US4019961A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Analytical enzymatic determination
Assignee: Roche Diagnostics GmbH
•
Filing: 1975-03-10
•
Granted: 1977-04-26
•
Expires: 1995-03-10
Abstract
Abstract An analytical process for enzymatic determination of a substrate in biological fluids. The fluid sample is reacted first in the absence of the specific kinase but in the presence of an NADH - regenerating enzymatic system with the reagents needed for the reaction sequence; then the enzymes are separated from the reaction solution, and thereafter the necessary enzymes are added to the filtrate and NADH consumption is measured.
Inventors
- 👤 Sigmar Klose
- 👤 August Wilhelm Wahlefeld
- 👤 Alexander Hagen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 09:20
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/61; C12Q1/26; C12Q1/32; C12Q1/34; C12Q1/44; C12Q1/48; G; G01; G01N; G01N2333/00; G01N2333/90; G01N2333/902; G01N2333/904; G01N2333/91; G01N2333/912; G01N2333/91205; G01N2333/9121; G01N2333/914; G01N2333/916; G01N2333/918
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
136 words
In an analytical process for enzymatic determination of a substrate in a biological fluid by phosphorylation of a substrate with ATP with the liberation of ADP, reaction of ADP with phosphoenol pyruvate in the presence of pyruvate kinase with the formation of pyruvate, hydrogenation of the pyruvate with NADH in the presence of lactate dehydrogenase, the improvement which comprises, reacting the biological fluid substrate, in the absence of its specific kinase but in the presence of a NADH-regenerating enzymatic system which is the specific dehydrogenase and a substrate for the dehydrogenase with adenoisine triphosphate, phosphoenol pyruvate, and NADH, separating the enzymes from the reaction mixture, adding the specific kinase for the substrate, pyruvic kinase and lactate dehydrogenase to the liquid product free of enzymes, measuring the NADH consumption, thereby measuring only the amount of phosphorylated substrate.
Claim 2
Dependent Claim
136 words
In an analytical process for enzymatic determination of a substrate in a biological fluid by phosphorylation of a substrate with ATP with the liberation of ADP, reaction of ADP with phosphoenol pyruvate in the presence of pyruvate kinase with the formation of pyruvate, hydrogenation of the pyruvate with NADH in the presence of lactate dehydrogenase, the improvement which comprises, reacting the biological fluid substrate, in the absence of its specific kinase but in the presence of a NADH-regenerating enzymatic system which is the specific dehydrogenase and a substrate for the dehydrogenase with adenoisine triphosphate, phosphoenol pyruvate, and NADH, separating the enzymes from the reaction mixture, adding the specific kinase for the substrate, pyruvic kinase and lactate dehydrogenase to the liquid product free of enzymes, measuring the NADH consumption, thereby measuring only the amount of phosphorylated substrate.
Claim 3
Dependent Claim
16 words
The process of claim 1 wherein the NADH-specific dehydrogenase is alcohol dehydrogenase, and a substrate therefor.
Claim 4
Dependent Claim
11 words
The method of claim 2 wherein the substrate is an alcohol.
Claim 5
Dependent Claim
10 words
The process of claim 3 wherein the alcohol is ethanol.
Claim 6
Dependent Claim
25 words
The process of claim 1 wherein the substrate is glycerol and the specific kinase added to the liquid product free of enzymes is glycerol kinase.
Claim 7
Dependent Claim
17 words
The process of claim 1 wherein the substrate is the reaction product of a prior enzymatic reaction.
Claim 8
Dependent Claim
20 words
The process of claim 6 wherein the substrate is glycerol and a fatty acid, which are enzymatically obtained using lipase.
Claim 9
Dependent Claim
13 words
The process of claim 7 wherein the substrate is obtained using also esterase.
Claim 10
Dependent Claim
16 words
The process of claim 8 wherein glycerol is formed and the specific kinase is glycerol kinase.
Claim 11
Dependent Claim
11 words
The process of claim 1 wherein the separation is by dialysis.
Claim 12
Dependent Claim
32 words
The process of claim 1 wherein the separation of the enzymes is carried out by means of a semi-permeable membrane selective for low molecular weight substances which prevents the passage of enzymes.
Claim 13
Dependent Claim
29 words
The process of claim 1 wherein the biological fluid containing the substrate also contains dissolved bilirubin through the series of reactions, which bilirubin is separated from the reaction mixture.
Claim 14
Dependent Claim
11 words
The process of claim 1 wherein the substrate is a triglyceride.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1980-04-25 | 1980-10-25 | 1981-04-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1984-04-25 | 1984-10-25 | 1985-04-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1988-04-25 | 1988-10-25 | 1989-04-26 | $7700.00 | Window Closed / Lapsed |