Branched Chain A-Ketoacid Dehydrogenase to Thiamine and Thiamine Pyrophosphate

Pediat. Res. 12: 235-238 (1978) Branched chain amino acids thiamine maple syrup urine disease vitamin responsiveness mitochondrial membranes In Vivo and in Vitro Response of Human Branched Chain a-Ketoacid Dehydrogenase to Thiamine and Thiamine Pyrophosphate DEAN J. DANNER,'27' FRANCES B. WHEELER, SANDRA K. LEMMON, AND LOUIS J. ELSAS I1 Department of Pediatrics, Division of Medical Genetics, Emory University School of Medicine, Atlanta, Georgia, USA Summary lability at 37"; and failure to respond to added NAD+, CoASH, and MgZ+. In a homozygous affected patient with maple syrup urine We propose that "excess" thiamine led to increased available disease, pharmacologic doses of thiamine lowered urinary excre- thiamine pyrophosphate which stabilized the branched chain a- tion of branched chain a-ketoacids and stimulated branched ketoacid dehydrogenase, decreased biologic turnover, increased chain a-ketoacid dehydrogenase (BCKAD) in his peripheral enzyme specific activity and produced in vivo tolerance to blood leukocvtes. Suvvlementation of his branched chain ami- branched chain aminoacids in these patients with maple syrup noacid restricted diei hth 100 mglday of thiamine eliminated urine disease. recurrent episodes of ketoacidosis.~heseclinical responses were Speculation studied in vitro using mitochondria1 inner membranes prepared from his cultured skin fibroblasts and those from another By studying the partially purified normal and mutant branched thiamine-responsive patient from Canada. BCKAD in both chain a-ketoacid dehydrogenases from cultured human fibro- mutant cell lines had similarities to normal enzyme including: blasts, direct in vitro effects of thiamine pyrophosphate can be identical apparent K,,, value for thiamine pyrophosphate; similar measured and related to in vivo clinical responses. This should heat inactivation profdes which were slowed by the presence of improve and extend the treatment and management of patients thiamine pyrophosphate; and stimulation above basal activity by with maple syrup urine disease and provide a method for study thiamine pyrophosphate. Differences in the enzymes included: of other mutant human enzymes located in the mitochondrial decreased apparent V,,, for thiamine pyrophosphate; increased membrane. INTRODUCTION The patient was dismissed from the facility and instructed to continue daily oral doses of 100 mg thiamine. After three years on thiamine therapy. Thiamine responsive maple syrup urine disease (ffiUD) was first described the patient was again admitted to the Clinical ~eseirrchFacility for reeval- in 1971. Oral loading with vitamin B decreased previously elevated plasma uation. Thiamine above Recommended Dietary Allowance level was removed from branched chain amino acid concentratiAns in a presumably homozygous affected his diet for three weeks and urinary branched chain a-ketoacid excretion and Canadian female child (22). In a preliminary study we reported a similar peripheral'white blood cell BCKAD activity was assayed. Bath 24 hr urinary effect from high doses of oral thiamine on two homozygous affected brothers excretion and BCKAD activity on admission was the same as day 28 of theinitial (6). MSUD results from a epecific decrease in the activity of branched chain study and remained at thia level throughout the three week test period. The a-ketoacid dehydrogenase (BCKAD) which oxidatively decarboxylates a-ketoiso- patient's monthly ketoacidosis of unknown cause disappeared and for six caproate (KIc), a-keto-0-methylvalerate (KMV) and a-ketoisovalerate (KIV), years the patient has remained stable on the restrictive diet.supplemented the transaminated products of leucine, isoleucine and valine respectively with thiamine at 100 mg/day and has required no further hospitalizations for (9.25). BCKAD is thought to be a multienzyme complex composed of a decarboxy- treatment of metabolic acidosis. lase, a transacylase and lipoamide oxidoreductase in which thiamine pyrophosphate (TPP) is a cofactor for the first component (5.24). Intracellular con- Since the activity of the dehydrogenase was reflected in cultured skin centration of this cofactor can most likely be increased by oral loading since fibroblasts, this tissue was used to investigate the mechanisms producing thiamine kinase is active in the intestine and most other tissues of man (15, the observed clinical responses to high doses of thiamine. Isolated inner 17.18.23). mitochondrial membranes provided a partially purified enzyme preparation which eliminated complicating variables in branched chain a-ketoacid decarboxyla- Vitamin responsive inborn errors of metaboliam have been recognized for a tion auch as membrane transport of substrates and cofactors and transamina- number of years and our understanding of the mechanism by which these res- tion of aminoacid precursors. Data of Johnson and Connelly with beef liver ponses are realized continues to advance (1.12.13,16,19.20.22). Classically. mitochondria suggested that the branched chain a-ketoacid dehydrogenase was holoenzyme function was augmented by mass action through increased coenzyme localized on the outside of the inner membranes, facing the intramitochon- production and binding to apoenzyme. In a more recently postulated mechanism, drial space (11). When the outer membrane was removed by digitonin, treat- the presence of "excess" coenzyme decreased the degradation of holoenzyme ment, properly oriented inner membranes were obtained (21). Thus, the enzyme (12.13). This mechanism was invoked as the means by which 'ITP improved BCKAE was made directly'accessible to the substrate and cofactors. activity (4,6). Supraphysiologic ingestion of thiamine increased BCKAD activity in normal adult liver by 2 fold, but required three weeks, the approx- When these membrane preparations were incubated at 37' without exogenou imate biological half life of mitochondrial turnover (4). Because BCKAD is cofactors followed by a 15 minute assay, 14c0 release from the a-ketoacids expressed incultured human skin fibroblasts, and a decreased activity is obser- became dependent upon the addition of cofacto?a during the assay. The ved in cells from MSLRI patients, thia tissue was used to study in vitro the most important cofactor to eliminate was thiamine pyrophosphate since the effect of TPP (2-6.9). Here we report the effect of oral loading with thiamine fall to baseline activity did not occur if this cofactor alone were added on urine concentrations of the branched chain a-ketoacids and on enzyme func- to the buffer (5). The left panel in figure 2 shows that BCKAD from normal tion in isolated white blood cells from a single homozygous affected patient. fibroblasts fell to basal activity after 90 minutes. Without exogenous co- We also report studies on the effect of TPP on BCKAD activity in isolated mi- factors during the assay, activity was only 3% of that found after 5 minutes tochondrial inner membranes from fibroblasts cultured from this thiamine res- of incubation. The 5 minute time point reflects the temperature equilibra- ponsive patient, the Canadian patient and normal controls. tion period used in all experiments and was used as our zero time point. When all four cofactors were present during the 15 minute assay, full res- METHOLX ARD MATERIALS toration of activity was possible. The right panel describes a similar effect with mutant enzyme. In contrast to normal enzyme, after 90 minutes Following detailed description of the research project and obtaining it was only possible to restore 34% of the mutant enzyme activity by the informed parental consent, a homozygous affected EUD patient was admitted addition of cofactors. Nl restoration of CO release was possible at the to the Clinical Research Facility at Emory University where continuous 30 minute time point with these preparations &om the mutant. Thus, the supervision of his diet was possible. This patient's synthetic diet was mutant enzyme complex appeared more sensitive to increasing duration of restricted in branched chain amino acids and supplemented with Recommended preincubation reflecting an increaaed lability at 37'. Dietary Allowances of all vitamins including 5 mg/day of thiamine. After 1 week of stabilization, the study was begun.. After no added thiamine, the Cofactors were then tested singly and in combination for their ability diet was then supplemented with 50. 100 and 150 mg of thiamine-HC1. respec- to restore decarboxylating activity to the proteins in inner mitochondrial tively for each of three succeeding weeks. A 24 hour urine was collected on membranes. All possible combinations were tested but only the results of day 1.3. and 7. and 10 ml of heparinized blood was collected after day 3 of the most pertinent data are reported in Table I. Ninety minutes of incu- each of four weekly periods. White blood cella were prepared from the bation was used to lower CO release to basal level. Again, note that the unclotted blpod using the polyvinyl pyrollidone method (20). These cells absence of thiamine pyropho$hate during this prein$ybation was necessary to were used fresh for assay of the BCKAD activity. attain basal activity (5). When NAD+, CoASH and Mg were present during preincubation and assay. BCKAD from normal cells was maintained at 18 fold Punch skin biopsy was used to obtain a primary culture of fibroblasts above basal level. This was not seen for enzyme from the mutant cells. from this patient (Mutant A). Fibroblasts cultured from the previously When thiamine pyropbosphate'was added along with the

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