A new assay method for pyruvate dehydrogenase in platelets [1] using in situ generation of [1-¹⁴C]pyruvate from [1-¹⁴C]lactate has been further developed for the use in cultured human fibroblasts and amniotic fluid cells.

A very low blank rate was found compared to the method using [1-¹⁴C]pyruvate as substrate and an activity of 20 times the blank was obtained in normal fibroblasts and amniotic fluid cells. The pyruvate dehydrogenase was linear with time and protein concentration. The activity was greatly decreased by addition of non-radioactive pyruvate to the assay mixture and by preincubation with ATP. The cofactor requirement was similar to pyruvate dehydrogenase from other sources. Normal activities in cultured human fibroblasts and amniotic fluid cells were 13.9 pkat/mg protein (n = 15) and 21.7 pkat/mg protein (n = 10), respectively. The method was found to be very reliable and makes the diagnosis of pyruvate dehydrogenase deficiency possible in easily accessible tissue such as cultured fibroblasts. Prenatal diagnosis may also be a possibility.