Mechanism of ascorbic acid interference in biochemical tests that use peroxide and peroxidase to generate chromophore.
BACKGROUND
Ascorbic acid interferes negatively in peroxidase-based tests (Trinder method). However, the precise mechanism remains unclear for tests that use peroxide, a phenolic compound and 4-aminophenazone (4-AP). We determined the chemical mechanism of this interference, by examining the effects of ascorbic acid in the reaction kinetics of the production and reduction of the oxidized chromophore in urate, cholesterol, triglyceride and glucose tests. Reaction of ascorbic acid with the Trinder method constituents was also verified.


RESULTS
Ascorbic acid interfered stoichiometrically with all tests studied. However, it had two distinct effects on the reaction rate. In the urate test, ascorbic acid decreased the chromophore formation with no change in its production kinetics. In contrast, in cholesterol, triglyceride and glucose tests, an increase in the lag phase of color development occurred. Of all the Trinder constituents, only peroxide reverted the interference. In addition, ascorbic acid did not interfere with oxidase activity nor reduce significantly the chromophore formed.


CONCLUSIONS
Peroxide depletion was the predominant chemical mechanism of ascorbic acid interference in the Trinder method with phenolics and 4-AP. Distinctive effects of ascorbic acid on the reaction kinetics of urate, cholesterol, glucose and triglyceride might be due to the rate of peroxide production by oxidases.
Martinello
Flávia
F
Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café, s/n, 14040-903 Ribeirão Preto, SP, Brazil.
Luiz da Silva
Edson
E
D000675:Ampyrone / Q000737:chemistry; D001205:Ascorbic Acid / Q000737:chemistry*; D001786:Blood Glucose / Q000032:analysis; D002784:Cholesterol / Q000097:blood* / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007700:Kinetics; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D009195:Peroxidase / Q000737:chemistry*; D010636:Phenols / Q000737:chemistry; D013237:Stereoisomerism; D013997:Time Factors; D014280:Triglycerides / Q000097:blood* / Q000737:chemistry; D014527:Uric Acid / Q000097:blood* / Q000737:chemistry
D001786:Blood Glucose; D010636:Phenols; D014280:Triglycerides; D000675:Ampyrone; D014527:Uric Acid; D002784:Cholesterol; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D009195:Peroxidase; D001205:Ascorbic Acid