The in vitro incorporation of acetate- 14C into the lipids of new born rat calvaria

Abstract

Recent in vitro studies have demonstrated the incorporation of various radioactive substrates into lipids of newborn rat calvaria and bone cell cultures. While a variety of lipid classes became labeled, the systems failed to demonstrate any appreciable synthesis of fatty acids or cholesterol from radioactive glycerol, glucose, serine, or orthophosphate. It was therefore decided to employ acetate as a labeled precursor to determine the synthetic capacity of calvaria for these two lipid classes. Calvaria were incubated for up to 4 hr with acetate- 14C, demineralized, and extracted with chloroform/methanol. The lipids were chromatographed on silicic acid impregnated papers and identified by various enzymatic and chemical tests. Radioactive lipids were located on chromatograms by autoradiography for counting purposes. The results demonstrated that calvaria have the ability to synthesize free fatty acids and cholesterol. Approximately 5% of the incorporated acetate- 14C was found in each of these two lipids. Other lipid classes also became labeled, but phospholipase A and sodium methoxide treatment showed their radioactivity to be due to esterified fatty acids. As with glycerol- 14C and glucose- 14C studies, lecithin and triglycerides were the two more heavily labeled substances and accounted for some 35% of lipid radioactivity, respectively. Phosphatidyl ethanolamine, phosphatidyl serine, and cardiolipin were labeled to a smaller extent. The radioactivity of all labeled lipids increased throughout a 4-hr incubation time.