In this direct colorimetric procedure, serum triglycerides are hydrolyzed by lipase, and the released glycerol is assayed in a reaction catalyzed by glycerol kinase and L-alpha-glycerol-phosphate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is monitored in the presence of horseradish peroxidase with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogenic system. The high absorbance of this chromogen system at 510 nm affords useful results with a sample/reagent volume ratio as low as 1:150, and a blank sample measurement is not needed. A single, stable working reagent is used; the reaction is complete in 15 min at room temperature. The standard curve is linear for triglyceride concentrations as great as 13.6 mmol/L. Average analytical recovery of triglycerides in human sera is 100.1%, and within-run and between-run precision studies showed CVs of less than or equal to 1.6 and less than or equal to 3.0%, respectively. The method is suitable for automation.

Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide.

The discovery of oxysterols as the endogenous liver X receptor (LXR) ligands and subsequent gene targeting studies in mice provided strong evidence that LXR plays a central role in cholesterol metabolism. The identification here of a synthetic, nonsteroidal LXR-selective agonist series represented by T0314407 and T0901317 revealed a novel physiological role of LXR. Oral administration of T0901317 to mice and hamsters showed that LXR activated the coordinate expression of major fatty acid biosynthetic genes (lipogenesis) and increased plasma triglyceride and phospholipid levels in both species. Complementary studies in cell culture and animals suggested that the increase in plasma lipids occurs via LXR-mediated induction of the sterol regulatory element-binding protein 1 (SREBP-1) lipogenic program.

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Role of LXRs in control of lipogenesis

Cholesterol and Bile Acid Metabolism Are Impaired in Mice Lacking the Nuclear Oxysterol Receptor LXRα

We describe an enzymatic method for rapid, precise measurement of serum triglycerides with use of sample:reagent ratios as large as 1:200. Hydrolysis of triglycerides is catalyzed by lipase to produce glycerol and free fatty acids. The glycerol generated is then phosphorylated by adenosine 5'-triphosphate in the presence of glycerol kinase. Oxidation of the resulting glycerol 3-phosphate to produce hydrogen peroxide is catalyzed by L-alpha-glycerophosphate oxidase. An intense red chromogen is produced by the peroxidase-catalyzed coupling of 4-aminoantipyrene and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate with hydrogen peroxide. This sensitive chromogen system not only permits use of unusually small sample volumes, it also facilitates a linear response to serum triglyceride concentrations up to at least 10 g/L while displaying good Ringbom (measure of accuracy) characteristics.

A peroxidase-coupled method for the colorimetric determination of serum triglycerides.

Reproducibility and validity of dietary patterns assessed with a food-frequency questionnaire

We describe a novel method for determining serum triglycerides, in which an enzymatic hydrolysis replaces the more commonly used saponification procedure. Under the conditions of the assay, the enzymatic hydrolysis can be completed in less than 10 min by the combined action of a microbial lipase and a protease. We have been able to demonstrate complete hydrolysis of triglycerides by thin-layer chromatography of the reaction products, by recovery of glycerol from sera of known triglycerides content, and by comparison of triglyceride assays on a number of sera assayed by our method vs. the AutoAnalyzer procedure. The hydrolysis is directly coupled to the enzymatic determination of glycerol, and is followed through absorbance changes at 340 nm. The assay is simple, rapid, and requires only 50 µl or less of sample. Because the enzymes used do not release glycerol from other compounds in serum, the hydrolysis can be considered specific for triglycerides.

Quantitative Determination of Serum Triglycerides by the Use of Enzymes

GLYCEROL ESTERS PRESENT IN AQUEOUS MEDIA, SUCH AS SERUM TRIGLYCERIDE AND MILK FAT, ARE ASSAYED BY COMPLETE ENZYMATIC HYDROLYSIS USING BOTH A LIPASE AND A PROTEASE, WHEREUPON THE LIBERATED GLYCEROL IS DETERMINED. A NUMBER OF ALTERNATIVE ENZYMATIC PROCEDURES ARE PROVIDED FOR THE GLYCEROL ASSAY, ALL IN THE ORGINAL AQUEOUS MEDIUM, SUCH AS CONVERSION TO GLYCEROL-1-PHOSPHATE WITH GLYCEROL KINASE; CONVERSION OF ATP TO ADP BY THE SAME RECTION FOLLOWED BY CONVERSION OF THE ADP PLUS PHOSPHOENOLPYRUVATE USING PYRUVATE KINASE TO ATP AND PYRUVATE ION FOLLOWED BY DETERMINING THE LATTER; AND ALTERNATIVELY CONVERTING THE PYRUVATE ION SO FORMED WITH NADH USING LACTATE DEHYDROGENASE TO LACTATE ION AND NAD AND DETERMINING THE LATTER BY OPTICAL MEASUREMENT.

Harold David

Papers

Quantitative Determination of Serum Triglycerides by the Use of Enzymes

Triglyceride hydrolysis and assay