Lipoprotein Phospholipids Research Articles

Studies on Dissolving States of Phospholipids and Analytical Method of Lipoproteins

Studies on Dissolving States of Phospholipids and Analytical Method of Lipoproteins

A Procedure for the Determination of High-Density Lipoprotein Choline-Containing Phospholipids

A procedure for the determination of serum high-density lipoprotein choline-containing phospholipids is described. The choline-containing phospholipids represent 91-97% of the total serum and high-density lipoprotein phospholipids, which have previously been determined in relation to liver disease, neoplastic disorders, diabetes mellitus and atherosclerosis. The enzymatic assay is quick, simple and precise. No interference was found from the precipitating agents used to isolate the high-density lipoprotein fraction. Several serum samples were assayed for high-density lipoprotein cholesterol and choline-containing phospholipids. From this preliminary data there would appear to be a correlation between these two lipid classes.

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).

Effects of diuretic and propranolol on plasma lipoprotein lipids

Ticrynafen (TCNF), a nonthiazide diuretic, has been reported to be nonhyperlipidemic. To define the effects of these drugs on plasma lipoproteins, experiments were performed in hypertensive subjects after placebo therapy, 4 wk after therapy with either hydrochlorothiazide (HCTZ) or TCNF, 3 mo after diuretic with propranolol, and 1 mo after therapy with propranolol alone. Plasma lipoproteins were separated by ultracentrifugation and the lipid fractions isolated by extraction and silicic acid thin-layer chromatography. Plasma low-density lipoprotein (LDL) total cholesterol fell and high-density lipoprotein (HDL) total cholesterol rose in subjects receiving TCNF. TCNF had no effect on plasma low-density lipoprotein (VLDL) triglyceride or phospholipid. There was no significant changes in LDL or HDL total cholesterol in subjects on HCTZ. HCTZ tended to increase plasma VLDL triglyceride and phospholipid. The addition of propranolol to either diuretic had no effect on LDL or HDL total cholesterol but increased VLDL triglyceride, especially in subjects on HCTZ. Propranolol alone had no effect on any of the lipids measured.

Turnover and uptake by organs of radioactive serum high-density lipoprotein cholesteryl esters and phospholipids in the rat in vivo.

The serum decay of rat serum high-density lipoprotein (HD lipoprotein), labelled biosynthetically with (32)P in the phospholipid or with (3)H in the cholesteryl ester moiety, was measured in rats after partial hepatectomy or sham operation. The serum decay of (3)H-labelled HD lipoprotein cholesteryl esters was biexponential. In sham-operated rats the t((1/2)) values for the rapid phase and the slow phase were 0.2+/-0.1h and 4.2+/-0.4h (means+/-s.e.m.) respectively. After removal of two-thirds of the liver the t((1/2)) value of the rapid phase did not change (0.1+/-0.1h), whereas the t((1/2)) value of the slow phase increased to 5.7+/-0.8h. Partial hepatectomy hardly changed extrahepatic tissue radioactivities, whereas the percentage of the injected dose recovered in the liver 6h after injection decreased from 34.0+/-1.9% before to 13.5+/-1.6% after partial hepatectomy. The (32)P-labelled HD lipoprotein phospholipids showed a rapid monoexponential decay from serum with t((1/2)) values of 0.71+/-0.3h and 1.48+/-0.11h after sham operation or partial hepatectomy respectively. The tissue (32)P radioactivities in the shamoperated rats, measured 1h after injection, were 46.0+/-1.7% (liver), 1.7+/-0.3% (adipose tissue), 3.7+/-1.2% (skeletal muscle) and 3.0+/-0.0% (erythrocytes) of the injected dose. Only the value for liver was affected by partial hepatectomy and decreased to 16.7+/-3.8%. In a previous publication [Van Tol, Van Gent, Van't Hooft & Vlaspolder (1978) Atherosclerosis29, 439-448] we showed in a highly comparable experimental setting that the turnover rates of HD apolipoproteins A and C in vivo are not influenced by removal of two-thirds of the liver. From the present study it is clear that the removal rates of radioactive HD lipoprotein cholesteryl esters and HD lipoprotein phospholipids from serum in vivo are decreased by partial hepatectomy. The results indicate the possibility of partly separate metabolic pathways of HD apolipoproteins A and C, HD lipoprotein cholesteryl esters and HD lipoprotein phospholipids. The phospholipids and cholesteryl esters of HD lipoprotein are metabolized predominantly by the liver. Possible mechanisms for the hepatic uptake and metabolism of HD lipoprotein cholesteryl (esters) and phospholipids are discussed.

Lipoprotein lipids in renal transplant recipients of different pre-transplant etiology of renal disease A comparison of male and female patients

Serum and lipoprotein lipids have been compared in male and female transplant recipients with glomerulonephritis or analgesic nephropathy as etiology of pre-transplant renal disease, and a number of differences were observed. 1. (1) Serum cholesterol and phospholipid levels were elevated in glomerulonephritis and female analgesic nephropathy, but not in male analgesic nephropathy patients. 2. (2) Glomerulonephritis patients had normal low density lipoprotein (LDL) cholesterol levels whereas these were elevated in female and depressed in male analgesic nephropathy patients. 3. (3) LDL phospholipid, on the other hand, was normal in male and elevated in female transplant recipients irrespective of etiology of pre-transplant renal disease, while high density lipoprotein (HDL) phospholipid levels were elevated in female glomerulonephritis patients only. Mild hypertriglyceridemia and a tendency to increased HDL cholesterol were observed in all patients. These results provide further evidence for the complexity of lipoprotein lipid abnormalities in renal disease.

Function of hepatic triglyceride lipase in lipoprotein metabolism

Rat hepatic triglyceride lipase (H-TGL) was purified from liver tissue extracts by affinity chromatography on Sepharose 4B with covalently linked heparin. The purified rat H-TGL exhibited the properties previously described for this enzyme. Enzyme protein was injected into rabbits for anti-H-TGL antibody production. Antirat-H-TGL did not react against rat lipoprotein lipase (LPL) but inhibited H-TGL-activity both in vitro and in vivo greater than 90%. These antibodies were injected into rats and lipoprotein analyses were performed over a 36-hr period. It could be shown that inactivation of H-TGL by anti-H-TGL gamma-globulins in vivo led to an increase in total triglyceride concentration from 70 mg/dl to 230 mg/dl due to an increase in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) triglycerides 4 hr after antibody injection; a marked increase in high density lipoprotein (HDL) phospholipid concentration was observed with almost no change in HDL-cholesterol and HDL-triglycerides. This study documents the ability of antirat-H-TGL-gamma-globulins to inhibit H-TGL in vitro and in vivo. Furthermore, the inhibition of triglyceride removal in vivo demonstrated that this enzyme together with LPL is responsible for the catabolism of VLDL-triglyceride.

Determination of high-density lipoprotein phospholipids in serum.

I describe a method for measuring high-density lipoprotein phospholipids. Magnesium chloride and dextran sulfate are used to precipitate all low-density and very-low-density lipoproteins. The supernate contains only high-density lipoproteins, the phospholipid concentration of which is determined by an enzymic method. The precision of the method (CV) is 2.35% (10 repeated assays), and the mean value for HDL-phospholipids was 1006 (SD 248) mg/L for 30 apparently healthy subjects. I used electrophoresis and enzymic color development to confirm the presence of HDL-phospholipids. Results are compared with those obtained by an ultracentrifugation method.

On the metabolic function of heparin-releasable liver lipase

Intravenous administration of specific antibody against heparin-releasable liver lipase (liver lipase) induced a 75% inhibition of the enzyme activity in situ . Administration of the antibody resulted in an increase of high density lipoprotein (density range 1.050–1.13 g/ml; HDL 2) phospholipid levels (20% after 1 h; 54% after 4 h). Short-term (1 h) treatment with antibody had no significant effect on any of the other lipoprotein components. After long-term (4 h) treatment the free cholesterol level of HDL 2 and all components in the very low density lipoprotein (VLDL) + intermediate density lipoprotein (IDL) fraction were elevated (1.5–2.0 fold). In the low density lipoprotein (LDL) fraction only the phospholipid level was affected (increased by 72%). All lipid components in the HDL 3 fraction were decreased by the antibody treatment, but this decrease was only statistically significant for the cholesterolesters. The rate of removal of iodine-labeled high density lipoprotein (HDL) and LDL from serum was not affected by the antibody treatment. These results suggest that liver lipase may promote phospholipid removal in vivo and show that a lowering of liver lipase in situ has profound consequences for serum lipoprotein metabolism.

Apolipoprotein cii enhances hydrolysis of monoglycerides by lipoprotein lipase, but the effect is abolished by fatty acids

Lipoprotein lipase is an enzyme that hydrolyzes triglycerides and phospholipids in lipoproteins. Several aspects of its mode of action are not well understood. One is why fatty acids strongly inhibit the activity [ 11. Another is why monoglycerides do not accumulate during hydrolysis of triglycerides, although the rate of monoglyceride hydrolysis has been reported to be substantially lower than the rate of triglyceride hydrolysis [2-41. We report here that under appropriate experimental conditions monoglyceride hydrolysis is enhanced several-fold by apolipoprotein CII and reaches rates exceeding those for triglyceride hydrolysis. We also demonstrate that fatty acids interfere with and at higher concentrations completely abolish the lipolysis-promoting effects of the cofactor protein. This previously unrecognized effect of fatty acids must be a major factor in their inhibition of acylglycerol hydrolysis.

Determination of cholesterol and phospholipid concentrations in high-density lipoproteins by heparin-manganese chloride precipitation

Cholesterol as well as phospholipids in high-density lipoproteins (HDL) have been measured in 60 serum samples after heparin-manganese chloride precipitation of the apolipoprotein B containing lipoproteins. The values obtained correlated well with values from ultracentrifugal separation (slope 1.11 for cholesterol, 0.92 for phospholipids). Furthermore, there was a significant correlation between the low-density lipoprotein (LDL) cholesterol estimated by the formula of Friedewald et al. and the ultracentrifugally separated LDL cholesterol (slope 1.02). By the additional determination of HDL phospholipids with a precipitation procedure, further information about the “antiatherogenic” is easily available.

Comparison of the phospholipase activity of bovine milk lipoprotein lipase against rat plasma very low density and high density lipoprotein

The hydrolytic activity of a lipoprotein lipase from bovine milk against triacylglycerol and phosphatidylcholine of rat plasma very low density lipoprotein was determined and compared to that against phosphatidylcholine of high density lipoprotein. 85–90% of the triacylglycerol in very low density lipoprotein were hydrolyzed to fatty acids and 25–35% of the phosphatidylcholine to lysophosphatidylcholine. High density lipoprotein phosphatidylcholine was only minimally susceptible to the enzyme. Even with high amounts of enzyme and prolonged incubation periods, lysophosphatidylcholine generation did not exceed 2–4% of the original amounts of labeled phosphatidylcholine in the high density lipoprotein. We conclude that phospholipids in high density lipoprotein are not substrates for the phospholipase activity of this lipoprotein lipase. These observations suggest that factors other than the presence of apolipoprotein C-II and of glycerophosphatides are of importance for the activity of lipoprotein lipases.

Complete exchange of phospholipids between microsomes and plasma lipoproteins mediated by liver phospholipid-exchange proteins

It is known that cholesterol and phospholipids are in dynamic equilibrium and spontaneously exchange both in vitro and in vivo between individual classes of plasma lipoproteins [l-4] . Spontaneous transfer of phospholipids has also been shown between lipoproteins and erythrocytes [5],mitochondria [6] ,liver microsomes [7] , liver slices [8] and cells in culture [9]. In these examples of phospholipid exchange it is presumed that transfer occurs upon formation of a collision complex. Evidence for a protein-facilitated transfer of lipoprotein phospholipids in serum is controversial [7,10]. On the other hand, eukaryotic cells contain proteins designated as phospholipid exchange proteins (PLEP) that do facilitate the transfer of phospholipids from one membrane to another [ 1 I-131 . A purified PLEP from beef liver shows a highly specific affinity for phosphatidylcholine (PC-PLEP [ 141). Two proteins have been isolated from bovine cerebral cortex which stimulate both the transfer of phosphatidylinositol (PI) and PC (PI-PLEP [IS] ). Recently, exchange proteins have been isolated from rat liver [ 161 and hepatoma [ 171 which lack specificity in the transfer of phospholipid. The purpose of the present study was to determine whether and if so, to what extent, purified PCand PI-exchange proteins catalyze the transfer of phospholipids between rat liver microsomes and the individual classes of lipoproteins. It will be shown that the protein-mediated transfer of labeled PC and PI from microsomes to very low density lipoproteins (VLDL), LDL and HDL was 5-lo-fold higher than

Rapid colorimetric methods for quantitative lipoprotein-X determination without electrophoresis

Two procedures for quantitative determination of the cholestasis-characterizing lipoprotein-X are described. Both procedures are based on ether-extractability of phospholipids which are relatively loosely bound in lipoprotein-X. The presence of cetyltrimethylammonium bromide at adequate concentration allowed complete ether extraction of phospholipids from lipoprotein-X even at low concentration, without affecting the phospholipids of normal lipoproteins. In one modification phospholipids were extracted into ether ionpaired with a bilirubin conjugate (e.g. Woodward's K conjugate) the concentration of which was then estimated in the ether extract. In the second modification phospholipids were directly determined in the ether extract. Calibration was done either with a lipoprotein-X concentrate or with a clear sonicated lecithin sol added to normal serum. The results obtained by both methods are in a good agreement with the results by agar gel electrophoresis.

Age-related changes in cholesterol and bile acid metabolism in rats.

Age-related changes in serum and liver cholesterol, phospholipid and triglyceride levels, serum lipoproteins, biliary secretion of cholesterol, phospholipid and bile acids, and fecal excretion of sterols and bile acids were examined in Sprague-Dawley and Wistar strain male rats, 7 to 106 weeks in age. Serum and liver lipid levels increased with age in both strains but the liver phospholipid level remained unchanged. The proportion of serum alpha-lipoprotein increases and that of beta- and prebeta-lipoproteins slightly decreased. Cholesterol and phospholipids in low density lipoprotein and cholesterol in high density lipoprotein fractions also increased with age. Bile flow and biliary secretion of cholesterol and bile acids decreased in aged rats, but when they were expressed in terms of units per rat they were almost constant without regard to age. Pool size, synthesis, secretion, and turnover frequency of bile acids also did not change when they were expressed per rat, though 7-week-old rats showed a low value for turnover frequency. Biliary secretion of phospholipid, however, increased in aged rats. Biliary secretion of chenodeoxycholic and alpha-muricholic acids decreased but that of cholic and hyodeoxycholic acids increased. Daily excretion of feces and fecal neutral sterols decreases in aged rats but the excretion of bile acids remained constant regardless of age. The ratio of coprostanol and cholesterol in the total sterols was not affected. Fecal lithocholic, beta-muricholic and P10 (probably omega-muricholic) acids were decreased with age but the other bile acid components were not changed or were slightly increased.

Replacement of endogenous phospholipids in rat plasma lipoproteins during intravenous infusion of an artificial lipid emulsion

Male Wistar rats, weighing 250–300 g, were intravenously infused (0.33 ml/h per 100 g body weight) with an artificial emulsion of soybean triacylglycerols and egg yolk phospholipids (Intralipid) for various periods of time ranging from a few hours to a few days. About 0.5 h after the infusion, the animals were sacrificed, various classes of plasma lipoproteins were isolated by centrifugation, and the total lipid profiles of the fractions were determined by high temperature gas chromatography. The lipoproteins collected after 24 h of infusion were subjected to a complete analysis of the molecular species of the various lipid classes. It was shown that continuous intravenous infusion of Intralipid, despite rapid apparent clearance of the visible lipid particles, resulted in a gradual accumulation of exogenous phosphatidylcholine and sphingomyelin, free cholesterol, and plant sterols in the plasma, which displaced the endogenous phospholipids and sterols from the various lipoproteins. At 24 h, about 70% of the endogenous phospholipid of all the plasma lipoproteins was replaced by egg yolk phospholipids of the Intralipid. Under the same conditions, no displacement nor exchange was observed of endogenous cholesteryl esters for exogenous triacylglycerols in any of the lipoprotein classes. Intralipid infusion for periods up to 3 days results in a limited further displacement of endogenous phospholipids by exogenous lipids in the plasma lipoproteins, but a complete substitution was not achieved because of a continued secretion of lipoproteins containing endogenous phospholipids. The infusion of Intralipid led to a significant increase in the proportion of the phospholipid and free sterol in all lipoproteins, which decreased the calculated diameter of the particles by about one-half. Only minor changes were seen in the lipid to protein ratios of the lipoproteins and in their apoprotein composition.

Effect of Clofibrate and Calcium in Type II A Hyperlipoproteinaemia

In type II A hyperlipoproteinaemia, treatment with cholestyramine (12 g), calcium clofibrate (2 g) and a combination of calcium clofibrate and calcium carbonate (2 + 2 g) reduced the cholesterol concentration. The combination of calcium and clofibrate was at least as effective as cholestyramine. Calcium clofibrate decreased very low density and low density lipoprotein cholesterol, triglycerides and phospholipids, whereas no changes were found in high density lipoproteins. Cholestyramine decreased low density lipoprotein lipids.

Kinetic study of the action of snake venom phospholipase A2 on human serum high density lipoprotein 3.

The hydrolysis of the phospholipids of intact human serum high density lipoprotein 3 (HDL3) by pure alpha-phospholipase A2 from Crotalus adamanteus was studied by pH-stat titration. The enzyme quantitatively hydrolyzed phosphatidylcholine and phosphatidylethanolamine and left sphinogomyelin intact, yielding a stable and water-soluble modified HDL. Lysophospholipids and free fatty acids, the products of hydrolysis, remained in the lipoprotein. When 1 mol of defatted bovine serum albumin/mol of substrate phospholipids was added to the reaction mixture, up to 60% of the fatty acids and 85% of the lysophospholipids were removed from the modified lipoprotein. The immunological reactivity of the hydrolyzed HDL remained unaltered in both the presence and absence of albumin. The changes in the physical properties of the lipoprotein during hydrolysis were rather small, the most notable being an increase in the hydrated density and in the electrophoretic mobility in alkaline buffers. The hydrolysis followed an apparent first order time course with product inhibition (KI) and yielded values of kcat/Km = 7 X 10(5 M(-1)s(-1) and KI congruent to 1 X 10(-4) M. Addition of albumin to the reaction mixture relieved the product inhibition without any alteration of the kinetic parameters. High concentrations of albumin protected some of the substrate phospholipids from hydrolysis, presumably through complexation to the lipoprotein. The Arrhenius plot for the experimental first order rate constant in the absence of albumin (kexp = kcat (KI/Km)) was linear between 15 degrees and 47 degrees, indicating the absence of any phospholipid phase transitions and yielding an activation energy of 15.2 kcal/mol. From the accessibility of the HDL phospholipids to phospholipase A2 one concludes that the phosphatidylcholine and phosphatidylethanolamine are located at, or are in rapid equilibrium with, the surface of this lipoprotein. It also appears that these phospholipids are not essential for maintaining the supramolecular properties of the lipoprotein in vitro. Thsu the study of the modified Hdl should provide valuable information concenring the structure and function of this lipoprotein particularly with regard to the role played by shiingomyelin.

Phospholipid composition and acyltransferase activity of lamellar bodies isolated from rat lung

The role of the lamellar body of the type II pneumocyte in the synthesis and storage of the phospholipids of the surfactant lipoprotein lining the alveolar surface has been investigated. Electron microscopy has been used to establish the purity of the isolated lamellar body, microsomal, and mitochondrial fractions. Additional proof of lamellar body purity was obtained by enzyme marker studies. The phospholipid:protein ratio of each of the above fractions was determined as well as that of surfactant lipoprotein isolated from rat lung. Lamellar body phospholipid:protein ratio was highest, 3.7 μmol of lipid phosphorus/mg of lung protein. The phospholipid composition of the lamellar body fraction was found to be similar to that of the isolated surfactant lipoprotein. Lamellar body phosphatidylcholine and phosphatidylglycerol each contained over 90% saturated fatty acids. The lamellar body fraction was found to possess significant acyltransferase activity between [1- 14C]palmitoyl-CoA and phosphatidylcholine. This activity was somewhat higher than in the microsomal fraction and much greater than in the mitochondrial fraction. The activity in all fractions was stimulated by Ca 2+ and Mg 2+. [1- 14C]oleoyl-CoA did not serve as an effective acyl donor. When 1-palmitoyl-2-lysophosphatidylcholine was used as the acceptor molecule and [1- 14C]palmitoyl-CoA the donor, acyltransferase activity was increased over that found with phosphatidylcholine as donor in all fractions. The microsomal fraction had the greatest activity and the lamellar body fraction the least. The data obtained support the hypothesis that the lamellar body is involved in the synthesis and storage of the phospholipids of the surfactant lipoprotein complex.

The Visibly Fluorescent Phospholipids of Serum Lipoproteins

The blusih-white fluorescence exhibited by lipoproteins from human serum has been investigated. It appears that high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low density lipoprotein (VLDL) all exhibit this fluorescence with excitation maximum at about 360 nm and fluorescence maximum at about 450 nm. The fluorescence resides in the phospholipid fraction of the lipoproteins and is not located in the fatty acids, since the water-soluble deacylated phospholipid fraction exhibited the same fluorescence. Upon electrophoresis of this fraction at pH 6.5 the fluorescent material carried a negative charge and moved a distance similar to glycerophosphoserine and glycerophosphoinositol, which both carry one negative charge. This negatively charged fluorescent product is ninhydrine negative, and gives a positive reaction for phosphate esters and 1,2-glycols. The detailed structure of the fluorescent product is not yet known. It is suggested that reactive aldehydes are involved in its formation from the aminophosphatides. Its possible relationship to tissue peroxidation is discussed.