Pediat. Res. 6: 875-879 (1972) Hyperammonemia methylmalonyl-CoA liver methylmalonyl-CoA racemase methylmalonic aciduria propionate Methylmalonyl Go enzyme A Racemase Defect: Another Cause of Methylmalonic Aciduria ELLEN SONG KANG'221, PHILIP J. SNODGRASS, AND PARK S. GERALD Department of Medicine, Division of Clinical Genetics, Children's Hospital Medical Center and Gastrointestinal Unit, Department of Medicine, Peter Bent Brigham Hospital and Harvard Medical School, Boston, Massachusetts, USA Extract Metabolism of 14C-propionate and methylmalonate was severely curtailed in fibro- blasts cultured from an infant with massive transient hyperammonemia (1370 ^g/100 ml), severe metabolic acidosis, and excretion of large amounts of methylmalonic acid (580 mg at 24°). Metabolism of succinate was normal. Metabolism of methylmalonate was not enhanced by the addition of excessive amounts of the cofactor, 5'-deoxyadenosylcobalamin (DBCC). The DBCG content of the liver was within normal limits. Homogenates of liver and fibroblasts metabolized methylmalonate approximately one-half as well as control samples when tritiated racemic methylmalonyl coenzyme A (CoA) was added. Inasmuch as L-methylmalonyl-CoA and not D-methylmalonyl- CoA is the substrate for the enzyme, methylmalonyl-CoA mutase, which converts L-methylmalonyl-CoA to succinyl-CoA, this indicates that the mutase was intact. Mitochondrial homogenate from liver, in contrast to normal samples, did not in- corporate tritium during the metabolism of synthetic methylmalonyl-CoA, which in- dicates that activity of racemase was deficient. Activities of the urea cycle enzymes were low but not rate limiting. Speculation A diet low in isoleucine, threonine, methionine, and valine may offer a rational thera- peutic approach to other affected patients. The hyperammonemia observed resembles that reported in propionyl-CoA carboxylase deficiency and Reye's syndrome, which raises the possibility of a common denominator in these several disorders. Introduction since the latter responds to the administration of phar- Excretion of massive amounts of methylmalonic acid macologic doses of vitamin B12 [13]. Although it has not been observed in patients re- in urine in the absence of vitamin B12 deficiency has been described in disease states of genetic origin in ported previously, excessive excretion of methylma- infants and children. This abnormality has been as- lonic acid might also occur with a defect of methylmal- cribed to a deficiency of methylmalonyl coenzyme A onyl-CoA racemase (EC 5.1.99.1), the enzyme which mutase (EC 5.4.99.2) activity in the patients re- catalyzes the step before the mutase reaction. ported to date either because of a defect of the apoen- In the normal state in the metabolism of propionate zyme or a defective synthesis of the specific vitamin B12 to succinate, the enzyme, propionyl-CoA carboxylase, cofactor required by this enzyme. Clinically, these two carboxylates propionyl-CoA to methylmalonyl-CoA. forms of methylmalonic aciduria are distinguishable Inasmuch as methylmalonyl-CoA contains an asymmet- 875 876 KANG ET AL. CH, CH, Laboratory Methods OH2 ATP AMP CH2 Ammonia was determined by a microdiffusion method HSCoA PPi | 1 _—--•• COOH > COSCoA [3]. propionate propionyl -CoA Methylmalonic acid in blood, urine, and tissues was measured by the following modification of the ATP /HCO3 method of Giorgio and Plaut [5]. The column was washed with 50 ml H2O and 15 ml 0.5 N HC1 before (Biotin) VADP carboxylase Vi elution of methylmalonic acid with 2 X 15 ml 0.5 N HC1. COOH Then samples were cooled in ice immediately after heat- ing before the addition of alkali. CH3-CH Skin biopsies were obtained from the patient, his COSCoA parents, and two normal infants (circumcision). d-methylmalonyl-CoA Ca) Explants were grown in Dulbecco's modified Eagle racemase | Medium or nutrient mixture Flo [15] which contained CH2COOH COOH I 10% fetal calf serum and 100 units penicillin/ml and — HC-CH,, CH2COSCoA 100 mg streptomycin/ml. Vitamin B12 was not detecta- CDBCC) I succinyl CoA mutase COSCoA ble in the Eagle's Medium and was over 5,000 pg/ml I—methylmalonyl-CoA (b) in medium F10 [16]. Fig. 1. Propionic acid metabolism in animal tissues. ATP: Adeno- Fibroblasts were grown to confluence in 60 mm sine 5'-triphosphate. HSCoA: Coenzyme A thioester. AMP: Adeno- diameter disposable Petrie dishes, harvested with sine 3'-monophosphate. PPi: Pyrophosphate. COSCoA: Coenzyme 0.05% trypsin which contained 200 mg ethylenedia- A carbonyl thioester. ADP: Adenosine 5'-diphosphate. Pi: Ortho- minetetracetic acid/1000 ml, washed twice with phos- phosphate. DBCC: 5'-Deoxyadenosylcobalamin. (a) and (b): Forms a and b, see the text. phate-buffered saline, and centrifuged at 100 X g for 1.5 min at room temperature after each wash. Cell counts were obtained immediately before each experi- ric carbon, two isomeric forms of this compound are ment. Fibroblast homogenates were prepared by rapid possible. The steric form resulting from the carboxyla- alternation of freezing and thawing at 37° five times in tion of propionyl-CoA, designated form a, must first be 1-2 volumes phosphate-buffered saline. converted to its optical enantiomorph by methylmal- For study of propionate metabolism, cells or homog- onyl-CoA racemase, before it can be converted to suc- enates were incubated in 1 ml Krebs phosphate buffer, cinate by the isomerase [10]. Defective racemase activ- pH 7.4, containing Na propionate-3-14C, 0.5 ^Ci (0.01 ity could therefore result in accumulation of methyl- HIM); 2-methyl-14C malonic acid, 0.25 ^Ci (0.06 HIM); malonyl-CoA (form a) which would result in the in- succinic-1,4-14C acid, 0.5 ^Ci (0.08 HIM) at 37°. Cells or creased excretion of methylmalonic acid after non- homogenates were incubated 3 hours for the pro- enzymatic cleavage of the thioester from the acid. (Fig. pionate and methylmalonate and 2 hours for the suc- cinate experiments. (In the methylmalonate experi- ments addition of 2.5 to 12 /xg DBCC did not cause a 14 Case History significant increase in CO2 evolved.) Unlabeled sub- strate was added and CO2 was released with 6 N H2- A newborn infant believed to have such a deficiency in 14 racemase activity has been observed recently. This full SO4. The CO2 evolved was collected in 20% KOH, added to Liquiflor before radioactivity was determined term male infant seemed to be normal until 2.5 days by scintillation spectrometry [17]. of age when he developed progressively severe meta- Liver obtained 1 hr after death was kept frozen at — bolic acidosis, obtundation, and coma. Hyperammone- 80° until analysis. Control livers for racemase, mutase, mia was noted and treated with four exchange transfu- and urea cycle enzymes were obtained from a neonate sions and other supportive measures. Despite these who died of hyaline membrane disease (several hours therapeutic attempts he failed to rally and on the 11th after death), an infant with an ornithine carbamyl day succumbed to overwhelming Klebsiella sepsis. De- transferase (OTC) deficiency (1 hr postmortem), a 3- tails of the clinical course and other studies of this year-old child with cystinosis (12 hr postmortem), and infant will be presented elsewhere [7]. a neonate who died of aortic atresia (12 hr postmor- Methylmalonyl-CoA racemase defect 877 Table I. Metabolism of labeled propionate, methylmalonate, tem). Liver was homogenized in 2 volumes 0.25 M su- 1 crose in the dark. The supernatant obtained after cen- and succinate of cultured fibroblasts trifuging at 5,000 X g for 1 hr at 4° was used. 2-Methyl-»C Succinic- Na propionate- malonic acid, l,4-"Cacid, u I4 For the methylmalonyl-CoA studies, liver and fibro- 3-"C, m^M «CO2 mjuM COs m/iU CO2 evolved/108 evolved/10s evolved/108 blast homogenates were incubated for 20 min with 20 cells/3 hr at 37° cells/3 hr cells/2 hr [tM Tris-sulfate buffer, pH 7.3, which contained 3H- at 37° at 37° methylmalonyl-CoA, 0.2 ^Ci; DBCC, 12 Mg/1.4-2.7 mg Control subject P 37 29 2.0 protein. Alkaline hydrolysis, extraction, and separa- Control subject IP 42 24 tion of methylmalonic and succinic acids were per- Patient 0.15 0.25 1.7 formed as described by Cardinale et al. [2]. The acids Patient's father 24 Patient's mother 10 were eluted with 0.1 N HC1 and added to Aquasol; the radioactivity was then measured by scintillation spec- 1 Each value represents the average of two to four assays. trometry. 2 Neonate, circumcision skin. Mitochondria were isolated from liver tissue accord- Table II. 1 ing to the method of Hogeboom [6] except that sus- Metabolism of labeled methylmalonate of liver 2-Methyl-14C malonic acid, m^ pension and sedimentation of mitochondria were per- metabolized/mg protein/3 hr formed once. The sediment was resuspended in 0.25 M sucrose and subjected to rapid, alternating freeze-thaw- Control subject P 2.12 ing 10 times. Homogenates containing 0.9-1.6 mg pro- Control subject IIZ 2.13 tein were added to 7 /xM unlabeled synthetic, racemic Patient 0.15 3 methylmalonyl-CoA, 50 pi water, and 0.6 mCi H2O 1 Each value represents the average of two to four assays. and were incubated at 30° for 25 min. A sucrose blank 2 Neonate, hyaline membrane disease. without protein was also centrifuged to measure spon- 3 Ornithine carbamylase transferase deficiency. taneous racemization. Salt-free neutralized hydroxyl- Table III. Metabolism of tritiated racemic methylmalonyl amine, 1 HIM [8] was added and the mixture was al- 1 lowed to stand at room temperature for 30 min. Subse- coenzyme A of cultured fibroblasts and liver Succinate-!H recovered from quent procedures were carried out at 4° according to protein (homogenized cells) the method of Mazumder et al. [11] except that the at 37°, dpm/mg/20 min supernatant was made 1.0 N with respect to NH OH 4 Fibroblasts before application to a column of Dowex 1-X10 in the Control subject2 404 chloride form.