Stanley Fowler

The subcellular biochemistry of human arterial lesions: I. Biochemical constituents and marker enzymes in diseased and unaffected portions of human aortic specimens

Abstract Atheromatous lesions (including media beneath) and uninvolved media from neighboring areas of the same vessel were dissected from 23 human aortas obtained at autopsy. After tissue slices were taken for histological assessment, homogenates were prepared and the levels of DNA, protein, hydroxyproline, free and esterified cholesterol, and eight enzymes known to be associated with various subcellular organelles [ N -acetyl-β-glucosaminidase, β-galactosidase, cathepsin C, acid cholesteryl esterase ( lysosomes ); malate dehydrogenase ( mitochondria ); neutral α-glucosidase ( endoplasmic reticulum ); 5′-nucleotidase ( plasma membrane ); catalase (“microperoxisomes”)] were compared between lesions and uninvolved media preparations. No difference was found in the contents of the two samples in DNA, protein, malate dehydrogenase, neutral α-glucosidase, or 5′-nucleotidase. In contrast, the lesions were significantly enriched in catalase activity (67%) and in each of the acid hydrolases (55 to 126%) as well as in free (300%) and esterified (822%) cholesterol, based on DNA, compared to uninvolved media from the same vessels. Increases in both catalase and acid hydrolases correlated significantly with increases in total, free and esterified cholesterol contents of the preparations. Changes in the acid hydrolase specific activities were also correlated significantly with each other. Increased catalase and acid hydrolase activities in human atheromas are similar to previous findings in aortas of experimental animals fed a cholesterol-rich diet and suggest the participation of peroxisomes and lysosomes in the cellular response to lipid accumulation in atherosclerosis. Because the analyses were conducted on aortas taken at autopsy, modelexperiments on aortas of rabbit cadavers were also employed to determine the extent to which postmortem changes may have affected the levels of enzyme activities and chemical constituents surveyed. These studies showed only minor changes occur during the first 48 hr after death. Investigations of the latency of N -acetyl-β-glucosaminidase in postmortem human and rabbit aortas indicated that lysosomes of arterial cells remain intact for a considerable time after death.

An automated method for the fluorescamine reaction using a peristaltic pump

Abstract An automated method is described for the measurement of amino acids and proteins by the fluorescamine reaction. Isopropanol is used as solvent for the fluorescamine, and all reagents are pumped by a peristaltic pump.