Composition used for the determination of beta-hydroxybutyric acid and a method for preparing the said composition

• US 4,803,158 A
• Filed: 10/23/1986
• Issued: 02/07/1989
• Est. Priority Date: 03/08/1983
• Status: Expired due to Fees

• Current Assignee: Kabushiki Kaisha Kyoto Daiichi Kagaku, Kabushiki Kaisha Sanwa Kagaku Kenkyusho
• Original Assignee: Kabushiki Kaisha Kyoto Daiichi Kagaku, Kabushiki Kaisha Sanwa Kagaku Kenkyusho
• Inventors: Harano, Yutaka, Shigeta, Yukio, Takahaski, Yoshinori, Yamada, Shigeki
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Saunders, David A.
• Application Number:
  US06/922,178
• Time in Patent Office: 838 Days
• Field of Search: 422/57, 435/26, 435/805, 435/25, 427/2
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: Y10S 435/805 Test papers

Abstract

This invention relates to a composition for determining β-hydroxybutyric acid, usable for doctors, nurses, and patients themselves simply and quickly without use of special instruments, and also relates to a method for preparing the said composition.

The composition of the invention oxidizes β-hydroxybutyric acid under alkaline conditions and the presence of nicotineamide adenine dinucleotide (NAD) by β-hydroxybutyric acid dehydrogenase, and the produced reduction-type NAD (NADH) reduces tetrazolium salt through an electron carrier to produce formazan developing color. The composition permits accurate measurement quickly and simply, and has excellent conservative stability, because it is a uniform solid-phase composition prepared by applying reagents necessary for the reaction of film impermeable to water together with a natural or synthesized film forming polymer. The invention further provides a method for preparing the said composition by applying, on a supporter impermeable to water, W/O emulsion which is formed by dispersing alkaline buffer, β-hydroxybutyric acid dehydrogenase, NAD, electron carrier, and tetrazolium salt in an organic solvent insoluble in water together with a natural or synthesized film forming polymer.

Claims

1. A test indicator consisting essentially of:

(a) a composition for determining beta-hydroxybutyric acid in a liquid, said composition consisting essentially of a single pH buffer for maintaining pH within the range of 7 to 9, beta-hydroxybutyric acid dehydrogenase, NAD (Nicotinamide adenine dinucleotide), diaphorase, and nitro-TB and a binder comprising a natural or synthesized film-forming polymer; and

(b) a water-impermeable substrate on which said composition is applied;

said indicator being a one-layered indicator on said substrate.

2. A method for preparing a test indicator for determining beta-hydroxybutyric acid in a liquid comprising:

(a) uniformly applying on a water-impermeable substrate a water-in-oil emulsion said emulsion being prepared by dispersing a first solution in a second solution, said first solution being prepared by dissolving in water a single pH buffer for maintaining pH within the range of 7 to 9, beta-hydroxybutyric acid dehydrogenase, NAD (Nicotinamide adenine dinucleotide), diaphorase and nitro-TB and a binder comprising at least one natural or synthetic film-forming polymer;

said second solution being prepared by dissolving at least one natural or synthetic film-forming polymer into a water-insoluble organic solvent; and

(b) drying said emulsion.

3. The method of claim 2 wherein said nitro-TB is the last ingredient to be dissolved during preparation of said first solution.