US3898130A - Rapid enzymatic hydrolysis of triglycerides - Google Patents

Rapid enzymatic hydrolysis of triglycerides Download PDF

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US3898130A
US3898130A US451735A US45173574A US3898130A US 3898130 A US3898130 A US 3898130A US 451735 A US451735 A US 451735A US 45173574 A US45173574 A US 45173574A US 3898130 A US3898130 A US 3898130A
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lipase
combination
acid
candida
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Stanley K Komatsu
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American Hospital Supply Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
    • C11C1/045—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis using enzymes or microorganisms, living or dead
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/921—Candida

Definitions

  • US. Pat. No. 3,703,591 discloses that the preliminary step, that of hydrolyzing the triglycerides to form glycerol, may be carried out enzymatically using a mixture of a lipase and a protease. While it has long been known that certain lipases may alone be used to hydrolyze triglycerides, and that the enzymatic activity might possibly be enhanced by the presence of a bile salt, prior procedures using only lipases (with or without bile salts) have been unsatisfactory because of the long duration of the hydrolysis step and because even then incomplete hydrolysis is the usual result.
  • an important aspect of this invention lies in the discovery that a complete and surprisingly rapid hydrolysis of triglycerides may be achieved by using a combination consisting only of certain lipases along with a bile salt, thereby dispensing with the requirement of including a proteolytic enzyme such as chymotrypsin as one of the reactants. More specifically, 100 percent hydrolysis may be accomplished in 3 to 5 minutes using a combination of Candida lipase and pancreatic lipase with a bile salt such as sodium taurodeoxycholate. The glycerol so produced may then be assayed by any of a number of known methods, one such method having already been described above.
  • the enzymatic hydrolysis of triglycerides by the coactive lipases be undertaken in the presence of the components of the three additional reaction systems represented in the equations given above so that all of such reactions may be undertaken simultaneously in a single operative procedure or, if desired, in a two step procedure in which one of the com- 0 ponents needed for the conversion of glycerol, such as the glycerol kinase, is added to all of the other components after hydrolysis has occurred and an initial reading of optical density has been made. Since the various components necessary for the enzymatic conversion of glycerol.
  • the combination of lipases and bile salt may be part of an assay mixture which includes one or more of those components known for use in the enzymatic conversion of glycerol and the concurrent oxidation of NADH in a colorimetric or spectrophotometric test.
  • the system responsible for the enzymatic hydrolysis of triglycerides comprises a coactive mixture of pancreatic lipase, a microbial lipase, and a bile salt. All three components are essential for effective hydrolysis.
  • pancreatic lipases having activity within the range of about 10 to 100 lipase units per milligram (mg), and preferably within the range of 20 to units per mg, are believed suitable, an example being the pancreatic lipase sold under the designation PL3 by Worthington Chemical Company, of Freehold, New Jersey.
  • the microbial lipase is more specifically a Can dida lipase which may, for example, be obtained from the cultured broth of Candida cylindracea.
  • Candida lipase should have activity within the range of 30 to 800 lipase units per mg, and preferably within the range of 200 to 800 lipase units per mg.
  • Other Candida lipases are believed to be equally effective when used in combination with the pancreatic lipase and bile salt of the triglyceride-hydrolyzing system.
  • pancreatic and Candida lipases While both pancreatic and Candida lipases must be present, it has been found that the proportional amounts of those constituents, measured in terms of lipase units, may be varied considerably in accordance with selected time requirements for completion of hydrolysis.
  • One lipase unit of activity is the amount sufficient to release one micromole of acid per minute at 25C. from an olive oil emulsion containing gum acacia and 15 mg per milliliter (ml) sodium taurocholate at a pH of about 8.0.
  • Such definition of a lipase unit, and the procedure on which it is based, are well established and are disclosed more fully in Worthington Enzyme Manual, p. 63 (1972).
  • the amount of pancreatic lipase in the reaction mixture should be at least 0.14 lipase units for each microliter of body fluid (blood serum or plasma) having a triglyceride value within the range of 0 to 500 mg per ml (mg percent) in order to achieve complete hydrolysis within 12 minutes.
  • the amount of Candida lipase in the mixture should be at least 0.28 units, and the amount of bile salt should be at least 0.002 mg, for each microlitcr of body fluid. Where shorter reaction times are required or desired, the amounts of such constituents must be increased.
  • pancreatic lipase units 0.54 Candida lipase units, and 0.02 mg of bile salt, are required for each microliter of body fluid.
  • the values for hydrolyzing 50 microliters of serum or plasma having a triglyceride value of O 500 mg percent within 12 minutes
  • a maximum time period for completion of hydrolysis in a clinical test has been arbitrarily set at approximately 12 minutes. It is to be understood, however, that in other tests where longer time periods are more acceptable, lower concentrations or amounts of the respective lipases and bile salt may be used.
  • a bile salt is an essential component of the system. While alkali metal salts of taurocholic, taurochenodeoxycholic or taurodehydrocholic acid may be used, particularly effective results have been achieved with alkali metal salts of taurodeoxycholic acid.
  • the salt of taurodeoxycholic acid in admixture with Candida lipase and pancreatic lipase under optimum conditions, has been found to produce faster results at lower concentrations than the other bile salts.
  • pancreatic and Candida lipases reacts with the triglycerides in body fluids to produce complete hydrolysis in periods as short as 3 minutes.
  • a bile salt preferably the salt of taurodeoxycholic acid
  • Such a combination of reactants may be used in any test requiring the rapid and complete hydrolysis of triglycerides.
  • the process and product of this invention may, for example, be used in conjunction with a complete triglyceride assay including the three glyceroldetermining reactions described at the beginning of this application and well known in the prior art. All of the components required for the complete colorimetric determination of triglycerides in body fluids may be premixed and lyophilized to provide a stable reagent set for clinical use.
  • a reagent suitable for practicing this invention may Bile Salt (Na) 0 to 500 mg percent, to the above reaction mixture.
  • the optical density is measured at 340 nm. Thereafter, 10 units of glycerol kinase is added and the mixture is again incubated at 25C. to 37C. for another 5 minutes. The optical density is again determined at 340 nm, and the difference in optical densities is proportional to the triglyceride content after appropriate adjustment, using conventional clinical laboratory procedures, for whatever blank reaction is produced.
  • Example 2 The procedure of Example I was performed using the same reactants, proportions, and conditions, except that a purified lipase obtained from the cultured broth of Candida cylindracea nov. sp. was substituted for the Candida lipase of the first example. Complete hydrolysis of the triglycerides of the sample were obtained within 5 minutes in the same manner as set forth in Example 1.
  • EXAMPLE 3 Several reagent combinations were prepared in accordance with Example 1 except that a variety of bile salts were used. The results were tabulated below. Each reagent combination contained 0.1 mg Candida lipase (activity of units), 3.0 mg pancreatic lipase (activity 180 units), and the amount of bile salt indicated. Times are given in minutes for completion percent) of hydrolysis of serum samples having triglycerides values of 100 mg percent (i.e., 100 mg per 100 ml water) and 280 mg percent.
  • An enzymatic process for rapidly liberating glycerol from its esterifled form as a fatty acid ester in an aqueous fluid comprising the step of mixing said fluid with a combination of Candida lipase, pancreatic lipase, and a bile salt selected from the group consisting of the alkali metal salts of taurodeoxycholic, taurocholic, taurochenodeoxycholic, and taurodehydrocholic acids.
  • pancreatic lipase in said combination provides at least 0.14 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
  • pancreatic lipase provides at least 1.2 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
  • a reagent combination for the rapid hydrolysis of fatty acid esters to liberate glycerol therefrom comprising a mixture of Candida lipase, pancreatic lipase, and a bile salt selected from the group consisting of the alkali metal salts of taurodeoxycholic, taurocholic, taurochenodeoxycholic, and taurodehydrocholic acids.
  • pancreatic lipase has at least 7 lipase units for each milliliter of reagent combination.
  • pancreatic lipase has at least 60 lipase units for each milliliter of reagent combination.
  • pancreatic lipase has an activity of about 10 to 100 lipase units per milligram.
  • pancreatic lipase has an activity of approximately 20 to lipase units per milligram.

Abstract

A process and product for rapidly liberating glycerol from its esterified form as a fatty acid ester, for example, when present in an aqueous media such as serum, wherein a combination of a pancreatic lipase and a microbial lipase, particularly Candida lipase, are mixed with the fatty acid ester in the presence of a bile salt.

Description

ilnited States Patent Komatsu Aug. 5, 1975 RAPID ENZYMATIC HYDROLYSIS OF TRIGLYCERIDES Stanley K. Komatsu, Laguna Hill, Calif.
Inventor:
Assignee: American Hospital Supply Corporation, Evanston, 111.
Filed: Mar. 18, 1974 Appl. N0.: 451,735
U.S. Cl 195/30; 195/63; 195/103.5 R Int. Cl Cl2d 13/02 Field of Search 195/30, 63
References Cited UNITED STATES PATENTS 11/1972 Bucolo et a1 195/103.5 R
OTHER PUBLICATIONS Alford et al., Journal of Lipid Research, Vol. 5, pp. 390-394, July 1964.
Primary ExaminerAlvin E. Tanenholtz Attorney, Agent, or FirmDawson, Tilton, Fallon & Lungmus [5 7 ABSTRACT 30 Claims, No Drawings RAPID ENZYMATIC HYDROLYSIS OF TRIGLYCERIDES BACKGROUND AND SUMMARY Glycerol Kinase (l) Glycerol ATP Glycerol Phosphate ADP Pyruvate Kinase (2) ADP Phosphoenolpyruvate ATP yruvate Lactic (3) Pyruvate NADH Lactate NAD Dehydrogenase in this combination of reactions, 1 mole of NADH is oxidized for each mole of glycerol phosphorylized; therefore, a change in the optical density at 340 nm is a direct measure of the amount of glycerol in the assay.
US. Pat. No. 3,703,591 discloses that the preliminary step, that of hydrolyzing the triglycerides to form glycerol, may be carried out enzymatically using a mixture of a lipase and a protease. While it has long been known that certain lipases may alone be used to hydrolyze triglycerides, and that the enzymatic activity might possibly be enhanced by the presence of a bile salt, prior procedures using only lipases (with or without bile salts) have been unsatisfactory because of the long duration of the hydrolysis step and because even then incomplete hydrolysis is the usual result.
Therefore, an important aspect of this invention lies in the discovery that a complete and surprisingly rapid hydrolysis of triglycerides may be achieved by using a combination consisting only of certain lipases along with a bile salt, thereby dispensing with the requirement of including a proteolytic enzyme such as chymotrypsin as one of the reactants. More specifically, 100 percent hydrolysis may be accomplished in 3 to 5 minutes using a combination of Candida lipase and pancreatic lipase with a bile salt such as sodium taurodeoxycholate. The glycerol so produced may then be assayed by any of a number of known methods, one such method having already been described above.
Other objects and advantages of the invention will be apparent as the specification proceeds. Additional references disclosing the state of the art are: R. G. H. Morgan and N. E. Hoffman, Biochim. Biophys. Acta, 248:143 (1971); R. L. Ory, J. Kiser and P. A. Pradel, Lipids, 4:261 (1968); H. Brockerhoff, 1. Bio. Chem. 246:5828 (1971); P. Desnuclle, Enzymes, 7:575 (1972); patent 2,527,305.
DESCRIPTION It is preferred that the enzymatic hydrolysis of triglycerides by the coactive lipases be undertaken in the presence of the components of the three additional reaction systems represented in the equations given above so that all of such reactions may be undertaken simultaneously in a single operative procedure or, if desired, in a two step procedure in which one of the com- 0 ponents needed for the conversion of glycerol, such as the glycerol kinase, is added to all of the other components after hydrolysis has occurred and an initial reading of optical density has been made. Since the various components necessary for the enzymatic conversion of glycerol. have already been indicated and are well known in the prior art, and since the proportions of such components are also well known, a detailed discussion herein is believed unnecessary. it is believed sufficient to state that the combination of lipases and bile salt may be part of an assay mixture which includes one or more of those components known for use in the enzymatic conversion of glycerol and the concurrent oxidation of NADH in a colorimetric or spectrophotometric test.
The system responsible for the enzymatic hydrolysis of triglycerides comprises a coactive mixture of pancreatic lipase, a microbial lipase, and a bile salt. All three components are essential for effective hydrolysis. A variety of pancreatic lipases having activity within the range of about 10 to 100 lipase units per milligram (mg), and preferably within the range of 20 to units per mg, are believed suitable, an example being the pancreatic lipase sold under the designation PL3 by Worthington Chemical Company, of Freehold, New Jersey. The microbial lipase is more specifically a Can dida lipase which may, for example, be obtained from the cultured broth of Candida cylindracea. Such Candida lipase should have activity within the range of 30 to 800 lipase units per mg, and preferably within the range of 200 to 800 lipase units per mg. Other Candida lipases are believed to be equally effective when used in combination with the pancreatic lipase and bile salt of the triglyceride-hydrolyzing system.
While both pancreatic and Candida lipases must be present, it has been found that the proportional amounts of those constituents, measured in terms of lipase units, may be varied considerably in accordance with selected time requirements for completion of hydrolysis. One lipase unit of activity is the amount sufficient to release one micromole of acid per minute at 25C. from an olive oil emulsion containing gum acacia and 15 mg per milliliter (ml) sodium taurocholate at a pH of about 8.0. Such definition of a lipase unit, and the procedure on which it is based, are well established and are disclosed more fully in Worthington Enzyme Manual, p. 63 (1972).
More specifically, the amount of pancreatic lipase in the reaction mixture should be at least 0.14 lipase units for each microliter of body fluid (blood serum or plasma) having a triglyceride value within the range of 0 to 500 mg per ml (mg percent) in order to achieve complete hydrolysis within 12 minutes. On the same basis, the amount of Candida lipase in the mixture should be at least 0.28 units, and the amount of bile salt should be at least 0.002 mg, for each microlitcr of body fluid. Where shorter reaction times are required or desired, the amounts of such constituents must be increased. Thus, for complete hydrolysis within 3 to 5 minutes, at least 1.2 pancreatic lipase units, 0.54 Candida lipase units, and 0.02 mg of bile salt, are required for each microliter of body fluid. Stated differently, in a reagent combination having a volume of 1 milliliter, the values (for hydrolyzing 50 microliters of serum or plasma having a triglyceride value of O 500 mg percent within 12 minutes) should be at least 7 pancreatic lipase units, 14 Candida lipase units, and 0.1 mg bile salt; or at least 60 pancreatic lipase units, 27 Candida lipase units, and 1.0 mg bile salt (for the hydrolysis of 50 microliters of such serum or plasma within 3 to 5 minutes).
Since speed in completing an assay may be important, particularly in connection with clinical diagnostic tests, a maximum time period for completion of hydrolysis in a clinical test has been arbitrarily set at approximately 12 minutes. It is to be understood, however, that in other tests where longer time periods are more acceptable, lower concentrations or amounts of the respective lipases and bile salt may be used.
As previously indicated, a bile salt is an essential component of the system. While alkali metal salts of taurocholic, taurochenodeoxycholic or taurodehydrocholic acid may be used, particularly effective results have been achieved with alkali metal salts of taurodeoxycholic acid. The salt of taurodeoxycholic acid, in admixture with Candida lipase and pancreatic lipase under optimum conditions, has been found to produce faster results at lower concentrations than the other bile salts.
As indicated above, and as illustrated more fully by the examples set forth hereinafter, the combination of pancreatic and Candida lipases, in admixture with a bile salt, preferably the salt of taurodeoxycholic acid, reacts with the triglycerides in body fluids to produce complete hydrolysis in periods as short as 3 minutes. Such a combination of reactants may be used in any test requiring the rapid and complete hydrolysis of triglycerides. The process and product of this invention may, for example, be used in conjunction with a complete triglyceride assay including the three glyceroldetermining reactions described at the beginning of this application and well known in the prior art. All of the components required for the complete colorimetric determination of triglycerides in body fluids may be premixed and lyophilized to provide a stable reagent set for clinical use.
EXAMPLE I A reagent suitable for practicing this invention may Bile Salt (Na) 0 to 500 mg percent, to the above reaction mixture.
Following incubation for approximately 5 minutes at a temperature between 25C. to 37C., the optical density is measured at 340 nm. Thereafter, 10 units of glycerol kinase is added and the mixture is again incubated at 25C. to 37C. for another 5 minutes. The optical density is again determined at 340 nm, and the difference in optical densities is proportional to the triglyceride content after appropriate adjustment, using conventional clinical laboratory procedures, for whatever blank reaction is produced.
EXAMPLE 2 The procedure of Example I was performed using the same reactants, proportions, and conditions, except that a purified lipase obtained from the cultured broth of Candida cylindracea nov. sp. was substituted for the Candida lipase of the first example. Complete hydrolysis of the triglycerides of the sample were obtained within 5 minutes in the same manner as set forth in Example 1.
EXAMPLE 3 Several reagent combinations were prepared in accordance with Example 1 except that a variety of bile salts were used. The results were tabulated below. Each reagent combination contained 0.1 mg Candida lipase (activity of units), 3.0 mg pancreatic lipase (activity 180 units), and the amount of bile salt indicated. Times are given in minutes for completion percent) of hydrolysis of serum samples having triglycerides values of 100 mg percent (i.e., 100 mg per 100 ml water) and 280 mg percent.
TIMES REQUIRED FOR COMPLETION OF HYDROLYSIS WITH ENZYME COMBINATIONS INCLUDING DIFFERENT BILE SALTS Time min.)
Amount (mg) Time (min.)
for 100 mg7zv for 280 mg7z Sample Sample l Taurocholic 6.0 20
(2) Taurodeoxycholic 0.3 l2 l.() 7 7 (3) Tuurochenodcoxycholic l.5 14 3.0 12
(4) Taurodchydrocholic 6.0 l3 24.0 13
While in the foregoing an embodiment of the invention has been disclosed in considerable detail for purposes of illustration, it will be understood that many of those details may be varied without departing from the spirit and scope of the invention.
1 claim:
1. An enzymatic process for rapidly liberating glycerol from its esterifled form as a fatty acid ester in an aqueous fluid, comprising the step of mixing said fluid with a combination of Candida lipase, pancreatic lipase, and a bile salt selected from the group consisting of the alkali metal salts of taurodeoxycholic, taurocholic, taurochenodeoxycholic, and taurodehydrocholic acids.
2. The process of claim 1 in which said acid is taurocholic acid.
3. The process of claim 1 in which said acid is taurodeoxycholic acid.
4. The process of claim 1 in which said acid is taurochenodeoxycholic acid.
5. The process of claim 1 in which said acid is taurodchydrocholic acid.
6. The process of claim 1 in which said bile salt is a sodium salt.
7. The process of claim 1 in which said Candida lipase is obtained from a culture of Candida cylindracea.
8. The process of claim 1 in which said Candida lipase in said combination provides at least 0.28 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
9. The process of clairn 8 in which said Candida lipase provides at least 0.54 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
10. The process of claim 1 in which said pancreatic lipase in said combination provides at least 0.14 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
11. The process of claim 10 in which said pancreatic lipase provides at least 1.2 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
12. The process of claim 1 in which said combination includes at least 0.002 milligrams of said bile salt for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
13. The process of claim 12 in which said combination includes at least 0.02 milligrams of said bile salt for each microliter of aqueous fluid having a triglyceride value of O to 500 mg percent.
14. A reagent combination for the rapid hydrolysis of fatty acid esters to liberate glycerol therefrom, comprising a mixture of Candida lipase, pancreatic lipase, and a bile salt selected from the group consisting of the alkali metal salts of taurodeoxycholic, taurocholic, taurochenodeoxycholic, and taurodehydrocholic acids.
15. The combination of claim 14 in which said acid is taurocholic acid.
16. The combination of claim 14 in which said acid is taurodeoxycholic acid.
17. The combination of claim 14 in which said acid is taurochenodeoxycholic acid.
18. The combination of claim 14 in which said acid is taurodehydrocholic acid.
19. The combination of claim 14 in which said bile salt is a sodium salt.
20. The combination of claim 14 in which said Candida lipase is obtained from a culture of Candida cylindracea.
21. The combination of claim 14 in which said Candida lipase has at least 14 lipase units for each milliliter of reagent combination.
.22. The combination of claim 21 in which said Candida lipase has at least 27 lipase units for each milliliter of reagent combination.
23. The combination of claim 14 in which said pancreatic lipase has at least 7 lipase units for each milliliter of reagent combination.
24. The combination of claim 23 in which said pancreatic lipase has at least 60 lipase units for each milliliter of reagent combination.
25. The combination of claim 14 in which at least 0.1 milligrams of bile salt are provided in each milliliter of reagent combination.
26. The combination of claim 25 in which at least 1.0 milligrams of bile salt are provided for each milliliter of reagent combination.
27. The reagent combination of claim 14 in which said pancreatic lipase has an activity of about 10 to 100 lipase units per milligram.
28. The combination of claim 27 in which said pancreatic lipase has an activity of approximately 20 to lipase units per milligram.
29. The reagent combination of claim 14 in which said Candida lipase has an activity of approximately 30 to 800 lipase units per milligram.
30. The reagent combination of claim 29 in which said Candida lipase has an activity of approximately 200 to 800 lipase units per milligram.

Claims (30)

1. AN ENZYMES PROCESS FOR RAPIDLY LIBERATING GLYCEROL FROM ITS ESTERIFIED FORM AS A FATTY ACID ESTER IN AN AQUEOUS FLUID COMPRISING THE STEP OF MIXING SAID FLUID WITH COMBINATION OF CANDIDA LIPASE PANCREATIC LIPASE AND A BILE SALT SELECTED FROM THE GROUP CONSISTING OF THE ALKALI METAL SALTS OF TAURODEOXYCHOLIC RAUROCHOLIC TAUROCHENODNOYCHLIC AND TAURODEHYDROCHOLIC ACIDS.
2. The process of claim 1 in which said acid is taurocholic acid.
3. The process of claim 1 in which said acid is taurodeoxycholic acid.
4. The process of claim 1 in which said acid is taurochenodeoxycholic acid.
5. The process of claim 1 in which said acid is taurodehydrocholic acid.
6. The process of claim 1 in which said bile salt is a sodium salt.
7. The process of claim 1 in which said Candida lipase is obtained from a culture of Candida cylindracea.
8. The process of claim 1 in which said Candida lipase in said combination provides at least 0.28 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
9. The process of claim 8 in which said Candida lipase provides at least 0.54 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
10. The process of claim 1 in which said pancreatic lipase in said combination provides at least 0.14 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
11. The process of claim 10 in which said pancreatic lipase provides at least 1.2 units of lipase activity for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
12. The process of claim 1 in which said combination includes at least 0.002 milligrams of said bile salt for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
13. The process of claim 12 in which said combination includes at least 0.02 milligrams of said bile salt for each microliter of aqueous fluid having a triglyceride value of 0 to 500 mg percent.
14. A reagent combination for the rapid hydrolysis of fatty acid esters to liberate glycerol therefrom, comprising a mixture of Candida lipase, pancreatic lipase, and a bile salt selected from the group consisting of the alkali metal salts of taurodeoxycholic, taurocholic, taurochenodeoxycholic, and taurodehydrocholic acids.
15. The combination of cLaim 14 in which said acid is taurocholic acid.
16. The combination of claim 14 in which said acid is taurodeoxycholic acid.
17. The combination of claim 14 in which said acid is taurochenodeoxycholic acid.
18. The combination of claim 14 in which said acid is taurodehydrocholic acid.
19. The combination of claim 14 in which said bile salt is a sodium salt.
20. The combination of claim 14 in which said Candida lipase is obtained from a culture of Candida cylindracea.
21. The combination of claim 14 in which said Candida lipase has at least 14 lipase units for each milliliter of reagent combination.
22. The combination of claim 21 in which said Candida lipase has at least 27 lipase units for each milliliter of reagent combination.
23. The combination of claim 14 in which said pancreatic lipase has at least 7 lipase units for each milliliter of reagent combination.
24. The combination of claim 23 in which said pancreatic lipase has at least 60 lipase units for each milliliter of reagent combination.
25. The combination of claim 14 in which at least 0.1 milligrams of bile salt are provided in each milliliter of reagent combination.
26. The combination of claim 25 in which at least 1.0 milligrams of bile salt are provided for each milliliter of reagent combination.
27. The reagent combination of claim 14 in which said pancreatic lipase has an activity of about 10 to 100 lipase units per milligram.
28. The combination of claim 27 in which said pancreatic lipase has an activity of approximately 20 to 80 lipase units per milligram.
29. The reagent combination of claim 14 in which said Candida lipase has an activity of approximately 30 to 800 lipase units per milligram.
30. The reagent combination of claim 29 in which said Candida lipase has an activity of approximately 200 to 800 lipase units per milligram.
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Cited By (19)

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US4011045A (en) * 1975-02-14 1977-03-08 Bonderman Dean P Turbidity reduction in triglyceride standards
US4012287A (en) * 1975-11-18 1977-03-15 Dr. Bruno Lange Gmbh Method and reagent for the quantitative analysis of triglycerides
US4045297A (en) * 1975-12-15 1977-08-30 Monsanto Company Triglycerides determination method
US4056442A (en) * 1976-06-01 1977-11-01 The Dow Chemical Company Lipase composition for glycerol ester determination
US4178285A (en) * 1978-12-20 1979-12-11 Felts James M Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system
US4179334A (en) * 1976-08-19 1979-12-18 Eastman Kodak Company Hydrolysis of protein-bound triglycerides
US4241178A (en) * 1978-01-06 1980-12-23 Eastman Kodak Company Process and composition for the quantification of glycerol ATP and triglycerides
US4259440A (en) * 1979-05-21 1981-03-31 Miles Laboratories, Inc. Hydrolysis and assay of triglycerides
US4264589A (en) * 1978-12-20 1981-04-28 Felts James M Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system
US4275151A (en) * 1977-02-03 1981-06-23 Eastman Kodak Company Hydrolysis of protein-bound cholesterol esters
US4275152A (en) * 1977-02-03 1981-06-23 Eastman Kodak Company Hydrolysis of protein-bound cholesterol esters
US4309502A (en) * 1980-06-30 1982-01-05 Beckman Instruments, Inc. Enzymatic assay for glycerol and triglycerides and a reagent for use therein
US4322496A (en) * 1980-04-17 1982-03-30 Eastman Kodak Company Inhibition of lactate oxidase
US4343897A (en) * 1979-02-05 1982-08-10 Boehringer Mannheim Gmbh Reagent for the determination of lipase and process for preparing same
US5162201A (en) * 1987-04-01 1992-11-10 Toyo Jozo Co., Ltd. Analytical method making use of monoglyceride lipase
US5273898A (en) * 1986-10-17 1993-12-28 Noro Nordisk A/S Thermally stable and positionally non-specific lipase isolated from Candida
WO2003040091A2 (en) * 2001-11-05 2003-05-15 Novozymes A/S Fat splitting process
US8268305B1 (en) 2011-09-23 2012-09-18 Bio-Cat, Inc. Method and compositions to reduce serum levels of triacylglycerides in human beings using a fungal lipase
CN105506052A (en) * 2015-11-23 2016-04-20 浙江理工大学 A method of improving catalyzed resolution properties of free lipase in an organic phase

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CA1163908A (en) * 1980-10-01 1984-03-20 Shyun-Long Yun Method for eliminating turbidity in a biological fluid and reagent therefor

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US4186251A (en) * 1973-03-01 1980-01-29 Miles Laboratories, Inc. Composition and method for determination of cholesterol
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4011045A (en) * 1975-02-14 1977-03-08 Bonderman Dean P Turbidity reduction in triglyceride standards
US4012287A (en) * 1975-11-18 1977-03-15 Dr. Bruno Lange Gmbh Method and reagent for the quantitative analysis of triglycerides
US4045297A (en) * 1975-12-15 1977-08-30 Monsanto Company Triglycerides determination method
US4056442A (en) * 1976-06-01 1977-11-01 The Dow Chemical Company Lipase composition for glycerol ester determination
US4179334A (en) * 1976-08-19 1979-12-18 Eastman Kodak Company Hydrolysis of protein-bound triglycerides
US4275152A (en) * 1977-02-03 1981-06-23 Eastman Kodak Company Hydrolysis of protein-bound cholesterol esters
US4275151A (en) * 1977-02-03 1981-06-23 Eastman Kodak Company Hydrolysis of protein-bound cholesterol esters
US4241178A (en) * 1978-01-06 1980-12-23 Eastman Kodak Company Process and composition for the quantification of glycerol ATP and triglycerides
US4264589A (en) * 1978-12-20 1981-04-28 Felts James M Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system
US4178285A (en) * 1978-12-20 1979-12-11 Felts James M Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system
US4343897A (en) * 1979-02-05 1982-08-10 Boehringer Mannheim Gmbh Reagent for the determination of lipase and process for preparing same
US4259440A (en) * 1979-05-21 1981-03-31 Miles Laboratories, Inc. Hydrolysis and assay of triglycerides
US4322496A (en) * 1980-04-17 1982-03-30 Eastman Kodak Company Inhibition of lactate oxidase
US4309502A (en) * 1980-06-30 1982-01-05 Beckman Instruments, Inc. Enzymatic assay for glycerol and triglycerides and a reagent for use therein
US5273898A (en) * 1986-10-17 1993-12-28 Noro Nordisk A/S Thermally stable and positionally non-specific lipase isolated from Candida
US5162201A (en) * 1987-04-01 1992-11-10 Toyo Jozo Co., Ltd. Analytical method making use of monoglyceride lipase
WO2003040091A2 (en) * 2001-11-05 2003-05-15 Novozymes A/S Fat splitting process
WO2003040091A3 (en) * 2001-11-05 2003-11-27 Novozymes As Fat splitting process
US8268305B1 (en) 2011-09-23 2012-09-18 Bio-Cat, Inc. Method and compositions to reduce serum levels of triacylglycerides in human beings using a fungal lipase
US9555083B2 (en) 2011-09-23 2017-01-31 Bio-Cat, Inc. Methods and compositions to reduce serum levels of triacylglycerides in human beings using a fungal lipase
CN105506052A (en) * 2015-11-23 2016-04-20 浙江理工大学 A method of improving catalyzed resolution properties of free lipase in an organic phase

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FR2318925A1 (en) 1977-02-18

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