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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.

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 choelstyramine 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.

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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.

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.

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.

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