An improved assay of 3-hydroxy-3-methylglutaryl-CoA reductase activity in Reuber H35 hepatoma cells without microsomes isolation.

The optimal conditions for measuring 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity in Reuber H35 hepatoma cells are described in this paper. Cells in the exponential phase of growth were lysed by incubation with Brij 97 detergent for 30 min. We used imidazole buffer supplemented with EDTA and leupeptine, two inhibitors of proteases. Disrupted cells were then centrifuged at 12,000 g. Although microsomes are usually reported as enzyme preparations for measuring HMG-CoA reductase, our data showed that hepatoma cells may be used without previous isolation of microsomes. The 12,000 g supernatant showed similar levels of total and specific activities to those found in the microsomal fraction obtained after 105,000 g centrifugation. The soluble fraction showed less than 10% of reductase activity. Reductase activity from Reuber H35 hepatoma cells increased proportionally to the reaction time from 30 to 90 min and to the amount of protein added in a range of 50-500 micrograms. Our modified method was very sensitive and reproducible, because very low specific activity (about 15-100 pmol min-1 per mg protein) could be quantified in different assay conditions obtaining similar values.

Metabolism of palmitic and docosahexaenoic acids in Reuber H35 hepatoma cells.

In this work, we have modified the fatty acid composition of Reuber H35 hepatoma cells by supplementation of the culture medium with a saturated (palmitic) or a polyunsaturated (docosahexaenoic) acid. These fatty acids were incorporated into total lipids and phospholipids of hepatoma cells. Palmitic acid readily increased the percentage of its monounsaturated derivative (16:1 n-7). When both fatty acids were supplemented at the same concentration, the percentage of docosahexaenoic acid in the total lipids and phospholipids of Reuber H35 cells increased more than that of palmitic acid. Although the levels of 16:0 increased, the addition of docosahexaenoic acid to the culture medium decreased the percentages of monoenoic acids. From our results, it can be concluded that palmitic and docosahexaenoic acids modify the fatty acid composition of Reuber H35 hepatoma cells. The profound changes induced by docosahexaenoic acid, especially those in the phospholipid fraction, may be of great interest given the main role of these components in the regulation of chemical and physical properties of biological membranes and/or membrane systems.

Regulation of mevalonate 5-pyrophosphate decarboxylase in HeLa cells. Inhibition of enzymatic protein synthesis by serum lipoproteins.

Mevalonate 5-pyrophosphate decarboxylase (EC 4.1.1.33) has been considered as a secondary site of regulation of cholesterogenesis. Because of this, we have studied the regulation of decarboxylase in HeLa cells by serum lipoproteins in the cell culture medium. A first group of experiments was performed with cells grown in Eagle's medium with 10% foetal calf serum. The specific activity of decarboxylase was increased when whole foetal calf serum was replaced with lipoprotein-poor serum. This increase was clearly reduced in the presence of cycloheximide. Addition of serum lipoproteins to a medium containing lipoprotein-poor serum led to a clear decrease in the decarboxylase activity. An identical decrease was observed after the addition of lipoproteins alone or in combination with cycloheximide. These results suggest for the first time that the effect of serum lipoproteins on decarboxylase activity should be a decrease in the rate of enzymatic protein synthesis, and corroborate the important role of reactions other than those catalysed by 3-hydroxy-3-methylglutaryl-CoA reductase in the regulation of cholesterogenesis.

Oxygen free radicals and human disease.

Oxygen free radicals are very reactive molecules which can react with every cellular component. They are normally produced in organisms being involved in various biologic reactions. However, too high levels of these partially-reduced O2 species can give rise to functional and morphologic disturbances in cells. There is evidence to implicate oxygen free radicals as important pathologic mediators in many human disease processes.

Effect of phenylalanine derivatives on the main regulatory enzymes of hepatic cholesterogenesis.

Phenylalanine, phenylpyruvate and phenylacetate produced a considerable inhibition of chick liver mevalonate 5-pyrophosphate decarboxylase while mevalonate kinase and mevalonate 5-phosphate kinase were not significantly affected. Phenolic derivatives of phenylalanine produced a similar inhibition of decarboxylase activity than that found in the presence of phenyl metabolites. The degree of inhibition was progressive with increasing concentrations of inhibitors (1.25-5.00 mM). Simultaneous supplementation of different metabolites in conditions similar to those in experimental phenylketonuria (0.25 mM each) produced a clear inhibition of liver decarboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. To our knowledge, this is the first report on the in vitro inhibition of both liver regulatory enzymes of cholesterogenesis in phenylketonuria-like conditions. Our results show a lower inhibition of decarboxylase than that of reductase but suggest an important regulatory role of decarboxylase in cholesterol synthesis.

A procedure for the simultaneous determination of lipid and protein in biomembranes and other biological samples.

Very small sample sizes frequently become the limiting factor in biochemical and biomembrane studies in which routine quantification of protein and bulk lipids are required. The procedure described here allows the simultaneous determination of protein and lipid without initial, multiple aliquots. The method is based on the quantitative precipitation of proteins from a defined hexane/isopropanol mixture. The liquid phase resulting after decanting and concentrating to dryness can then be used to assay the lipid content directly. Quantitative assay of protein can be achieved after resuspension of the pelleted material by addition of sodium dodecyl sulfate (0.1%) and deoxycholate (1%). The method is also applicable to other types of lipid- and protein-containing samples with a broad range of protein/lipid ratios and lipid compositions, as they occur, for example, in serum lipoproteins.

The effects of feeding a saturated fat-rich diet on enzymes of cholesterol metabolism in the liver, intestine and aorta of the hamster.

The effects of two dietary treatments on parameters of cholesterol metabolism were studied. Hamsters were maintained on diets containing 2% (w/w) cholesterol or 20% (w/w) hydrogenated coconut oil for 4 weeks. Both diets induced a hypercholesterolaemia. The effects of the two treatments on hepatic and intestinal acyl-CoA:cholesterol acyltransferase activity and 3-hydroxy-3-methylglutaryl-CoA reductase activity were measured. As expected, cholesterol feeding stimulated cholesterol esterification and inhibited cholesterol synthesis. Saturated fat-feeding had no effect on cholesterol synthesis but markedly inhibited cholesterol esterification in both liver and intestine. The diet-induced hypercholesterolaemia was strongly correlated with an increase in acyl-CoA:cholesterol acyltransferase activity in the activity. In contrast, the hypercholesterolaemia induced by feeding either of the two diets tended to increase aortic uptake of cholesterol and hence acyl-CoA:cholesterol acyltransferase activity. We suggest that the changes in cholesterol esterification correlate well with the expected flux of cholesterol into each tissue.

A procedure for eliminating interferences in the lowry method of protein determination.

A modification of the Lowry assay for the quantitative protein measurement in the presence of interfering materials has been developed. The method is based on a precipitation with a single-phase hexane:isopropanol solvent system and later resuspension of protein pellets with sodium dodecyl sulfate and deoxycholate. The new procedure eliminates the interference caused by Triton X-100, phospholipids, or dithiothreitol providing yields higher than 95% and seems to be especially suitable for protein determination on membrane preparations in samples with small volumes and/or very low protein concentrations.