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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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.Analytical Chemistry Insights 02/2008; 3:45-60.
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ABSTRACT: We investigated the influence of docosahexaenoic acid ethyl ester (DHA-EE) on 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity in the brains of adult and aged mice. Male mice (Crlj:CD-1) were fed diets containing 3% lard plus 2% linoleic acid ethyl ester (LA-EE), or 2% DHA-EE, for 3 months. The brain HMG-CoA reductase activity of 8-month-old (adult) mice was not significantly influenced by dietary intake of DHA-EE. However, in 18-month-old (aged) mice, its activity was enhanced with dietary intake of DHA-EE. Brain HMG-CoA reductase activity and brain cholesterol content significantly increased with age. Hepatic HMG-CoA reductase activity and the cholesterol content of both adult and aged mice were reduced in DHA-EE diet groups, compared with LA-EE diet groups. The DHA percentages of brain and liver microsomal fractions increased with the intake of DHA-EE in adult and aged mice. These results suggest that DHA may enhance brain HMG-CoA reductase activity in aged mice.The Journal of Nutritional Biochemistry 08/2007; 18(7):488-94. · 4.55 Impact Factor
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ABSTRACT: We investigated the effects of a high-cholesterol (HC) diet administered long term (25 or 55 weeks) on metabolic disorders including hepatic damage in mice. The mice were fed the HC diet (15 % milk fat, 1.5 % cholesterol and 0.1 % cholic acid, w/w) for 25 or 55 weeks. Body and adipose tissue weights were similar to those of mice fed a control diet. Consumption of the HC diet long term resulted in hypercholesterolaemia, hepatic steatosis and gallstones. In addition, focal nodular hyperplasia (FNH) and mild fibrosis of the liver developed in all mice fed the HC diet for 55 weeks. Plasma levels of monocyte chemoattractant protein (MCP)-1 were elevated, and the level of hepatic platelet-derived growth factor (PDGF)-B protein was increased in mice fed the HC diet compared with those fed the control diet. Thus, it seems likely that the liver fibrosis and FNH caused by the long-term consumption of a HC diet may be partly due to an elevation of plasma MCP-1 and hepatic PDGF expression.The British journal of nutrition 10/2009; 103(3):378-85. · 3.45 Impact Factor