Spectrofluorometric Determination of Kynurenic Acid with Horseradish Peroxidase in the Presence of Hydrogen Peroxide

Abstract

The catalytic activity of horseradish peroxidase (HRP) in the presence of hydrogen peroxide has been investigated for the fluorescent derivatization of kynurenic acid under conditions with no exposure to light. Non-fluorescent kynurenic acid was converted into a fluorescent compound (Ex: 367 nm, Em: 470 nm) with HRP in the presence of hydrogen peroxide, and the optimum conditions of this fluorescent derivatization were investigated. Moreover, this fluorescent derivatization was developed for a spectrofluorometric determination of trace amounts of kynurenic acid by measuring the fluorescence intensity of the fluorescent compound. The calibration curve obtained was linear from 1.0 to 10.0 nmol of kynurenic acid in a 1.0 mL sample solution. The relative standard deviation at 5.0 nmol of kynurenic acid was 5.71% (n = 5). By adjusting the bandwidths for both the excitation and emission to 15 nm, the calibration curve was also linear in the range between 0.1 to 1.0 nmol of kynurenic acid in a 1.0 mL sample solution. This method was applied to the fluorometric determination of trace amounts of kynurenic acid in the control sera.

Fluorometric Determination of Quinolinic Acid Using the Catalytic Activity of Horseradish Peroxidase

“…However, kynurenic acid showed severe interference because kynurenic acid was converted into a fluorescent compound with HRP under the conditions in this study, as described in the previous report. 22) Other substances and ions except for EDTA showed almost no interference.…”

“…22) The results indicated that the catalytic activity of HRP in the presence of H 2 O 2 is useful for the derivatization of a nonfluorescent substrate into a fluorescent compound. Accordingly, in the present study, we selected nonfluorescent QA as a substrate of HRP and investigated whether QA could be converted into a fluorescent compound by the catalytic activity of HRP in the presence of excess H 2 O 2 , and whether QA could be determined by measuring the fluorescence intensity of the reaction product.…”

“…In a previous study, 22) we attempted to determine whether the catalytic activity of horseradish peroxidase (HRP) in the presence of excess H 2 O 2 was usable for the fluorescent derivatization of a nonfluorescent substrate (AH 2 ) into a fluorescent compound according to the following reaction (1), which is routinely applied in the determination of trace amounts of H 2 O 2 in clinical and environmental analyses. 23,24) …”

Fluorescent Derivatization of Xanthurenic Acid and Nicotinic Acid with Horseradish Peroxidase in the Presence of Excess Hydrogen Peroxide

“…1: R2 and R3, COOH), a tryptophan metabolite, were converted into a fluorescent compound by the catalytic activity of HRP in the presence of excess H2O2. 6,7 Moreover, we demonstrated that this fluorescent derivatization could be applied to the determination of trace amounts of kynurenic acid and quinolinic acid in vital fluids. 6,7 Accordingly, in this study, we investigated whether other tryptophan metabolites, such as xanthurenic acid (XA), nicotinic acid (NA), 3-hydroxyanthranilic acid, and picolinic acid ( Fig.…”

“…6,7 Moreover, we demonstrated that this fluorescent derivatization could be applied to the determination of trace amounts of kynurenic acid and quinolinic acid in vital fluids. 6,7 Accordingly, in this study, we investigated whether other tryptophan metabolites, such as xanthurenic acid (XA), nicotinic acid (NA), 3-hydroxyanthranilic acid, and picolinic acid ( Fig. 1), will be converted into a fluorescent compound by the catalytic activity of HRP in the presence of excess H2O2.…”