Thin-layer Chromatographic assay for HMG-CoA reductase and mevalonic acid
Department of Biochemistry, Purdue University, Lafayette, Indiana 47907 USA Analytical Biochemistry
(Impact Factor: 2.22).
11/1969; 31(1):383-90. DOI: 10.1016/0003-2697(69)90279-6
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.
Available from: PubMed Central
- "Table 1 summarizes the previously described methods for the quantification of MVA in the liver (enzyme assay), plasma or urine. The primary methods for assaying HMGCR activity have utilized a RI technique that measures the radioactivity in [14C]MVA produced from [14C]HMG-CoA (Shapiro et al. 1969; Goldfarb and Pitot, 1971; Shefer et al. 1972). The methods have been used for the direct determination of enzyme activity but they are not applicable to the quantification of plasma or urinary MVA. "
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ABSTRACT: For more than a decade, we have developed stable isotope dilution mass spectrometry methods to quantify key intermediates in cholesterol and bile acid biosynthesis, mevalonate and oxysterols, respectively. The methods are more sensitive and reproducible than conventional radioisotope (RI), gas-chromatography (GC) or high-performance liquid chromatography (HPLC) methods, so that they are applicable not only to samples from experimental animals but also to small amounts of human specimens. In this paper, we review the development of stable isotope dilution mass spectrometry for quantifying mevalonate and oxysterols in biological materials, and demonstrate the usefulness of this technique.
Available from: Maria Luz Fernandez
- "The activity of microsomal HMG-CoA reductase (E.C. 184.108.40.206) was measured in hepatic microsomes as described by Shapiro et al. . HMG-CoA reductase activity was expressed as pmol of [14C] mevalonate produced per min per mg microsomal protein. "
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ABSTRACT: Microsomal transfer protein inhibitors (MTPi) have the potential to be used as a drug to lower plasma lipids, mainly plasma triglycerides (TG). However, studies with animal models have indicated that MTPi treatment results in the accumulation of hepatic TG. The purpose of this study was to evaluate whether JTT-130, a unique MTPi, targeted to the intestine, would effectively reduce plasma lipids without inducing a fatty liver.
Male guinea pigs (n = 10 per group) were used for this experiment. Initially all guinea pigs were fed a hypercholesterolemic diet containing 0.08 g/100 g dietary cholesterol for 3 wk. After this period, animals were randomly assigned to diets containing 0 (control), 0.0005 or 0.0015 g/100 g of MTPi for 4 wk. A diet containing 0.05 g/100 g of atorvastatin, an HMG-CoA reductase inhibitor was used as the positive control. At the end of the 7th week, guinea pigs were sacrificed to assess drug effects on plasma and hepatic lipids, composition of LDL and VLDL, hepatic cholesterol and lipoprotein metabolism.
Plasma LDL cholesterol and TG were 25 and 30% lower in guinea pigs treated with MTPi compared to controls (P < 0.05). Atorvastatin had the most pronounced hypolipidemic effects with a 35% reduction in LDL cholesterol and 40% reduction in TG. JTT-130 did not induce hepatic lipid accumulation compared to controls. Cholesteryl ester transfer protein (CETP) activity was reduced in a dose dependent manner by increasing doses of MTPi and guinea pigs treated with atorvastatin had the lowest CETP activity (P < 0.01). In addition the number of molecules of cholesteryl ester in LDL and LDL diameter were lower in guinea pigs treated with atorvastatin. In contrast, hepatic enzymes involved in maintaining cholesterol homeostasis were not affected by drug treatment.
These results suggest that JTT-130 could have potential clinical applications due to its plasma lipid lowering effects with no alterations in hepatic lipid concentrations.
Available from: Koichi Honke
- "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 220.127.116.11) (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. "
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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.
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