British Journal of Haematology, 1976, 32, 243.
Role of Vitamin B,, in Folate Coenzyme Synthesis JANET
PERRY, M. LUMB,M. LAUNDY,E. H. REYNOLDS AND I. CHANARIN
Medical Research Council Clinical Research Centre, Harrow, Middlesex (Received 7 July 1975; acceptedfor publication Ig July 1975) SUMMARY. Normal red cells in man were found to contain predominantly folate pentaglutamates with smaller amounts of tetra- and hexapolyglutamates. There was no change in the type of polyglutamate present in red cells from patients with vitamin B,, deficiency and primary folate deficiency. In contrast to the fall in red cell polyglutamate concentration in vitamin B,, deficiency, there was a marked fall in short-chain folates in early folate deficiency (treated non-anaemic epileptics) and a fall in both short chain and long chain polyglutamates in patients with severe folate deficiency and megaloblastic anaemia. These differences in folate distribution within cells exclude a primary failure to transport metliylfolate into cells as the lesion in vitamin Biz deficiency. The failure of folate polyglutamate synthesis in vitamin B, deficiency arises either from a failure to provide the proper substrate for polyglutamate synthesis or to a direct requirement for vitamin B, for polyglutamate synthesis. Folate in body fluids is 5-methyltetrahydrofolate. It has one glutamic acid residue and is the form in which folate is absorbed from the gut and transported to cells (Chanarin & Perry, 1969). The form in cells is largely a polyglutamate with up to six glutamic acid residues attached to the folate molecule. Evidence is accumulating that it is this longer chain folate which is the active coenzyme form. In Cfostridiu Curthoys & Rabinowitz (1972) showed that the binding of formyltetrahydrofolatetriglutamateto formyltetrahydrofolate synthetase is IOO fold greater than that of the monoglutamate, and Kisliuk et a1 (1974) using thymidylate synthetase from L. casei demonstrated that tri- and hexaglutamyltetrahydrofolate were three times better substrates for the enzyme than the equivalent monoglutamyl derivative. Both groups of workers postulated a regulation of folate metabolism by the products of these reactions. Two recent reports concern mammalian cell lines, one describing the effectiveness of dihydropteroylpolyglutamates as substrates for mammalian dihydrofolate reductase (Coward et al, 1974), and the other a hamster ovary cell line that could not form polyglutamates, and was shown to have an absolute requirement for adenosine, thymine and glycine (McBurney& Whitmore, 1974), compounds whose synthesis is folate dcpendent. In vitamin BIZdeficiency the reduced red cell folate concentration (Cooper & Lowenstein, 1964; Hansen, 1964) is due to a lowering of polyglutamyl folates only, the concentration of the shorter chain folates remaining the same as in control subjects (Jeejeebhoy et al, 1965; Chanarin eta!, 1974). W e have now studied the effect ofvitamin BIZtherapy on polyglutamyl folate concentrations, determined the glutamyl chain length of red cell folates in patients Correspondence: Dr I. Chanarin, Department of Haematology, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ. G
243
244
Janet Perry et a1
with both vitamin Blz and folate deficiency, and in normal subjects, and measured the distribution of short chain and long chain polyglutamates in anticonvulsant-treated epileptic patients. These results are discussed in relation to current views on the action of vitamin BIZ. MATERIALS, METHODS AND PATIENTS STUDIED The shorter chain and longer chain glutamyl folates in red blood cells were measured as previously described (Chanarin et al, 1974), and the results expressed as p g folate per litre of packed red cells. Observations were made on 42 patients with epilepsy, half of whom were macrocytic, receiving treatment with anticonvulsant drugs. These data will be presented in more detail elsewhere. Red blood cells were prepared for chromatography by a modification of the method of Shin et a1 (1974) in which the red cells were washed with buffered saline, pH 7.0, prior to being added to the boiling solution of ascorbate. To determine the type of folate compound in human red cells, the extracts were assayed microbiologically using Lactobacillus casei (NCIB8oIo), Streptococcus faecalis (NCIB6459) and Pediococctrs cerevisiae (NCIB7837). Folate activity was detected with L. casei only, even when aseptic addition techniques were used, indicating the presence of 5-methyltetrahydrofolate only. The concentrated lysates, together with marker compounds which were high specific activity tritiated 5-methyltetrahydrofolate polyglutamates of known chain length, were applied to an 0.9 x 50 cm column of DEAE cellulose (Whatman DE 52), and eluted with potassium phosphate buffer, pH 6. The buffer concentration was varied by a gradient consisting of 140 ml of 0.1 M phosphate in the mixing chamber and 500 ml of 1.0 M phosphate in the reservoir. Both solutions contained 0.2 M mercaptoethanol. 5 ml fractions were collected, and each fraction was assayed microbiologically (L. c m i ) both pre- and post-incubation with human plasma (0.1 ml plasma per rnl of column fraction). Gammaglutamyl carboxypeptidase in the plasma rapidly converts polyto mono-glutamyl folates which are then available as growth factors for the assay organism. Tritium activity was determined by adding 0.1 ml of each fraction to Bray’s scintillation fluid (Bray, 1960), whch was then counted in a Wallac-LKB liquid scintillation counter. Counts per minute were converted to disintegrations per minute (dpm) by the use of the external standard channels ratio method. The effect of vitamin BIZtherapy on polyglutamyl folate concentration was determined in five patients with pernicious anaemia. Column chromatography of red cell lysates was done in five normal subjects, four patients with pernicious anaemia (both pre and post vitamin B,, therapy) and one patient with severe folate deficiency. RESULTS When vitamin Blz was given to patients with pernicious anaemia the red cell folate concentration increased rapidly, and this increase was found to be due to a rapid restoration of polyglutamyl folates, with no change in the short-chain glutamyl folate levels. The five patients on whom sequential red cell folate estimations were done all showed a similar pattern, and a typical response to vitamin BIZtherapy is shown in Fig I. Chromatography of red cell lysates from six normal subjects showed almost identical
Vitamin B,, a d Folate Coenzyme
245
-
0
10 Days
20
FIG I. The effect of vitamin Blz administration on the red cell polyglutamyl folate concentration in pernicious anaemia. -, PteGlu 4-6; - - -, PteGlu 1-3.
elution patterns. The major component was methyltetrahydropentaglutamate, with a lesser quantity of tetra- and hexa-glutamates, and small peaks of what are presumably mono, diand tri-glutamates (Fig 2 ) . In vitamin B,, deficiency the pattern of polyglutamyl folates was similar to that found in normal subjects, methyl pentaglutamate again being the major fraction. Red cells taken from a patient with pernicious anaemia after 4 weeks of BIZtherapy showed increased amounts of the shorter chain polyglutamates which probably represent the folates formed after the first few days of vitamin BI2 administration (Fig 3). Severely folatedeficient red cells also gave an elution pattern similar to that found in normal red cells (Fig 4). 5CH, H4 PteGlu,
t
Fraction no.
FIG2. DEAE celIulose chromatography of an extract of normal red celIs assayed for foIate with L. casei. The labelled peaks are those whose identity was confirmed by co-chromatography with standard marker compounds.
Janet Perry et a1
PA after B,, therapy
5CH,
, PteGlu,
5CH, H, PteGlu,
PA before B,, therapy
I
Fraction na.
FIG 3 . DEAE cellulose chromatography of red cell extracts (assayed for folate with L. cusei) from a patient with pernicious anaemia before, and 4 weeks following, vitamin B, therapy.
Fraction no.
FIG4. DEAE cellulose chromatography of a red cell extract (assayed for folate with L. casei) from a severely folate deficient patient.
Vitamin B,, and Folate Coenzyme
247
TABLE I. Mean red blood cell folate concentrations in controls, vitamin B,, deficiency, anticonvulsanttreated epileptics and folate-deficient megaloblastic anaemia (pg/l.) Folute form jCH,H4PteGh
Controls (3 I ) Vitamin B1, deficiency (11) Treated epileptics (41) Folate deficiency (7)
(SEM)
5CH3H4PteGlu4-
86
(6.3)
203
85
(14.0)
108
19.5
(1.51)
21
(4.8)
160 48
(SEM) (15.68) (26.2) (11.1) (7.1)
In treated epileptics there was a marked reduction in the level of short chain folates in red cells without a significant fall in long chain compounds (Table I). In severe folate deficiency associated with a megaloblastic anaemia there is a marked reduction in both forms of folate. DISCUSSION In pernicious anaemia folate metabolism is abnormal. This is associated with a reduced level of polyglutamyl folates in cells in vitamin B,, deficiency as compared to normal subjects (Chanarin et al, 1974). There is a rapid restoration of red cell polyglutaniyl folate concentrations following B,, therapy in deficient subjects. These data indicate that vitamin B,, is required, either directly or indirectly, for the synthesis of 4-6-glutamyl folates. The reduction in red ceI1 shorter-chain folate in primary folate deficiency is not present in vitamin B,, deficiency. This implies that in vitamin B,, deficiency there is a specific failure to make polyglutamate which is not present in primary folate deficiency. Further these data indicate that the lack of folate coenzyme in vitamin Biz deficiency cannot be due only to failure to transport methyltetrahydrofolate into cells since the folate is handled differently once it is inside the vitamin B,, deficient erythroblast. Failure of methylfolate to enter red cells (Das & H o a r a n d , 1970; Tisman & Herbert, 1973 ; Tisman et al, 1975) is probably secondary to a failure to utilize methyltetrahydrofolate for polyglutamate synthesis within the erythroblast. There remain two alternative explanations for the role of vitamin B,, in folate polyglutamate synthesis. (I) There is a failure to utilize methyltetrahydrofolate for this purpose. This implies that methyltetrahydrofolate itself cannot serve as the substrate for polyglutamate synthesis. In the vitamin B,, deficient sheep labelled methyltetrahydrofolate was incorporated into polyglutamate (Gawthorn & Smith, 1973). However, in the rat liver Spronk (1974) has claimed that only tetrahydrofolate could be used. A fuller account of this work has not yet appeared. Lavoie et al(1974) failed to find labelled polyglutamate in lymphocytes when lowspecific activity methylfolate labelled in the methyl group was given. If methylfolate cannot be utilized then the lesion in vitamin B,, deficiency within the cell may lie in the demethylation of methylfolate. This was the implied lesion in the methylfolate trap hypothesis via thc homocysteine-methionine pathway (Herbert&Zalusky, 1962).There is new evidence that the methyl group in methylfolate can be removed in the absence of vitamin B,, by converting the methyl to formaldehyde. This occurs in brain and in platelets (Meller et al, 1975). It is
248
lanet Perry et a1
thus incorrect that the methyl in methylfolate can be removed only as part of homocysteineinethionine reaction and other pathways may be concerned. ( 2 ) There may be a more direct role for vitamin BIz in polyglutamate synthesis. ACKNOWLEDGMENT
We wish to thank Dr K. U. Beuhring for the kind gift of tritiated methyl polyglutamates. REFERENCES BRAY,G.A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analytical Biochemistry, I, 279. CHANARIN, I. &PERRY, J. (1969) Evidence for reduction and methylation of folate in the intestine during normal absorption. Lancet, ii, 776. CHANARIN,I., PERRY,J. & LUMB,M. (1974) The biochemical lesion in vitamin-B deficiency in man. Lancet, i, 1251. COOPER, B.A. & LOWENSTEIN, L. (1964) Relative folate deficiency of erythrocytes in pernicious anemia and its correction with cyanocobalamin. Blood, 24, 502. COWARD,J.K., PARAMESWARAN, K.N., CASHMORE, J.R. (1974) 7,8-Dihydropteroyl A.R. & BERTINO, oligo-y-L-glutamates: synthesis and kinetic studies with purified dihydrofolate reductase from mammalian sources. Biochemistry, 13, 3899. CURTHOYS, N.P. & RABINOWITZ, J.C. (1972) Formyltetrahydrofolate synthetase. Binding of folate substrates and kinetics of the reverse reaction. Journal of Biological Chemistry, 247. 1965. DAS,K.C. & HOFFBRAND, A.V. (1970) Studies offolate uptake by phytohaemagglutinin-stimulated lymphocytes. British Journal of Haematology, 19, 203. GAWTHORN, J.M. & SMITH,R.M. (1973) The synthesis of pteroylpolyglutamates by sheep liver enzymes in vitro. Biochemical Journal, 136, 295. HANSEN,H.A. (1964) On the Diagnosis of Folic Acid Deficiency. Almqvist & Wiksell, Stockholm. H E R B ~ TV., & ZALUSKY, R. (1962) Interrelations of vitamin B12 and folk acid metabolism: folic acid clearance studies.Journal of Clinical Investigation, 41, 1263. JEEJEEBHOY, K.N., PATHARE, S.M.& NORONHA, J.M.
(1965) Observations on conjugated and unconjugated blood folate levels in megaloblasticanemia and the effects of vitamin Blz. Blood, 26, 354. KISLIUK, R.L., GAUMONT, Y. & BAUGH,C.M. (1974) Polyglutamyl derivatives of folate as substrates and inhibitors of thymidylate synthetase. Journal of Biological Chemistry, 249, 4100. LAVOIE, A., TRIPP,E. & HOFFBRAND, A.V. (1974) The effect of vitamin B12 deficiency on methylfolate metabolism and pteroylpolyglutamate synthesis in human cells. Clinical Science and Molecular Medicine, 47, 617. MCBURNEY, M.W. & WHITMORE,G.J. (1974) Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells. Cell, a, 173. MELLEX,E., ROSENGARTEN, H., FRIEDHOFF, A.J., STEBBINS, R.D. & SILBER, R. (1975) 5-Methyltetrahydrofolic acid is not a methyl donor for biogenic amines: enzymatic formation of formaldehyde. Science, 187,171. SHIN, Y.S., BUEHRING,K.U. & STOKSTAD, E.L.R. (1974) Studies of folate compounds in nature. Folate compounds in rat kidney and red blood cells. Archives ofBiochemistry and Biophysics, 163, 211. SPRONK, A.M. (1974) Tetrahydrofolate polyglutamate synthesis in rat liver. Federation Proceedings, 32, Abstr. 1398, p. 471. V. (1973) B12dependence of TISMAN,G. & HERBERT, cell uptake of serum folate: an explanation for high serum folate and cell folate depletion in B12 deficiency. Blood, 41,465. TISMAN,G., W u , S-J.G., SAFIRE, G.J. & RODRIQUEZ, E. (1975) The methylfolate trap hypothesis. Lancet, i, 1184.
Role of Vitamin B,, in Folate Coenzyme Synthesis JANET
PERRY, M. LUMB,M. LAUNDY,E. H. REYNOLDS AND I. CHANARIN
Medical Research Council Clinical Research Centre, Harrow, Middlesex (Received 7 July 1975; acceptedfor publication Ig July 1975) SUMMARY. Normal red cells in man were found to contain predominantly folate pentaglutamates with smaller amounts of tetra- and hexapolyglutamates. There was no change in the type of polyglutamate present in red cells from patients with vitamin B,, deficiency and primary folate deficiency. In contrast to the fall in red cell polyglutamate concentration in vitamin B,, deficiency, there was a marked fall in short-chain folates in early folate deficiency (treated non-anaemic epileptics) and a fall in both short chain and long chain polyglutamates in patients with severe folate deficiency and megaloblastic anaemia. These differences in folate distribution within cells exclude a primary failure to transport metliylfolate into cells as the lesion in vitamin Biz deficiency. The failure of folate polyglutamate synthesis in vitamin B, deficiency arises either from a failure to provide the proper substrate for polyglutamate synthesis or to a direct requirement for vitamin B, for polyglutamate synthesis. Folate in body fluids is 5-methyltetrahydrofolate. It has one glutamic acid residue and is the form in which folate is absorbed from the gut and transported to cells (Chanarin & Perry, 1969). The form in cells is largely a polyglutamate with up to six glutamic acid residues attached to the folate molecule. Evidence is accumulating that it is this longer chain folate which is the active coenzyme form. In Cfostridiu Curthoys & Rabinowitz (1972) showed that the binding of formyltetrahydrofolatetriglutamateto formyltetrahydrofolate synthetase is IOO fold greater than that of the monoglutamate, and Kisliuk et a1 (1974) using thymidylate synthetase from L. casei demonstrated that tri- and hexaglutamyltetrahydrofolate were three times better substrates for the enzyme than the equivalent monoglutamyl derivative. Both groups of workers postulated a regulation of folate metabolism by the products of these reactions. Two recent reports concern mammalian cell lines, one describing the effectiveness of dihydropteroylpolyglutamates as substrates for mammalian dihydrofolate reductase (Coward et al, 1974), and the other a hamster ovary cell line that could not form polyglutamates, and was shown to have an absolute requirement for adenosine, thymine and glycine (McBurney& Whitmore, 1974), compounds whose synthesis is folate dcpendent. In vitamin BIZdeficiency the reduced red cell folate concentration (Cooper & Lowenstein, 1964; Hansen, 1964) is due to a lowering of polyglutamyl folates only, the concentration of the shorter chain folates remaining the same as in control subjects (Jeejeebhoy et al, 1965; Chanarin eta!, 1974). W e have now studied the effect ofvitamin BIZtherapy on polyglutamyl folate concentrations, determined the glutamyl chain length of red cell folates in patients Correspondence: Dr I. Chanarin, Department of Haematology, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ. G
243
244
Janet Perry et a1
with both vitamin Blz and folate deficiency, and in normal subjects, and measured the distribution of short chain and long chain polyglutamates in anticonvulsant-treated epileptic patients. These results are discussed in relation to current views on the action of vitamin BIZ. MATERIALS, METHODS AND PATIENTS STUDIED The shorter chain and longer chain glutamyl folates in red blood cells were measured as previously described (Chanarin et al, 1974), and the results expressed as p g folate per litre of packed red cells. Observations were made on 42 patients with epilepsy, half of whom were macrocytic, receiving treatment with anticonvulsant drugs. These data will be presented in more detail elsewhere. Red blood cells were prepared for chromatography by a modification of the method of Shin et a1 (1974) in which the red cells were washed with buffered saline, pH 7.0, prior to being added to the boiling solution of ascorbate. To determine the type of folate compound in human red cells, the extracts were assayed microbiologically using Lactobacillus casei (NCIB8oIo), Streptococcus faecalis (NCIB6459) and Pediococctrs cerevisiae (NCIB7837). Folate activity was detected with L. casei only, even when aseptic addition techniques were used, indicating the presence of 5-methyltetrahydrofolate only. The concentrated lysates, together with marker compounds which were high specific activity tritiated 5-methyltetrahydrofolate polyglutamates of known chain length, were applied to an 0.9 x 50 cm column of DEAE cellulose (Whatman DE 52), and eluted with potassium phosphate buffer, pH 6. The buffer concentration was varied by a gradient consisting of 140 ml of 0.1 M phosphate in the mixing chamber and 500 ml of 1.0 M phosphate in the reservoir. Both solutions contained 0.2 M mercaptoethanol. 5 ml fractions were collected, and each fraction was assayed microbiologically (L. c m i ) both pre- and post-incubation with human plasma (0.1 ml plasma per rnl of column fraction). Gammaglutamyl carboxypeptidase in the plasma rapidly converts polyto mono-glutamyl folates which are then available as growth factors for the assay organism. Tritium activity was determined by adding 0.1 ml of each fraction to Bray’s scintillation fluid (Bray, 1960), whch was then counted in a Wallac-LKB liquid scintillation counter. Counts per minute were converted to disintegrations per minute (dpm) by the use of the external standard channels ratio method. The effect of vitamin BIZtherapy on polyglutamyl folate concentration was determined in five patients with pernicious anaemia. Column chromatography of red cell lysates was done in five normal subjects, four patients with pernicious anaemia (both pre and post vitamin B,, therapy) and one patient with severe folate deficiency. RESULTS When vitamin Blz was given to patients with pernicious anaemia the red cell folate concentration increased rapidly, and this increase was found to be due to a rapid restoration of polyglutamyl folates, with no change in the short-chain glutamyl folate levels. The five patients on whom sequential red cell folate estimations were done all showed a similar pattern, and a typical response to vitamin BIZtherapy is shown in Fig I. Chromatography of red cell lysates from six normal subjects showed almost identical
Vitamin B,, a d Folate Coenzyme
245
-
0
10 Days
20
FIG I. The effect of vitamin Blz administration on the red cell polyglutamyl folate concentration in pernicious anaemia. -, PteGlu 4-6; - - -, PteGlu 1-3.
elution patterns. The major component was methyltetrahydropentaglutamate, with a lesser quantity of tetra- and hexa-glutamates, and small peaks of what are presumably mono, diand tri-glutamates (Fig 2 ) . In vitamin B,, deficiency the pattern of polyglutamyl folates was similar to that found in normal subjects, methyl pentaglutamate again being the major fraction. Red cells taken from a patient with pernicious anaemia after 4 weeks of BIZtherapy showed increased amounts of the shorter chain polyglutamates which probably represent the folates formed after the first few days of vitamin BI2 administration (Fig 3). Severely folatedeficient red cells also gave an elution pattern similar to that found in normal red cells (Fig 4). 5CH, H4 PteGlu,
t
Fraction no.
FIG2. DEAE celIulose chromatography of an extract of normal red celIs assayed for foIate with L. casei. The labelled peaks are those whose identity was confirmed by co-chromatography with standard marker compounds.
Janet Perry et a1
PA after B,, therapy
5CH,
, PteGlu,
5CH, H, PteGlu,
PA before B,, therapy
I
Fraction na.
FIG 3 . DEAE cellulose chromatography of red cell extracts (assayed for folate with L. cusei) from a patient with pernicious anaemia before, and 4 weeks following, vitamin B, therapy.
Fraction no.
FIG4. DEAE cellulose chromatography of a red cell extract (assayed for folate with L. casei) from a severely folate deficient patient.
Vitamin B,, and Folate Coenzyme
247
TABLE I. Mean red blood cell folate concentrations in controls, vitamin B,, deficiency, anticonvulsanttreated epileptics and folate-deficient megaloblastic anaemia (pg/l.) Folute form jCH,H4PteGh
Controls (3 I ) Vitamin B1, deficiency (11) Treated epileptics (41) Folate deficiency (7)
(SEM)
5CH3H4PteGlu4-
86
(6.3)
203
85
(14.0)
108
19.5
(1.51)
21
(4.8)
160 48
(SEM) (15.68) (26.2) (11.1) (7.1)
In treated epileptics there was a marked reduction in the level of short chain folates in red cells without a significant fall in long chain compounds (Table I). In severe folate deficiency associated with a megaloblastic anaemia there is a marked reduction in both forms of folate. DISCUSSION In pernicious anaemia folate metabolism is abnormal. This is associated with a reduced level of polyglutamyl folates in cells in vitamin B,, deficiency as compared to normal subjects (Chanarin et al, 1974). There is a rapid restoration of red cell polyglutaniyl folate concentrations following B,, therapy in deficient subjects. These data indicate that vitamin B,, is required, either directly or indirectly, for the synthesis of 4-6-glutamyl folates. The reduction in red ceI1 shorter-chain folate in primary folate deficiency is not present in vitamin B,, deficiency. This implies that in vitamin B,, deficiency there is a specific failure to make polyglutamate which is not present in primary folate deficiency. Further these data indicate that the lack of folate coenzyme in vitamin Biz deficiency cannot be due only to failure to transport methyltetrahydrofolate into cells since the folate is handled differently once it is inside the vitamin B,, deficient erythroblast. Failure of methylfolate to enter red cells (Das & H o a r a n d , 1970; Tisman & Herbert, 1973 ; Tisman et al, 1975) is probably secondary to a failure to utilize methyltetrahydrofolate for polyglutamate synthesis within the erythroblast. There remain two alternative explanations for the role of vitamin B,, in folate polyglutamate synthesis. (I) There is a failure to utilize methyltetrahydrofolate for this purpose. This implies that methyltetrahydrofolate itself cannot serve as the substrate for polyglutamate synthesis. In the vitamin B,, deficient sheep labelled methyltetrahydrofolate was incorporated into polyglutamate (Gawthorn & Smith, 1973). However, in the rat liver Spronk (1974) has claimed that only tetrahydrofolate could be used. A fuller account of this work has not yet appeared. Lavoie et al(1974) failed to find labelled polyglutamate in lymphocytes when lowspecific activity methylfolate labelled in the methyl group was given. If methylfolate cannot be utilized then the lesion in vitamin B,, deficiency within the cell may lie in the demethylation of methylfolate. This was the implied lesion in the methylfolate trap hypothesis via thc homocysteine-methionine pathway (Herbert&Zalusky, 1962).There is new evidence that the methyl group in methylfolate can be removed in the absence of vitamin B,, by converting the methyl to formaldehyde. This occurs in brain and in platelets (Meller et al, 1975). It is
248
lanet Perry et a1
thus incorrect that the methyl in methylfolate can be removed only as part of homocysteineinethionine reaction and other pathways may be concerned. ( 2 ) There may be a more direct role for vitamin BIz in polyglutamate synthesis. ACKNOWLEDGMENT
We wish to thank Dr K. U. Beuhring for the kind gift of tritiated methyl polyglutamates. REFERENCES BRAY,G.A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analytical Biochemistry, I, 279. CHANARIN, I. &PERRY, J. (1969) Evidence for reduction and methylation of folate in the intestine during normal absorption. Lancet, ii, 776. CHANARIN,I., PERRY,J. & LUMB,M. (1974) The biochemical lesion in vitamin-B deficiency in man. Lancet, i, 1251. COOPER, B.A. & LOWENSTEIN, L. (1964) Relative folate deficiency of erythrocytes in pernicious anemia and its correction with cyanocobalamin. Blood, 24, 502. COWARD,J.K., PARAMESWARAN, K.N., CASHMORE, J.R. (1974) 7,8-Dihydropteroyl A.R. & BERTINO, oligo-y-L-glutamates: synthesis and kinetic studies with purified dihydrofolate reductase from mammalian sources. Biochemistry, 13, 3899. CURTHOYS, N.P. & RABINOWITZ, J.C. (1972) Formyltetrahydrofolate synthetase. Binding of folate substrates and kinetics of the reverse reaction. Journal of Biological Chemistry, 247. 1965. DAS,K.C. & HOFFBRAND, A.V. (1970) Studies offolate uptake by phytohaemagglutinin-stimulated lymphocytes. British Journal of Haematology, 19, 203. GAWTHORN, J.M. & SMITH,R.M. (1973) The synthesis of pteroylpolyglutamates by sheep liver enzymes in vitro. Biochemical Journal, 136, 295. HANSEN,H.A. (1964) On the Diagnosis of Folic Acid Deficiency. Almqvist & Wiksell, Stockholm. H E R B ~ TV., & ZALUSKY, R. (1962) Interrelations of vitamin B12 and folk acid metabolism: folic acid clearance studies.Journal of Clinical Investigation, 41, 1263. JEEJEEBHOY, K.N., PATHARE, S.M.& NORONHA, J.M.
(1965) Observations on conjugated and unconjugated blood folate levels in megaloblasticanemia and the effects of vitamin Blz. Blood, 26, 354. KISLIUK, R.L., GAUMONT, Y. & BAUGH,C.M. (1974) Polyglutamyl derivatives of folate as substrates and inhibitors of thymidylate synthetase. Journal of Biological Chemistry, 249, 4100. LAVOIE, A., TRIPP,E. & HOFFBRAND, A.V. (1974) The effect of vitamin B12 deficiency on methylfolate metabolism and pteroylpolyglutamate synthesis in human cells. Clinical Science and Molecular Medicine, 47, 617. MCBURNEY, M.W. & WHITMORE,G.J. (1974) Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells. Cell, a, 173. MELLEX,E., ROSENGARTEN, H., FRIEDHOFF, A.J., STEBBINS, R.D. & SILBER, R. (1975) 5-Methyltetrahydrofolic acid is not a methyl donor for biogenic amines: enzymatic formation of formaldehyde. Science, 187,171. SHIN, Y.S., BUEHRING,K.U. & STOKSTAD, E.L.R. (1974) Studies of folate compounds in nature. Folate compounds in rat kidney and red blood cells. Archives ofBiochemistry and Biophysics, 163, 211. SPRONK, A.M. (1974) Tetrahydrofolate polyglutamate synthesis in rat liver. Federation Proceedings, 32, Abstr. 1398, p. 471. V. (1973) B12dependence of TISMAN,G. & HERBERT, cell uptake of serum folate: an explanation for high serum folate and cell folate depletion in B12 deficiency. Blood, 41,465. TISMAN,G., W u , S-J.G., SAFIRE, G.J. & RODRIQUEZ, E. (1975) The methylfolate trap hypothesis. Lancet, i, 1184.
