US4784945A
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Method for determining lipids
Assignee: Wayne State University
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Filing: 1986-10-30
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Granted: 1988-11-15
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Expires: 2006-10-30
Abstract
Abstract A method for determining lipids using thin layer chromatography and multiple enzymatic staining is described. The method uses a hydrolase enzyme, an oxidase enzyme and a peroxidase enzyme in the presence of a chromogen precursor to form an oxidized chromogen stain. The method is particularly useful in determining phospholipids from animal and human tissue and/or fluids.
Inventors
- 👤 Joseph D. Artiss
- 👤 Dennis Bozimowski
- 👤 Robert J. McEnroe
- 👤 Bennie Zak
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 12:20
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/60; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S436/00; Y10S436/907
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
166 words
A method for the determination of mixed lipids in a liquid which comprises: (a) providing a thin layer chromatographic plate having a stationary phase as the thin layer which separates the mixed lipids; (b) applying the liquid containing the mixed lipids to the stationary phase on the plate so that the mixed lipids separate into separated bands of individual lipids upon migration with a mobile phase; (c) reacting the individual lipids with an aqueous solution comprising a hydrolase enzyme, an oxidase enzyme, a peroxidase enzyme and chromogen precursor(s), wherein the hydrolase enzyme catalyzes ester hydrolysis products from the lipids, the oxidase enzyme produces hydrogen peroxide by reaction with the hydrolysis products which in turn reacts in the presence of the peroxidase enzyme with the chromogen percursor(s) to form an oxidized chromogen complex(es) which is water insoluble so that the chromogen complex(es) remains in the band on the stationary phase; and (d) determining the individual lipids in the thin stationary phase by means of the chromogen complex(es).
Claim 2
Independent Claim
166 words
A method for the determination of mixed lipids in a liquid which comprises: (a) providing a thin layer chromatographic plate having a stationary phase as the thin layer which separates the mixed lipids; (b) applying the liquid containing the mixed lipids to the stationary phase on the plate so that the mixed lipids separate into separated bands of individual lipids upon migration with a mobile phase; (c) reacting the individual lipids with an aqueous solution comprising a hydrolase enzyme, an oxidase enzyme, a peroxidase enzyme and chromogen precursor(s), wherein the hydrolase enzyme catalyzes ester hydrolysis products from the lipids, the oxidase enzyme produces hydrogen peroxide by reaction with the hydrolysis products which in turn reacts in the presence of the peroxidase enzyme with the chromogen percursor(s) to form an oxidized chromogen complex(es) which is water insoluble so that the chromogen complex(es) remains in the band on the stationary phase; and (d) determining the individual lipids in the thin stationary phase by means of the chromogen complex(es).
Claim 3
Dependent Claim
42 words
The method of claim 1 wherein the mixed lipids comprise a cholesterol-fatty acid ester which is hydrolyzed to cholesterol and a fatty acid with the hydrolase enzyme and wherein the cholesterol is oxidized by the oxidase enzyme to produce the hydrogen peroxide.
Claim 4
Dependent Claim
12 words
The method of claim 2 wherein the oxidase enzyme is cholesterol oxidase.
Claim 5
Dependent Claim
13 words
The method of claim 2 wherein the hydrolase enzyme is cholesterol ester hydrolase.
Claim 6
Dependent Claim
49 words
The method of claim 1 wherein the mixed lipids are selected from a phosphatidylcholine and a phosphatidylglycerol which are hydrolyzed by the hydrolase enzyme to a phosphatidic acid and choline or glycerol and wherein the choline or glycerol are oxidized by an oxidase enzyme to produce the hydrogen peroxide.
Claim 7
Dependent Claim
12 words
The method of claim 5 wherein the hydrolase enzyme is phosphatidylcholine phosphatidohydrolase.
Claim 8
Dependent Claim
12 words
The method of claim 6 wherein the oxidase enzyme is choline:oxygen 1-oxidoreductase.
Claim 9
Dependent Claim
53 words
The method of claim 1 wherein the mixed lipids comprises a triglyceride which is hydrolyzed by the hydrolase enzyme to fatty acids and to glycerol and wherein the glycerol is phosphorylated with ATP in the presence of glycerol kinase and then oxidized with glycero-3-phosphate oxidase as the oxidase enzyme to produce hydrogen peroxide.
Claim 10
Dependent Claim
47 words
The method of claim 1 wherein the mixed lipids comprises phosphatidylglycerol which is hydrolyzed by phospholipase D to produce glycerol which is in turn phosphorylated by ATP in the presence of glycerol kinase and then oxidized with glycero-3-phosphate oxidase as the oxidase enzyme to produce hydrogen peroxide.
Claim 11
Dependent Claim
16 words
The method of claim 1 wherein the chromogen is coupled with peroxidase as the peroxidase enzyme.
Claim 12
Dependent Claim
32 words
The method of claim 10 wherein the chromogen precursor is mixed 4-aminoantipyrine and 2,4-dichloro-1-naphthol which react in the presence of hydrogen peroxide and the peroxidase enzyme to form the oxidized chromogen complex(es).
Claim 13
Dependent Claim
20 words
The method of claim 1 wherein the mixed lipids comprise a cholesterol fatty acid, a phosphatidyl choline and a triglyceride.
Claim 14
Dependent Claim
33 words
The method of claim 1 wherein the mixed lipids comprise sphingomyelin which is hydrolyzed to phosphoceramide and choline and wherein the choline is oxidized by the oxidase enzyme to produce the hydrogen peroxide.
Claim 15
Dependent Claim
11 words
The method of claim 13 wherein the oxidase enzyme is choline:1-oxidoreductase.
Claim 16
Dependent Claim
21 words
The method of claim 1 wherein the chromogen complex(es) is determined with a densitometer measuring light absorbance by the chromogen complex(es).
References Cited (Backward Citations)
US4680259A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-11-15 | 1992-05-16 | 1992-11-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-11-15 | 1996-05-16 | 1996-11-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-11-15 | 2000-05-16 | 2000-11-15 | $7700.00 | Window Closed / Lapsed |