Deranged B 12 metabolism: Studies of fibroblasts grown in tissue culture

Abstract

Studies are reported of fibroblasts cultured from the skin of a patient with abnormalities of sulfur amino acid and of methylmalonic acid metabolism. These cells are unable to grow as do normal cells if methionine in the medium is replaced by homocystine. Addition of hydroxy-B 12, 1 μg/ml, to the medium allowed the cells of the patient to grow slowly. Extracts of the cells contained a normal concentration of cystathionine synthase, but an abnormally low concentration of holoenzyme for N 5-methyltetrahydrofolate methyltransferase activity. N 5-Methyltetrahydrofolate methyltransferase apoenzyme was present, as indicated by assay in the presence of appropriate B 12-containing compounds. Growth of control cells in media containing hydroxy-B 12, 1 μg/ml, led to a several-fold increase in total methyltransferase activity and a decrease of dependence of activity upon in vitro supplementation with B 12-containing compounds. Growth of the patient's cells under these conditions led to formation of a small, but detectable, amount of holoenzyme. However, the major part of the methyltransferase activity in extracts of his cells grown on hydroxy-B 12 remained dependent upon in vitro addition of a B 12-containing compound. The patient's cells converted propionate-1- 14C to 14CO 2 at a slow rate compared to control cells. During incubation with propionate-1- 14C his cells accumulated abnormally great amounts of methylmalonate- 14C but failed to form the usual amounts of radioactive succinate, lactate, glutamate, or aspartate. Growth of the patient's cells with supplemental hydroxy-B 12 in the medium partially restored the rate at which these cells converted propionate-1- 14C to 14CO 2. The specificity of this restorative effect as well as the effects of variation in concentration and time of exposure to hydroxy-B 12 are reported. All the available data are consistent with the hypothesis that cells from the patient are deficient in their ability to accumulate coenzymatically active derivatives of B 12 with resultant deficits in the activities of N 5-methyltetrahydrofolate methyltransferase and methylmalonyl-CoA isomerase, the two enzymes known in mammals to depend upon B 12 derivatives.