Thin-layer Chromatographic assay for HMG-CoA reductase and mevalonic acid

ArticleinAnalytical Biochemistry 31(1):383-90 · November 1969with21 Reads
Impact Factor: 2.22 · DOI: 10.1016/0003-2697(69)90279-6 · Source: PubMed
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.

    • "...zed 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 determina..."
      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.
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Feb 2008 · Analytical Chemistry Insights
    0Comments 3Citations
    • "...MG-CoA reductase (E.C. 1.1.1.34) was measured in hepatic microsomes as described by Shapiro et al. [23]. HMG-CoA reductase activity was expressed as pmol of [ 14 C] mevalonate produced per min per mg mic..."
      The activity of microsomal HMG-CoA reductase (E.C. 1.1.1.34) was measured in hepatic microsomes as described by Shapiro et al. [23]. HMG-CoA reductase activity was expressed as pmol of [ 14 C] mevalonate produced per min per mg microsomal protein.
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Sep 2005 · BMC Cardiovascular Disorders
    0Comments 68Citations
    • "...ride transfer protein (Wetterau et al., 1992), cholesterol and free fatty acid synthesis (Shapiro et al., 1969), lysosomal acid lipase (LAL, EC 3.1.1.13) (Ishii et al., 1995; Merkel et al., 1999), and both mito..."
      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 3.1.1.13) (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.
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Mar 2001 · Glycobiology
    W Wang W Wang W Li W Li Y Ikeda Y Ikeda +12 more authors... J I Miyagawa J I Miyagawa
    0Comments 23Citations
    • "...r 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase was measured as previously described by Shapiro et al. (1969) and modified by Heller & Strewsbury (1976). ..."
      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).
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Jan 2001 · British Journal Of Nutrition
    0Comments 48Citations
    • "...on 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 meth..."
      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).
    [Show abstract] [Hide abstract] 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.
    Preview · Article · Jul 1999 · Journal of Nutrition
    P T Chan P T Chan W P Fong W P Fong YL Cheung YL Cheung +2 more authors... Y Huang Y Huang
    0Comments 165Citations
    • "...ride transfer protein (Wetterau et al., 1992), cholesterol and free fatty acid synthesis (Shapiro et al., 1969), lysosomal acid lipase (LAL, EC 3.1.1.13) (Ishii et al., 1995; Merkel et al., 1999), and both mito..."
      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 3.1.1.13) (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.
    Full-text · Article ·
    W. Wang W. Wang W. Li W. Li Y. Ikeda Y. Ikeda +2 more authors... J.I. Miyagawa J.I. Miyagawa
    0Comments 1Citation
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