Quantitative determination of glucose utilizing enzyme cascade system

• US 5,213,966 A
• Filed: 09/25/1989
• Issued: 05/25/1993
• Est. Priority Date: 09/26/1988
• Status: Expired due to Fees

• Current Assignee: Konelab Corporation
• Original Assignee: Kone Oyj
• Inventors: Jokela, Hannu, Vuorinen, Pauli, Harmoinen, Aimo
• Primary Examiner(s): Moskowitz, Margaret
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/411,879
• Time in Patent Office: 1,338 Days
• Field of Search: 435/4, 435/5, 435/14, 435/34, 435/15, 435/18, 435/25, 435/26, 436/501, 436/68, 436/95, 436/164, 436/815
• US Class Current: 435/14
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10T 436/144444 Glucose

Abstract

A procedure is disclosed for the determination of glucose in a biological liquid, as well as a reagent mixture for use in conjunction with the procedure. The biological liquids concerned are especially blood, blood components, urine and spinal fluid. The procedure is based on a two-phase enzymatic reaction, the first phase of which comprises transforming glucose with the aid of ATP (adenosine-5-triphosphate) and hexokinase into glucose-6-phosphate and the second phase of which comprises transforming glucose-6-phosphate with the aid of NAD and glucose-6-phosphate dehydrogenase into 6-phosphogluconate. ATP, NAD and the enzymes mentioned are included in a suitably buffered reagent mixture, which can be added as a single dose to the glucose-containing liquid sample. The reaction is monitored kinetically by measuring the absorption caused by the NADH produced in the second phase twice within approximately one minute from the initiation of the first reaction phase. The glucose content of the sample can be calculated from the measured change of absorbance.

Claims

1. A process for quantitative determination of glucose in a biological liquid, comprising the steps:

a) adding to the liquid a mixture of reagents comprising ATP in a concentration in a range of 15 to 20 mmol/1, hexokinase, NAD and glucose-6-phosphate dehydrogenase, to initiate a two phase enzymatic reaction, including

a first phase in which glucose-6-phosphate reacts with NAD and glucose-6-phosphate dehydrogenase to form 6-phosphogluconate and NADH,

said ATP in said concentration range being present in an amount sufficient that only a small portion thereof reacts with glucose in said first phase, and a remainder thereof is present during said second phase, said ATP remainder causing deceleration of said second phase;

b) monitoring the kinetics of said reaction by measuring the light absorbance of said NADH twice within a period not exceeding about one minute from the initiation of said reaction; and

c) calculating the glucose concentration in the liquid from the absorbance measurements taken during said period.

2. A process according to claim 1, wherein the absorption measurements are performed within a period of from 35 to 50 seconds from the initiation of the reaction.

3. A process according to claim 1, wherein the biological liquid is blood or a blood component.

4. A process according to claim 1, wherein the biological liquid is a whole-blood hemolysate.

5. A process according to claim 1, wherein the hexokinase in the reaction mixture has an activity at least approximately 2340 U/l.

6. A process according to claim 1, wherein the glucose-6-phosphate dehydrogenase in the reaction mixture has an activity at least approximately 2340 U/l.

7. A process according to claim 1, wherein the concentration of ATP in the reaction mixture is approximately 17 mmol/l.

8. A process according to claim 1, wherein reagent mixture is added in a single dose to a glucose-containing sample to be analyzed.

9. A reagent mixture for quantitative determination of glucose according to claim 1, which comprises a buffered water solution containing ATP, NAD, hexokinase and glucose-6-phosphate dehyyrogenase, said solution being suitable for being added in a single dose to each glucose-containing sample to be analyzed.

10. A reagent mixture according to claim 9, having an ATP concentration approximately 17 mmol/l, a NAD concentration approximately 5 mmol/l, a hexokinase activity approximately 2500 U/l and a glucose-6-phosphate dehydrogenase activity approximately 2500 U/l.