0031-3998/85/1912-1288$02.00/0 PEDIATRIC RESEARCH Vol. 19, No. 12, 1985 Copyright O 1985 International Pediatric Research Foundation, Inc. Printed in U.S.A.

Comparison of Folic Acid Coenzyme Distribution Patterns in Patients with Methylenetetrahydrofolate Reductase and Methionine Synthetase Deficiencies

E. REGULA BAUMGARTNER, E. L. R. STOKSTAD, H. WICK, J. E. WATSON, AND GABRIELLA KUSANO Department of Pediatrics, University of Basel, 4005, Basel, Switzerland [E.R.B., H. W.J and Department of Nutritional Sciences, University of California, Berkeley, California, 94720 [E.L.R.S., J.E. W., G.K.]

ABSTRACT. Folic acid coenzyme distribution patterns dent 5-methyl-THF: homocysteine methyltransferase (EC were examined in the liver and kidney of two patients with 2.1.1.13) (methionine synthetase), designated cbl C and cbl D homocystinuria due to different inborn errors of metabo- mutants (1 1). In addition to homocystinuria these patients also lism affecting the remethylation of homocysteine to methi- have methylmalonic aciduria whereas folate levels in serum and onine. One patient, with severe mental retardation (and red blood cells are normal. The clinical symptoms vary widely; death at 3%yr), had greatly reduced levels of methylene- they include in the more severely affected patients (cbl C) failure tetrahydrofolic acid (THF) reductase in fibroblasts as well to thrive, developmental retardation, megaloblastic anemia (12- as in liver and kidney. Chromatographic separation of 19, and often early death (16). folate coenzymes in liver showed an abnormal pattern with We have followed two patients, one with each disorder (17). THF as the main component and almost no methyl-THF They illustrate the clinical and biochemical consequences of but total folate was normal. The other patient, who was defects at the point of interaction between folate and cobalamin dystrophic, microcephalic, and had megaloblastic anemia metabolism, namely at the transfer of the 5-methyl group from died at age 4 months. He had reduced levels of methionine 5-methyl-THF to homocysteine to produce methionine and synthetase in liver and kidney due to a defect of intracel- THF. The vascular changes resulting from homocystinemia have lular cobalamin metabolism. Chromatographic analysis of been discussed extensively (12, 18). The two disorders with his tissues showed methyl-THF to be the principal folate disturbances in different sites of folate metabolism vary greatly form and a markedly reduced total folate. These results in their clinical manifestations. It is to be expected that a defi- support the "methyl-THF trap" hypothesis and offer in- ciency of methylene-THF reductase would result in a decreased formation with respect to the possible therapy of these two proportion of methyl-THF relative to other folate coenzyme disorders. (Pediatr Res 19: 1288-1292,1985) forms. This has already been shown in fibroblast studies (10). On the other hand, malfunction of the methionine synthetase Abbreviations will lead to an accumulation of 5-methyl-THF since the meth- ylene-THF reductase reaction is essentially irreversible: i.e. re- THF, tetrahydrofolic acid duced folates are trapped as a deadend derivative and the indi- methyl-THF, methyltetrahydrofolic acid vidual becomes functionally folate deficient. This has been de- methylene-THF, methylenetetrahydtofolic acid scribed as the "methyl-THF trap" hypothesis (19-21). In order to test this assumption and to elucidate the discrep ancy in the clinical symptoms we have studied the folate coen- zyme distribution patterns in liver and kidney of these two Methylene-THF reductase (EC 2.2.2.68) deficiency, an inborn patients. error of folate metabolism, has been fairly well studied (1-8). This disorder is associated with a wide spectrum of neurologic CASE REPORTS abnormalities, often with mental retardation and rarely also with psychotic symptoms. The severity of the neurologic dysfunction Patient G.P. This patient was the second child of healthy, correlates with the degree of enzyme deficiency (9). Biochemi- consanguinous parents. His older brother was healthy. He was cally, the key findings are moderate homocystinuria and homo- born at term after an uneventful pregnancy and delivery. Weight, cystinemia accompanied by low or normal methionine levels length, and head circumference were at the 50th percentile. He and low folate levels in serum and red cells. Abnormal folate showed some dysmorphisms such as epicanthus, broad root of distribution has been observed in fibroblast cultures of these the nose, and syndactilism of the 2nd and 3rd toes. According patients (10). to his mother he was doing well during the first 6 months. At Homocystinuria with hypomethioninemia is also a key finding the age of 9 months he was unable to sit or to crawl. The in patients with a congenital defect of cobalamin coenzyme pediatrician was consulted only at the age of 1 yr and microce- synthesis leading to malfunction of the methyl-cobalamin depen- phaly, muscular hypotonia, and a pathological EEG were noted. Progressive neurologic deterioration led to his admission at Received May 21, 1984; accepted August 1, 1985. the age of 18 months. Weight was on the 50th, length on the Address reprint requests to Dr. R. Baumgartner, Department of Pediatrics University of Basel, Postfach 4005, Basel, Switzerland. 90th, but head circumference far below the 3rd percentile. The Supported in part by U.S. Public Health Service Grant AM-08171 from the boy had no coordinate eye movements, could not sit, and did National Institutes of Health. not even recognize his mother. He had muscular hypotonia and 88 DEFECTS IN METHk'L-THF METABOLISM 1289 minor motor seizures. The EEG was severely disorganized by pm; from controls A.J. 22 h pm, F.T. 9 h pm, E.H. 7% h pm slow background rhythms and sharp and slow wave complexes. and stored frozen at -60" C until analyzed. Exposure to artificial His mental age was 2 months. He had no evidence of ectopia light was carefully minimized during handling of the tissue and lentis, marfanoid features, or bone deformities. extraction procedures. Tissues (0.5 to 1.5 g depending on tissue The metabolic screening program revealed homocystinuria being studied) were homogenized with 9 volumes of ice cold 0.0 1 which was confirmed by ion exchange chromatography (99 M phosphate buffer pH 7.0 containing 1.0% mercaptoethanol. pmo1/24 h). In addition there was cystathioninuria (37.8 pmoll The homogenate was centrifuged at 20,000 x g for 30 min at 4" 24 h), elevated plasma homocysteine (35.6 pmol/liter) but very C. Duplicate 100 p1 samples of the supernatant (equivalent to 10 low concentrations of plasma methionine (4-12 pmol/liter). mg tissue) were used in the methionine synthetase and 200 p1 Methylmalonic acid was not detectable in urine. Serum folate samples in the methylene THF reductase assays. The extracts of concentrations were abnormally low (2 ng/ml, normal 6-20 ng/ kidney and liver used for the enzyme assays contained approxi- ml; Lactobacillus casei assay). The blood count was normal and mately 100 mg protein (Lowry method) per g tissue. The re- there were no megaloblastic alterations. The diagnosis of meth- mainder of the extract was frozen and stored at - 10" C for 1- 10 ylene-THF reductase deficiency was suspected. Treatment with days until folate distribution determinations were made. high doses of oral folic acid (5 mg/day) and hydroxocobalamin Methylene-THF reductase determination. The activity of this intramuscularly normalized the blood folate levels but had no enzyme in liver and kidney was determined by the "reverse clinical nor biochemical effect. The boy died unexpectedly at the direction" method of Kutzbach and Stokstad (22), in which age of 3% yr from bronchopneumonia. The histopathologic I4CH3-THFis enzymatically converted to I4C-formaldehydewith findings have been reported previously (18). Methylene-THF menadione as the electron acceptor. reductase deficiency was confirmed by enzyme assay in the Methionine synthetase determination. This was measured by patient's fibroblasts (2% of control values) (10). Addition of the method of Sauer and Jaenicke (23) in which cyanocobolamin coenzyme FAD did not stimulate the activity significantly (Wong was added and anaerobiosis maintained by dithiothreitol in a PWK, unpublished data). On the other hand, the activity of the nitrogen atmosphere. two BI2-dependent enzymes, methionine synthetase (Wong Folate distribution by column chromatatography. The frozen PWK, unpublished data), and MMA-CoA mutase (Baumgartner pH 7.0 extract was thawed, adjusted to pH 4.7 with HC1, 1% ER, unpublished data) in fibroblasts, were found to be normal. ascorbate added, the mixture heated to 100" C for 7 min, cooled, Cystathionine synthetase activity measured in liver was also centrifuged at 2000 rpm at room temperature, and the superna- normal (Baumgartner ER, unpublished data). tant incubated at 37" C for 3 h with a purified hog kidney Patient S.B. This case has been reported in detail elsewhere conjugase preparation (24). A sample equivalent to 200 mg of (12, 13, 17). S.B., first child of unrelated parents, was born at liver was chromatographed on a 9 x 200 mm QAE Sephadex term after an uncomplicated pregnancy with a birth weight and column using an exponential salt gradient containing 2% mer- head circumference in the 10th percentile and length in the 25th captoethanol as described by Chan (25). 2000 cpm (2 ng) of I4C- percentile. A younger brother is healthy. Neonatal hyperbiliru- methyl-THF was added to the extract before application to the binemia was treated with phototherapy. At 3 months of age he column to serve as a radioactive marker. Both the samples was admitted because of feeding difficulties, failure to thrive, and applied to the column and the column fractions were assayed progressive anemia. He presented with severe dystrophy (weight microbiologically with both L. casei (ATCC 4569) and strepto- below the 3rd percentile) and microcephaly (head circumference coccus faecalis (ATCC 8043) by the method of Tamura et al. far below 3rd percentile). His neurologic status, however, was (26). The samples were inoculated directly (without heat sterili- normal. He had hepatomegaly, hemolytic anemia, hematuria, zation) and incubated 18 h at 37" C. Under these conditions proteinuria, and slight uremia (52 mg/dl). The peripheral blood THF gave the same response as oxidized folates or as 5-formyl- smear showed hyperchromia, anisocytosis, pyknocytosis, and THF which was used as a standard. marked hypersegmentation of the neutrophils. The bone marrow revealed discrete megaloblastic changes and a great number of RESULTS giant promyelocytes. The results of the enzyme assays are given in Table 1 and the Methylmalonic aciduria and homocystinuria were detected by differential microbiologic assays are reported in Table 2. The the metabolic screening program. These findings together with folate coenzyme distribution patterns in liver for G.P., S.B. low methionine levels and the presence of cystathionine pointed (patients), and J.A. (control) are presented in Figure 1. The liver to a defect in BIZmetabolism. Exogenous cobalamin deficiency, enzyme values (Table 1) show that G.P. has almost no methyl- malabsorption, as well as abnormal transport of B12were ex- ene-THF reductase activity in either liver or kidney, but had cluded by normal total serum cobalamins (285 pg/ml, normal normal methionine synthetase levels. S.B. who exhibited homo- 200- 1000 pg/ml, Euglena gracilis) and normal transcobalamins. cystinuria and morphologic signs of a maturation defect in Serum folate (5.1 ng/ml, normal 4-16 ng/l, L. casei assay) and hemopoiesis had normal levels of methylene-THF reductase but red cell folate (490 ng/ml, normal 150-500 ng/ml) were also low levels of methionine synthetase in the liver and especially in normal. the kidney. The presence of a considerable residual activity in Treatment with blood transfusions, reduction of protein in- the liver of S.B. (about 25% of the values for metabolically take, and pharmocologic doses of cyanocobalamin intramuscu- normal 4-month-old infants reported herein) correlates well with larly resulted in a marked decrease of homocystine and methyl- the surprisingly high, almost normal concentrations of methyl- malonate levels, but had no effect on the clinical course. Re- cobalamin found in the patient's liver (13). This finding may be peatedly hemolysis occurred and the boy died after two acute the result of the B12 treatment which also had a clear biochemical episodes of heart failure 4 wk after admission at the age of 4 effect on the urinary methylmalonate and homocystine excretion months. (13). It is interesting to note that the B12 therapy had no effect in The diagnosis of a congenital disorder of defective synthesis of the kidney both in respect to cobalamin and methionine synthe- both cobalamin-coenzymes (probably cbl C mutant according to tase levels (methyl cbl and methionine synthetase amounting to Rosenberg's nomenclature) (1 1) was confirmed by assaying approximately 3% of control values) (13). The three "normal methyltransferase and methylmalonyl-CoA mutase in postmor- controls" with no homocystinuria or other evidence of disturbed tem liver and kidney and by analysis of tissue cobalamins (1 3). folate of BI2 metabolism had methionine synthetase activities ranging from 122 to 327 nmol/h/g tissue. These are comparable MATERIALS AND METHODS with previously reported values by various investigators as com- Tissue extraction methods. Liver and kidney samples were prised in Table 1. Notably the highest activity was found in the obtained at autopsy (from patients G.P. 12 h pm and S.B. 1 h liver of the youngest child (12 days old) which is in agreement 1290 BAUMGARTNER ET AL. Table 1. Tissue levels of methionine synthetase and methylenetetrahydrofolate reductase* Methionine synthetase Methylene-THF reductase (nmol g-I. h-') (nmol gL'.h-I) Patient Diagnosis or (age at death) cause of death Liver Kidney Liver Kidney Patients G.P. Reductase 409 190 2 2 (3% Y) deficiency S.B. cbl-C mu- 30 16 1430 925 (4 mo) tant

Controls A.J. Heart failure 112 (4 mo) F.T. Appendicitis 130 (4V2 mo) E.H. Cerebral he- 327 (1 2 days) morrhagy

Reference values Kalnitsky et a1.t (27) 1 day-10 mo 64 k 6 (36) Koblin et al. (31) Adults 414+ 77(6) Narisawat (7) Adults Newborns Baumgartner et a1.t (13) Adults 219 + 76 (14) * Enzyme activities are given in nmollg tissuelh. t The values from the literature are recalculated from nmol/mg protein/hour assuming that 100 mg of protein would correspond to 1 g of tissue as is the case in the extraction method used by us and by Sauer et al. (32). Mean values k SD or the range are indicated with the number of controls in parentheses.

Table 2. Estimation of folates in liver and kidney by dzferential microbiologic assay Liver Kidney L. casei S. faecalis L. casei S. faecalis Name Diagnosis (g/g) (g/g) %CHsTHp (g/g) (g/g) %CH3THFC Patients G.P. Reductase deficiency 2.90 2.70 7 1.71 1.38 20 S.B. cbl C mutant 2.05 0.95 54 0.26 0.10 62 Controls A.J. 3.95 1.23 69 1.44 0.52 64 F.T. 3.98 1.86 5 3 0.70 0.42 40 E.H. 4.17 1.92 54 1.30 0.55 58 * L. casei assay-S faec. assayll. casei assay x 100. with the data of Kalnitsky et al. (27) who observed that the Examination of the differential microbiologic assay (Table 2) specific activity of methionine synthetase decreased during de- shows that both the liver and kidney of G.P. (methylene-THF velopment. reductase deficiency) had low levels of methyl-THF. L. casei The diminished methylene-THF reductase levels in liver and values represent both methyl-THF and nonmethyl forms such kidney of patient G.P. are consistent with the greatly reduced as THF, 10-formyl-THF,and 5-formyl-THF. S.faecalis responds levels of this enzyme in cultured fibroblasts in this same patient to all folate forms with the exception of methyl-THF. The as reported by Rosenblatt et al. (10). Here the reductase values difference between these two assays represents methyl-THF. The of cultured fibroblasts of G.P. were 0.16 nmol/mg protein or percentage of methyl-THF relative to total folates as measured about 2% of normal control values. This is similar to the very by L. casei was 7% in the liver and 20% in the kidney. These low levels of reductase in both the liver and kidney which were values are considerably lower than those of normal controls (53 less than 1% of control values. to 69% in liver and 40 to 62% in kidney). In S.B. with the defect in cobalamin coenzyme synthesis, on By column chromatography, the folate distribution pattern in the other hand, methylene-THF reductase activity was higher the liver of G.P. (Fig. 1A) shows no L. casei activity in the than in the three controls. This finding might be explained by methyl-THF position as identified by a 14CH3THFmarker. The the impaired methyl transferase activity which leads to dimin- L. casei activity and S. faecalis activity of the THF peak were ished methionine and hence S-adenosylmethionine synthesis. essentially the same. The distribution pattern indicating the Low levels of S-adenosylmethionine are known to result in a loss absence of methyl-THF in the liver of G.P. is consistent with the of feedback inhibition of methylene-THF reductase (22). very low levels of methylene-THF reductase and the low level of DEFECTS IN METHYGTHF METABOLISM

LIVER G.f? LIVER S.B. METH. SYNTH. 80 DEFICIENCY - - a - -60 w, 6060 a 4 - 40 L I - 20 20 20 > 0 0

2 0 30 40 50 60 20 30 40 50 ' 60 $ LIVER A.J. r NORMAL I

2 0 30 4 0 5 0 6 0 FRACTION NUMBER (4.5ml) Fig. 1. Chromatographic separation of conjugase treated liver extracts on QAE Sephadex column (19 x 200 mm). The folate concentrations were determined in the fractions by L. casei (L.c.)and S. faecalis (SJ) assay. For details see "Materials and Methods." methyl-THF as measured by differential microbiologic assay. are the preferred substrates for polyglutmate synthetase (30), may The distribution pattern for the control liver from A.J. shows have resulted in the low concentrations of total folates in tissues approximately 70% methyl-THF (Fig. 1C) which agrees with the of the patient. This is in accordance with the reduced liver folate value of 69% methyl-THF as measured by microbiologic assay. levels observed in vitamin BI2-deficientrats (21). The accumu- The liver of S.B. (deficient in methionine synthetase due to lation of methyl-THF and the decrease in the total folate pool in abnormal cobalamin metabolism) contained mainly (85%) patient S.B. support the "methyl-THF trap" hypothesis. methyl-THF as measured by chromatographic separation (Fig. In contrast, our other patient, G.P. with methylene-THF re- 1B). This may be compared with 54% methyl-THF as measured ductase deficiency, had normal nonmethyl folates but a reduced by differential microbiologic assay. The value obtained by chro- methyl-THF. Accordingly, he exhibited no megaloblastic anemia matographic separation could be regarded as more reliable since and was not dystrophic. His total folates in liver and kidney were the "% methyl-THF value is obtained by difference between normal indicating normal polyglutamate synthesis. the L. casei and S. faecalis values and therefore subject to more The major differences in clinical presentation of these two error. Nevertheless, the results of microbiologic assay indicate a patients with different disorders affecting remethylation can thus decrease in total folate (L. casei activity) and nonmethyl folates be explained by their respective folate coenzyme patterns: meth- (S.faecalis activity) in tissues of S.B. especially in the kidney. ylene-THF reductase deficiency can be related to methyl-THF This indicates a disturbance of polyglutamate synthesis and a deficiency and the defect in cobalamin coenzyme synthesis to functional deficiency. functional folate deficiency. Concerning therapeutic implications, treatment with folic or DISCUSSION folinic acid may be beneficial for patients with reductase defi- To our knowledge, this is the first time that folate coenzyme ciency if their enzymatic defect is leaky and thus might be levels have been measured in liver and kidney of a patient (S.B.) partially overcome by high substrate concentrations. In addition, with a congenital defect in cobalamin coenzyme synthesis. The vitamin BIZ(hydroxocobalamin) is indicated to enhance methi- clinical and biochemical consequences of this disorder are similar onine synthetase activity, and thereby, THF and methionine to these of exogenous vitamin BIZdeficiency (28). The elevated (and hence S-adenosylmethionine) formation. Treatment with methyl-THF levels in the methionine synthetase-deficientpatient betaine is another possibility to improve remethylation of hom- (S.B.) is analogous to B12deficiency in which folate is trapped as ocysteine. Vitamin B6 should always be given to stimulate cys- methyl-THF. Methyl-THF is unavailable for all folate-dependent tathionine synthetase activity and thus reduce homocysteine enzymes except the BIZ-dependentmethionine synthetase (19- levels. An ultimate goal of the therapy in this defect, however, is 2 1, 29). The resulting reduction of nonmethyl folate coenzyme to increase methyl-THF concentrations in the cells. Whether this forms such as THF and methylene-THF led to a functional folate can be achieved by supplementation with methyl-THF is not yet deficiency in our patient S.B. Reduction of methylene-THF, known. Further studies concerning transport and degradation of which is essential for DNA synthesis, was clinically reflected by methyl-THF in tissues, especially in the brain, are required. the megaloblastic anemia and dystrophy. Reduced availability of Patients with defective methionine synthetase due to a cobalamin THF and other tetrahydrafolates such as 10-formyl-THF, which C or D mutation generally respond both clinically and biochem- 1292 BAUMGARTNER ET AL. ically to pharmacologic doses of hydroxocobalamin. Additional homocystinuria and methylmalonic aciduria. 11. Biochemical investigations. B6 Helv Paediatr Acta 34:483-449 treatment with folinic acid, betaine, and vitamin may be 14. Carmel R, Bedros AA, Mace JW, Goodman SI 1980 Congenital methylmalonic necessary in patients with incomplete response to hydroxocoba- aciduria-homocystinuria with megaloblastic anemia: Observations on re- lamin. sponse to hydroxocobalamin and on the effect of homocysteine and methi- onine on the deoxyuridine suppression test. Blocd 55:570-579 15. Dillon MJ, England JM, Gompertz, Goodey PA, Grant DB, Hussein HA-A, Acknowledgment. The authors thank Dr. Y. S. Shin for helpful Linnell JC, Matthews DM, Mudd SH, Newns GH, Seakins JWT, Uhlendorf discussion of our results. BW, Wise LI 1974 Mental retardation, megaloblastic anaemia, methylma- Ionic aciduria, and abnormal homocysteine metabolism due to an error in REFERENCES BIZmetabolism. Clin Sci Molec Med 4743-61 16. Levy HL, Mudd SH, Schulman JD, Dreyfus PM, Abeles RH 1970 A derange- 1. Mudd Sh, Uhlendorf BW, Freeman JM, Finkelstein JD, Shih VE 1972 Hom- ment in BIZmetabolism associated with homocystinemia, cystathioninemia, ocystinuria associated with decreased methylenetrahydrofolatereductase ac- hypomethioninemia and methylmalonic aciduria. Am J Med 48:390-397 tivity. Biochem Biophys Res Commun 46:905-912 17. Baumgartner ER, Schweizer K, Wick H 1977 Different congenital forms of 2. Freeman JM, Finkelstein D, Mudd SH 1975 Folate-responsive homocystinuria defective remethylation in homocystinuria: clinical, biochemical and mor- and "schizophrenia." A defect in methylation due to deficient methylene- phologic studies. Pediatr Res 1I: 10 15(abstr) tetrahydrofolate reductase activity. N Engl J Med 292:491-496 18. Baumgartner ER, Wick H, Ohnacker H, hobst A, Mauer R 1980 Vascular 3. Kanwar YS, Manaligod JR, Wong PWK 1976 Morphologic studies in a patient lesions in two patients with congenital homocystinuria due to different with homocystinuria due to 5, 10-methylenetetrahydrofolate reductase defi- defects of remethylation. J Inherited Metabl Dis 3:101-103 ciency. Pediatr Res 10:598-609 19. Noronha JM, Silverman M 1962 On folic acid, vitamin Biz methionine and 4. Wong PWK, Hmby M, Weiss EB, Diamond E 1977 Folic acid nonresponsive formiminoglutamate metabolism. In: Heinrich HC (ed) Vitamin BIZand homocystinuria due to methylenetertrahydrofolate reductase deficiency. Pe- Intrinsic Factor. 2nd Emopean Symposium. Verlag, Stuttgart, pp 728-736 diatrics 59749-756 20. Herbert V, Zalusky RR 1962 Interrelations of vitamin BIZ and folic acid 5. Narisawa K, Wada Y, Saito T, Suzuki H, Kudo M, Arawaka Ts, Katsushima metabolism: folic acid clearance studies. J Clin Invest 41:1263-1276 N, Tsuboi R 1977 Infantile type of homocystinuria with N5*10-methylene- 2 1. Gawthorne JM, Stokstad ELR 197 1 The effect of vitamin B12and methionine tetrahydrofolate reductase defect. Tohoku J Exp Med 121:185-194 on folic acid uptake by rat liver. Proc Soc Exp Biol Med 13642-46 6. Rosenblatt DS and Cooper BA 1979 Methylenetetrahydrofolate reductase 22. Kutzbach C, Stokstad ELR 1967 Feedback inhibition of methylenetetrahydro- deficiency: clinical and biochemical correlations. In: Botez MJ, Reynolds folate reductase in rat liver by S-adenosyl methionine. Biochem Biophys EH (eds) Folic acid in Neurology, Psychiatry and Internal Medicine. Raven Acta 139:2 17-220 Press, New York, pp 385-390 23. Sauer HJ, Jaenicke L 1972 Einfacher Test zur Messung der Methionip- 7. Narisawa K 1979 Brain damage in the infantile type of 5, lo-methylenetetra- synthetase-(MSFAktivitatund seine Anwendungsmoeglichkeitenin der Kli- hydrofolate reductase deficiency. In: Botez MI, Reynolds FH (eds) Folic nik. Klin Wochenschr 50:986-990 Acid in Neurology, Psychiatry and Internal Medicine. Raven Press, New 24. Brody T, Watson JE, Stokstad ELR 1982 Folate pentaglutamate and folate York, pp 391-400 hexaglutamate mediated one-carbon metabolism. Biochemistry 2 1:276-282 8. Harpey J-P, Rosenblan DS, Cooper BA, Moel GL, Roy C, Lafourcade J 1981 25. Chan MM-S, Stokstad ELR 1980 Metabolic responses of folic acid and related Homocystinuria caused by 5,lO-methylenetetra-hydrofolatereductase defi- compounds to thyroxine in rats. Biochim Biophys Acta 632244-253 ciency: A case in infant responding to methionine, folinic acid, pyridoxine, 26. Tamura T, Shin YS, Williams MA, Stokstad EIR 1972 Lactobacillus casei and vitamin BIZtherapy. J Pediatr 98:275-278 response to pteroylpolyglutamates. Anal Biochem 49:5 17-521 9. Boss GR, Erbe RW 1982 Decreased rates of methionine synthesis by methylene 27. Kalnitsky A, Rosenblatt D, Zlotkin S 1982 Differences in liver folate enzyme tetrahydrofolate reductasedeficient fibroblasts and lymphoblasts. J Clin patterns in premature and full term infants. Pediatr Res 16:628-631 Invest 67: 1659- 1664 28. Higginbottom MC, Sweetman L, Nyhan WL 1978 A syndrome of methylalonic 10. Rosenblatt DS, Cooper BA, Lue-Shing S, Wong PWK, Berlow S, Narisawa K, aciduria, homocystinuria, megaloblastic anemia and neurologic abnormali- and Baumgartner ER 1979 Folate distribution in cultured human cells. ties in a vitamin B12deficient breastfed infant of a strict vegetarian. N Engl Studies on 5, 10-CH2-H4 Pte Glu reductase deficiency. J Clin Invest 63: 1019- J Med 299:3 17-323 ----1025 29. Vidal AJ, Stokstad, ELR 1974 Urinary excretion of 5-methyletrahydrofolate 1 I. Rosenberg LE 1983 Disorders of propionate and methylmalonate metabolism. and liver Sadenosylmethionine levels in rats fed a vitamin B12deficient diet. In: Stanbury JB, Wynwrden JB, Fredrickson DS. Goldstein JL, Brown MS Biochim Biophys Acta 362245-257 (eds) The Metabolic is for Inherited Disease, 5th edn. McGraw-Hill, New 30. Cichowicz DJ, Foo SK, Shane B 1981 Folylpoly-y-glutamate synthesis by York, pp 474-497 bacteria and mammalian cells. Mole Cell Biol 39:209-228 12. Baumgartner ER, Wick H, Maurer R, Egli N, Steinmann B 1979 Congenital 3 1. Koblin DD, Waskell L, Watson JE, Stokstad ELR, Eger EI 1982 Nitrous oxide defect in interacellular cobalamin metabolism resulting in homocystinuria inactivates methionine synthetase in human liver. Anesth Analg 61:75-78 and methylmalonic aciduria, I. Case report and histopathology. Helv Pae- 32. Sauer HJ, howell JN, Jaenicke L 1973 Bedeutung und Regulation der Coba- diatr Acta 34:465482 laminabhaengigen Methionin-Synthetase und der Betain:Homocystein- 13. Baumgartner ER, Wick H, Linnell JC, Gaul1 GE, Bachmann C, Steinmann B Methyltransferase im Stofiechsel des Methionins und der Tetrahydrofol- 1979 Congenital defect in intracellular cobalamin metabolism resulting in saeure bei der Ratte. Res Exp Med 160: 17 1-180