CA1050908A - Hydrolysis of triglycerides with combination of lipases - Google Patents
Hydrolysis of triglycerides with combination of lipasesInfo
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- CA1050908A CA1050908A CA230,635A CA230635A CA1050908A CA 1050908 A CA1050908 A CA 1050908A CA 230635 A CA230635 A CA 230635A CA 1050908 A CA1050908 A CA 1050908A
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- lipase
- combination
- acid
- candida
- units
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Abstract
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 (EC 3.1.1.3) and a microbial lipase (EC 3.1.1.3), particularly Candida lipase (EC 3.1.1.3), are mixed with the fatty acid ester in the presence of a bile salt.
Description
5~9~
Background and Summary An effective enzymatic test for the determination of serum triglycerides turns mainly on the development of a rapid and complete process for the hydrolysis of the tri-glycerides, since various procedures for measuring the glycerol formed by such a reaction are well known. Thus, as described by F.H. Kreutz in Klin. Wochshr. 40:362 (1962), glycerol may be readily determined by combining the following reactions:
Glycerol Kinase (1) Glycerol + ATP ~ Glycerol Phosphate ~ADP
(EC 2.701.30) Pyruvate Kinase
Background and Summary An effective enzymatic test for the determination of serum triglycerides turns mainly on the development of a rapid and complete process for the hydrolysis of the tri-glycerides, since various procedures for measuring the glycerol formed by such a reaction are well known. Thus, as described by F.H. Kreutz in Klin. Wochshr. 40:362 (1962), glycerol may be readily determined by combining the following reactions:
Glycerol Kinase (1) Glycerol + ATP ~ Glycerol Phosphate ~ADP
(EC 2.701.30) Pyruvate Kinase
(2) ADP -~ Phosphoenolpyruvate ~ -- > ATP ~ Pyruvate (EC 2.7.1.40) Lactic
(3) Pyruvate ~ NADH - --- - --------- ----~ Lactate -~ NAD
Dehydrogenase (EC 1.1.1.27) In this combination of reactions, one mole of NADH
is oxidized for each mole of glycerol phosphorylized; there-fore, a change in the optical density at 340 nm is a direct ; 20 measure of the amount of glycerol in the assay.
U.S. Patent 3,703,591 discloses that the prelimin~ry step, that of hydrolyzing the triglycerides to form glycerol, may be carried out enzymatically using a mixture of a l:ipase (EC 3.1.1.3) and a protease (EC 3.4.21.). While it has long been known that certain lipases (EC 3.1.1.3) may alone be used to hydrolyze triglycerides, and that the enæymatic ac-tivity might possibly be enhanced by the presence of a bile salt, prior procedures using only lipases (EC 3.1.1.3) (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 r~h/ I f - ~ ~5~
hydrolysis of -triglycerides rnay be achieved by using a com- :
bination consisting only of ccrtain lipases (I~C 3.1.1.3) along with a bile salt, thereby dispensing with the requirement of including a proteoly-tic enzyme such as chymotrypsin (EC 3.4.
21.1) as one of the reactan-ts. More specifically, 100 per-cent hydrolysis may be accomplished in 3 t:o 5 minutes using a combination of Candida lipase (EC 3.1.1.3) and pancreatic lipase (EC 3.1.1.3) with a bile salt such as sodi.um tauro-deoxycholate. 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 objeets and advantages of the invention will be apparent as the specification proceeds. Additional ref~ ~ ~ -.~ erenees diselosing the state of the art are: R.G.H. Morgan ::
ancl N.E. Hoffman, Bioehim. Biophys. Acta, 2~8:1~3 (1971);
R.I,. Ory, ~. Kiser and P.~. Pardel, Lipids, 4:261 (:L968);
H. Broekerhoff, J. Bio. Chem. 246:5828 (1971); P. Desnuelle, Enzymes, 7:575 (1972~; U.S. patent 2,527,305.
Deseription . :.
--It is preferred that enzymatic hydrolysis of tri- .
glycer3des by the coactive lipases (EC 3.1.1.3) be undertaken in the presence of the eomponents of the three additional reaction systems representecl in the equat.ions.given above so that all of such reactions may be undertaken simultaneously in a single operative procedure or, if desired, in a two step proeedure in which one of the components needed for the con-version of glycerol, such as the glycerol kinase (EC 2.7.1.30), is added to all of the other components after hydrolysis has occurred and an initial readlng of optical density has been macde. Sinee the varlous components necessary for the enzy-matic conversion of glycerol have alreacly been indicated and are well known in the prior art, and since the proportions of ' ' : ~.
- 2 - ..
ph/..................... ~.
,, . . ~ . ;.. . . . .
Sl~908 such components are also well known, a detailed discussion herein is believed unnecessary. It is believed sufficient to state that the combination of lipases (EC 3.1.1.3) and bile salt may be part of an assay mixture which includes one or more of those components known for use in the enzymatic con-version of glycerol and the concurrent oxidation of NADH in a colorimetric or spectrophotometric test.
The system responsiblP for the enzymatic hydrolysis of triglycerides comprises a coactive mixture of pancreatic lipase (EC 3.1.1.3), a microbial lipase ~EC 3.1.1.3), and a bile salt. A11 three components are essential for effective hydrolysis. A variety of pancreatic lipases lEC 3.1.1.3) having activity within the range of about 10 to 100 lipase units per milligram (mg), and preferably within the range of 20 to 80 units per mg, are believed suitable, an example being the pancreatic lipase ~EC 3.1.1.3) sold under the de-signation "PL3" by Worthington Chemical Company, of Freehold, New Jersey. The microbial lipase (EC 3.1.1.3) is more speci-fically a Candida lipase (EC 3.1.1.3) which may, for example, be obtained from the cultured broth of Candida cylindracea.
Such Candlda lipase (EC 3.1.1.3) 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 ~EC 3.1.1.3) are believed to be equally ef~
fective when used in combination with the pa~creatic lipase (EC 3.1.1.3) and bile salt of the triglyceride-hydrolyæing system.
While both pancreatic and Candida lipases (EC 3.
1.1.3) must be presentt it has been found that the proportional amounts of those constituents, measured in;terms of lipase units, may ~e varied considera~ly in accordance with selected time requirements for completion of hydrolysis. One lipase ., I .
` ~Sl)9~
unit of activity is tlle amount sufficient to release one mic-romole of acid per minute at 25 degrees C. from an olive oil emulsion containing gum acacia and 15 mg per milliliter (ml) sodium taurochola-te at a pH of about 8Ø Such definition of a lipase unit, and the procedure on which it is based, : are well established and are disclosed more fully in Worth-ington Enzyme Manual, p. 63 (1972).
More specifically, the amount of pancreatic lipase : (EC 3.1.1.3) in the reaction mixture should be at least .~ 10 0.14 lipase units for each microliter of body fluid ~blood serum or plasma) having a triglyceride value within the ..
ran~e of 0 to 500 mg per 100 ml (mg%) in order to achieve :
complete hydrolysis within 12. minutes. On the same basis, the :
: amount of Candida lipase (EC 3.1.1.3) in the mixture should . ..
be at least 0.23 units, and the amount of bile salt should b~ at least 0.002 mg, for each microliter of body fluid.
Where shorter reaction times are required or desired, the . amounts of such constituents must be increased. Thus, for -compl.ete hydrolysis within 3 to 5 minutes, at least 1.2 ' 20 pancreatic lipase (EC 3.1.1.3? units, .54 Candida lipase (EC 3.1.1.3) units, and 0.02 mg of bile salt, are required for each microliter of body fluid. Stated differently, in a reagent combination havin~ a volume of one. milliliter, the values (for hydrolyzing 50 microliters of serum or plasma having a triglyceride value of 0 - 500 mg ~ within 12 min-.utes) should he at least 7 pancreatic lipase (EC 3.1.1.3) units, 14 Candida lipase ~C 3,1.1.3~ units, and 0.1 mg bile salt; or at least 60 pancreatic lipase (EC 3.1.1~3) .
units, 27 Candida lipase (EC 3~1.1.3) units, and 1~0 mg bile salt (for the hydrolysis of 50 microliters of such ser-um or plasma within 3 to 5 minutes)............................. .
~Since speed in compl~tlng an ass~y may be important, : ,., : ph/
''~
, , . ., , , ,: .
~9(~8 particularly in connection with clinical diagnostic tests, a maximum time period for completion o~ hydrolysis in a clinical test has been arbitrarily set at approximately 12 minu-tes. It is to be understood, however, that in other ; tests where longer time periods are more acceptable, lower concentrations or amounts oE the respecti~e lipases and bile salt may be used.
As previously indicated, a bile salt is an essen-tial component of the system. While alka:Li 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 (EC 3.1.1.3) and pancreatic l:ipase (EC 3.1.1.3) under opt:imuln cond:itions, has ~een ~ound to produce faster results at lo~er 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 ~EC 3.1.1.3) in admixture with a bile salt, preferably the salt of taurodeoxycholic acid, reacts with the triglycerides in body fluids to produce com-plete hydrolysi 6 in periods as short as 3 minutes. Such a combination of reactants may be used i.n any test requi.ring the rapia and complete hydrolysis of triglycerides. The process and product of this in~ention may, for example, be used in conjunction with a complete triglyceride assay including the three glycerol-determining reackions described at the be-ginning of this application and well known in the prior art.
.
All of the components required for the complete colorimetric 3~ de-termination of triglycerides in body fluids may be premixed and lyophilized -~o provide a stable reagent set for clinical use.
`:
:
~' ph/J,,~
, . . . : . . : , : .
Example 1 .
~ reagent suitable for practi.cing this invention may be prepared by making the following 3 ml reaction mixture Pancreatic lipase (EC 3.1.1.3) [Worthington PL3~, 18Q
lipase units Candida lipase (EC 3.1.1.3) [Worthington], 80 lipase units Sodium taurodeoxycholic acid, 3mg LDH, 10 International Units (IU) PyruYate kinase, 10 IU
NADH, 0.75 micromoles Phosphoenolpyruvate, 1.5 micromoles ATP disodium, 0.5 micromoles Magnesium chloride, 0.0067 M
Potassium phosphate buffer, 0.1 M, pH 7~0 The assay is carried out by adding an ali~uot of ' ' liquid containing the triglyceride to be assayed, such as 50 microliters of serum or plasma with triglyceride values of 0 to 500 mg ~, to the above reaction mixture. Following incubation for approximately 5 minutes at a temperature be-tween 25 degrees C. to 37 degrees C., the optical density is 20 measured at 340 nm. Thereafter, 10 units of glycerol kinase ~ (EC 2.7.1.30) is added and the mixture is again incubated at ; 25 degrees C. to 37 degrees C. for another 5 minutes. The optical density is again determined at 340 nm, and the dif-ference in optical densities is proportional to the triglyceride content after appropriate adjustment, using conventional clini-cal laboratory procedure$, for whatever blank reaction is pro-duced.
. Example 2 ; Th~ procedure of Example 1 was performed using the 30 same reactants, proportions, and conditions, except that a purified lipase (EC 3.1.1.3) obtained from the cultured broth of Candida cylindracea nov. sp. was substituted for the Candida lipase (EC 3.1.1.3) of the first example. Complete hydxolysis of the triglycerides of the sample were obtained within 5 min- .
utes in the same manner as set forth in Example 1.
Examp.le 3 Several reagent combinations were parepare:d in ac-~ordance with Example 1 except that a variety of bile salts ph/ ' - ~s~
were used. The results were tabulated below. Each reagent combination contained 0.1 mg Candida lipase (EC 3.1.1.3) (ac-tivity of 80 units), 3.0 mg pancreatic lipase (EC 3.1.1.3) ~activity 180 units), and the amount of bile salt indicated.
Times are given in minutes for completion (100%) of hydrolysis of serum samples having triglycerldes values of 100 mg percent (i.e., 100 mg per 100 ml water) and 280 mg percent.
TI~S REQVIRED FOR COMPLETION OF
HYDROLYSIS WITH ENZYME COMBINATIONS
INCLUDING ~IFFERENT BILE SA~TS
Bile Salt (Na)Amount (mg) Time (min.) Time (min) for lOOmg % for 280mg %
Sample_ Sample (1) Taurocholic 6.0 20 ---12.0 14 -~-18.~ 10 ---24.0 5 12 30,0 5 10 (2) Taurodeoxycholic 0.3 12 1.0 7 1.5 5 6 3.0 4 5 - (3) Taurochenodeoxycholic 1.5 -- 14 3,0 -- 12 6.0 3 10 (4~ Taurodehydrocholic 24 0 13 ---30.0 10 ---While in the foregoing an embodiment of the invention has been disclosed in considerable detail for purposes Oe illustration, it will be understood that many of those details may be varied without departing from the spirit and scope of the invention.
' 7 :
: .
.
Dehydrogenase (EC 1.1.1.27) In this combination of reactions, one mole of NADH
is oxidized for each mole of glycerol phosphorylized; there-fore, a change in the optical density at 340 nm is a direct ; 20 measure of the amount of glycerol in the assay.
U.S. Patent 3,703,591 discloses that the prelimin~ry step, that of hydrolyzing the triglycerides to form glycerol, may be carried out enzymatically using a mixture of a l:ipase (EC 3.1.1.3) and a protease (EC 3.4.21.). While it has long been known that certain lipases (EC 3.1.1.3) may alone be used to hydrolyze triglycerides, and that the enæymatic ac-tivity might possibly be enhanced by the presence of a bile salt, prior procedures using only lipases (EC 3.1.1.3) (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 r~h/ I f - ~ ~5~
hydrolysis of -triglycerides rnay be achieved by using a com- :
bination consisting only of ccrtain lipases (I~C 3.1.1.3) along with a bile salt, thereby dispensing with the requirement of including a proteoly-tic enzyme such as chymotrypsin (EC 3.4.
21.1) as one of the reactan-ts. More specifically, 100 per-cent hydrolysis may be accomplished in 3 t:o 5 minutes using a combination of Candida lipase (EC 3.1.1.3) and pancreatic lipase (EC 3.1.1.3) with a bile salt such as sodi.um tauro-deoxycholate. 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 objeets and advantages of the invention will be apparent as the specification proceeds. Additional ref~ ~ ~ -.~ erenees diselosing the state of the art are: R.G.H. Morgan ::
ancl N.E. Hoffman, Bioehim. Biophys. Acta, 2~8:1~3 (1971);
R.I,. Ory, ~. Kiser and P.~. Pardel, Lipids, 4:261 (:L968);
H. Broekerhoff, J. Bio. Chem. 246:5828 (1971); P. Desnuelle, Enzymes, 7:575 (1972~; U.S. patent 2,527,305.
Deseription . :.
--It is preferred that enzymatic hydrolysis of tri- .
glycer3des by the coactive lipases (EC 3.1.1.3) be undertaken in the presence of the eomponents of the three additional reaction systems representecl in the equat.ions.given above so that all of such reactions may be undertaken simultaneously in a single operative procedure or, if desired, in a two step proeedure in which one of the components needed for the con-version of glycerol, such as the glycerol kinase (EC 2.7.1.30), is added to all of the other components after hydrolysis has occurred and an initial readlng of optical density has been macde. Sinee the varlous components necessary for the enzy-matic conversion of glycerol have alreacly been indicated and are well known in the prior art, and since the proportions of ' ' : ~.
- 2 - ..
ph/..................... ~.
,, . . ~ . ;.. . . . .
Sl~908 such components are also well known, a detailed discussion herein is believed unnecessary. It is believed sufficient to state that the combination of lipases (EC 3.1.1.3) and bile salt may be part of an assay mixture which includes one or more of those components known for use in the enzymatic con-version of glycerol and the concurrent oxidation of NADH in a colorimetric or spectrophotometric test.
The system responsiblP for the enzymatic hydrolysis of triglycerides comprises a coactive mixture of pancreatic lipase (EC 3.1.1.3), a microbial lipase ~EC 3.1.1.3), and a bile salt. A11 three components are essential for effective hydrolysis. A variety of pancreatic lipases lEC 3.1.1.3) having activity within the range of about 10 to 100 lipase units per milligram (mg), and preferably within the range of 20 to 80 units per mg, are believed suitable, an example being the pancreatic lipase ~EC 3.1.1.3) sold under the de-signation "PL3" by Worthington Chemical Company, of Freehold, New Jersey. The microbial lipase (EC 3.1.1.3) is more speci-fically a Candida lipase (EC 3.1.1.3) which may, for example, be obtained from the cultured broth of Candida cylindracea.
Such Candlda lipase (EC 3.1.1.3) 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 ~EC 3.1.1.3) are believed to be equally ef~
fective when used in combination with the pa~creatic lipase (EC 3.1.1.3) and bile salt of the triglyceride-hydrolyæing system.
While both pancreatic and Candida lipases (EC 3.
1.1.3) must be presentt it has been found that the proportional amounts of those constituents, measured in;terms of lipase units, may ~e varied considera~ly in accordance with selected time requirements for completion of hydrolysis. One lipase ., I .
` ~Sl)9~
unit of activity is tlle amount sufficient to release one mic-romole of acid per minute at 25 degrees C. from an olive oil emulsion containing gum acacia and 15 mg per milliliter (ml) sodium taurochola-te at a pH of about 8Ø Such definition of a lipase unit, and the procedure on which it is based, : are well established and are disclosed more fully in Worth-ington Enzyme Manual, p. 63 (1972).
More specifically, the amount of pancreatic lipase : (EC 3.1.1.3) in the reaction mixture should be at least .~ 10 0.14 lipase units for each microliter of body fluid ~blood serum or plasma) having a triglyceride value within the ..
ran~e of 0 to 500 mg per 100 ml (mg%) in order to achieve :
complete hydrolysis within 12. minutes. On the same basis, the :
: amount of Candida lipase (EC 3.1.1.3) in the mixture should . ..
be at least 0.23 units, and the amount of bile salt should b~ at least 0.002 mg, for each microliter of body fluid.
Where shorter reaction times are required or desired, the . amounts of such constituents must be increased. Thus, for -compl.ete hydrolysis within 3 to 5 minutes, at least 1.2 ' 20 pancreatic lipase (EC 3.1.1.3? units, .54 Candida lipase (EC 3.1.1.3) units, and 0.02 mg of bile salt, are required for each microliter of body fluid. Stated differently, in a reagent combination havin~ a volume of one. milliliter, the values (for hydrolyzing 50 microliters of serum or plasma having a triglyceride value of 0 - 500 mg ~ within 12 min-.utes) should he at least 7 pancreatic lipase (EC 3.1.1.3) units, 14 Candida lipase ~C 3,1.1.3~ units, and 0.1 mg bile salt; or at least 60 pancreatic lipase (EC 3.1.1~3) .
units, 27 Candida lipase (EC 3~1.1.3) units, and 1~0 mg bile salt (for the hydrolysis of 50 microliters of such ser-um or plasma within 3 to 5 minutes)............................. .
~Since speed in compl~tlng an ass~y may be important, : ,., : ph/
''~
, , . ., , , ,: .
~9(~8 particularly in connection with clinical diagnostic tests, a maximum time period for completion o~ hydrolysis in a clinical test has been arbitrarily set at approximately 12 minu-tes. It is to be understood, however, that in other ; tests where longer time periods are more acceptable, lower concentrations or amounts oE the respecti~e lipases and bile salt may be used.
As previously indicated, a bile salt is an essen-tial component of the system. While alka:Li 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 (EC 3.1.1.3) and pancreatic l:ipase (EC 3.1.1.3) under opt:imuln cond:itions, has ~een ~ound to produce faster results at lo~er 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 ~EC 3.1.1.3) in admixture with a bile salt, preferably the salt of taurodeoxycholic acid, reacts with the triglycerides in body fluids to produce com-plete hydrolysi 6 in periods as short as 3 minutes. Such a combination of reactants may be used i.n any test requi.ring the rapia and complete hydrolysis of triglycerides. The process and product of this in~ention may, for example, be used in conjunction with a complete triglyceride assay including the three glycerol-determining reackions described at the be-ginning of this application and well known in the prior art.
.
All of the components required for the complete colorimetric 3~ de-termination of triglycerides in body fluids may be premixed and lyophilized -~o provide a stable reagent set for clinical use.
`:
:
~' ph/J,,~
, . . . : . . : , : .
Example 1 .
~ reagent suitable for practi.cing this invention may be prepared by making the following 3 ml reaction mixture Pancreatic lipase (EC 3.1.1.3) [Worthington PL3~, 18Q
lipase units Candida lipase (EC 3.1.1.3) [Worthington], 80 lipase units Sodium taurodeoxycholic acid, 3mg LDH, 10 International Units (IU) PyruYate kinase, 10 IU
NADH, 0.75 micromoles Phosphoenolpyruvate, 1.5 micromoles ATP disodium, 0.5 micromoles Magnesium chloride, 0.0067 M
Potassium phosphate buffer, 0.1 M, pH 7~0 The assay is carried out by adding an ali~uot of ' ' liquid containing the triglyceride to be assayed, such as 50 microliters of serum or plasma with triglyceride values of 0 to 500 mg ~, to the above reaction mixture. Following incubation for approximately 5 minutes at a temperature be-tween 25 degrees C. to 37 degrees C., the optical density is 20 measured at 340 nm. Thereafter, 10 units of glycerol kinase ~ (EC 2.7.1.30) is added and the mixture is again incubated at ; 25 degrees C. to 37 degrees C. for another 5 minutes. The optical density is again determined at 340 nm, and the dif-ference in optical densities is proportional to the triglyceride content after appropriate adjustment, using conventional clini-cal laboratory procedure$, for whatever blank reaction is pro-duced.
. Example 2 ; Th~ procedure of Example 1 was performed using the 30 same reactants, proportions, and conditions, except that a purified lipase (EC 3.1.1.3) obtained from the cultured broth of Candida cylindracea nov. sp. was substituted for the Candida lipase (EC 3.1.1.3) of the first example. Complete hydxolysis of the triglycerides of the sample were obtained within 5 min- .
utes in the same manner as set forth in Example 1.
Examp.le 3 Several reagent combinations were parepare:d in ac-~ordance with Example 1 except that a variety of bile salts ph/ ' - ~s~
were used. The results were tabulated below. Each reagent combination contained 0.1 mg Candida lipase (EC 3.1.1.3) (ac-tivity of 80 units), 3.0 mg pancreatic lipase (EC 3.1.1.3) ~activity 180 units), and the amount of bile salt indicated.
Times are given in minutes for completion (100%) of hydrolysis of serum samples having triglycerldes values of 100 mg percent (i.e., 100 mg per 100 ml water) and 280 mg percent.
TI~S REQVIRED FOR COMPLETION OF
HYDROLYSIS WITH ENZYME COMBINATIONS
INCLUDING ~IFFERENT BILE SA~TS
Bile Salt (Na)Amount (mg) Time (min.) Time (min) for lOOmg % for 280mg %
Sample_ Sample (1) Taurocholic 6.0 20 ---12.0 14 -~-18.~ 10 ---24.0 5 12 30,0 5 10 (2) Taurodeoxycholic 0.3 12 1.0 7 1.5 5 6 3.0 4 5 - (3) Taurochenodeoxycholic 1.5 -- 14 3,0 -- 12 6.0 3 10 (4~ Taurodehydrocholic 24 0 13 ---30.0 10 ---While in the foregoing an embodiment of the invention has been disclosed in considerable detail for purposes Oe illustration, it will be understood that many of those details may be varied without departing from the spirit and scope of the invention.
' 7 :
: .
.
Claims (30)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An enzymatic 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 a combination of Candida lipase (EC 3.1.1.3), pancreatic lipase (EC 3.1.1.3), 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 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 (EC 3.1.1.3) is obtained from a culture of Candida cylindracea.
8. The process of Claim 1 in which said Candida lipase (EC 3.1.1.3) 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 (EC 3.1.1.3) provides at least 0.54 units of lipase activity for each microliter of aqueous fluid having a tri-glyceride value of 0 to 500 mg percent.
10. The process of Claim 1 in which said pan-creatic lipase (EC 3.1.1.3) 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 per-cent.
11. The process of Claim 10 in which said pan-creatic lipase (EC 3.1.1.3) provides at least 1.2 units of lip-ase 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 com-bination 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 com-bination 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 hydroly-sis of fatty acid esters to liberate glycerol therefrom, comprising a mixture of Candida lipase (EC 3.1.1.3), pancreatic lipase (EC 3.1.1.3) 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 (EC 3.1.1.3) is obtained from a culture of Candida cylindracea.
21. The combination of Claim 14 in which said Candida lipase (EC 3.1.1.3) has at least 14 lipase units for each milliliter of reagent combination.
22. The combination of Claim 21 in which said Candida lipase (EC 3.1.1.3) has at least 27 lipase units for each milliliter of reagent combination.
23. The combination of Claim 14 in which said pancreatic lipase (EC 3.1.1.3) has at least 7 lipase units for each milliliter of reagent combination.
24. The combination of Claim 23 in which said pancreatic lipase (EC 3.1.1.3) 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 (EC 3.1.1.3) has an activity of about 10 to 100 lipase units per milligram.
28. The combination of Claim 27 in which said pancreatic lipase (EC 3.1.1.3) has an activity of approxi-mately 20 to 80 lipase units per milligram.
29. The reagent combination of Claim 14 in which said Candida lipase (EC 3.1.1.3) has an activity of approximately 30 to 800 lipase units per milligram.
30. The reagent combination of Claim 29 in which said Candida lipase (EC 3.1.1.3) has an activity of approximately 200 to 800 lipase units per milligram.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA230,635A CA1050908A (en) | 1975-07-02 | 1975-07-02 | Hydrolysis of triglycerides with combination of lipases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA230,635A CA1050908A (en) | 1975-07-02 | 1975-07-02 | Hydrolysis of triglycerides with combination of lipases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1050908A true CA1050908A (en) | 1979-03-20 |
Family
ID=4103505
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA230,635A Expired CA1050908A (en) | 1975-07-02 | 1975-07-02 | Hydrolysis of triglycerides with combination of lipases |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1050908A (en) |
-
1975
- 1975-07-02 CA CA230,635A patent/CA1050908A/en not_active Expired
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