Thin-layer chromatographic assay for HMG-CoA reductase and mevalonic acid
ABSTRACT An improved TLC system for the isolation and quantitation of mevalonic acid is described. This method has been applied to a variety of biological systems. The identification of the biological product as mevalonic acid has been confirmed by rechromatography with mevalonic acid standards. The procedure is simple, reproducible, and suitable for large numbers of HMG-CoA reductase assays. The system equals available GLC methods in sensitivity and is decidedly faster, particularly if large numbers of analyses are to be performed. Using a purified bacterial HMG-CoA reductase we obtained excellent agreement between a spectral assay and the TLC method presented. Prior extraction of mevalonic acid may be omitted if less exact results will suffice. Although the method is proposed for assay of HMG-CoA reductase, it is applicable to isolation and quantitation of mevalonolactone from a variety of biological systems.
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- "In an attempt to determine the mechanism of lipid accumulation, we examined the activities of microsomal triglyceride transfer protein (Wetterau et al., 1992), cholesterol and free fatty acid synthesis (Shapiro et al., 1969), lysosomal acid lipase (LAL, EC 18.104.22.168) (Ishii et al., 1995; Merkel et al., 1999), and both mitochondrial and peroxisomal β-oxidation (Otto and Ontko, 1978; Lazarow, 1981) in the livers of the transgenic mice, compared with their wild littermates. "
ABSTRACT: The alpha1,6 fucosyltransferase (alpha1,6 FucT) catalyzes the transfer of a fucose from GDP-fucose to the innermost GlcNAc residue of N-linked glycans via an alpha1,6 linkage. alpha1,6 FucT was overexpressed in transgenic mice under the control of a combined cytomegalovirus and chicken beta-actin promoter. Histologically numerous small vacuoles, in which lipid droplets had accumulated, were observed in hepatocytes and proximal renal tubular cells. Electron microscopic studies showed that the lipid droplets were membrane-bound and apparently localized within the lysosomes. Cholesterol esters and triglycerides were significantly increased in liver and kidney of the transgenic mice. Liver lysosomal acid lipase (LAL) activity was significantly lower in the transgenic mice compared to the wild mice, whereas LAL protein level, which was detected immunochemically, was increased, indicating that the specific activity of LAL was much lower in the transgenic mice. In all of the transgenic and nontransgenic mice examined, the activity of liver LAL was negatively correlated with the level of alpha1,6 FucT activity. As evidenced by lectin and immunoblot analysis, LAL was found to be more fucosylated in the transgenic mice, suggesting that the aberrant fucosylation of LAL causes an accumulation of inactive LAL in the lysosomes. Such an accumulation of inactive LAL could be a likely cause for a steatosis in the lysosomes of the liver and kidney in the case of the alpha1,6 FucT transgenic mice.Glycobiology 03/2001; 11(2):165-74. DOI:10.1093/glycob/11.2.165 · 3.14 Impact Factor
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- "Assays of 3-hydroxy-3-methylglutaryl-CoA reductase Liver microsomes were isolated according to Erickson et al. (1977). The activity of liver 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase was measured as previously described by Shapiro et al. (1969) and modified by Heller & Strewsbury (1976). "
ABSTRACT: The present study was designed to study the mechanisms by which dietary conjugated linoleic acids (CLA) decrease serum cholesterol. Hamsters were fed a semi-synthetic diet containing 1 g cholesterol/kg diet with or without supplementation with 20 g linoleic acid (LA) and 20 g CLA/kg diet. After 8 weeks, serum fasting total cholesterol (TC) and triacylglycerol (TG) were significantly lower in the LA-supplemented and CLA-supplemented groups compared with those of the control (CTL) hamsters. In contrast to LA, CLA significantly lowered hepatic cholesterol but it increased the level of adipose tissue cholesterol, suggesting that the hypocholesterolaemic mechanism of CLA is different from that of LA. CLA decreased the activity of intestinal acyl CoA:cholesterol acyltransferase (ACAT) whereas LA had no effect on this enzyme. Consequently, CLA supplementation increased the faecal excretion of total neutral sterols, but it had no or little effect on the faecal acidic sterols. If the ACAT is associated with cholesterol absorption, the part of mechanisms by which CLA decreases serum cholesterol may involve down-regulation of intestinal ACAT activity.British Journal Of Nutrition 01/2001; 84(6):935-41. DOI:10.1017/S0007114500002579 · 3.34 Impact Factor
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- "Liver micosomes were isolated according to Erickson et al. (1977). The activity of liver HMG-CoA-R was measured as previously described by Shapiro et al. (1969) and modified by Heller and Strewsbury (1976). The activity of liver FAS was measured using the methods described by Nepokroeff et al. (1975). "
ABSTRACT: These studies were designed to test the hypolipidemic activity of green tea epicatechins (GTE) isolated from jasmine green tea. In Experiment 1, three groups of hamsters were given a semisynthetic diet containing 200 g lard/kg and 1 g cholesterol/kg for 4 wk. The control group received distilled water, and the other two groups received either 15 g/L green tea water extract (GTWE) or 5.0 g/L GTE solution. Both the GTWE and GTE groups had lower concentrations of serum total cholesterol (TC) and triacylglycerols (TG) than the controls (P < 0.05). In Experiment 2, four groups of hamsters received tap water as the drinking fluid, but they were given the same high fat and cholesterol diet supplemented with 0 (control), 1.1, 3.4 or 5.7 g GTE/kg diet. The hypolipidemic effect of jasmine GTE was dose dependent. In Experiment 3, the time-course of changes in serum TC and TG was monitored in hamsters given the high fat diet supplemented with 5.7 g GTE/kg in comparison with that of controls. The hypolipidemic effects of dietary GTE were evident after feeding for 2 wk. Dietary supplementation of GTE did not affect liver fatty acid synthase. However, GTE-supplemented hamsters had higher fecal excretions of total fatty acids, neutral sterols and acidic sterols compared with the control group. In Experiment 4, hamsters were fed nonpurified diet; the control group drank distilled water, and the GTE group drank distilled water containing 5.0 g GTE/L. No differences in activities of 3-hydroxy-3-methyl glutaryl coenzyme A reductase and intestinal acyl CoA:cholesterol acyltransferase were observed. This study suggests that the hypolipidemic activity of GTE is not due to inhibition of synthesis of cholesterol or fatty acid but is most likely mediated by its influence on absorption of dietary fat and cholesterol.Journal of Nutrition 07/1999; 129(6):1094-101. · 4.23 Impact Factor