A peroxidase-coupled method for the colorimetric determination of serum triglycerides.
01 Mar 1983-Clinical Chemistry (American Association for Clinical Chemistry)-Vol. 29, Iss: 3, pp 538-542
TL;DR: 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.
Abstract: 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.
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References
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TL;DR: A novel method for determining serum triglycerides, in which an enzymatic hydrolysis replaces the more commonly used saponification procedure, which is simple, rapid, and requires only 50 µl or less of sample.
Abstract: 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.
3,113 citations
"A peroxidase-coupled method for the..." refers background or methods or result in this paper
...Bucolo and David (4) incorporated bovine serum albumin into their reagent, to bind the released fatty acids, but we find a-cyclodextrin to be much superior for this purpose....
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...The totally enzymatic methods currently are widely used because of their inherent simplicity, amenability to automation, and the excellent correlation of results with those by the chemical methods (4)....
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...The reaction sequence is similar to others described previously (4, 5) up to the point where L-a-glycerophosphate oxidase catalyzes the generation of hydrogen peroxide from glycerol 3-phosphate....
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...6 L Eultraviolet method (4)1 of a lipemic serum....
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...Determination of serum triglycerides has progressed from totally chemical methods (1, 2) through partly chemicalpartly enzymatic methods in which alkaline saponification is used to hydrolyze the triglycerides followed by an enzymatic determination of the generated glycerol (3), to totally enzymatic methods in which lipases are used for the hydrolysis step (4,5)....
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TL;DR: In this direct colorimetric procedure, serum triglycerides are hydrolyzed by lipase, and the released glycerol is assayed in a reaction catalyzed by glycersol kinase and L-alpha-glycerol-phosphate oxidase in a system that generates hydrogen peroxide.
Abstract: 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.
2,758 citations
TL;DR: A microprocedure for the direct determination of triglyceride concentrations in biologic specimens is presented and depends on the quantitative removal of phosphatides from the sample and the subsequent determination of esterified glycerol.
1,744 citations
"A peroxidase-coupled method for the..." refers methods in this paper
...Determination of serum triglycerides has progressed from totally chemical methods (1, 2) through partly chemicalpartly enzymatic methods in which alkaline saponification is used to hydrolyze the triglycerides followed by an enzymatic determination of the generated glycerol (3), to totally enzymatic methods in which lipases are used for the hydrolysis step (4,5)....
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TL;DR: A new direct colorimetric procedure for uric acid assay in serum or urine is described, utilizing a 3,5-dichloro-2-hydroxybenzene sulfonic acid/4-aminophenazone chromogenic system in the presence of horseradish peroxidase and uricase from Aspergillus flavus, affording useful results with sample/reagent volume ratios as low as 0.025.
Abstract: A new direct colorimetric procedure for uric acid assay in serum or urine is described, utilizing a 3,5-dichloro-2-hydroxybenzene sulfonic acid/4-aminophenazone chromogenic system in the presence of horseradish peroxidase and uricase from Aspergillus flavus. This chromogen system has a high absorptivity, affording useful results with sample/reagent volume ratios as low as 0.025. The procedure is applicable to serum, plasma, or diluted urine. A single working reagent is used; the reaction is complete in less than 15 min at room temperature. The red dye formed is measured at 520 nm; a blank sample measurement is not needed. The standard curve for the method is linear for uric acid concentrations up to 1500 mumol/L. Average analytical recovery of uric acid in human sera and urine exceeded 99%; within-run and between-run precision studies showed CV's of less than or equal to 1.2 and less than or equal to 2.2%, respectively. The new procedure correlated well with the uricase/catalase and uricase/ultraviolet methods. The method is suitable for automation.
921 citations
"A peroxidase-coupled method for the..." refers methods in this paper
...Attempts to prevent this interaction by including potassium ferrocyanide (10, 17, 18) or amidopyrine (19) in the reagent were unsuccessful....
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Book•
01 Jan 1954
TL;DR: In this article, the authors present a course to make the student understand the advanced instrumentation available for chemical analysis, and the student would be able to choose the instrument needed for analysis.
Abstract: Course Educational Objectives: To make the student understand the advanced instrumentation available for chemical analysis. Course Outcomes: After studying this course the student would be able to choose the instrument needed for analysis. UNIT-I (12 Lectures) AN INTRODUCTION TO INSTRUMENTAL METHODS: Terms Associated With Chemical Analysis, Classification Of Instrumental Techniques, A Review Of The Important Considerations In Analytical Methods, Basic Functions of Instrumentation, Important Considerations in Evaluating an Instrumental Method.
867 citations