Test system and procedure for the determination of NAD (P) H

• US 4,629,697 A
• Filed: 12/23/1983
• Issued: 12/16/1986
• Est. Priority Date: 12/24/1982
• Status: Expired due to Fees

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Helger, Roland, Limbach, Berthold
• Primary Examiner(s): Marantz, Sidney
• Assistant Examiner(s): Foulke, Cynthia Lee
• Application Number:
  US06/564,866
• Time in Patent Office: 1,089 Days
• Field of Search: 435/10, 435/12, 435/25, 435/26, 435/805, 435/810, 422/56, 436/904
• US Class Current: 435/26
• CPC Class Codes: C12Q 1/008 for determining co-enzymes or co-factors, e.g. NAD, ATP
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/904 Oxidation - reduction indicators

Abstract

A test system having an extended range of measurement and an appropriate procedure for the determination of NAD(P)H or of substrates or enzymes which react to form or consume NAD(P)H in fluids is provided. The test system contains, at one and the same time, several substances acting independently of one another as electron acceptors with respect to NAD(P)H and having different electrochemical potentials. Addition of the test system to the sample solution gives rise to different end products which can be analytically differentiated and which are evaluated visually or by other techniques of measurement.

Claims

1. A test system useful for providing an extended range of measurement for the determination of NADH, NADPH, NAD⁺ or NADP⁺, or of a substrate or enzyme which reacts with the formation or consumption of NADH or NADPH, comprising effective amounts of at least two redox indicators which, independently of each other, act as electron acceptors with respect to NAD(P)H and which each have different electrochemical potentials.

2. A test system of claim 1, wherein the redox indicators have a standard potential of -0.3 to +0.5.

3. A test system of claim 1, wherein the redox indicators have a standard potential of -0.3 to +0.3.

4. A test system of claim 1 further comprising an effective amount of a buffer which maintains a pH of 4.5-8.0.

5. A test system of claim 1 further comprising an effective amount of a buffer which maintains a pH of 5.0-7.0.

6. A test system of claim 5 wherein the buffer is a phosphate buffer, a citrate buffer, 2-N-morpholinoethanesulphonic acid sodium salt, bis(2-hydroxy-ethyl) aminotris(hydroxymethyl)methane, or piperazine-N,N'"'"'-bis(2-ethanesulphonic acid) dipotassium salt.

7. A test system of claim 1 further comprising an effective amount of an electron transfer agent.

8. A test system of claim 7 wherein the electron transfer agent is, phenazine methosulphate, phenazine ethosulphate, methoxyphenazine methosulphate, Meldola'"'"'s blue, diaphorase or a mixture thereof.

9. A test system of claim 1 wherein each redox indicator is dichlorophenol-indophenol or a tetrazolium salt.

10. A test system of claim 1 wherein each redox indicator is dichlorophenol-indophenol, 3-(4-iodo- phenyl)-2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide.

11. A test system of claim 1 further comprising a background dyestuff.

12. A test system of claim 11 wherein the background dyestuff is titanium yellow.

13. A test system of claim 1 wherein the redox indicators are dichlorophenol-indophenol and 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride.

14. A test system of claim 1, wherein said indicators are impregnated within an absorbent material.

15. A method for the determination of NADH, NADPH, NAD⁺ or NADP⁺ in an aqueous sample solution, comprising treating the sample solution with at least two system compatible redox substances which act, independently of one another, as electron acceptors with respect to NADH or NADPH and which have different electrochemical potentials, whereby there are sequentially formed corresponding, different end products, and analytically measuring each end product.

16. A method of claim 15 wherein the NADH, NADPH, NAD⁺ or NADP⁺ moiety is derived from the presence of a substrate or enzyme which has reacted to form said moiety, and the measurement of said end products constitutes a determination of the substrate or enzyme.

17. A method of claim 15 wherein the measurement is conducted photometrically.

18. A method of claim 15 wherein the measurement is conducted visually.

19. A method of claim 15 wherein the determination is carried out in a buffered aqueous solution of a pH of 4.5-8.0.

20. An absorbent material impregnated with a test system useful for providing an extended range of measurement for the determination of NADH, NADPH, NAD⁺ or NADP⁺, or of a substrate or enzyme which reacts with the formation or consumption of NADH or NADPH, comprising effective amounts of at least two redox indicators which, independently of each other, act as electron acceptors with respect to NAD(P)H and which have different electrochemical potentials.