Effect of peroxyl radicals on lecithin/cholesterol acyltransferase activity in human plasma

The objective of this study was to determine whether subjecting human plasma to oxidant stress reduces the activity of lecithin/cholesterol acyltransferase (LCAT, EC 2.3.1.43). Plasma was incubated for 4h with 2.25–45 mM of 2,2′-azobis(2-amidinopropane)HCl (AAPH), a source of peroxyl radicals. A time- and concentration-dependent reduction of LCAT activity occurred, relative to control samples incubated in the absence of AAPH. Reduction of LCAT activity was disproportionate to elevation of thiobarbituric acid-reactive substances (TBARS) in the plasma. Added ascorbate was able to significantly prevent reduction of LCAT activity, but this effect was unrelated to blockage of TBARS formation by the antioxidant. The results suggest that LCAT activity can be down-modulated by oxidant stress, but not necessarily by lipid peroxidation.     

Inhibition of human lecithin cholesterol acyltransferase by monoterpenes

The lecithin-cholesterol acyltransferase activity of human plasma was found to be inhibited by Rowachol, a proprietary mixture of pure monoterpenes. Menthol, the major ingredient in Rowachol (32%), and a number of other monoterpenes were found to inhibit the enzyme independently. Concentrations of monoterpenes required to achieve 50% inhibition were of the same order of magnitude as the cholesterol concentration present in the reaction mixture.     

The influence of phthalate esters on human plasma lecithin/cholesterol acyltransferase

The effect of various phthalate esters on the lecithin/cholesterol acyltransferase activity in man was studied in vitro. The enzymatic activity was strongly reduced with all phthalates except for the dimethyl phthalate. The inhibition rate depends on the phthalate concentration and also on the carbon number of the alkyl groups of phthalates.     

Effect of ethanol on plasma triglycerides in male and female rats

A single large dose of ethanol was given to fasted rats and to rats fed a fat-free diet containing orotic acid. An increased plasma triglyceride concentration after ethanol feeding was consistently found in fasted male rats, while the results in fasted female rats varied between the experiments. The total rate of triglyceride secretion into the plasma in fasted rats was estimated as the Triton-induced hypertriglyceridemia 6–7.5 hr after ethanol feeding. The effect of ethanol on the triglyceride secretion from extrahepatic sources was estimated in the same way in rats, with the hepatic triglyceride secretion blocked by orotic acid. Ethanol enhanced the Triton-induced hypertriglyceridemia in both male and female fasted rats, but to a greater extent in male rats. Ethanol did not stimulate the extrahepatic triglyceride secretion during this period.     

Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

Lecithin cholesterol acyltransferase (LCAT) is an interfacialenzyme active on both high-density (HDL) and low-density lipoproteins(LDL). Threading alignments of LCAT with lipases suggest thatresidues 50–74 form an interfacial recognition site andthis hypothesis was tested by site-directed mutagenesis. The(56–68) deletion mutant had no activity on any substrate.Substitution of W61 with F, Y, L or G suggested that an aromaticresidue is required for full enzymatic activity. The activityof the W61F and W61Y mutants was retained on HDL but decreasedon LDL, possibly owing to impaired accessibility to the LDLlipid substrate. The decreased activity of the single R52A andK53A mutants on HDL and LDL and the severer effect of the doublemutation suggested that these conserved residues contributeto the folding of the LCAT lid. The membrane-destabilizing propertiesof the LCAT 56–68 helical segment were demonstrated usingthe corresponding synthetic peptide. An M65N–N66M substitutiondecreased both the fusogenic properties of the peptide and theactivity of the mutant enzyme on all substrates. These resultssuggest that the putative interfacial recognition domain ofLCAT plays an important role in regulating the interaction ofthe enzyme with its organized lipoprotein substrates.     

Synthesis of cholesterol and total lipid by male and female rats fed beef tallow or corn oil

Male and female weanling rats were fed diets containing 2 or 42% of calories as corn oil or 40% as beef tallow plus 2% as corn oil until they were 12 or 18 weeks of age. Incorporation of C¹⁴-acetate into lipids of serum and liver and concentration of lipids in serum, liver, and carcass at the end of these periods were determined. Net synthesis of noncholesterol lipid was repressed by changing the diet from 2% to 42% of calories from either dietary fat in both sexes and at both ages. Cholesterol net synthesis was enhanced 29-fold in males and 22-fold in females fed 42% corn oil compared to 2% corn oil to the age of 12 weeks. It was enhanced only 2.6-fold for males and 3.4-fold for females by 40% beef tallow plus 2% corn oil. At 18 weeks of age cholesterol synthesis in males fed 42% corn oil was 7.3 and in females 9.1 times the value for those fed 2% corn oil. At this age the values for rats fed 40% beef tallow plus 2% corn oil were 1.2 and 3.7 times those for 2% corn oil fed rats of the respective sexes.     

The influence of purines on plasma lecithin: Cholesterol acyltransferase activity in the rat

The activity of plasma lecithin: cholesterol acyltransferase (LCAT) in the rat is significantly inhibited in vitro by guanine, xanthine, and hypoxanthine. LCAT activity decreases with increase in xanthine concentration. The other two purines, adenine and uric acid, had no significant effect.     

Decreased lecithin: Cholesterol acyltransferase activity in the plasma of hypercholesterolemic pigs

Lecithincholesterol acyltransferase (LCAT) activity levels were determined, as function of plasma total cholesterol (TC) in 13 normocholesterolemic (TC<85 mg/dL) and in 28 hypercholesterolemic (TC>98 mg/dL) pigs. The normocholesterolemic group consisted of pigs that carried apo-B allelic genes other thanLpb ⁵ and orLpb ⁸. The hypercholesterolemic group consisted ofLpb ^5/x andLpb ^5/8 heterozygous andLpb ^5/5 homozygous animals. The data reported in this study show that the LCAT activity in the plasma of hypercholesterolemic (HC) pigs (79±43 units) was significantly lower (p<0.0005) compared to the normocholesterolemic controls (175±45 units). Furthermore, LCAT activity was positively correlated with TC in the normocholesterolemic group (r=+0.54; p<0.05), whereas it was negatively correlated with TC in the hypercholesterolemic group (r=−0.73; p<0.001). Additional data obtained from incubation experiments suggest that the lower LCAT activity in hypercholesterolemic pigs may be due, at least in part, to inhibition of LCAT activity by components found in the lipoprotein-deficient fractions of the plasma of hypercholesterolemic pigs.     

Reduced plasma lecithin cholesterol acyl transferase activity in rats fed iron-deficient diets

An iron-deficient diet containing no fat (FF?Fe) or containing either 14% hydrogenated coconut oil (HCNO?Fe) or 14% corn oil (CO?Fe) was fed to separate groups of rats for 10 weeks. In the control group, the corresponding iron-supplemented diets were fed FF+Fe, HCNO+Fe, CO+Fe. When rats were fed iron-deficient diets, their plasma lecithin cholesterol acyl transferase (LCAT) activity was significantly reduced as compared to controls. Their plasma also contained relatively more cholesteryl esters (CE) than free cholesterol (CH). In rats fed FF+Fe and CO+Fe diets, plasma contained similar levels of CE and CH. In those fed HCNO+Fe diet, plasma had 40% less CE than CH. Red cell CH content was significantly greater in the CO?Fe group. Iron deficiency, as indicated by low blood hemoglobin (Hb) and hematocrit (Hct) values, was also observed only in this group. The triglyceride and phospholipid contents of plasma in rats fed iron-deficient diets were significantly lower than of those in the control groups. Thus, changes in LCAT activity and CE/CH ratio in plasma showed the effect of iron-deficient diet consumption even before the blood Hb and Hct levels were reduced.     

Lung phosphatidylcholine and fasting in male and female rats

Lung from male and female rats fasted for 4 days were used. Phospholipid, phosphatidylcholine and its molecular species were analyzed in lungs from these rats and effects of fasting upon the biosynthesis of phosphatidylcholine in lungs from both sexes were determined using radioactive choline. The molecular species of phosphatidylcholine in both male and female rats did not differ with fasting except the monoenoic species. Incorporation of choline into phosphatidylcholine in both male and female rats significantly increased after fasting, but distribution of radioactivity in phosphatidylcholine yielded similar values in each group. These results suggest that the decrease of saturated phosphatidylcholine content after fasting may be not due to specific change in saturated phosphatidylcholine.     

Effects of site-directed mutagenesis on the serine residues of human lecithin: Cholesterol acyltransferase

Lecithin:cholesterol acyltransferase (LCAT) is a serine protease-type enzyme that esterifies cholesterol in human plasma and is activated by apolipoprotein A-I in high-density lipoproteins. LCAT contains 22 serine residues, including Ser 181, which is thought to be part of the catalytic site. In order to determine the importance of these serine residues in LCAT, we prepared six LCAT mutants: LCAT (Ser19→Ala), LCAT (Ser181→Gly), LCAT (Ser208→Ala), LCAT (Ser216→Ala), LCAT (Ser225→Ala) and LCAT (Ser383→Ala). We also replaced the adjacent asparagine residues in two additional mutants, LCAT (Ser19→Ala, Asn20→Thr) and LCAT (Ser383→Ala, Asn384→Thr), in order to ascertain the effect of the serines onN-glycosylation. The mutant complementary DNA (cDNA) were subcloned into a eukaryotic expression vector (pSG5) and expressed in COS-6 cells. By polymerase chain reaction analysis, LCAT-specific messenger RNA (mRNA) was found in all mutant and wild-type transfectants. Western blot analysis revealed LCAT-specific bands in media and lysates of the transfected cells. With two exceptions, the amounts of LCAT mass secreted by the transfectants were similar to that of the wild type (mean, 90% mass of wild type; range, 34–138%). Except for LCAT (Ser181→Gly), which was inactive, the specific activities of the remainder of the mutant enzymes were also similar (mean, 95% activity of wild type; range, 65–169%). These results indicate that Ser181 is part of the catalytic site and that stereoconservative substitutions for serines have minor effects on the synthesis, secretion and specific activities of human LCAT.     

Effect of isoessential fatty acid lipids from animal and plant sources on cholesterol levels in mature male rats

Isopolyunsaturated lipids isolated from plant and animal sources were included in the diets of mature male rats. Liver and blood serum cholesterol lowering effects were noted only in the lipid from the vegetable source. The authors suggest that the cholesterol lowering effect of vegetable oils is associated with the generally betaunsaturated triglycerides found therein. Journal Article No. 3758, Michigan Agricultural Experiment Station, East Lansing, Mich.     

Comparative specificity of plasma lecithin: Cholesterol acyltransferase from ten animal species

The molecular specificities of plasma lecithin:cholesterol acyltransferase (LCAT) from ten animal species have been compared. Using a reassembled high density lipoprotein containing a mixture of phosphatidylcholines, the relative rates of liberation of different species of cholesteryl ester were measured. All but two species of LCAT clustered according to one of three patterns of substrate specificity. The LCAT from six species, including human, did not transfer highly polyunsaturated fatty acyl chains. In addition, human LCAT transesterified saturated fatty acyl chains more effectively than unsaturated fatty acyl chains. We conclude that the structures of the active sites of the enzymes differ, and that this may be related to size constraints that prevent efficient binding of large bulky phosphatidylcholines.     

Hypercholesterolemia in rats: Combined effect of high cholesterol diet and female sex steroids

The influence of estradiol and a contraceptive steroid combination on plasma cholesterol was studied in female rats on both normal and high-cholesterol diets which did not contain thiouracil. The high-cholesterol diet resulted in moderate hypercholesterolemia without weight loss, even with prolonged feeding. Hypercholesterolemia was markedly accentuated in the presence of either endogenous or exogenous sex hormones.     

Effect of plant sterols,fatty acids and lecithin on cholesterol absorption in vivo in the rat

The inhibitory effect of plant sterols, fatty acids and lecithin on cholesterol intestnal absorption was studied in the unanesthetized rat using a single pass perfusion technique. Bile was excluded from the perfused intestine. Cholesterol absorption did not change following the additions of cholestanol, cholestanone, lanosterol, stigmasterol and β-sitosterol. A 3-fold increase in the molarity of cholestanol and β-sitosterol or the separate additions of the saturated short and medium chain fatty acids, butyric and octanoic, also did not change cholesterol absorption. The unsaturated long chain fatty acids, oleic, linoleic, linolenic and arachidonic, inhibited cholesterol absorption. Lecithin additions at concentrations of 0.1–1.5 mM caused a progressive, dose-related inhibition of cholesterol absorption. The inhibitory effect of these agents on cholesterol absorption is likely to have been caused by changes in cholesterol solubility in the micelle and shifts in the partition coefficient of cholesterol away from the cell membrane to the micelle.     

Distribution of cholesterol among its carriers in the bile of male and female hamsters

The distribution of cholesterol among its carriers was studied in the bile of male and female hamsters. Sasco hamsters (Sasco Inc., Omaha, NE) were fed a semipurified diet with 0.0% cholesterol and 4% butterfat (group 1, males; group 4, females); a semipurified diet with 0.3% cholesterol and 1.2% plamitic acid (group 2, males; group 5, females); and a semipirified diet with 0.3% cholesterol and 4% safflower oil (group 3, males; group 6, females). At the end of six weeks, gallstones were found only in male hamsters receiving both cholesterol and dietary fat (fatty acid) (incidence of cholesterol stones: 90% in group 2; 22% in group 3). The biliary cholesterol carriers were separated and isolated from the bile of the hamsters by gel filtration chromatography, using the method of Pattinson [Pattinson, N.R., Willis, K.E., and Frampton, C.M. (1991)J. Lipid Res. 32, 205–214]. In those male hamsters that formed cholesterol gallstones, significant amounts of cholesterol were present in the void volume which contained large cholesterol phospholipid vesicles (void volume vesicles) (23% in group 2 and 15% in group 3). Smaller cholesterol/phospholipid vesicles were eluted next (fractions 30–45) and contained 15% of biliary cholesterol in group 2 and 21% in group 3. The remainder of the cholesterol was associated with mixed cholesterol/phospholipid/bile salt micelles. The cholesterol/phospholipid ratio was larger in both the void volume vesicles and small vesicles (2.40 and 1.48 in group 2; 2.56 and 1.33 in group 3, respectively) compared to the micelles (about 0.3 in groups 2 and 3). In contrast, the bile of the female hasmters contained few vesicles (3% small vesciles in group 5) and the cholesterol/phospholipid ratio of these vesicles was lower (0.94). Hamsters fed cholesterol-free diets (groups 1 and 4) had no biliary cholesterol/phospholipid vesilces; and cholesterol was present in micelles. The results suggest that both the gender and the diet of the hamsters affected the distribution of biliary cholesterol between vesicles and micelles. The development of cholelithiasis in this animal model appears to depend on the rapid nucleation of cholesterol-rich phospholipid vesicles in bile.     

Effect of Castration and hormonal supplementation on cholesterol cholelithiasis in the male hamster

This study examined the effect of castration and dietary hormonal supplementation on cholesterol cholelithiasis in male hamsters. Animals fed a standard lithogenic diet developed cholesterol gallstones (17%) after 6 wk, while castrated hamsters did not form any stones. Addition of a synthetic androgen, methyltestosterone, to the lithogenic diet induced cholelithiasis in castrated animals (50%). The biles of normal and castrated-hormone supplemented hamsters had cholesterol saturation indices of 1.0 and 1.1, respectively, while the bile of the castrated animals remained unsaturated (0.6). The ratio of cholic acid/chenodeoxycholic acid in bile increased after castration, but returned to normal levels following hormonal supplementation. Biliary cholesterol carriers were separated by ultracentrifugation. Animals in the stone-forming groups (normal and castrated-hormone treated) had a significant proportion of their biliary cholesterol in vesicles (44 and 46%, respectively); castrated hamsters had less cholesterol in vesicle form (9%). The molar ratio of cholesterol/phospholipid in vesicles was reduced after castration (0.93 vs. 0.42) and increased by hormonal supplementation (1.89). In conclusion, when compared to normal male hamsters fed a standard lithogenic diet, castration reduced the cholesterol saturation of bile, lowered the vesicular/micellar ratio in bile, and inhibited cholesterol cholelithiasis. Dietary androgen supplementation increased the lithogenicity of bile, resulting in stone formation in castrated animals.     

Long-term effects of high-fat diets on peroxisomal β-oxidation in male and female rats

In weanling male rats a 4-fold increase of heart triacylglycerols was observed after three days on a high-fat diet containing partially hydrogenated fish oil (PHFO). In female rats this increase was only about 50%. No significant differences were observed between female and male rats in the fatty acid composition of the accumulated lipids. The initial level of peroxisomal β-oxidation activity was similar in male and female rats in both liver and heart. After three weeks of receiving high-fat diets, the rats showed a marked increase in peroxisomal β-oxidation activity with PHFO in the diet and less with soybean oil (SO), confirming previous studies with male rats. Catalase activity was similarly affected in hearts of both sexes. In male rats the levels of peroxisomal β-oxidation observed after three weeks of feeding on the high-fat diets were found to be maintained, both in liver and heart, during a feeding period of three months. The response to high-fat diets in females, however, seems to be further accentuated after three months of feeding, resulting in a capacity of peroxisomal β-oxidation in liver of about three times that of the male rats when calculated on a total body-weight basis.