US4499184A
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Analysis for total cholesterol using lecithin:cholesterol acyl transferase (LCAT)
Assignee: Clinical Diagnostic Systems Inc
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Filing: 1982-06-01
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Granted: 1985-02-12
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Expires: 2002-06-01
Abstract
Abstract Methods, compositions and elements for the analysis of total cholesterol in aqueous fluids containing cholesterol and cholesterol esters are disclosed. Cholesterol esters in the fluid samples are first converted to cholesterol by: (a) contacting a sample of the fluid with sufficient amounts of lysolecithin and an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity to convert substantially all the cholesterol esters to cholesterol in the sample, and (b) removing cholesterol from the aqueous fluid other than by conversion to cholesterol esters. The total cholesterol is quantitatively determined by measuring the amount of the removed cholesterol. For example, the amount is measured gravimetrically or by converting the cholesterol to a detectable product.
Inventors
- 👤 Charles T. Goodhue
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 13:28
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/10; C12N9/1025; C12N9/1029; C12Q; C12Q1/00; C12Q1/60; Y; Y10; Y10S; Y10S435/00; Y10S435/805
Claims Analysis
Total Claims: 38
Claim 1
Independent Claim
72 words
A method for determining total cholesterol in an aqueous fluid containing cholesterol esters and free cholesterol comprising: (a) contacting a sample of said fluid with sufficient amounts of lysolecithin and an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity to convert substantially all the cholesterol esters in said sample to cholesterol, (b) removing cholesterol from said sample other than by conversion to cholesterol esters, and (c) measuring the amount of said removed cholesterol.
Claim 2
Independent Claim
72 words
A method for determining total cholesterol in an aqueous fluid containing cholesterol esters and free cholesterol comprising: (a) contacting a sample of said fluid with sufficient amounts of lysolecithin and an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity to convert substantially all the cholesterol esters in said sample to cholesterol, (b) removing cholesterol from said sample other than by conversion to cholesterol esters, and (c) measuring the amount of said removed cholesterol.
Claim 3
Dependent Claim
13 words
The method of claim 1 wherein the source of said enzyme is serum.
Claim 4
Dependent Claim
14 words
The method of claim 1 wherein the source of said enzyme is a microorganism.
Claim 5
Dependent Claim
26 words
The method of claim 3 wherein said microorganism is selected from the group consisting of Staphylococcus aureus, Phycomyces blakesleeanus and a member of the family Vibrionaceae.
Claim 6
Dependent Claim
11 words
The method of claim 3 wherein said microorganism is Aeromonas hydrophila.
Claim 7
Independent Claim
76 words
A method for determining total cholesterol in an aqueous fluid containing cholesterol esters and free cholesterol comprising: (a) contacting a sample of said fluid with sufficient amounts of lysolecithin and an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity to convert substantially all the cholesterol esters in said sample to cholesterol, (b) removing cholesterol from said sample by conversion to a detectable product other than a cholesterol ester, and (c) measuring the amount of said detectable product.
Claim 8
Independent Claim
76 words
A method for determining total cholesterol in an aqueous fluid containing cholesterol esters and free cholesterol comprising: (a) contacting a sample of said fluid with sufficient amounts of lysolecithin and an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity to convert substantially all the cholesterol esters in said sample to cholesterol, (b) removing cholesterol from said sample by conversion to a detectable product other than a cholesterol ester, and (c) measuring the amount of said detectable product.
Claim 9
Dependent Claim
33 words
The method of claim 6 wherein said cholesterol is enzymatically reacted with oxygen to yield hydrogen peroxide, and hydrogen peroxide enzymatically converts a color-forming substance to a colored species as said detectable product.
Claim 10
Dependent Claim
47 words
The method of claim 6 wherein said cholesterol is enzymatically reacted with oxygen in the presence of cholesterol oxidase to yield hydrogen peroxide, and hydrogen peroxide converts a color-forming substance, in the presence of a material exhibiting peroxidative activity, to a colored species as said detectable product.
Claim 11
Independent Claim
82 words
A method for determining total cholesterol in an aqueous fluid containing cholesterol esters and free cholesterol comprising: (a) contacting a sample of said fluid with an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity, lecithin, and a hydrolyzing enzyme capable of hydrolyzing lecithin to lysolecithin, each in sufficient amounts to convert substantially all the cholesterol esters in said sample to cholesterol, (b) removing cholesterol from said sample other than by conversion to cholesterol esters, and (c) measuring the amount of said removed cholesterol.
Claim 12
Independent Claim
82 words
A method for determining total cholesterol in an aqueous fluid containing cholesterol esters and free cholesterol comprising: (a) contacting a sample of said fluid with an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity, lecithin, and a hydrolyzing enzyme capable of hydrolyzing lecithin to lysolecithin, each in sufficient amounts to convert substantially all the cholesterol esters in said sample to cholesterol, (b) removing cholesterol from said sample other than by conversion to cholesterol esters, and (c) measuring the amount of said removed cholesterol.
Claim 13
Dependent Claim
12 words
The method of claim 9 wherein said hydrolyzing enzyme is phospholipase A-2.
Claim 14
Dependent Claim
28 words
The method of claim 10 wherein said LCAT and phospholipase A-2 enzyme are derived from the microorganism Aeromonas hydrophila and said lecithin is provided by said aqueous fluid.
Claim 15
Dependent Claim
81 words
A composition for determining total cholesterol in an aqueous fluid containing free cholesterol and cholesterol esters comprising: (a) a reagent composition comprising sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and lysolecithin to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 16
Dependent Claim
81 words
A composition for determining total cholesterol in an aqueous fluid containing free cholesterol and cholesterol esters comprising: (a) a reagent composition comprising sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and lysolecithin to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 17
Dependent Claim
14 words
The composition of claim 12 wherein the source of said LCAT is a microorganism.
Claim 18
Dependent Claim
26 words
The composition of claim 13 wherein said microorganism is selected from the group consisting of Staphylococcus aureus, Phycomyces blakesleeanus and a member of the family Vibrionaceae.
Claim 19
Dependent Claim
11 words
The composition of claim 13 wherein said microorganism is Aeromonas hydrophila.
Claim 20
Dependent Claim
76 words
The composition of claim 12 wherein said means for chemical removal of cholesterol and said means for chemical conversion of said removed cholesterol is a second enzyme composition comprising cholesterol oxidase sufficient to convert said removed cholesterol to hydrogen peroxide, a color-forming substance sufficient to react with said hydrogen peroxide, and a material exhibiting peroxidative activity which catalyzes the reaction of said color-forming substance with hydrogen peroxide to produce a colored species as said detectable product.
Claim 21
Dependent Claim
87 words
A composition for determining total cholesterol in an aqueous fluid containing free cholesterol and cholesterol esters comprising: (a) an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity, lecithin, and a hydrolyzing enzyme capable of hydrolyzing lecithin to lysolecithin, each in sufficient amounts to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 22
Dependent Claim
87 words
A composition for determining total cholesterol in an aqueous fluid containing free cholesterol and cholesterol esters comprising: (a) an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity, lecithin, and a hydrolyzing enzyme capable of hydrolyzing lecithin to lysolecithin, each in sufficient amounts to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 23
Dependent Claim
12 words
The composition of claim 17 wherein said hydrolyzing enzyme is phospholipase A-2.
Claim 24
Dependent Claim
20 words
The composition of claim 17 wherein the source of said LCAT and phospholipase A-2 enzyme is the microorganism Aeromonas hydrophila.
Claim 25
Dependent Claim
76 words
The composition of claim 19 wherein said means for chemical removal of cholesterol and said means for chemical conversion of said removed cholesterol is a second enzyme composition comprising cholesterol oxidase sufficient to produce hydrogen peroxide from said removed cholesterol, a color-forming substance sufficient to react with said hydrogen peroxide, and a material exhibiting peroxidative activity which catalyzes the reaction of said color-forming substance with hydrogen peroxide to produce a colored species as said detectable product.
Claim 26
Dependent Claim
89 words
A composition for determining total cholesterol in an aqueous fluid containing cholesterol, cholesterol esters and lecithin comprising: (a) sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and a hydrolyzing enzyme capable of hydrolyzing lecithin to lysolecithin, each in sufficient amounts to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 27
Dependent Claim
89 words
A composition for determining total cholesterol in an aqueous fluid containing cholesterol, cholesterol esters and lecithin comprising: (a) sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and a hydrolyzing enzyme capable of hydrolyzing lecithin to lysolecithin, each in sufficient amounts to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 28
Dependent Claim
18 words
The composition of claim 21 wherein said LCAT and hydrolyzing enzyme are derived from the microorganism Aeromonas hydrophila.
Claim 29
Dependent Claim
67 words
A composition for converting cholesterol esters to cholesterol in an aqueous fluid containing cholesterol esters comprising: (a) a reagent composition comprising sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and lysolecithin to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, and (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters.
Claim 30
Dependent Claim
67 words
A composition for converting cholesterol esters to cholesterol in an aqueous fluid containing cholesterol esters comprising: (a) a reagent composition comprising sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and lysolecithin to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, and (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters.
Claim 31
Dependent Claim
79 words
A dry analytical element for determining total cholesterol in an aqueous fluid containing free cholesterol and cholesterol esters, said element comprising an absorbent layer, and a composition comprising: (a) a reagent composition comprising sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and lysolecithin to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, and (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters.
Claim 32
Dependent Claim
79 words
A dry analytical element for determining total cholesterol in an aqueous fluid containing free cholesterol and cholesterol esters, said element comprising an absorbent layer, and a composition comprising: (a) a reagent composition comprising sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and lysolecithin to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, and (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters.
Claim 33
Dependent Claim
101 words
A dry analytical element for determining total cholesterol in an aqueous fluid containing free cholesterol, cholesterol esters and lecithin, said element comprising an absorber layer, and a composition comprising: (a) sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and a hydrolyzing enzyme capable of hydrolyzing lecithin to lysolecithin, each in sufficient amounts to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 34
Dependent Claim
101 words
A dry analytical element for determining total cholesterol in an aqueous fluid containing free cholesterol, cholesterol esters and lecithin, said element comprising an absorber layer, and a composition comprising: (a) sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and a hydrolyzing enzyme capable of hydrolyzing lecithin to lysolecithin, each in sufficient amounts to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 35
Dependent Claim
19 words
An element as in claim 25 wherein said LCAT and hydrolyzing enzyme are derived from the microorganism Aeromonas hydrophila.
Claim 36
Dependent Claim
92 words
A dry analytical element for determining total cholesterol in an aqueous fluid containing free cholesterol and cholesterol esters, said element comprising an absorbent layer, and a composition comprising: (a) a reagent composition comprising sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and lysolecithin to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 37
Dependent Claim
92 words
A dry analytical element for determining total cholesterol in an aqueous fluid containing free cholesterol and cholesterol esters, said element comprising an absorbent layer, and a composition comprising: (a) a reagent composition comprising sufficient amounts of an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and lysolecithin to convert substantially all the cholesterol esters in a sample of said fluid to cholesterol, (b) means for chemical removal of cholesterol from said sample other than by conversion to cholesterol esters, and (c) means for chemical conversion of said removed cholesterol to a detectable product.
Claim 38
Dependent Claim
19 words
The dry analytical element of claim 27 wherein said absorbent layer is a spreading layer carried on a support.
References Cited (Backward Citations)
GB1501561A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-02-12 | 1988-08-13 | 1989-02-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-02-12 | 1992-08-13 | 1993-02-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-02-12 | 1996-08-13 | 1997-02-12 | $7700.00 | Window Closed / Lapsed |