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US4254222A Expired / Lapsed US ⏱️ 0.0 years remaining

Semi-quantitative assay of lactic acid and β-hydroxy butyrate

Assignee: Individual
•
Filing: 1978-07-19
•
Granted: 1981-03-03
•
Expires: 1998-07-19
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Abstract

Abstract There is provided a procedure for determining the concentration of a metabolic acid in a biological fluid. The method involves use of a tetrazolium salt, a pyridine nucleotide, an electron carrier, and an enzyme that is a dehydrogenase for the specific acid being assayed. These components are used to form an assay mixture; thereafter a quantity of the fluid to be assayed is combined with the assay mixture, so that there may commence a reaction in which the tetrazolium salt is changed to a formazan in an amount that is indicative of the concentration of the specific acid. In a preferred embodiment, the procedure permits assay of the concentration of beta-hydroxybutyrate, and the assay mixture includes components in the relative proportions of 0.8 micromoles 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT), 0.32 micromoles phenazine methosulfate (PMS), 1.5 micromoles nicotinamide adenine dinucleotide (NAD), and 0.75 International Units beta-hydroxybutyrate dehydrogenase, in a water solution buffered at a pH of approximately 8.5 with hydrogen phosphates of potassium together with glycine and sodium hydroxide and also containing alkylphenoxypolyethyoxyethanol. In another preferred embodiment, the procedure permits assay of lactic acid by means of a similar assay mixture utilizing 27.5 International Units of lactic dehydrogenase in place of the beta-hydroxybutyrate dehydrogenase used in the previously discussed embodiment. In the latter procedure a preferred embodiment of the assay mixture is buffered at a pH of approximately 9.6 in a glycine-sodium hydroxide buffer and also contains phenoxypolyethyoxyethanol.

Inventors

  • 👤 Oliver E. Owen

Bibliographic Details

Application Number —
Jurisdiction US
Data Source google_patents
Last Synchronized 2026-11-09 01:47
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/26; C12Q1/32; Y; Y10; Y10S; Y10S435/00; Y10S435/805; Y10S435/81

Claims Analysis

Total Claims: 10
Claim 1
Independent Claim 45 words
An assay mixture, for determining the concentration of beta-hydroxybutyrate in a biological fluid, such mixture comprising the following components in the approximate concentrations set forth: 1.1 m Molar tetrazolium salt; 0.4 m Molar electron carrier; 2.0 m Molar NAD; and 1 International Unit/ml beta-hydroxybutyrate dehydrogenase.
Claim 2
Dependent Claim 45 words
An assay mixture, for determining the concentration of beta-hydroxybutyrate in a biological fluid, such mixture comprising the following components in the approximate concentrations set forth: 1.1 m Molar tetrazolium salt; 0.4 m Molar electron carrier; 2.0 m Molar NAD; and 1 International Unit/ml beta-hydroxybutyrate dehydrogenase.
Claim 3
Dependent Claim 18 words
A mixture according to claim 1, wherein the tetrazolium salt is INT, and the electron carrier is PMS.
Claim 4
Dependent Claim 19 words
A mixture according to claim 1, wherein the tetrazolium salt is INT, and the electron carrier is Meldola Blue.
Claim 5
Dependent Claim 78 words
The mixture according to any of claims 2 or 3, such mixture further comprising components, in approximate concentrations as follows: (i) a mixture of 0.03 Molar potassium dihydrogen phosphate with 0.03 Molar potassium, monohydrogen phosphate in proportions to give in solution a buffer having a pH of approximately 8.5; and (ii) 2.5 g. glycine/l, 6.3 ml alkylphenoxypolyethyoxyethanol/l, and sufficient sodium hydroxide to give with the foregoing two components in solution a buffer having a pH of approximately 8.5.
Claim 6
Dependent Claim 47 words
An assay mixture for determining the concentration of lactic acid in a biological fluid, such mixture comprising the following components in the approximate concentrations set forth: 1.1 m Molar tetrazolium salt; 0.45 m Molar electron carrier; 2.1 m Molar NAD; and 39 International Units/ml of lactic dehydrogenase.
Claim 7
Dependent Claim 47 words
An assay mixture for determining the concentration of lactic acid in a biological fluid, such mixture comprising the following components in the approximate concentrations set forth: 1.1 m Molar tetrazolium salt; 0.45 m Molar electron carrier; 2.1 m Molar NAD; and 39 International Units/ml of lactic dehydrogenase.
Claim 8
Dependent Claim 18 words
The mixture according to claim 5, wherein the tetrazolium salt is INT, and the electron carrier is PMS.
Claim 9
Dependent Claim 19 words
A mixture according to claim 5, wherein the tetrazolium salt is INT and the electron carrier is Meldola Blue.
Claim 10
Dependent Claim 47 words
A mixture according to any of claims 6 or 7, such mixture further comprising components in approximate concentrations as follows: 5.3 g glycine/l, 7 ml alkylphenoxylpolyethyoxyethanol/l, and sufficient sodium hydroxide to give with the foregoing two components in solution a buffer having a pH of approximately 9.6.

References Cited (Backward Citations)

US3539453A Cited
US3528888A Cited
US3867258A Cited

USPTO Annuity & Maintenance Windows

Annuity Stage Window Opens Payment Deadline Surcharge Period Until Estimated Fee Status
3.5 Years (Year 4) 1984-03-02 1984-09-01 1985-03-03 $2000.00 Window Closed / Lapsed
7.5 Years (Year 8) 1988-03-02 1988-09-01 1989-03-03 $3760.00 Window Closed / Lapsed
11.5 Years (Year 12) 1992-03-02 1992-09-01 1993-03-03 $7700.00 Window Closed / Lapsed

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