The rapid and specific determination of picomole quantities of 8-aminolevulinate has been accomplished by its enzymatic conversion to uroporphyrinogen I and subsequent fluorometric detection of the oxidized uroporphyrin I. The coupled-enzyme assay was linear with time and protein concentration and required less than 3 h for 25 individual determinations. Under the standard assay conditions, 5-100 pmol of uroporphyrin I was reliably quantitated; these values corresponded to a range of δ-aminolevulinate synthase activities from 0.2 to 15 nmol/h/ml enzyme. The sensitivity of this method was comparable to the more time-consuming radiochemical determinations of δ-aminolevulinate synthase activity. The δ-aminolevulinate synthase activity of liver homogenates from uninduced rats was 8.7 U/g liver (37 °C).
Pulse-chase studies were performed to investigate the metabolism of phosphatidylethanolamine (PEA) in cultured fibroblasts from patients with mucolipidosis type IV (MLIV) and normal controls. When cultured cells were incubated with ^3 H-ethanolamine, 80-90% of the intracellular radioactivity was associated with PEA. Compared to the metabolism of ^3 H-PEA in normal cells, the phospholipid was retained in greater amounts and degraded more slowly in the MLIV fibroblasts. The ^3 H-PEA concentration in lysosomal preparations isolated by Percoli gradients was more than 3-fold greater in MLIV than in normal cells after 10 days chase. These studies indicate that PEA catabolism is deranged in MLIV and suggest that the primary metabolic defect causes abnormal phospholipid catabolism in the lysosomes of affected individuals.
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