Method for analyzing plural oxidizable components in a liquid

• US 4,725,539 A
• Filed: 02/28/1984
• Issued: 02/16/1988
• Est. Priority Date: 03/08/1983
• Status: Expired due to Fees

• Current Assignee: Oriental Yeast Company Limited (Nisshin Seifun Group Incorporated), Oriental Electronics Co., Ltd.
• Original Assignee: Oriental Yeast Company Limited (Nisshin Seifun Group Incorporated), Oriental Electronics Co., Ltd.
• Inventors: Arakawa, Nobuhiko, Oka, Osamu, Ohashi, Minoru, Numazawa, Kenichi, Utugi, Yoshio
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Deck, Randall E.
• Application Number:
  US06/584,347
• Time in Patent Office: 1,449 Days
• Field of Search: 435/4, 435/10, 435/14, 435/18, 435/21, 435/25, 435/22, 435/15
• US Class Current: 435/10
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: G01N 2333/9029 acting on -CH2- groups (1.17)
• CPC Class Codes: G01N 2333/90694 with oxygen as acceptor (1.7.3), e.g. uricase (1.7.3.3)
• CPC Class Codes: G01N 2333/91142 Pentosyltransferases (2.4.2)
• CPC Class Codes: G01N 27/30 Electrodes, e.g. test electrodes; Half-cells G01N27/414 takes precedence
• CPC Class Codes: G01N 27/327 Biochemical electrodes , e.g. electrical or mechanical details for in vitro measurements

Abstract

A new method of rapidly analyzing plural substances in the presence of biological catalyzers is disclosed. The method is practiced by way of the steps of injecting both pH buffer solution and specimen into a reaction cell, successively adding a plurality of enzymes to induce uptake reaction of dissolved oxygen, causing the plurality of substances to be subjected to selective oxidation in the stepwise manner, obtaining a stepdown curve of dissolved oxygen by automatically recording the oxidative process by means of a dissolved oxygen sensor, qualitatively determining each of the substances with reference to the kind of added enzymes and the order of their addition and quantitatively determining the same with reference to the extent of decrease in dissolved oxygen. Typically, oxidation of the substances is carried out by way of two or three or further more steps. The reaction cell for performing the method is equipped with a dissolved oxygen sensor on the one side wall and its upper portion is airtightly closed with a plug through which a fine bore is formed for the purpose of supply of buffer solution, specimen and enzymes.

Claims

1. A method for analyzing inosinic acid, inosine and hypoxanthine in a liquid containing them, which comprises the steps:

(a) adding a volume of the liquid and pH buffer solution at a pH of 10.5 to a reaction cell designed to prevent air from entering the cell, said reaction cell being equipped with an oxygen sensor connected to an automatic recording device,

(b) adding alkaline phosphatase enzyme to catalyze the hydrolysis of inosinic acid to inosine,

(c) adjusting the pH of said liquid to neutrality and adding xanthine oxidase enzyme to catalyze the oxidation of hypoxanthine to xanthine,

(d) recording oxygen sensor output,

(e) adding nucleoside phosphorylase enzyme to catalyze the conversion of inosine to hypoxanthine, the xanthine oxidase enzyme present catalyzing the oxidation of the thus formed hypoxanthine to xanthine,

(f) recording oxygen sensor output,

(g) adding a volume of the liquid identical to that added in step a, the nucleoside phosphorylase and the xanthine oxidase enzymes present catalyzing the conversion of the inosine added to hypoxanthine and the oxidation of the hypoxanthine added and that thus formed to xanthine,

(h) recording oxygen sensor output, and

(i) calculating the amount of hypoxanthine present from the decrease in oxygen sensor output in step d, calculating the amount of inosinic acid present from the decrease in oxygen sensor output in steps d plus f less the decrease in oxygen sensor output in step h, and calculating the amount of inosine present from the decrease in oxygen sensor output in step h less the decrease in oxygen sensor output in step d.

2. A method for analyzing hypoxanthine and xanthine in a liquid containing them, which comprises the steps:

(a) adding a volume of the liquid to a reaction cell designed to prevent air from entering the cell, said reaction cell being equipped with an oxygen sensor connected to an automatic recording device,

(b) adding xanthine oxidase to catalyze the oxidation of hypoxanthine and xanthine to uric acid,

(c) recording oxygen sensor output,

(d) adding uricase to catalyze the oxidation of uric acid to allantoin,

(e) recording the oxygen sensor output,

(f) calculating the ratio of decrease in oxygen sensor output in step (c) to decrease in oxygen sensor output in step (e), and

(g) determining the amounts of hypoxanthine and xanthine present in the liquid from a graph wherein ratios of decrease in oxygen sensor output in step (c) to decrase in oxygen sensor output in step (e) in mixtures containing known amounts of hypoxanthine and xanthine are plotted against the amounts of hypoxanthine and xanthine present in known mixtures.