EP0153872A2
Expired / Lapsed
EP
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Method for the determination of the reduced form of nicotinamide adenine dinucleotide
Assignee: Minaris Medical Co Ltd
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Filing: 1985-02-28
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Granted: —
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Expires: 2005-02-28
Abstract
Abstract A method is disclosed for the determination of NAD(P)H which comprises reacting the NAD(P)H with (1) peroxidase or thiol reductase and (2) diaphorase or an electron carrier, in the presence of a chromogen and determining the pigment thus formed. The chromogens used are compounds of the formula(I): …<CHEM>… wherein Y is hydrogen or hydroxyl; R1, R2 and R3 are each …<CHEM>… groups wherein Z is hydroxyl, amino, substituted amino, alkyl, substituted alkyl, alkenyl, aryl, substituted aryl, acyl, halogen, nitro, sulfo, carboxyl, or alkoxy; n is 0, 1, 2 or 3; provided that at least one Z in R1, R2 and R3 is hydroxyl, amino, or substituted amino. …<??>Also disclosed are reagents comprising the chromogen in admixture with the peroxidase or thiol oxide reductase and the diaphorase or electron carrier.
Inventors
- 👤 Norihito Aoyama
- 👤 Akira Miike
- 👤 Yoshaiki Shimizu
- 👤 Toshio Tatano
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-30 05:07
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/008; C12Q1/26; C12Q1/28; C12Q2326/00
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
153 words
A method for the determination of NAD(P)H in a sample which comprises reacting the NAD(P)H with a chromogen in the presence of (1) peroxidase or thiol oxide reductase and (2) diaphorase or an electron carrier, and determining the pigment thus formed, characterised in that the chromogen is a compound of the formula 1: wherein Y is hydrogen or hydroxyl; R l , R 2 and R 3 may be the same or different, and are groups represented by the following general formula (II), (III) or (IV): wherein Z (which may be the same or different) is hydroxyl, amino, substituted amino, alkyl, substituted alkyl, alkenyl, aryl, substituted aryl, acyl, halogen, nitro, sulfo, carboxyl or alkoxy; n is 0, 1, 2 or 3; provided that in at least one of the R l , R 2 and R 3 groups there is a Z substituent, that substituent being selected from hydroxyl, amino or substituted amino.
Claim 2
Independent Claim
153 words
A method for the determination of NAD(P)H in a sample which comprises reacting the NAD(P)H with a chromogen in the presence of (1) peroxidase or thiol oxide reductase and (2) diaphorase or an electron carrier, and determining the pigment thus formed, characterised in that the chromogen is a compound of the formula 1: wherein Y is hydrogen or hydroxyl; R l , R 2 and R 3 may be the same or different, and are groups represented by the following general formula (II), (III) or (IV): wherein Z (which may be the same or different) is hydroxyl, amino, substituted amino, alkyl, substituted alkyl, alkenyl, aryl, substituted aryl, acyl, halogen, nitro, sulfo, carboxyl or alkoxy; n is 0, 1, 2 or 3; provided that in at least one of the R l , R 2 and R 3 groups there is a Z substituent, that substituent being selected from hydroxyl, amino or substituted amino.
Claim 3
Dependent Claim
22 words
A method according to claim 1, wherein at least R l and R 2 are groups represented by the general formula (II).
Claim 4
Dependent Claim
27 words
A method according to claim 1 or 2, wherein the reaction is carried out in the presence of PMS, MPMS or Meldola's Blue as said electron carrier.
Claim 5
Dependent Claim
25 words
A method according to claim 1 or 2, wherein said method is carried out by using peroxidase and diaphorase as said components 1 and 2.
Claim 6
Independent Claim
34 words
A method according to any one of claims 1-4, wherein said determination is carried out photometrically by measuring the absorption of the reaction solution at a wavelength in the visible region of the spectrum.
Claim 7
Independent Claim
43 words
A method according to any one of claims 1-5 as applied to the determination of NAD(P)H produced in a sample by an enzymatic reaction involving an enzyme, a substrate and NAD, which is converted to NAD(P)H as a result of the enzymatic reaction.
Claim 8
Dependent Claim
23 words
A method according to claim 6, wherein said substrate is glucose-6-phosphate, galactose, lactic acid, alcohol, malic acid aldehyde, xanthine, glycerol or 3a-hydroxybile acid.
Claim 9
Dependent Claim
50 words
A reagent for the determination of NAD(P)H in a sample, characterised in that the reagent comprises, in admixture, a) a peroxidase or thiol oxide reductase, b) diaphorase or an electron carrier, and c) a chromogen of the formula I as defined in claim 1 or as modified by claim 2.
Claim 10
Dependent Claim
20 words
A reagent according to claim 8, wherein component b) is an electron carrier selected from PMS, MPMS and Meldola's Blue.
Claim 11
Dependent Claim
16 words
A reagent according to claim 8, wherein component a) is peroxidase and component b) is diaphorase.
Claim 12
Dependent Claim
33 words
A reagent according to claim 8, 9 or 10 additionally containing NAD and an enzyme capable of reacting with a substrate to be determined with the consequent conversion of said NAD to NAD(P)H.
References Cited (Backward Citations)
No citation records indexed.
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