Changes in the concentration of plasma

Changes in whole plasma and lipoprotein apoprotein concentrations were determined after a single in.jection of Triton WR 1339 into rats. Concentrations of apoproteins A-I (an activator of 1ecithin:cholesterol acyl transferase), arginine-rich apoprotein (ARP), and B apoprotein were measured by electroimmunoassay. The content of C-I I apoprotein (an activator of lipoprotein lipase) was estimated by the ability of plasma and lipoprotein fractions to promote hydrolysis of triglyceride in the presence of cow's milk lipase and also by isoelectric focusing on polyacrylamide gels. Apoproteins C-I1 and A-I were rapidly removed from high density lipoprotein (HDL) after Triton treatment and were recovered in the d 1.21 g/ml infranate fraction. A I was then totally cleared from the plasma within 10-20 hr after injection. Arginine-rich apoprotein was removed from HDL and also partially cleared from the plasma. The rise in very low density lipoprotein (VLDI,) apoprotein that followed the removal of apoproteins from HDL was mostly attributed to the B apoprotein, although corresponding smaller increases were observed in VLDL ARP and C apoproteins. The triglyceride:cholesterol, triglyceridep-otein, and B:C apoprotein ratios of VLDL more closely resembled nascent rather than plasma VLDL 10 hr after Triton injection. These studies suggest that the detergent may achieve its hyperlipidemic effect by disrupting HDL and thus removing the A-I and C-I1 proteins from a normal activating environment comprising VLDL, HDL, and the enzymes. The possible involvement of. intact HDL in VLDL catabolism is discussed in relation to other recent reports which also suggest that abnormalities of the VLDL-LDL system may be due to the absence of normal HDI,.-Ishikawa, T., and N. Fidge. Changes in the concentration of plasma lipoproteins and apoproteins following the administration of Triton WR 1339 to rats. ,/. Lipid Res. 1979. 20: 254-264. Supplementary key words electroimmunoassay . HDL disruption . activator protein triglyceride and cholesterol synthesis (4-7) and, more recently, the inhibitory action of the detergent has been usefully employed for investigating metabolic interrelationships between plasma lipoproteins (810). We felt that Triton WR 1339 may also prove useful for studying the involvement of apoproteins in triglyceride catabolism since any changes in the composition or concentration of these proteins may be related to the delay in triglyceride catabolism that follows administration of the detergent. Some of the factors that are known to be important in the catabolism of very low density lipoprotein are the peptide components of' high density lipoprotein, specifically the A-I and C-I1 apoproteins ( 1 1 ) . The first of these (A-I) activates 1ecithin:cholesterol acyl transferase, an enzyme which is considered responsible for the formation of most of the serum cholesterol esters (12) and which, in cooperation with lipoprotein lipase, is thought to take part in the conversion of VLDL to the cholesterol ester-rich low density lipoprotein (LDL). C-I1 peptide is a powerful activator of lipoprotein lipase (13) which is responsible for the removal of the VLDL triglyceride during its transformation into LDL. Because A-I is the major protein of HDL and most C-11 in the plasma of rats is bound to this lipoprotein (14), it is possible that HDL plays a prominent role in the catabolism of rat VLDL. In a recent study, Portman et al. (10) reported disturbances in HDL structure after injecting Triton into squirrel monkeys and, in preliminary experiments with the detergent, we noted marked changes in HDL apoprotein composition following Triton administration to rats. In order to study the effect of the detergent on apoSince the discovery that Triton WR 1339 causes hyperlipidemia in experimental animals (1) the detergent has had widespread use as a tool for studying lipid metabolism (2,3). Early investigators exploited its capacity to block lipid clearance for measuring rates of Abbreviations: IEF, isoelectric focusing; T M U , tetramethylurea; VLDL, very low density lipoprotein; LDI., low density lipoprotein; HDL, high density lipoprotein; ARP; arginine-rich protein; SDS, sodium dodecylsulfate; IDL, intermediate density lipoprotein. ' Send reprint requests to Dr. N. Fidge, Baker Medical Research Institute, Commercial Road, Prahan, Victoria, Australia 3 181. 254 Journal of Lipid Research Volume 20, 1979 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom proteins in more detail and, specifically, to investigate the role of HDL apoproteins on VLDL catabolism, we have measured changes in apoprotein concentrations of whole plasma and lipoproteins at various times after injecting Triton WR 1339 into rats. Monovalent antisera to several key rat apoproteins were used to quantitate these changes. METHODS AND MATERIALS Male Sprague-Dawley rats, 220-250 g and fed commercial rat pellets containing 18% protein, 5% fat, 6% fiber, and the usual trace elements and vitamins were used in this study. Prior to Triton injection, they were fasted overnight and the experiments were started between 10 and 11 AM. Triton WR 1339 (Sterling Pharmaceuticals, NSW, Australia) dissolved in 0.15 M NaCI, pH 7.4, (phosphate-buffered saline) was in-jected into the tail veins at a dose of 250 mg per kg body weight. Rats were then bled from the abdominal aorta, under light ether anesthesia, at various times after injection. Four animals were exsanguinated at each time point (see Results) and in each experiment four animals were also injected with 1 ml of 0.15 M NaCI, providing a control group for comparison with the ‘Triton group. The plasma obtained from each rat was dialyzed for 72 hr against phosphate-buffered saline, pH 7.4, containing 1 mM EDTA with several changes of dialysate, to remove Triton WR 1339 (9). Samples of plasma were kept for lipid and apoprotein assays and the remainder was used to separate lipoproteins. Isolation of lipoproteins Plasma lipoproteins were isolated as described before (15). The lipoprotein fractions isolated in these experiments were very low density lipoprotein (VLDL, d < 1.006 g/ml), intermediate density lipoprotein (IDL, d 1.006-1.019 g/ml), low density lipoprotein (LDL, d 1.0191.063 g/ml), high density lipoprotein (HDL, d 1.063-1.21 glml), and the infranate fraction (IF, d > 1.21). Lipoprotein fractions were dialyzed against phosphate-buffered saline, pH 7.4, and aliquots were removed for the quantitative determination of lipid and protein moieties as well as for electroimmunoassay. Portions of each lipoprotein fraction were also recentrifuged at their respective upper densities for compositional analysis and for separation of apoproteins on polyacrylamide gels.