Method of measuring the total ketone body and a sample reagent

• US 5,618,686 A
• Filed: 03/04/1994
• Issued: 04/08/1997
• Est. Priority Date: 03/08/1993
• Status: Expired due to Term

• Current Assignee: Nitto Boseki Company Limited
• Original Assignee: Nitto Boseki Company Limited
• Inventors: Sato, Yoshiro, Takekawa, Akiko, Katayama, Katsuhiro, Kojima, Ryo
• Primary Examiner(s): Kight, John
• Assistant Examiner(s): LEARY, LOUISE N
• Application Number:
  US08/205,797
• Time in Patent Office: 1,131 Days
• Field of Search: 435/26, 435/25, 435/14, 435/4, 435/148, 435/975, 436/130, 436/128, 436/127, 436/14, 436/63, 436/74, 436/43
• US Class Current: 435/26
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10T 436/104998 Glucose, ketone, nitrate standard or control
• CPC Class Codes: Y10T 436/11 Automated chemical analysis

Abstract

A method of measuring the total ketone body in a sample comprising converting acetoacetic acid in the sample to 3-hydroxybutyric acid in advance and subsequently converting the whole 3-hydroxybutyric acid including 3-hydroxybutyric acid existing in the sample originally to acetoacetic acid to lead the reduction of nicotinamide adenine dinucleotide according to the action of 3-hydroxybutyrate dehydrogenase and a sample reagent thereof is disclosed. According to this invention an assay of the total ketone body in samples can be carried out conveniently, highly precisely and quickly.

Claims

1. A method of assaying the total ketone body in a sample, which is applicable to an automatic analyzer and comprises the steps of:

(1) converting acetoacetic acid in the sample to 3-hydroxybutyric acid with the aid of 3-hydroxybutyrate dehydrogenase in the presence of reduced-type nicotinamide adenine dinucleotide, said reaction being conjugated with another reaction in which the resulting oxidized-type nicotinamide adenine dinucleotide is used as a coenzyme to form its reduced-type;

(2) converting both 3-hydroxybutyric acid originally existed in the sample and 3-hydroxybutyric acid converted by step (1) to acetoacetic acid with the aid of 3-hydroxybutyrate dehydrogenase and oxide-type nicotinamide adenine dinucleotide; and

(3) measuring the absorbance of reduced-type nicotinamide adenine dinucleotide formed by step (2).

2. A method according to claim 1 wherein said another enzyme reaction is carried out using an enzyme selected from the group consisting of isocitrate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, aldehyde dehydrogenase and glucose dehydrogenase, and with its substrate.

3. A method according to claim 2 wherein said enzyme is isocitrate dehydrogenase, and said substrate is citric acid.

4. A method according to claim 1 wherein in step (2), an excess amount of oxydized-type nicotinamide adenine dinucleotide is added to the reaction system, and the pH of the system is shifted to an alkaline region to convert both 3-hydroxybutyric acid originally existed in the sample and 3-hydroxybutyric acid previously converted from acetoacetic acid to acetoacetic acid.

5. A kit of reagent for assaying the total ketone body comprising:

(1) a buffering agent,

(2) 3-hydroxybutyrate dehydrogenase,

(3) reduced-type nicotinamide adenine dinucleotide,

(4) oxidized-type nicotinamide adenine dinucleotide,

(5) an enzyme capable of converting oxidized-nicotinamide adenine dinucleotide to reduced-type nicotinamide adenine dinucleotide with the conjugated reaction with 3-hydroxybutyrate dehydrogenase, and

(6) a substrate of the enzyme of the above (5).

6. A kit of reagent according to claim 5 further comprising an inhibitor of the enzyme of (5).