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

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