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/min) 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 CO2/10(6) cells/hr, respectively).
AdoCbl synthesis was normal (0.27 pg
AdoCbl/mg cells wet weight) in nonresponsive fibroblasts but was undetectable (less than 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.