Joyce Lebowitz

Studies of methylmalonyl coenzyme A carbonylmutase activity in methylmalonic acidemia. I. Correlation of clinical, hepatic, and fibroblast data.

Extract: Methylmalonyl-CoA carbonylmutase (mutase) activity was measured in fibroblast extracts from 15 patients with methylmalonic acidemia and in extracts of postmortem tissues from 6 of these children. Propionate oxidation and synthesis of 5′-deoxyadenosylcobalamin (AdoCbl, the vitamin B12 coenzyme that is part of the mutase holoenzyme) were measured in intact fibroblasts. Mutase activity was low in the absence of added AdoCbl in fibroblast extracts from both control subjects and patients. When the assay included supplemental AdoCbl, mutase activity increased in the control subjects (to 24.0 pmol succinate/mg protein/mini and in extracts from eight of the patients (20.8 pmol/mg protein/min), but showed almost no change in extracts from the other seven patients (0.16 pmol/mg protein/min). We have defined the eight fibroblast lines that showed normal mutase activity in the presence of AdoCbl as “responsive lines” and the other seven lines as “nonresponsive.” In the liver or kidney extracts of postmortem tissues, mutase activity responded to AdoCbl supplementation if fibroblast mutase activity from that patient had responded, and failed to respond if fibroblast activity failed to respond. Mean propionate oxidation in intact fibroblasts was much higher in control lines than in either responsive or nonresponsive lines (0.728 vs 0.097 vs 0.080 nmol CO2/106 cells/hr, respectively). AdoCbl synthesis was normal (0.27 pg AdoChl/mg cells wet weight) in nonresponsive fibroblasts but was undetectable (<0.005 pg/mg cells) in the responsive lines. Thus, the deficiency of mutase activity in responsive fibroblast lines is due to the failure to synthesize significant amounts of AdoCbl, whereas the deficiency in nonresponsive lines is due to some other abnormality, presumably a defect in the mutase apoenzyme.Speculation: Fibroblasts can be used to define whether patients with methylmalonic acidemia have an error of vitamin B12 metabolism if both mutase activity and AdoCbl synthesis are measured. The response of fibroblast mutase activity to the addition of AdoCbl reflects accurately the responsiveness of the enzyme in other tissues of the body. If an error of vitamin B12 metabolism is diagnosed with cultured fibroblasts, a prolonged trial of vitamin B12 therapy is warranted in the patient to seek evidence of in vivo responsiveness and clinical benefit from vitamin therapy.

Studies of methylmalonyl-coenzyme A carbonylmutase activity in methylmalonic acidemia: II. In vitro binding kinetics with adenosylcobalamin

Abstract Vitamin B 12 coenzyme binding to its apoenzyme in four control and four responsive methylmalonic acidemia (MM-emia) patients is described. Control and patient lines had similar K m values of 4.1 to 4.8 × 10 −8 m . These data confirm other studies that the responsive MM-emia patients described to date lack normal AdoCbl synthesis and that they exhibit normal apoenzyme binding to AdoCbl. Fibroblast mutase appears to bind its coenzyme tightly and has a K m range similar to measured values in purified enzyme.

Effect of Valine on Propionate Metabolism in Control and Hyperglycinemic Fibroblasts and in Rat Liver

Summary: Measurement of methylmalonyl-CoA mutase and propionyl-CoA carboxylase activities in lysates from fibroblasts derived from control, nonketotic hyperglycinemia, propionic acidemia, and both vitamin B12-responsive and -nonresponsive variants of methylmalonic acidemia showed only one abnormality: a 59% decrease in carboxylase activity in the nonketotic hyperglycinemic lysates (P < 0.01). When fibroblasts from all cell types were grown on valine-supplemented (24 mM) media, mutase activity was generally inhibited. As for carboxylase activity, control lines were inhibited 35% as compared to controls without valine and propionic acidemia activity was undetectable. On the other hand, carboxylase activity in both methylmalonic acidemia variants was increased 40% and nonketotic hyperglycinemia carboxylase activity was increased 80% (P < 0.01) when grown on valine-supplemented media. Isoleucine could not substitute for valine in producing increased carboxylase activity in these mutants.Glycine cleavage activity in fresh rat liver homogenates (11.1 μmol/mg protein/90 min) did not vary significantly when 24 mM valine was added to the reaction (9.9 μmol/mg protein/90 min). Therefore, the hyperglycinemia observed in both ketotic and nonketotic forms is probably not caused by a direct effect of valine on the glycine cleavage reaction.These data suggest that the presence of increased amounts of propionic acid in scrum or urine does not necessarily rule out the possibility of nonketotic hyperglycinemia due to the decreased activity of the carboxylase enzyme.Speculation: Decreased activity of the propionyl-CoA carboxylase enzyme in nonketotic hyperglycinemia fibroblasts suggests altered propionate metabolism in this mutation. Additionally, growth of nonketotic hyperglycinemia fibroblasts on high concentrations of valine results in greatly increased carboxylase activity. This effect of valine is also evident in methylmalonic acidemia lysates although no decrease in carboxylase activity in these mutants grown under normal conditions is observed. Although the valine effect is not presently understood, it may be possible that nonketotic hyperglycinemia can be diagnosed from fibroblasts grown in tissue culture based on the combination of these two findings: (1) decreased propionyl-CoA carboxylase activity in nonketotic hyperglycinemic lysates grown under normal culture conditions and (2) increased propionyl-CoA carboxylase activity in nonketotic hyperglycinemic lysates grown on valine-supplemented media.

Propionate metabolism in fetal livers of 15 to 19 weeks' gestation

Extracts of hepatic tissue obtained from saline-induced abortuses were analyzed for methylmalonyl CoA carbonylmutase (MM) and propionyl CoA carboxylase (PC) activity. MM activity was similar to control values, which suggests that abortion material may be used to confirm the prenatal diagnosis of methylmalonic acidemia. Confirmation of a presumptive propionic acidemia diagnosis is more tenuous due to the instability of PC and the possibility that saline may induce PC activity.

METHYLMALONYLCoA CAKBONYLMUTASE (MUTASE) ACTIVITY IN VIVO AND IN VITRO-FURTHER EVIDENCE OF GENETIC HETEROGENEITY

Patients with the genetic defect, methylmalonic acidemia (MM-emia), have vomiting, ketoacidosis, failure to thrive and a high mortality. Clinically, patients are usually classified by their in vivo response to vitamin B12 i.e. some tend to normalize biochemically whereas others do not. Cell free extracts of liver and/or skin fibroblasts were assayed for mutase activity by measuring succinate formation from methylmalonylCoA. Vitamin B12 coenzyme (DBCC) synthesis was measured by incorporation of Co57 precursor into DBCC. 5 patients, clinically unresponsive, had no in vitro mutase activity and synthesized DBCC normally. 4 additional patients had normal in vitro mutase activity and defective DBCC formation. However, only 2 of the 4 were responsive in vivo. Intact fibroblasts were studied for conversion of propionate-1-14C to 14CO2. Good correlation of CO2 production was noted in the 5 lines with no mutase activity but variable results were found in the other 4.Liver mutase activity accurately reflects fibroblast extract activity in any individual patient. Response by intact cells to B12 is variable in some patients. “Responsiveness” should be limited to the changes noted in vivo and should remain a clinical description rather than one to delineate possible mechanisms responsible for the defect.