CHOLESTERYL ESTER METABOLISM IN RABBITS: EFFECT OF CHOLESTEROL FEEDING AND CLOFIBRATE TREATMENT

摘要

One of the clinical manifestations of atherosclerosis is the drastic increase in serum cholesteryl esters. It is generally assumed that the increase in cholesteryl esters during atherogenesis may be the result of an increased lecithin cholesterol acyl transferase (LCAT) activity in serum. Since cholesteryl ester formation in serum is not only dependent on LCAT levels but also on serum lipoprotein composition, and since the lipoprotein composition is significantly altered during atherogenesis, LCAT level per se need not necessarily reflect the actual rate of cholesterol esterification in the intact system. Further, it is also possible that the increase in serum cholesteryl esters may be due to defective catabolism rather than accelerated synthesis.;The present study was aimed at determining the rates of serum cholesterol esterification as well as hydrolysis during the development of cholesterol-induced atherosclerosis in rabbits.;The elevation of cholesteryl esters in serum during cholesterol feeding could not be accounted for in terms of the LCAT reaction taking place in the serum. This is an apparent contradiction to a report by Wells & Rongone (Proc. Soc. Exp. Biol. Med., 130 661 (1969)), The difference in the assay conditions used in the two studies might have contributed to the contradictory results. In the present study the assay of Stokke and Norum (Scand. J. Clin. Lab. Invest. 27, 21 (1971)) was employed. The results provide a measure of the composite effect of the enzyme concentration and the substrate efficiency. This assay might give a better reflection of the overall reaction taking place in vivo.;No cholesteryl ester hydrolysis could be demonstrated in rabbit serum with any of the different preparations of (cholesteryl oleate) substrate used. In liver two cholesteryl ester hydrolysing activities were detected, one with an acid pH (4.5) optimum which has been reported previously and the other, detected for the first time in this study, with a neutral pH (7.4) optimum. The acid cholesterol esterase activity, which was the predominant cholesteryl ester hydrolysing activity (located in the lysosomes) in liver, was not found to be significantly affected by cholesterol feeding.;It was concluded that the increase in serum cholesteryl esters is neither the result of increased synthesis in serum nor the result of decreased acid cholesterol esterase activity in liver.;The neutral cholesterol esterase activity was stimulated by 2-mercaptoethanol and sodium taurocholate, while these reagents had no effect on the acid cholesterol esterase activity in the lysosomes. Freezing and thawing, on the other hand, enhanced the lysosomal acid esterase activity, while it slightly decreased the neutral activity in the microsomes. Sulfhydryl reagents like N-ethylmaleimide, p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid) at 1 mM concentrations, totally inhibited the microsomal esterase while N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) caused only a 15-30% inhibition of the lysosomal esterase activity. However, p-chloromercuribenzoate caused a 90% inhibition of the acid esterase. The inhibition of microsomal esterase by DTNB was reversed by excess of 2-mercaptoethanol. These results indicated that the two cholesterol esterase activities in rabbit liver are distinct and have different requirements for optimal activity.;The neutral cholesterol esterase was partially purified from the liver microsomes by extraction with taurocholate followed by precipitation with ammonium sulfate. The enzyme appeared to be quite unstable.;Although clofibrate is known to have a hypocholesteremic effect in human and rat serum, the present study showed that clofibrate did not have a similar hypocholesteremic effect in rabbits. It was also observed that serum cholesterol esterification and liver acid cholesterol esterase activity were not affected by clofibrate treatment.