H. Ramirez

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.

Different Developmental Patterns of 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Chick Tissues according to Their Role in Cholesterogenesis

The developmental pattern of microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity was different in liver, intestine and brain of neonatal chicks. Hepatic reductase activity sharply increased between 5 and 9 days after hatching. This pattern agrees with changes in acetate incorporation into non-saponifiable lipids by liver slices. Both enzyme activity and acetate incorporation seem to be related to the hepatic uptake of cholesterol from the yolk sac during the first days after hatching and to the new synthesis of cholesterol commencing in the second week. Changes in intestinal reductase suggest that the enzyme was less sensitive to the cholesterol in yolk sac. Brain reductase did not change within the age assayed (1-15 days), being independent from the levels of yolk sac or serum cholesterol during the later steps of myelination.

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.

Relationship between inhibition of 3-hydroxy-3-methylglutaryl-coa reductase by cholesterol feeding and short-term changes in membrane fluidity during neonatal development

Both 5% cholesterol feeding and fasting produced a decrease in the hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity, although certain diurnal variations remained during the second day of treatment. Supplementation of 5% cholesterol to the diet produced a significant increase in cholesterol content of hepatic microsomes, whereas no significant variations were observed after fasting. The phospholipid content of hepatic microsomes did not change by fasting. However, cholesterol feeding produced a clear decrease in microsomal phospholipids. After 7 hr of cholesterol feeding, an increase of nearly 3-fold in the cholesterol/lipidic phosphorus molar ratio was found. Fasting had no effect on this molar ratio. The changes observed by cholesterol feeding agree with a mechanism of regulation of hepatic reductase by alteration in membrane fluidity, a mechanism that would be already operative during the neonatal period.

Different thermal behavior of neonatal hepatic and cerebral 3-hydroxy-3-methylglutaryl-CoA reductase

The Arrhenius plots of hepatic and cerebral 3-hydroxy-3-methylglutaryl-CoA reductase activity were studied in neonatal chicks fed with a standard diet. Supplementation of the diet with 2% cholesterol from hatching has no effect on the thermal characteristics of the brain enzyme. The Arrhenius plot of brain reductase was practically similar to that found in control chicks. However, hepatic reductase was inhibited by cholesterol feeding. Dietary cholesterol increased the cholesterol/lipidic phosphorus molar ratio in liver microsomes, whereas no significant differences were observed in brain microsomes. These results are in agreement with the hypothesis that activity of hepatic reductase is regulated by the fluidity of microsomal membrane and show that cholesterol feeding does not alter the fluidity of microsomal membranes in neonatal chick brain having, thus, no effect on the thermal behavior of cerebral reductase.

Effect of Dietary Cholesterol and Cholestyramine on Developmental Pattern of 3-Hydroxy-3-Methylglutaryl-CoA Reductase

Supplementation of the diet with 2% cholesterol suppressed the increase observed in the hepatic and intestinal 3-hydroxy-3-methylglutaryl-CoA reductase activity from normally fed chicks during the first days after hatching. Cholestyramine feeding clearly increased both hepatic and intestinal reductase activities. In contrast, brain reductase did not show significant changes by cholesterol or cholestyramine feeding. Dietary cholesterol produced a clear increase in the cholesterol/lipidic phosphorus molar ratio of hepatic and intestinal microsomal membranes. However, this molar ratio did not change by cholestyramine feeding during postnatal development. Both dietary cholesterol and cholestyramine had practically no effect on the cholesterol/lipidic phosphorus molar ratio of brain microsomes. The relationship between the inhibition of reductase activity by dietary cholesterol and the increase of cholesterol/lipidic phosphorus molar ratio is in agreement with a mechanism of regulation of both hepatic and intestinal reductase by alterations of membrane fluidity, mechanism that would be already operative during the neonatal period.

Development of the diurnal rhythm of chick 3-hydroxy-3-methylglutaryl-CoA reductase.

Chick liver and intestine 3-hydroxy-3-methylglutaryl-CoA reductase did not show diurnal rhythm at hatching. Differences in activity between light and dark periods appeared during the first week and remained more or less constant between 10–14 days after hatching. Hepatic and intestinal reductase activities were maximal during the light period and minimal during the dark period. Amplitude of the rhythm was practically similar in both tissues (about 3-fold) although specific activities and differences between peak and nadir values were always higher in liver. Chick brain reductase did not show significant diurnal variations in the age range assayed.

Lipid composition of brain myelin from normal and hyperphenylalaninemic chick embryos

The influence of hyperphenylalaninemia on the lipid composition of brain myelin has been investigated in 19-day-old chick embryos. CNP-ase activity was used as myelin marker enzyme for myelin isolation. CNP-ase activity was significantly lower in hyperphenylalaninemic myelin when compared with control. No significant differences were observed after experimental treatment in the total lipid content of myelin as well as in the proportion of cholesterol:phospholipid:galactolipid. Nevertheless, a clear increase in the percentage of esterified cholesterol was found. No appreciable alterations were observed in the phospholipid composition of brain myelin from both control and hyperphenylalaninemic chick embryos. However, the ratio of unsaturated to saturated fatty acids in serine plasmalogen and sphingomyelin was considerably increased by this treatment. This ratio in choline and ethanolamine phosphatides from treated embryos did not differ from that of controls.

Synthesis of sterols by chick brain and liver during embryonic development.

The evolution throughout embryonic development of the rate at which acetate was converted into sterols was studied in chick brain and liver. Acetate incorporation (nmol/h/g tissue) was clearly higher in brain than in liver and sharply decreased with the age of embryo. Cholesterol and desmosterol were the major sterols formed from acetate by chick embryo brain, followed by lanosterol and squalene. No desmosterol was found in chick embryo liver, organ where cholesterol was the major sterol synthesized. In brain, the relative percentage of cholesterol increased throughout embryonic development reaching more than 50% at hatching, while the percentage of desmosterol decreased during the same period and represented at hatching only about 10-15% of the total nonsaponifiable fraction. The relative percentages of lanosterol and squalene did not change significantly throughout the period assayed. In liver, the percentage of cholesterol increased until 19 days but sharply decreased at hatching.

Development of lipidic composition of neonatal chick liver and intestine microsomes: changes induced by cholesterol feeding

Changes in cholesterol and phospholipid content of chick liver and intestine microsomes were studied throughout the two first weeks of life. Differences observed throughout postnatal development were mainly due to the free cholesterol. Cholesterol feeding resulted in a clear increase of the amounts of both free and esterified cholesterol. Phospholipid content of chick liver and intestine microsomes did not change significantly after hatching. Phosphatidylcholine and phosphatidylethanolamine were found to be the major phospholipids. Although the amount of each phospholipid could be affected by cholesterol feeding, its relative percentage did not change by this treatment.