased from the holoenzyme, much of it links to R-binder mainly from saliva, although R-binder is alsopresent in gastric and intestinal juice. Pancreatic en-zymes serve to release B12 from its R-binder link andpermit its uptake by intrinsic factor. In pancreatitisB12 absorption is impaired because of failure to de-grade the R-binder. Brugge eta!23 devised a suitableB12 absorption test in which the absorption of B12bound to R-binder is compared with absorption ofB12 bound to intrinsic factor. With controls, theSchilling test result with B12 and intrinsic factor was21-2%, and with R-binder, 16-2%. With pancreaticdisease the absorption of B12 with intrinsic factor was

Chanarin16-3% and with R-binder only 10%. The combina-tion is clearly of value in the diagnosis of chronic pan-creatic disease.

There remain unresolved problems in relation toB12 absorption in patients with atrophic gastritis andafter partial gastrectomy, where there is no clear cor-relation between the serum B12 concentration and theresults of standard B12 absorption tests.

Intrinsic factor, its assay, and its antibodyIntrinsic factor, necessary for the intestinal absorp-tion of B12, is secreted by the gastric parietal cell. Anassay of intrinsic factor was introduced simulta-neously by Ardeman etal5 and Abels etal in 1963.6The problem in developing such an assay is that gas-tric juice has at least three B12 binding proteins: in-trinsic factor, partially digested intrinsic factor, andR-binder. When labelled B12 is added to gastric juicesome binds to all three components, hence the bind-ing of B12 alone cannot be used as a measure of in-trinsic factor. Each of these binding proteins,however, can be prevented from taking up B12. Anintrinsic factor antibody present in serum in morethan half the patients with pernicious anaemia, andwhen added to gastric juice, will react with intrinsicfactor and prevent B12 uptake by intrinsic factor.Thus B12 binding is now due only to R-binder, andwhen this value is subtracted from the total B12 bind-ing of gastric juice, it is a measure of intrinsic factorcontent. One unit of intrinsic factor is the amountbinding I ng of B12.Cobinamide is a B12 analogue that lacks the ben-

zimidazole moiety. It binds firmly to R-binder but notat all to intrinsic factor. Addition of cobinamide togastric juice will block the R-binder, so that sub-sequent addition of labelled B12 will bind only to in-trinsic factor.24Assay of intrinsic factor has confirmed its virtual

disappearance in pernicious anaemia; there is a vastexcess present in healthy subjects. Only 1% of thenormal daily output is required for normal B12 ab-sorption.10 Finally, abnormal forms of intrinsic fac-tor have been shown in megaloblastic anaemia ininfancy-that is, these have bound labelled B12 buthave failed to potentiate its intestinal absorption.25

Detection of antibody to intrinsic factor is a usefulaid in diagnosis ofpernicious anaemia but, rarely, thisantibody can be present in patients with Graves' dis-ease, atrophic gastritis etc in the absence of perniciousanaemia.26 More than 40% of patients with perni-cious anaemia, however, do not have such an anti-body in serum but may have it in the gastric secretion,when it is likely to be an IgA immunoglobulin ratherthan a IgG as in serum. Even those who lack humoralantibody against intrinsic factor generally show evi-dence of cell mediated immunity against intrinsic fac-

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Megaloblastic anaemia, cobalamin, andfolate

tor." The importance of cell mediated immunity isshown by the high incidence of pernicious anaemia inpatients with acquired hypogammaglobulinaemia,30% of whom develop the disease.'0 These patientsare unable to form humoral antibodies. These intrin-sic factor antibodies are able to neutralise residualintrinsic factor secretion, or damage intrinsic factorsecreting cells and convert a just adequate B12 ab-sorption (present in simple atrophic gastritis) to aninadequate one so that a strong negative B,2 balancedevelops. This is soon followed by clinically overt per-nicious anaemia.

Bl2 transport proteins

R-protein (transcobalamins I and III) is present in allbody fluids and most cells. A physiological role forthese glycoproteins has not been identified. TC III inplasma is largely shed from leucocytes.

Transcobalamin II is the prime transport proteinfor B12. It has a molecular weight of 35000 daltonsand is produced by the liver, macrophages, and ilealenterocytes. It is essential for moving B,2 out of theileal enterocyte to plasma during B12 absorption andfor uptake of B12 by cells. There are specific receptorsfor TC II, and TC II is internalised into cells by endo-cytosis. In the absence of TC II a severe B12 deficientmegaloblastic anaemia develops, usually within sixweeks of birth, which has to be treated with very largedoses of B12 (1000 pg two to three times a week) sothat some enters cells by passive diffusion. Even B,2present in serum on R-binder in TC II deficiency can-not be used because of lack of the transport protein,so that paradoxically normal serum B12 concen-tration occurs concomitantly with severe tissue de-pletion of B 2. '

Folates

Assay of folate concentration with Lactobacillus caseiin serum and whole blood has provided data that aresimilar to those resulting from B12 assay. A lowserum folate concentration probably represents astate of negative folate balance and may be found inone third of all hospital patients. Red cells, on theother hand, contain some 30 times more folate thanserum. Folate is incorporated into the red cell in themarrow by the developing erythroblast, and there isno folate turnover by mature red cells. Thus folate islocked in the red cell for the duration of its life span.Change in red cell folate comes from release of redcells from the marrow of different folate content. Alow red cell folate represents established folatedeficiency, irrespective of whether the subject is nor-moblastic or megaloblastic. Unfortunately, isotopemethods used to assay folate have concentrated on

981

the trivial serum folate assay and not on the clinicallyimportant red cell assay.

Urinary Figlu excretion is too sensitive for diagno-sing folate deficiency for routine clinical practice.Like the serum folate concentration, abnormal resultsare found in one third of hospital patients, and it is nolonger used.The nature and function of intracellular folates

have been clarified to a large extent. The folate ana-logue that is absorbed from food28 and which circu-lates in body fluids is 5-methyltetrahydrofolate. Thereis some suggestive evidence for a membrane associ-ated transport protein that facilitates its entry intocells.29 Once in the cell, additional glutamic acid resi-dues are added to give a form with 5-glutamic acidresidues, although analogues with 4, 6, and 7 residuesare present. In this form the folate (now termed folatepolyglutamate) is retained in the cell and serves as theactive coenzyme in transfer of single carbon units forpurine, pyrimidine, and methionine synthesis. Cellsthat have lost the ability to make polyglutamates can-not grow unless the end products of folate metabo-lism such as purines are supplied.30The folate antagonist methotrexate is also taken

into the cell and is converted into a polyglutamate inwhich form it persists in the cell over a long period.Not all intracellular folate is polyglutamate andabout one third has one glutamic acid residue.

The deoxyuridine (dU) suppression test

This test, which was modified by Metz eta!3' from theoriginal account by Killman,32 assesses the manner inwhich marrow cells convert deoxyuridine into deoxy-thymidine. The single carbon unit required in this step(methylene or -CH2-) is supplied by 5, 10-methylenetetrahydrofolate. An aliquot of marrow cells is incu-bated with deoxyuridine. Normal cells will meetabout 95% of their thymidine requirements by syn-thesis from deoxyuridine. The results of the test areassessed by adding preformed [3H]thymidine. The re-maining 5% requirement for thymidine is met by us-ing this labelled compound.Marrow cells from megaloblastic patients are un-

able to methylate deoxyuridine as well as normalcells, and less than 90% is used and more than 10%[3H]thymidine is taken up into DNA. If B12 is addedto a further aliquot of marrow cells there is some cor-rection of the impairment in B12 deficient marrowsbut no correction with those deficient in folate. For-myltetrahydrofolate (such as folinic acid) will correctthe impairment in both B,2 and folate deficiency.The dU suppression test is a test for "megalo-

blastosis" and provides an indication of the nature ofthe deficiency when it is combined with addition ofB,2 or folate to the incubation mixtures. It is, how-

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982ever, time consuming, the manipulations taking thebetter part of a day.

Cobalamin-folate interrelations

The methylfolate trap hypothesis to explain B12 fol-ate interrelations was proposed just over 25 yearsago.8 9 Both B12 and folate are required for the syn-thesis of methionine (fig 2). The methyl group frommethylfolate is passed on to homocysteine to formmethionine, and B12 is an intermediary in this trans-fer. In the absence of B12 this pathway is interrupted.Two further observations led to the formulation ofthe methylfolate trap hypothesis.1 The thermodynamics of the reaction by whichmethylfolate is formed from methylenefolate (5, 10,CH2 folate-e 5-CH3 folate) is strongly shifted tothe right-that is, on the basis of in vitro studies itwas concluded that once methylfolate was formed itwas improbable that it could be oxidised back tomethylenefolate.2 In untreated pernicious anaemia the serum folate(largely methylfolate) is increased. It was thereforededuced that in B12 deficiency methylfolate could notbe used, it could not be oxidised back to methy-lenefolate, and as more folate accumulated in themethyl form a shortage of other folate analoguesemerged. It was consequently supposed that there wasimpaired transfer of formyl for purine synthesis andmethylene transfer for thymidine synthesis and thatthis led to interference with DNA synthesis and mega-loblastosis. The raised serum folate was cited as sup-porting methylfolate trapping.

These views have been difficult to test as patientswith untreated pernicious anaemia do not readilylend themselves to the sort of study required.The situation was transformed by the observation

that the anaesthetic gas nitrous oxide (N20) is acti-vated by organometallic complexes of which B12 isthe prime example.33 N20 is cleaved and at the sametime B12 is oxidised to an irreversibly inert form.N20, in fact, specifically inactivates the enzyme me-thionine synthetase, of which B12 is a coenzyme.34Exposure to N20 rapidly produces "B12 deficiency."In man this can lead to megaloblastic anaemia, neu-ropathy, and even death. Other species do not becomemegaloblastic, but some like the monkey and proba-bly fruit bats develop a neuropathy. All, however, de-velop very similar biochemical changes, and thesehave now been studied in some depth. It is now possi-ble to assess the validity of the premises on which themethylfolate trap hypothesis was formulated.

I DOES METHYLFOLATE TRAPPING ACCOUNTFOR THE KNOWN DEFECTS IN B12 DEFICIENCY?As the methyl group of methylfolate cannot be trans-

Chanarin

"C"..fH4 Folate H4 Folate B12 Homocysteine

(methionine)

Fig 2 A single carbon unit ("C") taken up byfolate isreduced ifnecessary, to -CH=, -CH2-, and ultimately-CH3 (methyl). The methyl group is passed on tohomocysteine toform methionine and B1 2 is a coenzyme inthis transfer.

ferred to homocysteine in B12 deficiency there is im-paired use of methylfolate. Thus in the dUsuppression test methylfolate did not correct the de-fect in B12 deficient marrow cells. Neither in marrowcells from pernicious anaemia nor from rats treatedwith N20, however, was tetrahydrofolate itself ableto correct the defect in thymidine synthesis, althoughformylfolate was fully active.35 This is quite an un-expected result from the point of view of the methyl-folate trap hypothesis as tetrahydrofolate is outsidethe trap.The same pattern of results emerged when the form

of folate used by liver for making folate poly-glutamate was studied. Normal rat liver used all fol-ate analogues equally for making the polyglutamate.When B12 was inactivated by N20, however, no poly-glutamate at all was made from methylfolate and tet-rahydrofolate, but normal amounts fromformylfolate.36

Failure to use tetrahydrofolate and the normal useof formyltetrahydrofolate are not predicted bymethylfolate trapping. Rather they suggest a role forB12 in the formylation of folate.

2 IS THE IN VITRO PREDICTION THATMETHYLFOLATE CANNOT BE OXIDISED BACK TOMETHYLENE AND FORMYLFOLATE CORRECT ININ VIVO?One of the two papers in 1961 that formulated themethylfolate trap theory also contained the seeds forits destruction.8 Noronha and Silverman found that1% methionine added to the diet and fed for 24 hoursto rats before sacrifice resulted in a considerable de-cline in the methylfolate concentrations in liver whileincreasing the formylfolate and tetrahydrofolate con-tent. This occurred in both control and B12 deficientrats. This implies oxidation of the methyl group toformyl and to carbon dioxide. This has beenconfirmed many times since. Brody etal37 showedthat this occurred within 30 minutes of an injection of

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Megaloblastic anaemia, cobalamin, andfolate 983

methionine and that the methyl group oxidation oc-curred on folate hexaglutamate.The amount of methionine needed to promote ox-

idation of the methyl group of methylfolate was aslittle as 0 5 pmol. S-adenosylmethionine was less ac-tive than methionine and seemed to be active onlythrough its further metabolism to methionine. Thusthe hepatic concentration of methylfolate is regulatedby that of methionine in both normal and B12deficient animals.

Other observations also point to in vivo oxidationof the methyl moiety of methylfolate. Methylfolatecan serve as the methyl donor in the methylation ofbiogenic amines.38 The mechanism entails the oxid-ation of the methyl group to formate, which in turn istransferred to the biogenic amine. The enzyme res-ponsible for the oxidation of the methyl group wasthe same one concerned with its formation-namely,methylenetetrahydrofolate reductase38

Furthermore, Thorndike and Beck39 reported thatthe methyl group of methylfolate was oxidised in anessentially similar manner by lymphocytes from bothnormoblastic controls and one patient with perni-cious anaemia.

3 WHAT IS THE PATHWAY FOR OXIDATION OFTHE METHYL GROUP OF METHYLFOLATE IN B12DEFICIENCY?Lumb et al40 showed that in B12 inactivated ratswhere the methyl group could not be transferred tohomocysteine, methyltetrahydrofolate itself was usedas the substrate for forming methylfolate-polyglutamate. When methylfolate labelled with ['4CJin the methyl group was used ["4C]-labelled poly-glutamates containing 3, 4, and 5 glutamic acid resi-dues were identified. As no polyglutamate with the[14CJ label having 6 glutamic acid was present it wasconcluded that the methyl group was oxidised fromthe hexaglutamate.

Methionine and cobalamin-folate function

Not only are both folate and cobalamin required formethionine synthesis, but many of the effects of B12deficiency in both man and rat are overcome to agreater or lesser degree by the provision of methi-onine.

In man methionine given to patients with perni-cious anaemia in relapse will abolish an abnormal uri-nary formiminoglutamic acid excretion and, used inappropriate dose, will correct the abnormal dU sup-pression test.4'

In fruit bats treated with N2042 and inmonkeys43 methionine will considerably improveneuropathy. In B12 inactivated rats methionine willrestore to normal folatepolyglutamate synthesis.36

Even more effective than methionine, is a derivativeof methionine via S-adenosylmethionine-namely,5-methylthioadenosine.36 This compound is furthermetabolised to methionine and formate. Methioninecan also yield formate by a second pathway-namely,oxidation of its methyl group.

How B12 deficiency affects folate metabolism

Methionine is the pivotal compound in B12 folate me-tabolism. Lack of B12 produces a shortage of methi-onine that is only partially alleviated by induction ofanother pathway to methylate homocysteine-namely, betaine methyltransferase whereby betainesupplies the methyl group to methylate homocysteineinstead of methylfolate. Lack of methionine has twoconsequences:1 Lack of single carbon units at the formate level ofoxidation, which arises from both oxidation of themethyl group of methionine and via S-adenosyl-methionine -+ decarboxylated S-adenosylmethionine

5-methylthioadenosine -+ 5-methylthioribosemethionine plus formate. Formate is required forpurine synthesis and for formation of formyl-tetrahydrofolate, which seems to be the preferred sub-strate for folate polyglutamate synthesis, and byfurther reduction into methylene for pyrimidine syn-thesis.2 The lack of S-adenosylmethionine, as indicated,may restrict supply of formate. It may interfere withgeneral transmethylation reactions, although a rolefor transmethylation in haemopoiesis has still to beshown. There is no fall in S-adenosylmethionine val-ues in the brain in fruit bats treated with N20 andhence impaired transmethylation in brains is unlikely.

Serine has been widely regarded as the main sourceof single carbon units largely because it is readilyavailable by synthesis from glucose. B12 inactivationdoes not affect the pathways by which serine can do-nate a single carbon unit. Nevertheless, large doses ofserine do not in any way ameliorate the effect of B12inactivation. This must cast doubt on the importanceof serine as a major single carbon unit donor.The hypothesis that has been advanced to explain

B12-folate interrelations-the formate-starvationhypothesis-postulates that B12 deficiency leads toshortage of methionine, and this in turn, to a shortageof active-formate derived from methionine.

References

I Mollin DL, Ross GIM. The vitamin B12 concentrations of serumand urine of normals and of patients with megaloblastic ana-emias and other diseases. J Clin Pathol 1952;5:129-39.

2 Chaiet L, Rosenblum C, Woodbury DT. Biosynthesis of radio-active vitamin B12 containing cobalt60. Science 1950;111:601-2.

3 Baker H, Herbert V, Frank 0, et al. A microbiologic method fordetecting folic acid deficiency in man. Clin Chem 1959;5:275-80.

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clearance in geriatric cases. Gerontology Clinics 1961;3:173-82.5 Ardeman S, Chanarin 1. Method for assay of human gastric in-

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6 Abels J, Bouma W, Nieweg HO. Assay of intrinsic factor withanti-intrinsic factor serum in vitro. Biochim Biophys Acta1963;71:227-9.

7 Luhby AL, Cooperman JM, Teller DN. Histidine metabolic load-ing test to distinguish folic acid deficiency from vit B12 in mega-loblastic anaemias. Proc Soc Exp Biol Med 1959;101:350-2.

8 Noronha JM, Silverman M. On folic acid, vitamin B12 methi-onine and formiminoglutamic acid metabolism. In: HeinrichHC, ed. Vitamin B1 2 und intrinsicfactor 2. Stuttgart: Enke, 1962:728-36.

9 Herbert V, Zalusky R. Interrelation of vitamin B12 and folic acidmetabolism: folic acid clearance studies. J Clin Invest 1962;41:1263-76.

10 Charanin I. The megaloblastic anaemias. 2nd ed. Oxford: Black-well Scientific Publications, 1979.

11 Raven JL, Robson MB, Morgan JO, Hoffbrand AV. Comparisonof three methods for measuring vitamin B12 in serum: Radio-isotope, Euglena gracilis and Lactobacillus leichmannii. Br JHaematol 1972;22:21-3 1.

12 Raven JL, Robson MB. Experience with a commercial kit for theradioisotopic assay of vitamin B12 in serum: the Phadebas B12test. J Clin Pathol 1974;27:59-65.

13 Green R, Newmark PA, Musso AM, Mollin DL. The use ofchicken serum for measurement of serum vitamin B,2 concen-tration by radioisotope dilution: description of method andcomparison with microbiological assay results. Br J Haematol1974;27:507-26.

14 Kolhouse JF, Kondo H, Allen NC, Podell E, Allen RH.Cobalamin analogues are present in human plasma and canmask cobalamin deficiency because current radioisotope dilu-tion assays are not specific for time cobalamin. N Engi J Med1978;299:785-92.

15 Muir M, Chanarin I. Separation of cobalamin analogues inhuman sera binding to intrinsic factor and to R-type vitamin B12binders. Br J Haematol 1983;54:613-21.

16 Chanarin I, Muir M. Demonstration of vitamin B12 analogues inhuman sera not detected by microbiological assay. Br J Hae-matol 1982;51:171-3.

17 Muir M, Chanarin I. Solid-phase vitamin B12 assays usingpolyacrylamide-bound intrinsic factor and polyacrylamide-bound R-binder. Br J Haematol 1983;53:423-35.

18 Mollin DL, Waters AH, Harriss E. Clinical aspects of the meta-bolic inter-relationships between folic acid and vitamin B12. In:Heinrich HC, ed. Vitamin B1 2 und intrinsic factor 2. Stuttgart:Enke, 1962:737-55.

19 Chanarin I, Waters DAW. Failed Schilling tests. Scand J Hae-matol 1974;12:245-8.

20 Dawson DW, Sawers AH, Sharman RK. Malabsorption of pro-tein bound vitamin B12. Br Med J 1984;288:675-8.

21 Doscherholmen A, McMahon J, Ripley D. Inhibitory effect ofeggs on vitamin B12 absorption: description of a simple oval-bumin "Co-vitamin B12 absorption test. Br J Haematol 1976;33:261-72.

22 Heyssel RM, Bozian RC, Darby WJ, Bell MC. Vitamin B12 turn-over in man. The assimilation of vitamin B12 from naturalfoodstuff by man and estimates of minimal dietary require-ments. Am J Clin Nutr 1966;18:176-84.

23 Brugge WR, Goff JS, Allen NC, Podell ER, Allen RH. Devel-opment of a dual label Schilling test for pancreatic exocrinefunction based on the differential absorption of cobalaminbound to intrinsic factor and R protein. Gastroenterology1980;78:937-49.

24 Begley JA, Trachtenberg A. An assay for intrinsic factor based onblocking of the R binder of gastric juice by cobinamide. Blood1979;53:788-93.

25 Katz M, Lee SK, Cooper BA. Vitamin B12 malabsorption due toa biologically inert intrinsic factor. N Engi J Med 1972;287:425-9.

26 Rose MS, Chanarin I, Doniach D, Brostoff J, Ardeman S.Intrinsic-factor antibodies in absence of pernicious anaemia. 3-7year follow-up. Lancet 1970;ii:9-12.

27 Chanarin I, James D. Humoral and cell-mediated intrinsic-factorantibody in pernicious anaemia. Lancet 1974;i:1078-80.

28 Chanarin I, Perry J. Evidence for reduction and methylation offolate in the intestine during normal absorption. Lancet 1969;ii:776-8.

29 Antony AC, Kane MA, Portillo RM, Elwood PC, Kolhouse JF.Studies of the role of particulate folate-binding protein in theuptake of 5-methyltetrahydrofolate by cultured human KB cells.J Biol Chem 1985;260:1491 1-17.

30 Taylor RT, Hanna ML. Folate-dependent enzymes in culturedChinese hamster cells: folylpolyglutmate synthesis and itsabsence in mutants autotrophic for glycine + adenosine +thymidine. Arch Biochem Biophys 1977;181:331-4.

31 Metz J, Kelly A, Swett VC, Waxman S, Herbert V. DerangedDNA synthesis by bone marrow from vitamin B12-deficienthumans. Br J Haematol 1968;14:575-92.

32 Killman S-A. Effect of deoxyuridine on incorporation of tritiatedthymidine: difference between normoblasts and megaloblasts.Acta Med Scand 1964;175:483-8.

33 Banks RGS, Henderson RJ, Pratt JM. Reactions of gases in solu-tion. III: Some reactions of nitrous oxide with transition-metalcomplexes. J Chem Soc 1968:2886-9.

34 Deacon R, Lumb M, Perry J, et al. Selective inactivation ofvitamin B12 in rats by nitrous oxide. Lancet 1978;ii:1023-4.

35 Deacon R, Chanarin 1, Perry J, Lumb M. The effect of folateanalogues on thymidine utilization by human and rat marrowcells and the effect of the deoxyuridine suppression test. Post-grad Med J 1981;57:61 1-6.

36 Perry J, Chanarin I, Deacon R, Lumb M. Chronic cobalamininactivation impairs folate polyglutamate synthesis in the rat. JClin Invest 1983;71:1183-90.

37 Brody T, Watson JE, Stokstad ELR. Folate pentaglutamate andfolate hexaglutamate mediated one-carbon metabolism. Bio-chemistry 1982;21:276-82.

38 Pearson AGM, Turner AJ. Folate-dependent 1-carbon transferto biogenic amines mediated by methylenetetrahydrofolatereductase. Nature 1975;258:173-4.

39 Thorndike J, Beck WS. Production of formaldehyde fromN5-methyl-tetrahydrofolate by normal and leukemic leukocytes.Cancer Res 1977;37:1 125-32.

40 Lumb M, Chanarin 1, Perry I, Deacon R. Turnover of the methylmoiety of 5-methyltetrahydropteroylglutamic acid in thecobalamin-inactivated rat. Blood 1985;66:1171-2.

41 Sourial NA, Brown L. Regulation of cobalamin and folate me-tabolism by methionine in human bone marrow cultures. ScandJ Haematol 1983;31:413-23.

42 Van der Westhuyzen J, Fernandes-Costa F, Metz J. Cobalamininactivation by nitrous oxide produces severe neurological im-pairment in fruit bats: protection by methionine and aggrevationby folates. Life Sci 1982;31:2001-10.

43 Scott JM, Dinn JJ, Wilson P, Weir DG. Pathogenesis of subacutecombined degeneration. A result of methyl group deficiency.Lancet 1981;ii:334-7.

Requests for reprints to: Dr I Chanarin, Northwick ParkHospital, Watford Road, Harrow, Middlesex HAI 3UJ,England.

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