Clinical Science and Molecular Medicine (1974) 47, 617-630.

THE EFFECT OF VITAMIN B i 2 DEFICIENCY ON METHYLFOLATE METABOLISM AND

PTEROYLPOLYGLUTAMATE SYNTHESIS IN HUMAN CELLS

A. LAVOIE, E. TRIPP AND A. V. HOFFBRAND

Department of Haematology, Royal Postgraduate Medical School, London

(Received 22 July 1974)

SUMMARY

1. The uptake of 14C from [methyl-14C]methyltetrahydrofolate was significantly reduced in the phytohaemagglutinin (PHA)-stimulated lymphocytes from nine patients with untreated pernicious anaemia compared with the uptake in seven normal subjects.

2. The uptake of I4C from [14C]methyltetrahydrofolate by the lymphocytes from seven of the patients with pernicious anaemia was consistently increased by addition of vitamin B,, in vitro.

3. The proportion of 14C taken up from [‘4C]methyltetrahydrofolate transferred to non-folate compounds was found to be significantly reduced in the PHA-stimulated lymphocytes from nine patients with untreated pernicious anaemia compared with the proportion transferred in the PHA-stimulated lymphocytes from seven normal subjects. Addition of vitamin BIZ in vitro consistently increased the transfer in vitamin B,,-deficient cells but had no consistent effect in normal cells. 4. Normal and vitamin B,,-deficient PHA-stimulated lymphocytes took up

[3H]folic acid and after 72 h incubation converted this largely into pteroylpoly- glutamate forms.

5. The proportion of labelled lymphocyte folate as pteroylpolyglutamate after incubation with [3H]folic acid was the same in vitamin B,,-deficient as in normal lymphocytes and the proportion of pteroylpolyglutamates formed in vitamin BIZ- deficient lymphocytes was unaffected by addition of vitamin B,, in uitro.

6. No radioactivity could be decteted in pteroylpolyglutamates after incubating normal PHA-stimulated lymphocytes with [14C]methyltetrahydrofolate for 72 h, suggesting that pteroylpolyglutamate forms of folate cannot be made directly from methyltetrahydrofolate.

7. These results are consistent with the ‘methyltetrahydrofolate trap’ hypothesis

Correspondence: Professor A. V. Hoffbrand, Department of Haematology, The Royal Free Hospital, Pond Street, Hampstead, London NW3 2QG.

617

618 A. Lauoie, E. Tripp and A. V. Hofbrand in vitamin B,, deficiency. It is suggested that reduced synthesis of pteroylpoly- glutamates reported by others in vitamin B,,-deficient cells may be secondary to the failure of removal of the methyl group from methyltetrahydrofolate rather than to a direct effect of vitamin B,, deficiency on the enzyme responsible for pteroylpolyglutamate synthesis.

8. Reduced entry of methyltetrahydrofolate into vitamin B,,-deficient cells may be secondary to failure of conversion of this compound into tetrahydrofolate.

Key words : methyltetrahydrofolate, folic acid, pteroylpolyglutamates, vitamin BIZ, phytohaemagglutinin-transformed lymphocytes.

A variety of disturbances of folate metabolism have been described in vitamin B,, deficiency. In humans, these include a high serum folate concentration (Waters & Mollin, 1961, 1963; Herbert & Zalusky, 1962), increased clearance of folic acid (PteGlu) but decreased clearance of 5-methyltetrahydrofolate (methyl-H,PteGlu, methyltetrahydrofolate) from plasma (Chanarin, Mollin & Anderson, 1958; Herbert & Zalusky, 1962; Nixon & Bertino, 1972), low erythrocyte folate (Hansen & Weinfeld, 1962; Cooper & Lowenstein, 1964; Hoffbrand, Newcombe & Mollin, 1966), low leucocyte folate (Hoffbrand & Newcombe, 1967), decreased transport of methyltetrahydrofolate into cells (Das & Hoffbrand, 1970a; Tisman & Herbert, 1973), and an increased ratio of free to total pteroylpolyglutamate content in mature erythro- cytes, implying decreased synthesis of pteroylpolyglutamate forms of folate (Jeejeebhoy, Pathare & Noronha, 1965; Chanarin, Perry & Lumb, 1974).

Decreased liver folate, decreased synthesis of pteroylpolyglutamate from folic acid and an excess of methylated compared to formylated folates has also been described in the livers of vitamin B,,-deficient experimental animals (Thenen & Stokstad, 1973 ; Smith & Osborne- White, 1973).

The only biochemical reaction known to require both vitamin B,, and folate in mammalian cells is the methylation of homocysteine to methionine (Stokstad & Koch, 1967; Huennekens, 1968; Weissbach & Taylor, 1968), and it has been suggested that the defects of folate meta- bolism in vitamin B,, deficiency are consequent on folate becoming 'trapped' as methyl- tetrahydrofolate through failure of this reaction (Noronha & Silverman, 1962; Herbert & Zalusky, 1962). In the present paper the results of studies of the transfer of the methyl group from labelled methyltetrahydrofolate to non-folate compounds and of pteroylpolyglutamate synthesis from methyltetrahydrofolate and from folic acid are reported. These studies provide direct evidence in favour of the methyltetrahydrofolate trap hypothesis in vitamin B, deficiency and may help to unify the observations of a methyltetrahydrofolate trap, reduced methyl- tetrahydrofolate transport into cells and reduced intracellular pteroylpolyglutamate formation into a single theory to explain the abnormalities of folate metabolism seen in patients with megaloblastic anaemia due to vitamin B, deficiency.

MATERIALS A N D M E T H O D S Incorporation of radioactivity from [methyl-14C]naethyltetrahydrofolate (['4C]methyl-H4PteGlu) into non-folate compounds

Lymphocyte cultures were set up from heparinized venous blood as previously described

Methylfolate metabolism in vitamin B, deficiency 619 (Das & Hoffbrand, 1970a), except that Triosill-Ficoll was used to separate lymphocytes and granulocytes (Boyum, 1968). Phytohaemagglutinin (€‘HA) (Wellcome) was added at time zero and the cultures were incubated for 72 h. [14C]Methyl-H4PteGlu (The Radiochemical Centre, Amersham, Bucks.; specific radioactivity 61 mCi/mmol), a 50:50 mixture of diastereoisomers epimeric about C-6, 0.5 pCi, was added to each of the 3 ml cultures. [14C]Methyl-H4PteGlu was dissolved in 0.2 moll1 2-mercaptoethanol and stored in aliquots at -70°C until the day of use. The purity was checked by chromatography on DEAE- cellulose of an aliquot of the batch and of the aliquot stored for the longest time; both showed over 95% purity (Corrocher, Bhuyan & Hoffbrand, 1972). In some experiments, 7.4 nmol (10 pg) of vitamin BIZ (cyanocobalamin) was also added in vitro at the start of the incubation period to half of the cultures. In one experiment, bone-marrow cells from a patient with untreated pernicious anaemia were incubated with [‘4C]methyl-H4PteGlu. The bone marrow culture was set up as described by Metz, Kelly, Chappinswett, Waxman & Herbert (1968), 0.5 pCi of [‘4C]methyI-H4PteGlu being added to 3 x lo6 bone-marrow cells in a total volume of 1 ml.

The cells were incubated at 37°C without shaking (lymphocytes for 72 h, bone marrow for 24 h). It is likely that some decomposition of [14C]methyl-H4PteGlu, and to a less extent of [3H]folic acid (see below), occurred during the 72 h incubation periods, which were used to achieve significant radioactivity counts in the cells. However, the decomposition products of these compounds do not chromatograph as folate or protein in the experiments listed below. Moreover, control experiments with [14C]methyl-H4PteGlu and C3H]folic acid deliberately allowed to decompose over weeks on the bench have both shown less than 10% uptake of radioactivity by normal PHA-stimulated lymphocytes over a 72 h period compared to experiments with chromatographically pure ( > 95%) materials. The cells were harvested by centrifugation three times in cold (4°C) TC 199 medium (Wellcome), suspended in cold 0.1 mol/l phosphate-citrate buffer, pH 4.6, containing 0.057 mol/l ascorbic acid and 0.2 mol/l 2-mercaptoethanol and sonicated. Polyglutamate forms of folate were deconjugated by incubation with 0.1 ml of fresh normal human plasma for 90 min since this completely con- verts the compounds into monoglutamate forms and the non-protein compounds were extracted by heat at 11 5°C for 5 min. The protein precipitate was washed twice in the phosphate-citrate buffer and the precipitate digested with 2 ml of Hyamine hydroxide (1 mol/l in methanol. Koch-LLight Laboratories). The proportion of 14C in folate and non-folate compounds in the combined washings and supernatant was measured as described by Nixon, Glutsky, Nahas & Bertino (1973) as follows: 1 ml was kept for radioactivity counting, the rest was poured through an ion-exchange column (AG-1, X8, Biorad; 2.5 cm x 0.5 cm). The eluate was collected and combined with a 3 ml wash of a 1 mmol/l solution of methionine. Folates were then eluted from the resin with 50 ml of 1 mol/l hydrochloric acid. The total radioactivity in each of the two eluates was then measured. Recovery of d.p.m. added to the AG-1 (X8) column ranged from 87 to 115% (mean 97%).

Incorporation of 3H-labelled folic acid into pteroylpolyglutamates In these experiments, PHA-stimulated lymphocytes were incubated for 72 h with tritiated

folic acid [3’,5’,9-3H]folic acid (31 Ci/mmol; The Radiochemical Centre), 2 Ci (64.5 pmol)/3 ml culture. The [3H]folic acid was made up in distilled water, stored in aliquots at -70°C and

620 A . Lavoie, E. Tripp and A . V. Hofbrand the purity checked by DEAE-cellulose column chromatography (Corrocher et al., 1972) on the initial sample and final aliquot (both of which showed over 95% purity). The cells were then washed three times with cold (4°C) TC 199 medium and resuspended in 0.1 mol/l potassium phosphate buffer, pH 8.5, containing 1 g of ascorbic acid/100 ml and 0.2 mol/l 2-mercaptoethanol, and heat-extracted at 115°C for 5 min. The labelled folates in the super- natant were treated with 4 ml of potassium permanganate solution (126 mmol/l) to digest folates at the C-9-N-10 bond before DEAE-cellulose column chromatography of the p - aminobenzoylglutamate fragments, according to the number of glutamate moieties remaining as described by Houlihan & Scott (1972).

TABLE 1. Haematological data for the ten patients with untreatedpernicious anaemia and seven control subjects

Pernicious anaemia (lymphocytes) 1

2 3 4 5 6 7 8 9

F/52 M/72 F/55 F/76 F/60 MI63 F/63 F/49 MI41

10.5 10.1 7.1 9.6 6.3 6.9 8.6 5.1 5.8

44.3 (60) 70.8 ( 96) 5.9 (8)

44.3 (60) 50-2 (68) 22.6 (32) 44.3 (60) 56.1 (76) 70.8 (96)

48.0 (21.1) 10.9 (4.8) 28.1 (12.4) 29.7 (13.1) 25.9 (1 1.4) 345 (15.2) 22.4 (9.9) 14.3 (6.3) 8.4 (3.7)

Bone marrow 10 M/58 7.3 20.7 (28) 12.7 (5.6)

Normal subject (lymphocytes) 1 MI38 14.2 460 (624) 15.0 (6.6)

2 M/31 15.0 236 (320) 17.7 (7.8) 3 M/28 13-5 218 (296) 11-8 (5-2) 4 F/28 12.7 361 (489) 22.9 (10.1) 5 F/33 13.6 213 (289) 8.4 (3.7) 6 F/26 12.5 282 (382) 21.5 (9-5) 7 M/30 14.5 314 (425) 18-6 (8.2)

Incorporation of radioactivity from ['4C]methyl- H4PteGlu into pteroylpolyglutamates Experiments were performed as described in the preceding paragraph, except that the cells

were incubated for 72 h with 0.5 pCi of ['4C]methyl-H4PteGlu/3 ml culture. After washing and heat-extraction the extract was poured through a DEAE-cellulose column and folates were eluted with a phosphate buffer gradient as described by Corrocher et al. (1972). The radioactivity in the eluates was measured.

Methy yolate metabolism in vitamin B, deficiency 621 Liquid-scintillation counting

The counting liquid consisted of Triton XlOO 500 ml, toluene 1 ml, POPOP (1,4-bis-(5- phenyloxazol-2-y1)benzene) 0.45 g, PPO (2,5-diphenyloxazole) 6 g, to 10 ml of which 1 ml of the sample was added. Counts were converted from c.p.m. into d.p.m. by use of an internal standard ([14C]toluene, 0.502 pCi of 14C/g; The Radiochemical Centre).

Subjects Studies were made on the lymphocytes of nine patients with untreated pernicious anaemia.

The marrow from a tenth patient was also studied (Table 1). The patients comprised four males and six females. Their haemoglobin concentrations ranged from 5.1 to 10.5 g/dl and all showed florid megaloblastic marrow appearances with adequate iron stores, and malabsorption of vitamin B,, corrected by intrinsic factor. The serum vitamin B12 con- centrations ranged from 5.9 to 70.8 pmol/l(8-96 ngll) and serum folate concentrations from 8-4 to 48 nmol/l (3.7-21-2 pg/1). Cells were also obtained from seven normal healthy adult volunteers, all with normal serum vitamin B, and folate and haemoglobin concentrations (Table 1).

RESULTS

Transfer of l4C from [‘4C]methyl-H4PteGlu to non-folate compounds There was significantly lower uptake of 14C from [14C]methyl-H4PteGlu by the vitamin

B,,-deficient lymphocytes compared with normal lymphocytes (P< 0.005; t-test) (Table 2). Addition of vitamin B,, in uitro significantly increased the uptake in the vitamin B,,-deficient cells (P<0-005; paired t-test) but had no consistent effect in the four normal cell samples tested. The proportions of 14C recovered as folate, protein and non-folate, non-protein compounds in the PHA-stimulated lymphocytes and bone marrow are also shown in Table 2. The proportion recovered as folate in the nine patients with pernicious anaemia ranged from 28.8 to 56.0% (mean 39.4%). This was significantly greater than the proportion of I4C recovered as folate in the lymphocytes from the seven normal subjects (range 9.1-26-0, mean 15.0%) (P<O-Ool). In the seven pernicious anaemia patients tested, addition of vitamin B,, in vitro significantly reduced both the amount and proportion of 14C remaining as folate (t-test for paired samples, P < O . O l and <0.05 respectively) and significantly increased the amount recovered as protein (t-test for paired samples, P<O.Ool). Vitamin B,, added in uitro had no consistent effect on the proportion of 14C recovered as non-folate, non-protein compounds in the vitamin B,,-deficient cells or on the proportion recovered as folate, protein, and non-folate, non-protein compounds in normal cells (Table 2).

Synthesis of pteroylpolyglutamate from [3H] folic acid: eflect of vitamin B,, deJiciency This was studied in the PHA-stimulated lymphocytes from four normal and four vitamin

B,,-deficient patients. The proportion of the incorporated radioactivity as pteroylpoly- glutamates in the four normal PHA-stimulated lymphocytes ranged from 85 to 92% (mean 88%). After KMnO, digestion, the p-aminobenzoylglutamate fragments chromatographed

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Methy yolate metabolism in vitamin B, deJiciency 623 just in front of KMn0,-treated synthetic pteroylhexaglutamic acid (Fig. 1). Labelled pteroylpolyglutamates chromatographing in a position identical with labelled compounds synthesized in normal cells were synthesized from [3H]folic acid in vitamin BIZ-deficient lymphocytes from all four patients, and the proportion of pteroylglutamate among the labelled cell folate was similar (range 84-93%, mean 87%) to normal and was not significantly altered by addition of vitamin B,, in vitro at the beginning of the 72 h cultures in any of the four cases (Fig. 1).

10 30 50 70 Fraction no.

FIG. 1. DEAE-cellulose column chromatography of untreated pernicious anaemia lymphocytes labelled with [3H]folic acid after treatment with 2% potassium permanganate (see the text). Typical results from one of four patients. (a) Results with vitamin B,, added at the start of culture; (b) results without added vitamin BIZ. 0, Radioactivity of hydrolysed labelled lympho- cyte folate; 0, extinction of hydrolysed synthetic pteroylhexaglutamic acid (PteGlu6) standard [prepared by Dr J. Perry, Northwick Park Hospital, by the method of Krumdieck & Baugh (196911.

Synthesis of pteroylpolyglutamates from [14C]methyZ-H4PteGlu In none of three experiments with normal lymphocytes and one experiment with vitamin

B1 ,-deficient lymphocytes from case 10 could I4C-labelled pteroylpolyglutamates be detected after incubation of the PHA-stimulated lymphocytes with [14C]methyl-H,PteGlu for 72 h. The radioactivity eluted entirely as a single peak corresponding to standard [14C]methyl- H,PteGlu (Fig. 2).

624 A . Lauoie, E. Tripp and A . V. Hoflbrand

0 I0 20 30 40 50 60 70 00 Fraction no.

FIG. 2. DEAE-cellulose column chromatography of normal lymphocytes labelled with [ 14CJ-

methyltetrahydrofolate. A single peak at tube 17 corresponding to standard ['4C]methyltetra- hydrofolate ([14C]methyl-H4PteG1u) was observed and no radioactivity was detected in the pteroyldi-, pteroyltri- or pteroylpoly-glutamate regions (tubes 26-75 ; Corrocher et d., 1972).

DISCUSSION

The results of these studies confirm previous reports that uptake of methyltetrahydrofolate by vitamin B,,-deficient cells is subnormal and can be corrected by addition of vitamin B,, in vitro (Das & Hoffbrand, 1970a; Tisman & Herbert, 1973). They also give direct support for the concept that vitamin B,, deficiency causes a trapping of folate in the methyltetra- hydrofolate form in human cells, as also shown by Chello & Bertino (1973) for L5178Y cells, and suggest how such a trap might explain reduced formation of other intracellular folates, including pteroylpolyglutamate forms, in vitamin B , , deficiency.

The studies of transfer of the methyl group from methyltetrahydrofolate to non-folate compounds show that this transfer is consistently decreased in the cells of vitamin B12- deficient patients compared with normal cells, and can be partly or completely corrected in vitamin B,, deficiency by addition of vitamin B,, in vitro. The transfer of the methyl group to protein was also consistently increased by addition of vitamin B,, in vitro, presumably owing to increased formation of methionine. The differences, though consistent and significant, were not great and might seem insufficient to explain a major disturbance of intracellular folate metabolism. However, the synthesis and activity of the enzyme 5,lO-methylenetetrahydrofolate reductase, which synthesizes 5-methyltetrahydrofolate, and of the enzyme 5-methyltetra- hydrofolate-homocysteine methyltransferase may be modified by a number of intracellular metabolites including homocysteine, methionine and S-adenosylmethionine (Kutzbach & Stokstad, 1967; Kamely, Littlefield & Erbe, 1973), and the effect of a block due to vitamin BIZ deficiency in conversion of homocysteine into methionine on the levels of these inter- mediates in human marrow and other cells may be important in determining how much tetrahydrofolate can be formed from methyltetrahydrofolate inside these cells. For instance, Thenen & Stokstad (1973) and Smith, Osborne-White & Gawthorne (1974) observed that methionine added to the diet substantially increased pteroylpolyglutamate synthesis in vitamin B,,-deficient rats and sheep respectively. Thenen & Stokstad (1973) attributed their results to conversion of methionine into S-adenosylmethionine, which then inhibited methylenetetra-

Methylfolate metabolism in vitamin B, , deficiency 625

hydrofolate reductase (Kutzbach & Stokstad, 1967) and so maintained folate in the tetra- hydrofolate and 10-formyltetrahydrofolate states. Noronha & Silverman (1962) originally showed that added dietary methionine does indeed reduce the proportion of 5-methylfolate derivatives in vitamin B,,-deficient rat liver and increases 10-formylfolate derivatives.

An alternative theoretical explanation for the results here could be proposed, based on the assumptions : (a) that human lymphocyte and bone-marrow cells take up significant amounts of the inactive as well as the biologically active diastereoisomer of methyltetrahydrofolate, (b) that lack of vitamin B,, only reduces uptake of the active diastereoisomer, and (c) that only the active isomer is capable of transferring its [14C]methyl group to protein. Weir, Brown, Freedman & Scott (1973) have shown that passive transfer of the inactive form of [14C]methyltetrahydrofolate may occur across the human small intestinal mucosa. The relative uptake of active and inactive isomers of methyltetrahydrofolate by human haemopoietic cells has not been studied and this theory therefore cannot be completely ruled out. I t is, however, a less attractive explanation for the present results on 14C transfer from [l4C]methy1tetra- hydrofolate, since in each case of pernicious anaemia studied, the absolute radioactivity counts recovered as folate fell on addition of vitamin BIZ.

Folate enters human marrow and other cells in vivo in the reduced monoglutamate form, methyltetrahydrofolate, which has been produced from ingested natural folates by the in- testinal mucosal cells (Perry & Chanarin, 1970). Over 90% of intracellular folate is, however, in the pteroylpolyglutamate form, methylated, formylated and with other single carbon atom substitutions. Cells must therefore be capable of converting monoglutamate forms of folate into polyglutamate forms. The results here suggest, however, that the methyltetrahydrofolate which enters the cell is not a direct substrate for pteroylpolyglutamate formation in human cells, since no [14C]methylated pteroylpolyglutamates could be detected from [methyl-14C]- methyltetrahydrofolate. The alternative explanation for the observation in Fig. 2, that methyl- ated pteroylpolyglutamates are formed directly from methyltetrahydrofolate and the [14C]- methyl groups are then rapidly and completely removed, seems unlikely in view of the high proportion of pteroylpolyglutamates which are methylated in mammalian cells (Corrocher et al., 1972; Thenen & Stokstad, 1973). It is more likely that a monoglutamate form of folate other than methyltetrahydrofolate is the natural substrate for pteroylpolyglutamate for- mation in human cells. Griffin & Brown (1964) showed that in Escherichia coli di- or tetra- hydrofolate is the preferred substrate for this reaction and it is probable that highly reactive tetrahydrofolate is the normal substrate in human cells for pteroylpolyglutamate formation. Chanarin et al. (1974) have postulated that vitamin B,, is needed directly for pteroylpoly- glutamate synthesis in human cells. The alternative hypothesis is suggested here that pteroyl- polyglutamate formation may be less than normal in vitamin BI2 deficiency, not because of direct involvement of vitamin B , in pteroylpolyglutamate synthesis but rather because the ‘methyltetrahydrofolate trap’ leads to reduced supply of tetrahydrofolate, the correct sub- strate for pteroylpolyglutamate formation (Fig. 3). The ability of vitamin B1 ,-deficient cells to synthesize pteroylpolyglutamates normally from folic acid shown here fits with the view that there is no intrinsic defect of polyglutamate formation in vitamin BIZ-deficient cells.

The twin observations of relative failure of vitamin BIZ-deficient cells to take up methyl- tetrahydrofolate with normal uptake of folic acid in vitro (Das & Hoffbrand, 1970a; Tisman & Herbert, 1973) and slow clearance in vivo of methyltetrahydrofolate but rapid clearance of folic acid (Chanarin ef al., 1958; Herbert & Zalusky, 1962; Nixon & Bertino, 1972) are con-

626 A . Lavoie, E. Tripp and A . V. Hoflrund sistent with the hypothesis that incorporation of folic acid into pteroylpolyglutamates is normal in human vitamin B, ,-deficient cells, whereas pteroylpolyglutamate formation from methyltetrahydrofolate is defective (Fig. 3). The normal monoglutamate pool of folate found in the cells of vitamin BIZ-deficient rats (Thenen & Stokstad, 1973) and humans (Chanarin et al., 1974) rather than an increased pool as might be expected on the ‘methyltetrahydrofolate trap’ hypothesis may be due to inability of cells to retain excess amounts of the principal monoglutamate form, 5methyltetrahydrofolate. Conversion of folates from monoglutamate into polyglutamate forms is probably necessary for retention in the cells (Brown, Davidson & Scott, 1974) and excess excretion of 5-methyltetrahydrofolate from cells in vitamin B,, defi- ciency could maintain the intracellular pool of monoglutamate constant.

PLASMA

methyl-HQteG

PteGlu-

CELLS

methyl-H,PteGlu 1 homocysteine -

I rnethionine t- BIZ* I

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H,PteGlu, -* etc.

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FIG. 3. Proposed site of block in pteroylpolyglutamate formation from methyltetrahydrofolate (methyl-H,PteGlu) in vitamin B I Z deficiency (*). PteGlu = folic acid; HzPteGlu = dihydro- folate; H,PteGlu = tetrahydrofolate; H4PteGI~i, o r 3, C t E . = tetrahydropteroyldi- or triglutarnate, etc.

For vitamin B ,-dependent methionine synthesis methyltetrahydrofolate, a monoglutamate, rather than the polyglutamate derivative, is likely to be the active coenzyme in mammalian systems (Guest, Friedman, Dilworth & Woods, 1964; Mangum, Murray & North, 1969). For other folate-requiring reactions, pteroylpolyglutainates may be the natural coenzymes and monoglutamates only useful models of these reactions (Blakley, 1957, 1959; Rabino- witz, 1960). The results of Shin, Williams & Stokstad (1972) and Houlihan & Scott (1972) have shown that pteroylpentaglutamates are the main forms in rat liver and our results confirm that pteroylpenta- and pteroylhexa-glutamates predominate in human lymphocytes

Methylfolate metabolism in vitamin B, dejiciency 627

(Lavoie & Hoffbrand, 1974, and unpublished observations). The predominance of these forms of folate in rapidly proliferating primitive cells as well as in resting cells in both humans and rats suggests that these pteroylpolyglutamates may form the natural folate coenzymes needed for reaction in purine and thymidine synthesis (Lavoie, Tripp, Parsa & Hoffbrand, 1975).

Thymidylate synthesis is defective in vitamin BIZ-deficient marrow and lymphocytes (Metz et al., 1968; Das & Hoffbrand, 1970b) and this can be corrected by vitamin B,, and folic acid, but not by methyltetrahydrofolate, and the defect can be made worse by methionine and reduced by homocysteine (Metz et al., 1968; Das & Hoffbrand, 1970b; Waxman, Metz & Herbert, 1969). These observations are consistent with the present hypothesis that vitamin B,, deficiency may cause reduced DNA synthesis by the sequence of reduced synthesis of the probable natural coenzyme for thymidylate synthetase, 510-methylenetetrahydropteroyl- penta- (or hexa-)glutamate, due to a lack of tetrahydrofolate for polyglutamate formation due to trapping of folate in the monoglutamate form of methyltetrahydrofolate (Fig. 3). Failure of serine-glycine interconversion (DeGrazia, Fish, Pollycove & Wallerstein, 1972 ; Ellegaard & Esmann, 1973), deranged purine metabolism (Herbert, Streiff, Sullivan & McGeer, 1964) and excessive excretion of formiminoglutamic acid (Spray & Witts, 1959; Chanarin, 1960; Kohn, Mollin & Rosenbach, 1961; Zalusky & Herbert, 1961) could all be due to lack of formation of the relevant pteroylpolyglutamate coenzymes, as also proposed by Chanarin et al. (1974). Reduced interconversion of pteroylmonoglutamate forms due to the ‘methyl- tetrahydrofolate trap’ could also deprive the cells of monoglutamate coenzyme forms where these are the natural coenzymes in human cells.

The failure of patients with vitamin BIZ deficiency to respond to physiological doses of folic acid (Marshall & Jandl, 1960) suggests defective utilization of folic acid itself in vitamin Blz deficiency. A number of explanations for this are possible, despite the normal pteroyl- polyglutamate formation from folic acid in vitamin BIZ-deficient cells shown here. It may be that conversion in vivo of the small doses of folic acid into methyltetrahydrofolate occurs in the intestine or liver with the subsequent inability of the vitamin B,,-deficient bone marrow to utilize methyltetrahydrofolate. Displacement of methyltetrahydrofolate from the liver by folic acid may also prevent the marrow cells from obtaining folic acid unchanged (Chanarin & McLean, 1967). Larger doses of folic acid cross the intestine and circulate partly unchanged, and these might be expected to enter the bone marrow as such and to produce a haemato- logical response after conversion inside cells into folate coenzymes. It may also be that vitamin B,, deficiency causes a disturbance in interconversion of folate coenzymes at the pteroyl- polyglutamate level, as suggested by the observation of Thenen & Stokstad (1973) in the rat, or has some other as yet unrecognized effect on pteroylpolyglutamate metabolism. Further measurements are now needed of the intracellular concentrations and turnover of specific folate monoglutamate and polyglutamate coenzymes in vitamin B, deficiency to investigate these possibilities.

ACKNOWLEDGMENTS

We thank Dr L. Al-Naib, Dr I. Chanarin, Dr B. I. Hoffbrand, Dr J. G. McSorley, Dr E. Offerman, Dr J. Parker-Williams, Dr P. D. Roberts and Dr Phyllida Roberts for assistance in studying the patients with pernicious anaemia, Dr J. Perry for the gift of pteroylhexa-

A . Lavoie, E. Tripp and A . V. HoJgirand glutamic acid and Mr J. 0. Morgan for the microbiological assays. A.L. is in receipt of a Research Fellowship from the Medical Research Council of Canada, E.T. and A.V.H. are in receipt of grants from the Leukaemia Research Fund and A.V.H. from the Medical Research Council.

REFERENCES

BLAKLEY, R.L. (1957) Interconversion of serine and glycine: some further properties of the enzyme system. Biochemical Journal, 65,342-348.

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