Enzymatic method and stabilized solutions for determining total cholesterol in human serum

• US 4,378,429 A
• Filed: 08/21/1981
• Issued: 03/29/1983
• Est. Priority Date: 08/23/1979
• Status: Expired due to Term

• Current Assignee: Ivan E. Modrovich
• Original Assignee: Ivan E. Modrovich
• Inventors: Modrovich, Ivan E.
• Primary Examiner(s): Castel, Benoit
• Application Number:
  US06/294,987
• Time in Patent Office: 585 Days
• Field of Search: 435/11, 435/19, 435/188, 435/25, 435/28, 435/810, 23/230 B, 252/408 R
• US Class Current: 435/11
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: C12Q 1/60 involving cholesterol
• CPC Class Codes: C12Q 2326/00 Chromogens for determinations of oxidoreductase enzymes
• CPC Class Codes: C12Q 2326/50 Phenols; Naphthols; Catechols
• CPC Class Codes: C12Q 2326/96 4-Amino-antipyrine

Abstract

Stabilized enzymes useful in the diagnostic assay of total cholesterol are prepared by dissolving a salt of cholic acid in a buffer solution providing a pH within the range of about 4 to about 9, to the solution is added a cholesterol esterase. The solution is then mixed with a polyhydroxy organic compound and TRITON-X-100. A cholesterol oxidase and a peroxidase are each dissolved in separate portions of buffer solution and introduced into the buffered solution containing the cholesterol esterase. 4-aminoantipyrine is then added to the solution. The resultant solution is a stabilized enzyme solution which can be used in the total cholesterol assay of a serum sample. The stabilized solution can be used in combination with a chromogen diluent solution which is made by dissolving phenol and TRITON-X-100 in water or a buffer solution. The combination of the stabilized enzyme solution and the chromogen diluent solution provides a solution which has utility in the spectrophotometric assay of total cholesterol.

Claims

1. A stabilized aqueous enzyme solution for use in the determination of total cholesterol which comprises dissolved in an aqueous buffer solution having a pH of about 4 to 9, a surfactant present in an amount up to about 2.5 percent by volume of solution, and an effective amount of a salt of cholic acid, cholesterol oxidase, cholesterol esterase and a polyhydroxy organic compound present in concentration of from about 7.5 percent by volume of solution to an amount insufficient to materially reduce the enzyme activity of the cholesterol esterase and cholesterol oxidase.

2. A stabilized aqueous enzyme solution as claimed in claim 1 in which an effective amount of oxidase is present.

3. A stabilized aqueous enzyme solution as claimed in claim 2 in which an effective amount of 4-aminoantipyrine is present.

4. A stabilized aqueous enzyme solution as claimed in claim 2 in which an effective amount of phenol is present.

5. A solution as recited in claim 3 wherein the 4-aminoantipyrine is provided in an amount from about 150 to about 450 mg per liter of total stabilized enzyme solution.

6. A solution as recited in claim 1 wherein the salt of cholic acid is sodium cholate.

7. A solution as recited in claim 1 wherein the salt of cholic acid is added in an amount up to about 15 g per liter of the stabilized enzyme solution.

9. An aqueous stabilized enzyme solution as claimed in claim 1 or 8 in which the polyhydroxy organic compound is present in a concentration of from about 7.5 to about 50 percent by volume.

10. A solution as claimed in claim 1 or 8 wherein the cholesterol esterase is from the microorganism pseudomonas fluorescens ATCC 21156.

11. A solution as claimed in claim 1 or 8 wherein the cholesterol oxidase is recovered from a microorganism selected from the group consisting of pseudomonas sp, nocardia erythropolis, and brevibacterium stero licum.

12. A solution as claimed in claim 1 or 8 wherein the aqueous buffer solution comprises potassium dihydrogen phosphate and sodium hydroxide and has a pH of from about 6 to 8.

14. A solution as recited in claim 1 or 8 wherein the cholesterol esterase is provided at least in an amount sufficient to complete the deesterification of cholesterol esters within a time of about ten minutes at a temperature of about 37°

C.

15. A solution as recited in claim 1 or 8 wherein the polyhydroxy compound is selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol.

16. A solution as recited in claim 1 or 8 wherein the surfactant is provided in an amount from about 0.1 to about 0.5 percent by volume of the stabilized enzyme solution.

17. A solution as recited in claim 1 or 8 wherein the cholesterol oxidase is produced from nocardia erythropolis and the pH of the aqueous buffer solution is from about 6 to about 8.

18. A solution as recited in claim 1 or 8 wherein the cholesterol oxidase is produced from pseudomonas sp and the pH of the buffer solution is about 4 to 7.

19. A solution as recited in claim 1 or 8 wherein the cholesterol oxidase is produced from brevibacterium stero licum and the pH of the buffer solution is about 5 to 7.

20. A solution as recited in claim 1 or 8 wherein the cholesterol oxidase is provided in a total amount of about 500 to about 750 IU/liter.

43. A solution as recited in claim 1, 8, 35 or 40 wherein the surfactant is octylphenoxy polyethoxy ethanol having an HLB value of 13.5.

44. A cholesterol analysis solution which comprises from about 4 to about 6 volumes of water combined with each volume of the stabilized aqueous-enzyme solution as claimed in claim 1, 8, 35 or 40.

8. An aqueous stabilized enzyme solution for use in the determination of total cholesterol which comprises dissolved in an aqueous buffer solution having a pH range of about 4 to 9, a surfactant present in an amount up to about 2.5 percent by volume of solution and an effective amount of sodium cholate, the cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine, phenol and a polyhydroxy organic compound in an amount of about 7.5 percent by volume of solution to an amount insufficient to materially reduce enzyme activity of said cholesterol esterase, cholesterol oxidase and peroxidase.

13. A solution as recited in claim 8 wherein the sodium cholate is added in an amount up to about 15 g per liter of the stabilized enzyme solution.

21. A two-solution total cholesterol assay kit formed of an aqueous enzyme concentrate solution and a chromogen diluent solution, in which:

(a) said aqueous enzyme concentrate solution comprises an aqueous buffer solution having a pH range of about 4 to 9, a surfactant and an effective amount of a salt of cholic acid, cholesterol esterase, cholesterol oxidase, 4-aminoantipyrine, and a polyhydroxy organic compound present in an amount of about 7.5 percent by volume of the enzyme concentrate solution to an amount insufficient to reduce enzyme activity of said cholesterol esterase, cholesterol oxidase and peroxidase in combined aqueous enzyme concentrate solution and chromogen diluent solutions; and

(b) said chromogen diluent solution comprises dissolved in an aqueous solution having a pH of about 4 to 9, a surfactant and an effective amount of phenol, the total amount of surfactant present being up to 2.5 percent by volume of the combined aqueous enzyme concentrate solution and chromogen diluent solution.

22. A kit as claimed in claim 21 in which the cholesterol esterase is from the microorganism psudomonas fluorescens ATCC 21156.

23. A kit as claimed in claim 21 wherein the cholesterol oxidase is recovered from a microorganism selected from the group consisting of pseudomonas sp, nocardia, erythropolis, and brevibacterium stero licum.

24. A kit as claimed in claim 21 in which each aqueous buffer solution comprises potassium dihydrogen phosphate and sodium hydroxide and has a pH of from about 6 to 8.

25. A kit as recited in claim 21 wherein the salt of cholic acid is added in an amount up to about 15 g per liter of aqueous enzyme concentrate.

26. A kit as claimed in claim 21 in which the salt of cholic acid is sodium cholate.

27. A kit as claimed in claim 21 in which the cholesterol esterase is provided at least in an amount sufficient to complete the deesterification of cholesterol esters within a time of about ten minutes at a temperature of about 37°

C.

28. A kit as claimed in claim 21 in which the polyhydroxy compound is selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol.

29. A kit as claimed in claim 21 in which the surfactant is provided in a total concentration of an amount of from about 0.1 to about 0.5 percent by volume of the aqueous enzyme solution and chromogen diluent.

30. A kit as claimed in claim 21 wherein the cholesterol oxidase is produced from nocardia erythropolis and the pH of the aqueous buffer solution is from about 6 to about 8.

31. A kit as recited in claim 21 wherein the cholesterol oxidase is produced from pseudomonas sp and the pH of the buffer solution is about 4 to 7.

32. A kit as recited in claim 21 wherein the cholesterol oxidase is produced from brevibacterium stero licum and the pH of the buffer solution is about 5 to 7.

33. A kit as recited in claim 21 wherein the cholesterol oxidase is provided in a total amount of about 500 to about 750 IU/liter of aqueous enzyme concentrate solution.

34. A kit as claimed in claim 21 in which the 4-aminoantipyrine is provided in an amount of from about 150 to about 450 mg per liter of aqueous enzyme concentrate solution.

51. A cholesterol analysis solution which comprises from about 4 to about 6 volumes of water combined with each volume of assay solution formed by admixing the stabilized enzyme concentrate solution and chromogen diluent solution of claims 21, 45 or 49.

57. A method as recited in claim 21, 45, 49, 52, 53, or 54 wherein the surfactant is octylphenoxy polyethoxy ethanol having an HLB value of 13.5.

35. A stabilized aqueous enzyme solution for use in the determination of total cholesterol which comprises dissolved in an aqueous solution having a pH of about 4 to 9, a surfactant present in an amount up to about 2.5 percent by volume of solution, a salt of cholic acid present in an amount up to about 15 grams per liter of solution, cholesterol oxidase present in an amount of from about 300 to 1,000 IU/l of solution, cholesterol esterase present in an amount up to about 1,000 IU/liter of solution, and a polyhydroxy organic compound present in concentration of from about 7.5 to about 50 percent by volume of solution.

36. A stabilized aqueous enzyme solution as claimed in claim 35 in which per oxidase is present in an amount of from about 150 KU to 300 KU per liter of solution.

37. A stabilized aqueous enzyme solution as claimed in claim 36 in which 4-aminoantipyrine is present in an amount of from about 150 to about 450 mg per liter of solution.

38. A stabilized aqueous enzyme solution as claimed in claim 36 in which an effective amount of phenol is present.

39. A solution as claimed in claim 35 wherein the aqueous buffer solution comprises potassium dihydrogen phosphate and sodium hydroxide and has a pH of from about 6 to 8.

41. A solution as recited in claim 35 or 40 wherein the polyhydroxy compound is selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol.

42. A solution as recited in claim 35 or 40 wherein the surfactant is provided in an amount from about 0.1 to about 0.5 percent by volume of the stabilized enzyme solution.

40. An aqueous stabilized enzyme solution for use in the determination of total cholesterol which comprises dissolved in an aqueous buffer solution having a pH range of about 4 to 9, a surfactant present in an amount up to about 2.5 percent by volume of solution, sodium cholate present in an amount up to about 15 grams per liter of solution, cholesterol esterase present in an amount up to about 1,000 IU per liter of solution, cholesterol oxidase present in an amount of from about 300 to 1,000 IU per liter of solution, peroxidase, present in an amount of from about 150 to 300 KU per liter of solution, 4-aminoantipyrine present in an amount of from about 150 to about 450 mg per liter of solution, an effective amount of phenol is present and a polyhydroxy organic compound in an amount of about 7.5 to about 50 percent by volume of solution.

45. A two-solution total cholesterol assay kit formed of an aqueous enzyme concentrate solution and a chromogen diluent solution, in which:

(a) said aqueous enzyme concentrate solution comprises an aqueous buffer solution having a pH range of about 4 to 9, a surfactant and a salt of cholic acid present in an amount up to 15 grams per liter of enzyme concentrate solution, cholesterol esterase present in an amount up to about 1,000 IU per liter of solution, cholesterol oxidase present in an amount of from about 300 to 1,000 IU per liter of solution, 4-aminoantipyrine present in an amount of from about 150 to about 450 mg per liter of solution, peroxidase present in an amount of from about 150 to 300 IU per liter of solution, and a polyhydroxy organic compound present in an amount of about 7.5 percent to about 50 percent by volume of the enzyme concentrate solution; and

(b) said chromogen diluent solution comprises dissolved in an aqueous buffer solution and having a pH of about 4 to 9, a surfactant and an effective amount of phenol, the total amount of surfactant present being up to 2.5 percent by volume of the combined aqueous enzyme concentrate solution and chromogen diluent solution.

46. A kit as claimed in claim 45 in which each aqueous solution comprises potassium dihydrogen phosphate and sodium hydroxide and has a pH of from about 6 to 8.

47. A kit as claimed in claim 45 in which the polyhydroxy compound is selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol.

48. A kit as claimed in claim 45 in which the surfactant is provided in a total concentration of an amount of from about 0.1 to about 0.5 percent by volume of the aqueous enzyme solution and chromogen diluent.

49. A kit including a stabilized enzyme concentrate solution and chromogen diluent solution for use in assaying total cholesterol in a sample comprising:

(a) a stabilized enzyme concentrate solution comprising an aqueous buffer solution providing a pH of about 4 to about 9 and containing an effective amount of each of sodium cholate, cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine, a surfactant, and a a polyhydroxy compound selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol; and

(b) a chromogen diluent solution comprising an aqueous solution containing an effective amount of phenol and a surfactant, the total of said surfactant provided being in an amount up to about 2.5 percent by volume of the total volume of the stabilized enzyme concentrate and chromogen diluent solution and said polyhydroxy organic compound being provided in an amount insufficient to materially reduce enzyme activity of said cholesterol esterase, cholesterol oxidase and peroxidase.

50. A kit as recited in claim 49 wherein the stabilized aqueous enzyme concentrate solution comprises 50 percent by volume of an aqueous buffer solution providing a pH of about 6.60, 50 percent by volume of glycerol, 10 mM/l±

2 percent by volume of sodium cholate, 1,000 IU/l of cholesterol esterase, 500 IU/l±

5 percent of cholesterol oxidase, 150,000 IU/l±

5 percent peroxidase, 1.5 percent by volume of surfactant, and 1.5 mM/l±

2 percent 4-aminoantipyrine and the chromogen diluent solution is aqueous and comprises 10 mM/l of phenol, 0.1 percent by volume of surfactant.

52. A kit for use in assaying total cholesterol in a sample comprising a stabilized aqueous enzyme concentrate solution which is diluted with water in a ratio of about one part enzyme solution to about four parts water prior to conducting an asay, the concentrate solution comprising about 50 percent by volume of an aqueous buffer solution providing a pH of about 6.60, about 50 percent by volume glycerol, 10 mM/l±

2 percent of sodium cholate, 1,000 IU/l±

5 percent of cholesterol esterase, about 1.5 percent by volume surfactant, 500 IU/l±

5 percent cholesterol oxidase, 150,000 IU/l±

5 percent peroxidase, 1.5 mM/l±

20 percent of 4-aminoantipyrine and 30 mM/l±

2 percent of phenol.

55. A kit as recited in claim 52, 53, or 54 wherein the cholesterol esterase is from psueodmonas fluroescens.

56. A kit as recited in claim 52, 53, or 54 wherein the cholesterol oxidase is from nocardia erythropolis.

58. A cholesterol analysis solution formed by combining from about 4 to about 6 volumes of water with each volume of the stabilized enzyme concentrate solution as claimed in claim 52, 53 or 54.

53. A kit for use in the oxygen consumption assay of total cholesterol in a sample comprising a stabilized aqueous enzyme solution comprising about 92.5 percent by volume of an aqueous buffer solution providing a pH of about 6.6, 10 mM/l±

2 percent of sodium cholate, about 1000 IU/l±

5 percent cholesterol esterase, 7.5 percent by volume glycerol, about 0.225 percent by volume surfactant, and about 1000 IU/l±

5 percent cholesterol oxidase.

54. A kit for use in the oxygen consumption assay of total cholesterol in a sample comprising an aqueous stabilized enzyme concentrate solution which can be diluted with water in a ratio of one part enzyme concentrate to four parts water, the stabilized enzyme concentrate solution comprising about 50 percent by volume of an aqueous buffer solution providing a pH of about 6.6, about 50 percent by volume glycerol 10 MM/l±

2 percent sodium cholate, and about 5333 IU/l±

5 percent cholesterol esterase, about 1.5 percent by volume surfactant, and about 6,666 IU/l cholesterol oxidase.

59. A method of forming a stabilized enzyme solution effective for use in the determination of total cholesterol, the method comprising the steps of:

(a) preparing a buffer solution having a pH range of about 4 to 9;

(b) dissolving a salt of cholic acid in a first portion of the buffer solution;

(c) dissolving cholesterol esterase in the first portion of the buffer solution;

(d) adding a polyhydroxy organic compound selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol to the first portion of the buffer solution;

(e) adding a surfactant to the first portion of the buffer solution;

(f) dissolving cholesterol oxidase in a second portion of the buffer solution and adding the dissolved cholesterol oxidase to the first portion of the buffer solution;

(g) dissolving peroxidase in a third portion of the buffer solution and adding the dissolved peroxidase to the first portion of the buffer solution; and

(h) dissolving 4-aminoantipyrine in the first portion of the buffer solution to form a stabilized enzyme solution for use in the determination of total cholesterol, said stabilized enzyme solution containing said polyhydroxy organic compound in an amount insufficient to reduce enzyme activity of said cholesterol esterase, cholesterol oxidase and peroxidase and said surfactant in an amount up to about 2.5 percent by volume of said stabilized enzyme solution.

60. A method as recited in claim 59, where the salt of cholic acid is sodium cholate.

61. A method as recited in claim 59, wherein the salt of cholic acid is added in an amount up to about 15 grams per liter of final volume of the stabilized enzyme solution.

64. A method as recited in claim 59, 62 or 63 wherein the peroxidase is from horseradish source.

67. A method as recited in claim 59, 62 or 63 in which the surfactant is provided in an amount of from about 0.1 to about 0.5 percent by volume of solution.

70. A method as recited in claim 59, 62, 63 or 69 wherein the cholesterol esterase is from the microorganism pseudomonas fluorescens ATCC 21156.

71. A method as recited in claim 59, 62, 63 or 69 wherein the cholesterol oxidase is recovered from a microorganism selected from the group consisting of pseudomonas sp, nocardia erythropolis, and brevibacterium stero licum.

72. A method as recited in claim 59, 62, 63 or 69 wherein the buffer solution comprises an aqueous solution comprising potassium dihydrogen phosphate and sodium hydroxide providing a pH range of about 6 to 8.

73. A method as recited in claim 59, 62, 63 or 69 wherein the cholesterol esterase is provided at least in an amount sufficient to complete the deesterification of cholesterol esters in a time less than about ten minutes at a temperature of about 37°

C.

74. A method as recited in claim 59, 62, 63 or 69 wherein the polyhydroxy compound is present in an amount up to about one-half of the volume of the enzymic solution.

75. A method as recited in claim 59, 62, 63 or 69 wherein the polyhydroxy compound is present in an amount from about 7.5 to about 50 percent by volume of the enzyme solution.

76. A method as recited in claim 59, 62, 63 or 69 wherein the surfactant is octylphenoxy polyethoxy ethanol having an HLB value of 13.5.

77. A method as recited in claim 59, 62, 63 or 69 wherein the cholesterol oxidase is produced from nocardia erythropolis and the pH of the buffer solution is from about 6 to about 8.

78. A method as recited in claim 59, 62, 63 or 69 wherein the cholesterol oxidase is produced from pseudomonas sp and the pH of the buffer solution is about 4 to 7.

79. A method as recited in claim 59, 62, 63 or 69 wherein the cholesterol oxidase is produced from brevibacterium stero licum and the pH of the buffer solution is about 5 to 7.

80. A method as recited in claim 59, 62, 63 or 69 wherein the cholesterol oxidase is provided in an amount of about 500 to 750 IU/liter.

62. A method of forming a stabilized enzyme solution effective for use in the determination of total cholesterol, the method comprising the steps of:

(a) preparing a buffer solution having a pH range of about 4 to 9;

(b) dissolving sodium cholate in a first portion of the buffer solution;

(c) dissolving cholesterol esterase in the first portion of the buffer solution;

(d) adding a polyhydroxy organic compound selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol to the first portion of the buffer solution;

(e) adding a surfactant to the first portion of the buffer solution;

(f) dissolving cholesterol oxidase in a second portion of the buffer solution and adding the dissolved cholesterol oxidase to the first portion of the buffer solution containing cholesterol esterase;

(g) dissolving peroxidase in a third portion of the buffer solution and adding the dissolved peroxidase to the first portion of the buffer solution containing cholesterol esterase;

(h) dissolving 4-aminoantipyrine in the first portion of the buffer solution containing cholesterol esterase; and

(i) adding phenol to the first portion of the buffer solution containing cholesterol esterase to form a stabilized enzyme solution for use in the determination of total cholesterol, said stabilized enzyme solution containing said polyhydroxy organic compound in an amount insufficient to materially reduce enzyme activity of said cholesterol esterase, cholesterol oxidase and peroxidase and said surfactant in an amount up to about 2.5 percent by volume of said stabilized enzyme solution.

63. A method of preparing a two-solution, total cholesterol assay kit formed of an enzyme concentrate solution and a chromogen diluent solution, the method comprising the steps of:

(a) preparing a buffer solution having a pH range of about 4 to 9;

(b) preparing an enzyme concentrate solution for the assay kit by dissolving sodium cholate, cholesterol esterase, a polyhydroxy organic compound selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol, and a surfactant in a first portion of the buffer solution, dissolving cholesterol oxidase in a second portion of the buffer solution and adding the dissolved cholesterol oxidase to the first portion of the buffer solution containing cholesterol esterase, dissolving peroxidase in a third portion of the buffer solution and adding the dissolved peroxidase to the first portion of the buffer solution containing cholesterol esterase, and dissolving 4-aminoantipyrine in the first portion of the buffer solution containing cholesterol esterase to form the enzyme concentrate solution; and

(c) preparing a chromogen diluent solution for the assay kit by dissolving phenol and a surfactant in a fourth portion of the buffer solution wherein said surfactant is added in an amount up to about 2.5 percent volume of the total volume of said enzyme concentration solution and said chromogen diluent solution, and said polyhydroxy organic compound is added in an amount insufficient to materially reduce the enzyme activity of said cholesterol esterase, cholesterol oxidase and peroxidase.

65. A method as recited in claim 63 where the salt of cholic acid is added in an amount up to about 15 g per liter of volume of combined enzyme concentrate solution and chromogen diluent solution.

66. A method as recited in claim 63 wherein the surfactant is provided in an amount from about 0.1 to about 0.5 percent by volume of the combined volume of enzyme concentrate solution and chromogen diluent solution.

68. A method as recited in claim 63 wherein the 4-aminoantipyrine is provided in an amount from about 150 to about 450 mg per liter of combined enzyme concentration solution and chromogen diluent solution.

69. A method of forming a stabilized enzyme solution effective for use in the oxygen consumption assay of total cholesterol, the method comprising the steps of:

(a) preparing a buffer solution having a pH range of about 4 to 9;

(b) dissolving a metal salt of cholic acid in a first portion of the buffer solution;

(c) dissolving cholesterol esterase in the first portion of the buffer solution;

(d) adding a polyhydroxy organic compound selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol to the first portion of the buffer solution;

(e) adding a surfactant to the first portion of the buffer solution in an amount up to about 2.5 percent by volume of the stabilized enzyme solution and wherein said polyhydroxy organic compound is present in an amount insufficient to materially reduce enzyme activity of said cholesterol esterase and cholesterol oxidase.