J Clin Pathol 1992;45:277-283 277 Occasional articles

Cobalamin and : Recent developments

I Chanarin, R Deacon, M Lumb, J Perry

Introduction of cobalamin neuropathy in New York in the New concepts in this field continue to develop absence of anaemia or macrocytosis. However, at the clinical level,within the laboratory and in the same incidence of neuropathy (68%) was the realm of basic research. The appreciation found in a London study of consecutive that the anaesthetic gas nitrous oxide (N20) patients with macrocytosis, megaloblastic specifically inactivates the cobalamin depen- marrows, and low serum cobalamin concentra- dent enzyme, synthetase, and tions.6 Routine serum cobalamin assays in produces megaloblastic anaemia in man, has patients presenting with neuropathy did not made it possible to perform extensive animal reveal additional cases with cobalamin studies on cobalamin-folate interrelations, and deficiency. Thus, although the same prevalence to test the methylfolate trap hypothesis in a of cobalamin neuropathy was found in these meaningful way. This has led to the develop- two studies, in the United Kingdom all the ment of alternative ideas as to how cobalamin patients were identified by a routine blood regulates folate metabolism, based on a large count, but in the USA only a proportion seem body of data obtained from study of animals in to have been identified by this means. Such which cobalamin was inactivated. Cobalamin patients were identified by a raised serum neuropathy, too, has been explored in greater MMA concentration. In a second report of 145 depth than has been possible hitherto. On the patients with serum cobalamin concentrations diagnostic side, considerable claims have been below 200 pg/ml Stabler et al7 found that 86 made for the measurement of serum methyl- responded to treatment with cobalamin, and malonic acid as an indicator of cobalamin 94% of these had raised serum MMA concen- deficiency. trations as did 14% of patients who did not respond to cobalamin. One third of these Serum and patients were haematologically normal. This cobalamin neuropathy group extended their studies to patients with An increased urinary excretion of methyl- serum cobalamin concentrations up to 300 pg/ malonic acid (MMA) is well established as a ml and found significant numbers with specific test for cobalamin deficiency. Although neuropathy, raised serum MMA concentra- the test seems to be specific, it is not an early tions, and response of the neuropathy to treat- sign, and some of the least anaemic patients, ment with cobalamin (Communication to the usually with higher though reduced serum FASEB Research Conference, Saxon's River, cobalamin concentrations, have normal MMA USA, August, 1990). excretion.' Treatment with cobalamin is fol- Attempts to identify undiagnosed pernicious lowed by a rapid restoration of a normal output anaemia, including neuropathy, in large of MMA in three to five days. A method for population groups have been made by a variety assay of MMA in serum2 has allowed MMA of means8 and none has indicated a pool of concentrations to be measured in the same missed patients. Thus claims to having dis- samples sent for cobalamin assay and it has covered a significant group ofpatients in whom been combined with measurement ofthe serum cobalamin deficiency has been overlooked, concentration. need to be looked at critically and demand more Plasma homocysteine is raised in most solid proof than has been offered. patients with megaloblastic anaemia due to The ability to confirm cobalamin deficiency either cobalamin or as impair- from a serum sample with a low cobalamin ment of the conversion of homocysteine to concentration by raised MMA concentration is methionine is common to both deficiencies.3 a potentially welcome advance. Apart from the Of 196 consecutive serum samples with difficulty of the assay, which will preclude its MRC Clinical cobalamin concentrations below 170 pg/ml, use by most laboratories, there are a number of Research Centre, 33% had both raised MMA and homocysteine loose ends that need to be tied. Northwick Park concentrations, 45% had normal concentra- The specificity of the assay has to be addres- Hospital, Harrow, Middlesex tions of both metabolites, and 22% an increase sed. Some eight of 59 (14%) of patients not I Chanarin in only one of these substances.4 All but two of responding to cobalamin also had increased R Deacon the patients with an increase in either MMA or MMA concentrations.7 Some several months, M Lumb had evidence of cobalamin or rather than days, elapsed before serum MMA J Perry homocysteine folate deficiency, although whether bone concentrations declined with cobalamin treat- Correspondence to: Dr I Chanarin, marrow aspirations, dU suppression tests, or ment (FASEB Conference, 1990). The decline 11 Fitzwilliam Avenue, Kew, cobalamin absorption tests were done, is not in urinary MMA after oral or Richmond, Surrey stated. represents the return to normal of the metabol- Accepted for publication 18 September 1991 Lindenbaum et al5 reported a high incidence ism not of MMA, but of MMA-coenzyme A. 278 Chanarin, Deacon, Lumb, Perry

MMA, given orally or intravenously, is homocysteine is a potent inhibitor of trans- excreted into urine in a similar way in both methylation reactions and its persistence in the controls and patients with cobalamin central nervous system suggests that it could deficiency.' Assay of serum MMA may interfere with methylation pathways. Weir et therefore not have the specificity or sensitivity al " found a significant increase in S-adenosyl- of urinary MMA excreted after a dose of a homocysteine concentrations in cobalamin physiological precursor. deficient pig brain and this lent further support Fourteen ofthe cobalamin responders repor- to their hypothesis that transmethylation was ted by Stabler et al' had normal Schilling tests, impaired. Direct assessment of methylation and some of the abnormal Schilling tests, as is was not attempted. often the case, could have been due to incom- There are, however, many data which plete urine collection. What was the clinical militate against the hypothesis of an impaired diagnosis in the apparently cobalamin deficient transmethylation. The concentration of S- patients who absorbed cobalamin normally? adenosylmethionine in cobalamin deficient The major evidence offered-that the raised brain remains normal or even raised.'2"1 There serum MMA concentration signified true was no impairment of labelled methyl group cobalamin deficiency-was the improvement incorporation into brain phospholipids in fruit of neuropathy in response to treatment with bats dying of cobalamin neuropathy, and no cobalamin. While some- might regard it as changes in synaptosomal and myelin lipid unethical to carry out a double blind trial with methylation.'4 Methylation of myelin basic cobalamin v placebo, at least the assessment of protein in cobalamin deficient fruit bats was no response should be done blind by a neurologist different from that in controls.'5 not familiar with the treatment that had been Viera-Makings et al 6 repeated the porcine given. Biased assessment ofminor neurological studies of Weir et al in fruit bats, a species that signs in the elderly can be misleading. has been widely used to study cobalamin Evaluation of the place of serum MMA in neuropathy. In cobalamin deficient fruit bats diagnosis requires assessment in fully charac- fatal neuropathy ensued but, unlike the case in terised case material. The clinical importance pigs, S-adenosylmethionine and S-adenosyl- ofa raised serum MMA needs to be assessed in homocysteine concentrations in brain relation to established criteria, including urin- remained unchanged from those in controls. ary MMA excretion after oral valine, marrow Thus the weight of evidence is that impaired morphology, and the dU supression test, as methylation is not the cause of cobalamin well as other criteria normally used in the neuropathy. The observations of Weir et al investigation of patients with megaloblastic remain of great interest. anaemia. Clinical implications of nitrous oxide Cobalmin neuropathy and methylation anaesthesia A substantial proportion of methionine in Although nitrous oxide has been in general use mammalian cells is converted into S-adenosyl- as an anaesthetic agent for over 100 years and methionine, the principal donor of methyl its safety is well established, the observation by groups in transmethylation reactions. The Lassen et al 7 that N20 inhalation given for five impairment of methionine synthesis in to six days to control spasms in tetanus cobalamin and folate deficiency has raised the produced severe, even fatal, megaloblastic possibility that lack of methionine may in turn anaemia was unexpected. Banks et al 8 showed result in lack of S-adenosylmethionine and that N20 reacted with transition-metal com- hence impairment of methylation. Substance plexes of which cobalamin in vivo was the was given to this view by the observation of prime example. N20 reacts with cob[I]alamin impaired choline synthesis in rats with in methionine synthetase; the N20 is cleaved cobalamin deficiency.9 Scott et al'0 found that and the active oxygen species produced cobalamin neuropathy in monkeys, induced by oxidises both the cobalt of cobalamin as well as inactivation of cobalamin by nitrous oxide the apoenzyme. Recovery after N20 requires (N20), was improved by feeding them meth- not only new cobalamin but further synthesis ionine. They suggested that impaired trans- of apoenzyme, taking three to four days. The methylation in nerve tissue was the explana- mutase enzyme, which does not have reduced tion. cobalamin, is not affected by N20 until Further indirect evidence came from the cobalamin stores fall, and there is no increase in neuropathy that developed in pigs exposed to urinary MMA excretion. N20. When S-adenosylmethionine donates its' Amess and his colleagues'9 noted that the methyl group it becomes S-adenosylhomocys- development of megaloblastic haemopoiesis teine. Normally the adenosine is removed and and an abnormal dU suppression test were the remaining homocysteine is recycled back to improved by the addition of cobalamin, in methionine by accepting a methyl group from patients given N20 after surgery, although either methylfolate or from betaine. In normal even exposure to the gas over 24 hours did not brain only the cobalamin dependent pathway produce significant clinical problems. using methylfolate as methyl donor, is present. Megaloblasts persist in the marrow for a fur- In cobalamin deficient brain neither pathway ther three days and giant granulocyte precur- is present. Thus there is no mechanism for sors are still present up to five to six days later recycling the homocysteine other than by after which hypersegmented neutrophils are transporting it to the liver where betaine first seen in the marrow and then in the methyltransferase is available. S-adenosyl- peripheral blood.20 Cobalamin andfolate: Recent developments 279

Intermittent N20 exposure is usually the widely accepted despite the absence of studies result of N20 abuse by dentists, operating to test the hypothesis which proved difficult to theatre technicians, and others. It leads to devise. Many observations were made that classic cobalamin neuropathy.2' It can occur were compatible with such a hypothesis but when N20 is given regularly even for as little as they were equally compatible with any other 15 minutes twice daily, to allow painful hypothesis that postulated interference of procedures to be done.2 folate function as a result of cobalamin Probably the most important effect of N20 deficiency. Nevertheless, belief in the exposure may be when it is used as an anaes- hypothesis was such that Dr J Bertino was able thetic agent in patients with cobalamin malab- to tell an international meeting on pteridines sorption (pre-pernicious anaemia, ileal disease, and at La Jolla, California, USA, that etc) when residual cobalamin stores are des- the methylfolate trap was not hypothesis; it was troyed. Impaired cobalamin absorption fact. prevents the replenishment of these cobalamin The proofrequired to validate a methylfolate stores from dietary cobalamin postoperatively, trap is: (1) evidence that in vivo (as well as in and megaloblastic anaemia or neuropathy vitro) methylene reductase operates only in one devlops several months later.2' direction; (2) that methylfolate is not metab- The effects of prolonged or repeated N20 olised in cobalamin deficiency; and (3) its exposure can largely be prevented by 30 mg sequestration, if this is the case, is such as to folinic acid given six hourly.24 curtail 1-C unit transfer. A few observations appeared that were at odds with the hypothesis. Methylfolate was given intravenously to patients with cobalamin Effect of cobalamin deficiency on folate deficiency in the expectation that, as it could metabolism not be used, it would be cleared from plasma at The diagnosis and treatment of patients with a slower rate than normal.'0 In fact, the disorders of cobalamin or folate metabolism opposite occurred. The removal of methylH4- generally poses few problems. The interrela- folate was more rapid than normal and was tion ofthese two trace vitamins continues to be most rapid in the most anaemic patients. the subject of debate. Methylfolate was found to be a methyl donor Folates are required for the transfer ofsingle in the methylation of biogenic amines such as carbon (1-C) units in the synthesis of three of dopamine. The mechanism proved to be oxida- the four bases of DNA (guanine, adenine, and tion of the methyl group of methylfolate to thymine), for methionine synthesis, as well as formaldehyde (methylene) and this was trans- for synthesis of other compounds.25 These 1-C ferred to the biogenic amine, the methylene units are formate (-CHO) required for purine again being reduced to methyl. Three groups synthesis, methylene (-CH2-) for thymidine isolated the enzyme responsible for oxidising synthesis, and the methyl (-CH3) for methio- the methyl group of methylfolate and agreed nine synthesis. that it was methylene reductase."" Methylene Cobalamin, with folate, is necessary for reductase can be readily made to go in the methionine synthesis, but cobalamin is not forbidden direction by provision of an electron directly involved in the synthesis of any of the acceptor and, indeed, the standard assay for bases needed for DNA. Yet in cobalamin methylene reductase is in the direction of deficiency synthesis ofall these bases is severely methyl into methylene. impaired.26 This is the result of a role Finally, Thorndike and Beck'4 reported that cobalamin has in the availability of 1-C units. the methyl group of methylfolate was oxidised in an essentially similar manner by lym- THE METHYFOLATE TRAP phocytes from normoblastic subjects and by Methylfolate arises by reduction of the -CH2- the lymphocytes from a patient with untreated group on CH2-H4folate to CH3H4folate. The pernicious anaemia. The likely reason (see enzyme involved is methylene H4folate reduc- below) is the provision of methionine in the tase. In vitro studies show that the reaction suspending medium which promotes prompt strongly favours methylfolate synthesis27 and methyl group oxidation. that the reaction does not go in the reverse direction (methylfolate to methylenefolate) to Accumulation ofmethylHj4olate in cobalamin any great extent. This forms the basis for the deficiency methylfolate trap hypothesis put forward in Inactivation of cobalamin by N20 leads to an 1962 by Herbert and Zalusky2' and by Noronha initial rise in the concentration of methylfolate and Silverman.' polyglutamate in tissues due to cessation of The hypothesis proposes that, as in transfer of the methyl group to homocysteine. cobalamin deficiency, the methyl group of After 12 hours, however, the methylfolate methylH4folate cannot be passed on to concentration starts to fall to well below nor- homocysteine to form methionine and, secon- mal. After five days 80% of folate, mainly dly, the methyl group cannot be oxidised back methylfolate, has disappeared from the liver to methylene to form methyleneH4folate: the and other tissues. The fall is the result of H4folate portion is immobilised or trapped. In considerable loss of methylfolate into the time an overall lack of free H4folate will inter- urine.'5 Although there is no accumulation of fere with other 1-C unit transfers and hence methylfolate, the data neither support nor depress thymidine and purine synthesis. This contradict a biochemical folate trap. Both hypothesis was intellectually appealing and was H4folate and smaller amounts of formyl- 280 Chanarin, Deacon, Lumb, Perry

Table 1 Utilisation of ["C] formate by bone marrow cellsfrom normal and cobalamin dietary intake or possibly excessive protein deficient rats catabolism, and in such circumstances syn- are shut down and methionine End product of Marrow cellsfrom: thetic pathways single carbon unit precursors including methylH4folate removed. metabolism Controls: Mean (SD) Cobalamin deficient: Mean (SD) In intact animals taking water only the half- Guanine-RNA 0 97 (0-52) (n = 7)t 0-27 (0 09) (n = 3) life of the methyl group on methylH4folate in -DNA 0-52 (0-28) (n = 6) Trace* (n = 3) rat liver is two hours.39 In cobalamin deficiency Adenine-RNA 1 51 (0-75) (n = 7) 0 97 (0 29) (n = 3) the mechanism -DNA 0-27 (0-12) (n = 6) Trace* (n = 3) the half-life is much longer and Thymine-DNA 2-60 (0-61) (n = 6) 0-72* (0-14) (n = 3) of disposal of the methyl group is different. Methionine 21-2 (11-2) (n = 5) 0* 44-6 (8-3) (n = 4) 3-3* (0-1) (n = 2) Intact fasting control animals not receiving Choline 59-2 (28 2) (n = 5) 14-1 (2-1) (n = 3) methionine pass on the methyl group of Proteint 735 (265) (n = 4) 228* (76) (n = 2) CO2t 0 54 (0-15) (n = 4) 0-07 (0-03) (n = 3) methylfolate to homocysteine. Fasting MethylH4folate 0 (n = 3) 0 (n = 3) cobalamin deficient animals use the methyl- FormylH4folate 0 (n = 3) 0 (n = 3) folate as a substrate for adding on *Significantly different from control value. residues (forming folate polyglutamate, the tExpressed as nmol formate/mg protein. active coenzyme). When six glutamic acid residues are present the methyl group is oxidised, releasing H4folate. Thus after giving H4folate are always detectable in cobalamin methylH4folate labelled in the methyl group deficient tissues. Disappearance of H4folate, to cobalamin deficient rats, labelled methyl- postulated in the methylfolate trap, cannot be H4folate pentaglutamate is detected in liver but demonstrated. not methylH4folate hexaglutamate.39 Deacon et al26 incubated bone marrow cells The available data do not lend any support to from control and cobalamin deficient rats with a methylfolate trap hypothesis. In addition [I4C]formate for one and a half hours and there is a considerable body of data that cannot measured the incorporation of this 1-C unit be explained by a methylfolate trap (see below). into purines, thymine, methionine, etc, by isolating these end products by high pressure FORMATE STARVATION HYPOTHESIS liquid chromatography. The utilisation of for- This view indicates that the role ofcobalamin is mate requires its uptake by H4folate to form in the supply of formylH4folate and, in par- formylH4folate and thereafter its transfer in ticular, in promoting the attachment offormate synthetic pathways (table 1). All the values to tetrahydrofolate. In the absence of with cobalamin deficient cells were lower than cobalamin, formate accumulates in tissues and those with control cells, indicating an overall is excreted in the urine. "Active" formate impairment of 1-C unit transfer. There was no normally arises in the course of intermediary demonstrable labelled formylH4folate, indicat- metabolism, particularly of methionine, and ing that the transfer of formate was rapid. this formate is readily linked to H4folate in both Furthermore, there is no detectable label as control and cobalamin deficient cells. The methylH4folate. The absence of ['4C]methyl- formate starvation hypothesis is based on a H4folate accumulation indicates that the trap- considerable body of data accumulated largely ping of methylfolate cannot be the explanation by study of cobalamin deficient rats exposed to for the overall failure of 1-C unit metabolism N20, and provides a satisfactory explanation with cobalamin deficient marrow cells (table 1). for virtually all the observations made in rela- This study is one of the few direct tests for tion to the effects of Cobalamin deficiency. methylfolate trapping and none was found. Metabolism ofMethylH4olate in cobalamin - 5-CH3H4PteGlu deficiency Methionine 100 gmol Noronha and Silverman29 found that when -- CHO-R-4PteGlu methionine was added to a rat diet in both I --y- H4PteGlu control and cobalamin deficient animals, there 0 - 70- a in liver and a rise in the .d'a was fall methylfolate 60- concentrations of formylfolate and H4folate. 0- This has been confirmed many times, usually (17 50- 0) giving methionine by injection. A parenteral 40- dose of methionine in an amount well within 30- the daily intake produces disappearance of 0) 90% of the methylfolate from liver within 15 4-c 20- minutes and a rise in formylH4folate (fig 1)."' 10- This occurs in both control and cobalamin IL O0 deficient animals. Methylfolate only reac- 6 1 2 3 4 cumulates in liver when the methionine con- centrations have fallen to base line. Thus there Hours after methionine is a profound difference between in vitro and in Figure 1 Rats were given 100 ,umol methionine vivo data. In intact animals reduc- intraperitoneally and thefolate analogues in the liver methylene isolated by high pressure liquid chromatography and tase readily works in both directions reducing assayed microbiologically. The injection of methionine methylene to methyl or oxidising methyl to wasfollowed by a pronouncedfall in the concentration of Methionine is a toxic aminoacid in methyl-H4folate and, at the same time, a rise informyl- methylene. H4-folate and unsubstituted H4folate. The data indicate excess and its concentration is rigidly con- that methionine led to oxidation of the methylgroup on trolled. Excess methionine can only arise from methyl-H4-folate toformyl and CO2. Cobalamin andfolate: Recent developments 281

Table 2 Synthesis offolate-polyglutamate in livers of rats givenfolate-monoglutamate formed in liver from six folate analogues labelled with either ['4C] or [3H] by control and Percentage offolate analogue in rat liver converted intofolate-polyglutamate cobalamin deficient rats. The control rats used all six folate analogues equally with about half Folate analogue Controls Cobalamin deficient the folate taken up by liver being converted into Folate 51 0 polyglutamate. The cobalamin deficient H4folate 55 0 animals were not able to use the first three Methyl-H4folate 42 0 10-formyl-H4folate 52 46 analogues at all, including H4folate itself, but 5,10-methenyl-H4folate 55 59 used the last three normally.4' These last three 5-formyl-H4folate 52 49 folate analogues all had a 1-C unit at the formate level of oxidation. The enzyme which adds glutamic acid residues to folate, folate Reversal of cobalamin deficiency by polyglutamate synthetase, is induced in formylHjolate (but not Hjolate) cobalamin deficient rats.42 There are two sets of observations indicating Thus the effect of cobalamin deficiency was that the effects of cobalamin deficiency are bypassed by providing formylH4folate. Fur- reversed by supplying folate carrying a 1-C thermore, the inability of cobalamin deficient unit at the formate level of oxidation. This is animals to use H4folate, or folate itself, which is available as folinic acid (5, formyl-H4folate). completely stable, is at odds with the methyl- All active folates are reduced-that is, unlike folate trap hypothesis because these analogues pteroyglutamic acid used pharmacologically, are outside the "trap" and should be used they have four additional hydrogens and are normally. called tetrahydrofolates or H4folates. H4folate The second set of circumstances in which is unstable unless protected by reducing agents formylH4folate but not H4folate bypasses the such as ascorbate. Its relative instability is one effects of cobalamin deficiency is in the of the criticisms that has been made in relation synthesis of thymidine. In this pathway a 1-C to these studies.' Thus H4folate (Sigma) has unit (methylene or -CH2-) from methylene- always been freshly reconstituted from the dry H4folate is added to deoxyuridine to form state in 1% ascorbate and used immediately. Its thymidine. Using marrow cells from patients identity has been confirmed by its spectro- with untreated pernicious anaemia,43 as well as photometric absorption. It is very unlikely that marrow from cobalamin deficient rats,44 the oxidation of H4folate was a factor in any of formylH4folate restored thymidine synthesis these results. completely but H4folate was relatively ineffec- The active folate coenzyme in vivo is not tive at the same dose. H4folate but H4folate polyglutamate. Thus The effectiveness of formylH4folate as intracellularly glutamic acid residues are added opposed to the ineffectiveness of H4folate as a to H4folate or to one ofits analogues so that the substrate suggests that cobalamin is concerned number increases from one up to seven. Table 2 with either the supply to or utilisation of shows the amount of folate polyglutamate formate by the folate coenzyme. Reversal of cobalamin deficiency by provision of 'CHO' formate by methionine The synthesis of methionine requires both Methionine cobalamin and folate. Over many years Stok- stad and his colleagues45 have shown that methionine reverses the effects of cobalamin deficiency induced by diet in rats. This has proved to be the case in cobalamin deficiency caused by N2O as well. Thus methionine Methylthio- restored folate polyglutamate synthesis in ribose cobalamin deficient rats4'; restored impaired S-adenosyl- thymidine synthesis'; prevented the develop- methionine ment of cobalamin neuropathy in cobalamin Adoo> deficient monkeys,47 in cobalamin deficient fruit bats,48 and in cobalamin deficient pigs"; restored formylation of H4folate in small gut segments from cobalamin deficient rats49; and reduced formininoglutamic acid excretion in cobalamin deficient rats50 and man.5' 52 Methylthic There are two ways in which methionine adenosin4 e provides "active" formate that the Decarbox)ylated bypasses S-adenosIa1- effects of cobalamin deficiency. Methylthioadenosine: In man between 05-1 P_emethionsinyl mmol of methionine is metabolised each day along the polyamine pathway via S-adenosyl- methionine in the synthesis of spermidine and (fig 2). The synthesis of these com- Polyamines pounds uses three of the carbons of methio- Figure 2 The pathway by which methionine yields an activeformate('CHO) unit. nine, leaving behind a methylthio- (CH3SH =) Ado = adenine. unit attached to ribose. The latter is recycled 282 Chanarin, Deacon, Lumb, Perry into methionine using the CH,SH = of the the biochemical lesion in cobalamin deficiency starting methionine, the remaining carbons is a failure to link formate to H4 folate. By coming from ribose. A 1-C unit as formate is contrast, formate produced in other pathways, released for each mole of methionine formed.53 such a formate arising from the metabolism of Methylthioadenosine was significantly more methionine along the polyamine path, is used effective than methionine itself in restoring normally by cobalamin deficient cells. The folate polyglutamate synthesis in the livers of term "active" formate has been used for this cobalamin deficient rats4' and restored normal form of formate. In biochemical terms the formylation of H4folate in cobalamin deficient difference between formate and "active" for- gut segments.49 mate is not clear. The data suggest that Methylthioadenosine labelled with ['4C] in cobalamin may have a role in the formation of the carbon converted to formate is a potent "active" formate but the necessary investiga- donor of 1-C units in purine synthesis by tions to explore this possibility have yet to be marrow cells from both control and cobalamin done. deficient rats.54 The formate released from The enzyme linking formate to H4folate is methionine via the polyamine pathway is formylH4folate synthetase. The enzyme is sufficient to meet man's formate requirements induced in cobalamin deficiency5758 and the (S Harvey-Mudd, personal communication). increased enzyme activity returns to normal Serine is a potent donor of 1-C units in in only after cobalamin deficiency has been vitro systems, but Deacon (unpublished corrected-for example, by returning animals observations) was unable to demonstrate use of breathing N20 to an air environment. the ,B carbon of serine for purine or thymidine synthesis in intact animals. It may be that in vivo serine is so rapidly metabolised along Folate in the prevention of neural tube other pathways that little is available to donate defects 1-C units. On the other hand, labelled formate Reports that folate (alone or with other was readily used as a 1-C unit in vivo. Formate vitamins) significantly reduced the incidence of via methionine may be the major source of 1-C neural tube defects when taken before or very units in vivo. early in pregnancy59 '" stimulated the setting up of a large trial. A total of 1817 women who had Oxidation of methylH4olate: A rise in the had a previous infant affected with a neural methionine concentration leads to rapid oxida- tube defect were randomly placed in one offour tion of the methyl group of methylH4folate to categories.6' Twenty seven infants were born methylene and formate and hence makes for- with neural tube defects, six in the group taking mate available (fig 1). Thus methionine makes 4 mg folic acid daily, and 21 in the groups not formate available by the release of "active" receiving folic acid. Folate was given before formate in the course of the synthesis of conception until the twelfth week of preg- polyamines from methionine, as well as nancy. The difference was highly significant. promoting the oxidation ofthe methyl group of Other vitamins had no protective effect. methylfolate to methylene and formate. This Earlier data6' had suggested that the women seems to be the explanation for the effect of having infants with neural tube defects did not methionine in reversing cobalamin deficiency. have evidence offolate deficiency. The effect on the embryo may be an example of localised Accumulation offormate in tissues in cobalamin folate deficiency, the supply of folate to the deficiency embryo being limited even in women with Direct measurement of endogenous formate apparently adequate folate nutrition, and it concentrations in liver, blood,26 and brain results in impaired cell division at this crucial (unpublished observations) show a striking time in the development of the embryo. An accumulation of formate in all these tissues in increase in folate concentrations in tissue fluids cobalamin deficient rats (table 3). Increased may overcome this failure oflocal folate supply. formate in the urine of cobalamin deficient rats has been reported.5556 The accumulation of formate in brain indicates that the same bio- chemical defect that is present in liver is present in the 1 Chanarin I, England JM, Mollin C, Perry J. Methylmalonic central nervous system. Thus it acid excretion studies. Br J Haematol 1973;25:45-53. seems likely that the same biochemical lesion is 2 Stabler SP, Marcell PD, Podell ER, Allen RH, Lindenbaum present in all tissues in J. Assay of methylmalonic acid in the serum of patients cobalamin deficiency. with cobalamin deficiency using capillary gas chromato- The accumulation of formate indicates that graphy-mass spectrometry. J Clin Invest 1986;77: 1686-12. 3 Stabler SP, Marcell PD, Podell ER, Allen RH, Savage DG, Table 3 Endogenousformate concentrations in rat Lindenbaum J. Elevation of total homocysteine in the blood, liver, and brain in cobalamin deficiency (nitrous- serum of patients with cobalamin or folate deficiency oxide detected by capillary gas chromatography-mass spec- exposure) trometry. J Clin Invest 1988;81:466-74. 4 Green R, Gatautis V, Jacobsen DW. Serum methylmalonic Formate acid (MMA) and homocysteine (HCY) are more specific Days ofN O tests than serum vitamin B,, for identifying true cobalamin exposure Blood (.ug/ml) Liver (pg/g) Brain (pg/g) (CBL) deficiency. Blood 1990;76(Suppl 1):33a. 5 Lindenbaum J, Healton EB, Savage DG, et al. Neuropsy- 0 23 (3)* 29 (8) 0 chiatric disorders caused by cobalamin deficiency in the 1 80 (18) 83 (18) 14 (14) absence of anemia or macrocytosis. N Engi J Med 2 73 (20) 75 (17) 26 (14) 1988;318:1720-8. 5 72 (12) 66 (8) 6 Shorvon SD, Carney MWP, Chanarin I, Reynolds EH. The neuropsychiatry of megaloblastic anaemia. Br Med J 1980;281:1036-8. *1 SD. 7 Stabler SP, Allen RH, Savage DG, Lindenbaum J. 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