C. Ehnholm

High density lipoprotein subfractions, apolipoprotein A‐I containing lipoproteins, lipoprotein (a), and cholesteryl ester transfer protein activity in alcoholic women before and after ethanol withdrawal

Abstract. We studied 11 female alcoholics before and after ethanol withdrawal of 2 weeks and 10 healthy normolipidaemic, nonalcoholic women of similar age. In alcoholic women the HDL2 mass was increased by 63% (P<0.01) on admission and normalized (P<0.01) during abstention. The concentrations of HDL3 cholesterol and its mass remained unchanged throughout the study. Consistently with the fall of HDL2 gradient gel electrophoresis analyses also demonstrated decrease of the cholesterol concentration of HDL2b and HDL2a (P<0.05) during alcohol withdrawal. On admission the apo A‐II concentration was increased by 48% (P < 0.01) and it was normalized (P< 0.001) during abstention. Among apo A‐I containing lipoproteins the most prominent change occurred in Lp A‐1: A‐11, which fell by 32% (P<0.01) during 1 weeks alcohol withdrawal. During abstention the lipoprotein (a) concentration increased in 10 out of 11 women. In patients cholesteryl ester transfer (CETP) activity increased by 35% (P<0.01) during 1 week of ethanol withdrawal. On admission post‐heparin plasma lipoprotein (LPL) and hepatic lipase activities were increased by 25% (P = NS); during 1 weeks abstention they both returned to the control level (P < 0.05–< 0.01). In conclusion, chronic alcoholic women display multiple changes of lipoprotein metabolism which are rapidly reversed during abstinence. In contrast to alcoholic men, studied previously by us using the same study design and methods, there was no significant elevation of HDL3 cholesterol and apo A‐I. The data suggest that alcohol interferes with several regulatory steps of HDL metabolism which are partly gender dependent.

A simple purification procedure for rat hepatic lipase

Lipoprotein lipase (LPL, EC 3.1 .1.3.) of the extrahepatic tissues is responsible for the hydrolysis of triacylglycerols transported in plasma chylomicrons and very low density lipoproteins [ 1,2]. This exoenzyme can be released into the circulation by intravenous injection of heparin or similar poiyanions [3]. In addition to the lipoprotein lipase, post-heparin plasma contains a triacylglycerol lipase of hepatic origin [4]. The hepatic lipase also hydrolyzes monoacyi~ycerols and phospholipids [5,6]. The role of the hepatic lipase in the lipoprotein metabolism is not known. It has been suggested that the enzyme could participate in the hydrolysis of the lipids in ‘retnnant’ lipoproteins [7]. The activity of the hepatic lipase is not lowered in hypertri~yceridaemia, and it does not correlate to the plasma level of triacylglycerol [ 11. Accordingly, the hepatic lipase cannot be the rate-lil~iting factor in plasma triacyl~ycerol removal. It has also been proposed that the hepatic lipase could mediate the breakdown of lipoprotein monoacylglycerols [8,9]. As the role of the hepatic lipase in lipoprotein metabolism is far from resolved, we felt it necessary to purify the enzyme in sufficient quantities to enable unambi~ous in vitro studies. We now describe a simple purification procedure for the rat hepatic lipase using heparin-containing liver perfusates as a starting material.

Effect of long-term antihypertensive and hypolipidemic treatment on high density lipoprotein cholesterol and apolipoproteins A-I and A-II

The concentrations of total serum cholesterol and triglycerides and serum HDL cholesterol, triglycerides and apoproteins A-I and A-II were measured in 119 men after 4 years of active participation in a multifactorial primary prevention trial of coronary heart disease. No difference was observed in total serum cholesterol, triglycerides, HDL lipids or apoproteins between the control subjects without medication and the men treated with antihypertensive drugs (beta-blockers alone or in combination with thiazides). The concentration of HDL cholesterol was significantly lower and that of apoprotein A-II significantly higher in the individuals treated with clofibrate than in the controls. On the other hand, the levels of both HDL cholesterol and apoprotein A-I were lower in the men treated with probucol than in the controls, whereas that of A-II was within the control limits. The ratio HDL cholesterol/apoprotein A-I was subnormal in all 3 groups treated with lipid-lowering drugs, as if the treatment had lowered the cholesterol saturation of the HDL fraction. The levels of HDL cholesterol and apoprotein A-I were negatively correlated with the length of the treatment in subjects treated with probucol but not in the other groups. These results suggest that in long-term use, probucol and possibly clofibrate lower both the concentration and the cholesterol/apoprotein ratio of the HDL fraction.

Lack of serum cholesterol-lowering effect of skimmed milk and butter milk under controlled conditions.

The effects of skimmed milk and butter milk on the plasma concentration of cholesterol, triglyceride and high density lipoprotein cholesterol were studied in voluntary male prisoners under carefully controlled conditions. No significant differences were observed in the serum lipid or lipoprotein levels between the groups ingesting the control diet and the diets containing 2.71 of skimmed milk or 2.01 of butter milk per day for 3 weeks.

Plasma high-density lipoproteins and hepatic microsomal enzyme induction

SummaryThe relationship between high-density lipoproteins (HDL) in plasma and hepatic structure and microsomal function has been investigated in 54 patients undergoing diagnostic liver biopsy. Plasma HDL cholesterol and major apoproteins were correlated with hepatic histology and microsomal enzyme activity assessed directly as liver cytochrome P-450 concentration and indirectly by plasma antipyrine clearance rate. HDL cholesterol, the concentrations of apoproteins A-I and A-II, the HDL cholesterol/total cholesterol ratio and cytochrome P-450 were low in subjects with moderate or severe hepatic fatty infiltration or cirrhosis when compared with the values for subjects with a normal liver. HDL cholesterol and apoprotein A-I and the HDL cholesterol/total cholesterol ratio were directly proportional to the amount of non-fatty parenchyma in the livers. Subjects with a normal liver undergoing treatment with enzyme-inducing drugs, such as phenytoin, phenobarbital and primidone, had higher HDL cholesterol, apoproteins A-I and A-II, HDL cholesterol/total cholesterol ratio, cytochrome P-450 and antipyrine clearance rate than subjects not receiving such therapy. Treatment with inducers appeared to have compensated for the effect of liver disease in lowering plasma HDL. In the entire population, and also in subjects not taking inducing drugs, when considered separately, plasma HDL cholesterol, apoproteins A-I and A-II and the HDL cholesterol/total cholesterol ratio were significantly correlated with cytochrome P-450 concentration. In subjects on enzyme inducers, HDL cholesterol and apoprotein A-I levels and the HDL cholesterol/total cholesterol ratio were proportional to the magnitude of the induction. Serum triglycerides were inversely proportional to the measures of liver microsomal enzyme activity. The lipoprotein pattern, high HDL cholesterol and apoproteins A-I and A-II, and high HDL cholesterol/total cholesterol ratio that accompany microsomal induction are characterized by a reduced risk of atherosclerotic vascular disease and a prolonged expectation of life. The plasma changes presumably reflect the effect of enzyme inducers, such as phenytoin and phenobarbital on hepatic lipids and proteins.

Human postheparin plasma hepatic lipase activity against triacylglycerol and phospholipid substrates

Recent evidence has suggested that the major physiological substrate of the heparin-releasable (postheparin plasma) hepatic lipase is HDL2-phospholipid. However, all the current assay methods for this enzyme are based on the use of triacylglycerol substrate. Even though both lipolytic activities of hepatic lipase are likely to be due to a single enzyme it is possible that the use of unphysiological lipid as a substrate may give misleading results. Therefore we did parallel assays of the activity of postheparin plasma hepatic lipase using triacylglycerol, monoacylglycerol and phospholipid substrates. The correlation coefficients between the three lipolytic activities were 0.95-0.98, indicating that identical results are obtained using any of these three lipids in the assay of postheparin plasma hepatic lipase.

Effect of parenteral hyperalimentation on serum lipoproteins and on lipoprotein lipase activity of adipose tissue and skeletal muscle

Abstract. This study reports on the effects of parenteral nutrition with glucose alone or in combination with Intralipidr̀ on heparin‐releasable lipoprotein lipase (LPL) activity of adipose tissue and skeletal muscle and on serum lipoproteins. Thirteen patients with postoperative hypercatabolism and nine patients with caloric malnutrition were studied. The average adipose tissue LPL activity increased 5‐fold during 4‐day glucose infusion (P < 0.001) and 7.4‐fold during Intralipidr̀ plus glucose infusion (P< 0.001). In contrast, no change occurred in the LPL activity of skeletal muscle. Glucose infusion caused a significant increase in VLDL and LDL triglyceride concentrations and the Intralipidr̀ plus glucose infusion was followed by a rise in LDL and HDL triglyceride concentrations. HDL cholesterol decreased by 26% (P < 0.01) during glucose and by 19% (P< 0.05) during Intralipidr̀ plus glucose. Apoprotein A I was very low already at the start of parenteral alimentation and it did not change during either nutrition. The HDL cholesterol and apoprotein A I and A II levels were each positively correlated with adipose tissue LPL activity before parenteral nutrition but not after it.

Uptake and degradation of rat chylomicron remnants, produced in vivo and in vitro, in rat hepatocyte monolayers.

1. The uptake of small and large chylomicrons in rat hepatocyte monolayer cultures was compared to the uptake of chylomicron remnants prepared either in vitro with pure milk lipoprotein lipase or in hepatectomized rats. 2. Small chylomicrons (Sf less than 400) markedly inhibited remnant uptake and were taken up more efficiently than large ones (Sf greater than 400), indicating that size may be an important factor for the rate of uptake. The Lineweaver-Burk analysis of the data indicated that the V values for the uptake of both small chylomicrons (Sf less than 400) and of remnants prepared either in hepatectomized rats or in vitro was significantly higher than for chylomicrons with Sf greater than 400, whereas the Km values for the different particles did not differ significantly. 3. Preincubation of chylomicrons with serum caused marked changes in their apolipoprotein composition. A loss of apolipoprotein A-I and an increase in apolipoprotein E content was observed by scanning of SDS-polyacrylamide gels. Th preincubation decreased, however, the subsequent uptake of the chylomicrons. In contrast, the uptake of remnants prepared in vivo, or in vitro with serum present, exceeded that of remnants prepared in vitro with albumin or fetal calf serum as the fatty acid acceptor. 4. The data thus indicate that both the decrease in size and the changes in the particle surface during lipolysis with serum present are likely to contribute to the differences seen in the rate of uptake between native chylomicrons and remnants in hepatocyte monolayers.

STIMULATION OF LIPOPROTEIN LIPASE ACTIVITY OF RAT ADIPOSE TISSUE AND POST-HEPARIN PLASMA BY N6-(PHENYLISOPROPYL)ADENOSINE

Adenosine has profound effects on adipose tissue metabolism. It is an inhibitor of 13-adrenergic stimulation of cyclic AMP accumulation [1,2] and lipolysis [2-101 in tire isolated rat fat cell. It also opposes the effects of adrenaline on phosphate uptake [ 11 ]. In vivo, tile nucleoside has been reported to lower plasma free fatty acid and glycerol concentrations [8]. The effects of adenosine are thought to be mediated by two membrane receptors, one requiring an intact ribose (R-site) and tile other one an intact purine moiety [ 12-14] (P-site). Adenosine is rapidly metabolized but its effects on the R-site [12-14] are shared by purine-substituted analogs such as N6-(phenylisopro pyl)adenosine that are not deaminated by the enzyme adenosine deaminase (EC 3.5.4.4) [15]. Lipoprotein lipase (EC 3.1.1.3.4) is the enzyme responsible for the hydrolysis and uptake of triglycerides from circulating chylomicrons and very low density lipoproteins [16-19] . The enzyme is thought to be located on the endothelial surface of the capillary bed of extrahepatic tissues and it can be released into the circulation by intravenous injection of heparin [ 16-20] . Another heparin-releasable ectoenzyme is probably located on the outer surface of the endothelial cells of the liver and it is therefore called hepatic lipase [20]. This enzyme has been suggested to hydrolyze high density lipoprotein phospholipids and triglycerides [21 ]. This work was undertaken to find out ifN6-(phenyl isopropyl)adenosine has effects on lipoprotein lipase activity and, consequently, on the uptake of circulating triglycerides into tissues. Male Lewis albino rats ( 150 -250 g body wt) were used. They were fed ad libitum with a standard chow until injected intraperitoneally with 50-300/al isotonic NaC1 with/without N6-(phenylisopropyl)adenosine, at which time food was withheld. At the times indicated, the animals were anaesthetized with ether and blood was drawn by cardiac puncture into heparinized syringes. For post-heparin plasma lipase assays, blood was drawn 2 min after intravenous injection ofheparin (2000 units/kg body wt). The blood was centrifuged and the resulting plasma was stored at 2 0 ° C until assayed. Free glycerol was estimated enzymatically by using the commercial kit no. 125 032 of BoehringerMannheim GmbH. Tile assays for porst-heparin plasma lipases and the antibody to rat hepatic lipase have been described in [22]. The method used to measure heparin-releasable lipoprotein lipase activity of adipose tissue will be presented in detail in the text. N6-(phenylisopropyl)Adenosine was a gift from Dr Harald Stork of Boehringer-Mannheim GmbH. Glyceroltri-[14C] oleate (49 mCi/mmol) was from the Radiochemical Centre (Amersham). Heparin was from Medica (ttelsinki). All other reagents were from Sigma (St Louis MO).

Studies on the effect of hepatectomy on pig post-heparin plasma lipases

Abstract The effect of different amounts of heparin injected intravenously in swine on lipoprotein lipase and hepatic lipase activities in post-heparin plasma was studied using an immunochemical method. After the injection of 50 I.U. of heparin/kg body weight the apparent half-life of lipoprotein lipase and hepatic lipase activity measurable in post-heparin plasma was 15 min. This was prolonged to more than 60 min after the injection of 1000 I.U./kg body weight. It is concluded that the higher the heparin dose injected the longer can lipolytic activities be measured in plasma. A possible explanation for these findings is that the amount of circulating heparin governs the distribution of lipoprotein lipase and hepatic lipase between an endothelial-bound form and a circulating form and thus determines the apparent ‘half-life’ of lipase activity measurable in plasma. The apparent half-life of radioactively labelled heparin in normal swine was not different from that observed in hepatectomized animals. After hepatectomy no immunoreactive hepatic lipase activity could be demonstrated in post-heparin plasma confirming our previous findings that the liver is the only source of hepatic lipase. To study the role of the liver in the clearance of plasma lipoprotein lipase activity after the administration of heparin normal and hepatectomized pigs were given 200 I.U./kg body weight followed by a heparin infusion of 100 I.U./ h per kg body weight. In the control pigs the heparin injection caused a rapid release of lipoprotein lipase and hepatic lipase activities. These activities were maintained in the circulation during the 3-h infusion at a level of about 60% of the levels measurable 30 min after the injection. In hepatectomized pigs the lipoprotein lipase activity rose during the infusion to about six times the activity recorded 30 min after heparin administration. From these experiments we conclude that after heparin injection the liver is involved in the clearance of post-heparin plasma lipolytic activity.