Method of polarographic analysis of lactic acid and lactate

• US 4,467,811 A
• Filed: 02/08/1982
• Issued: 08/28/1984
• Est. Priority Date: 08/02/1979
• Status: Expired due to Fees

• Current Assignee: Children's Hospital & Medical Center
• Original Assignee: Children's Hospital & Medical Center
• Inventors: Clark, Leland C. Jr.
• Primary Examiner(s): Padgett, Benjamin R.
• Assistant Examiner(s): Wallen, T. J.
• Application Number:
  US06/346,951
• Time in Patent Office: 932 Days
• Field of Search: 128/635, 128/637, 204/1 T, 204/1 R, 204/138, 204/160.1, 204/401-403
• US Class Current: 205/778
• CPC Class Codes: A61B 5/145 Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissuemeasuring of blood pressure or blood flow A61B5/02; non-radiation detecting or locating of foreign bodies in blood A61B5/06
• CPC Class Codes: A61B 5/1486 using enzyme electrodes, e.g. with immobilised oxidase
• CPC Class Codes: C12M 21/18 Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
• CPC Class Codes: C12M 29/04 Filters; Permeable or porous membranes or plates, e.g. dialysis
• CPC Class Codes: C12M 33/14 with filters, sieves or membranes
• CPC Class Codes: C12M 41/32 of substances in solution
• CPC Class Codes: C12Q 1/26 involving oxidoreductase

Abstract

A method of measuring lactic acid or lactate and derivatives thereof in liquids which is extremely versatile and is suitable for use in a number of areas such as the rapid measurement of lactic acid in whole blood, the ratio of lactic acid to pyruvic acid in whole blood, in-vivo measurement of lactic acid and the study of living lactic acid-producing cells. The acid or lactate is measured by reacting the lactic acid with lactic oxidase to produce pyruvate and H₂ O₂. The H₂ O₂ is then measured polarographically. The current produced is directly proportional to the lactate level. Preferably, the lactic oxidase is trapped between two semi-permeable membranes. One membrane is placed in contact with an electrolyte at the tip of an electrode and the second membrane contacts the liquid being tested.

Claims

1. The method of measuring lactic acid or derivatives thereof in a liquid and subsequently measuring pyruvate in said liquid comprising reacting said lactic acid or said derivatives thereof with lactic oxidase to produce pyruvate and hydrogen peroxide and measuring said hydrogen peroxide polarographically;

and

contacting said pyruvate with pyruvate oxidase, thereby producing acetic acid and hydrogen peroxide and measuring the produced hydrogen peroxide polarographically.

2. A method of measuring the ratio of lactic acid or derivatives thereof to pyruvic acid or derivatives thereof in a liquid comprising measuring the lactic acid in said liquid by reacting said lactic acid or derivative thereof with lactic oxidase, thereby producing hydrogen peroxide and pyruvate and measuring said hydrogen peroxide polarographically, and further comprising converting pyruvic acid in said liquid to lactic acid and subsequently measuring the lactic acid in said blood.

3. The method claimed in claim 2 wherein said liquid is whole mammalian blood.

4. The method claimed in claim 2 wherein said lactic oxidase is trapped between an inner and an outer membrane layer wherein said outer membrane layer separates said liquid from said enzyme and allows lactic acid or derivatives thereof to pass therethrough, and said inner membrane separates said enzyme from an electrolyte and allows hydrogen peroxide to pass therethrough.

5. The method claimed in claim 4 wherein said anode is separated from said inner membrane by a capillary layer of electrolyte.

6. The method claimed in claim 2 wherein said pyruvic acid in said liquid is converted to lactic acid by adding NADH and lactic dehydrogenase to said liquid.

7. A method of measuring the ratio of lactic acid or derivatives thereof to pyruvic acid or derivatives thereof in whole mammalian blood comprising measuring the lactic acid in said blood by reacting said lactic acid or derivatives thereof with lactic oxidase to produce pyruvate and hydrogen peroxide and measuring the hydrogen peroxide polarographically;

and

adding NADH and lactic dehydrogenase to said blood, thereby converting pyruvic acid or derivatives thereof in said blood to lactic acid and subsequently reacting the lactic acid with lactic oxidase to produce hydrogen peroxide and measuring the hydrogen peroxide polarographically.