CA1109373A - Method of determining lipase activity using a novel triglyceride reagent and method for preparing that reagent - Google Patents
Method of determining lipase activity using a novel triglyceride reagent and method for preparing that reagentInfo
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- CA1109373A CA1109373A CA277,628A CA277628A CA1109373A CA 1109373 A CA1109373 A CA 1109373A CA 277628 A CA277628 A CA 277628A CA 1109373 A CA1109373 A CA 1109373A
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- excipient
- triglyceride
- triolein
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- 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
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Abstract
ABSTRACT
An improved method of determining lipase activity in a sample by reacting the sample with a triglyceride emulsion and measuring the rate at which the triglyceride is hydrolyzed into fractions by the lipase in which the emulsion is formed by providing, as a reagent, a solid matrix of an excipient uniformly impregnated with the trigylceride, and the solid matrix is dissolved in an aqueous reaction mixture to release the triglyceride.
The reagent is prepared by forming a solution of the triglyceride with a solvent, mixing the solution with an excipient and evaporating the solvent to leave a solid matrix of the excipient impregnated with the triglyceride.
An improved method of determining lipase activity in a sample by reacting the sample with a triglyceride emulsion and measuring the rate at which the triglyceride is hydrolyzed into fractions by the lipase in which the emulsion is formed by providing, as a reagent, a solid matrix of an excipient uniformly impregnated with the trigylceride, and the solid matrix is dissolved in an aqueous reaction mixture to release the triglyceride.
The reagent is prepared by forming a solution of the triglyceride with a solvent, mixing the solution with an excipient and evaporating the solvent to leave a solid matrix of the excipient impregnated with the triglyceride.
Description
~3~
BACKGROUND OF T~IE INVENTION
1. Field of the Invention: The presen-t invention relates to a method for determining the lipase activity of a sample by reacting the sample with a triglyceride emulsion, and to a method of preparing the triglyceride reagent used to form the emulsion.
BACKGROUND OF T~IE INVENTION
1. Field of the Invention: The presen-t invention relates to a method for determining the lipase activity of a sample by reacting the sample with a triglyceride emulsion, and to a method of preparing the triglyceride reagent used to form the emulsion.
2. Discussion of the Prior Art: Lipase is an enzyme .
produced by the pancreas. Lipase activity in blood or blood serum is increased by pancreatitis and other diseases of the pancreas. A measure of lipase activity in blood or blood serum is, therefore, a useful diagnostic tool to physicians.
A traditional method of measurinq lipase activity is the turbidimetri.c method described ~y Shihabi and Bishop in Clinical Chemistry, 17, pp. 1150-1153 (1971), in which the decrease in absorbance oE an olive oil/water emulsion is related to lipase activity. Olive oil is a mixture of substances called collectively triglyceride. When mixed in aqueous solution, triglyceride forms an emulsion. Light incident on this emulsion is scattered by the oil droplets so that very little light is transmitted through the emul~
sion. Lipase hydrolyzes triglyceride into fragments which are soluble in the reaction mixture. As the triglyceride is hydrolyzed, light scattering by the emulsion is decreased and transmission of the light through the medium is increased.
It is currently believed that the lipase attacks the triglyceride at the liquid-oil interface so that the emulsified state of the triglyceride is important to the reaction. Important as it is, current techniques of forming the triglyceride emulsion are inadequate. Triglyceride emulsions formed in a homogenizer, such as a blender,or by a solvent/aqueous mixture, exhibit limited stability ~four to eight weeks). In addition, they are difficult to make and in general lack reproducibility. Both deficiencies limit the usefulness of the assay in automated analyzers.
SUMMARY OF T~IE INVENTION
The present invention provides a method for deter-mining the lipase activity of a sample by reacting the sample with an emulsion of a triglyceride in an aqueous reaction solution, and measuring the rate at which the triglyceride is hydrolyzed to soluble fragments. The emulsion is formed by providing as the triglyceride reagent, a solid matrix of an excipient uniformly impregnated with the triglyceride, the excipient being soluble in the aqueous reaction solution, and dissolving the solid matrix to release the triglyceride, as an emulsion, into thé aqueous reaction solution.
In the preferred embodiment, the excipient com-prises polyethylene glycol and a sugar or sugar alcohol such as mannitol, inositol, sorbitol, maltose, dextrose, lactose, dextran and mixtures thereof; and it is impreg-nated with triolein, one of the major triglycerides.
The invention also provides a process for pre-paring a reagent for use in conjunction with an aqueous reaction solution to determine the lipase activity of a sample, comprising the steps of:
(a) ~orming a liquid solution of at least one trigly-ceride (e.g., triolein) with a solvent for that triglyceride;
(b) mixing the liquid solution with a predetermined quantity of an excipient which is soluble in the aqueous reaction solution, the volume of solvent used to form the ~ ~ ~3~3~
liquid solution being chosen to substantially saturate the excipient; and (c) evaporating the solvent to leave a solid matrix of the excipient impregnated with the triglyceride, the amount of triglyceride used to form the liquid solution being chosen to give the desired concentration of trigly-ceride per weight of solid matrix.
DESCRIPTION OF THE PREFERRF,D EMBODIMEN~
The Eirst step in the process of preparing the triglyceride reagent for use in determining the lipase activity of a sample, particularly a sample of blood or blood serum, is to dissolve the triglyceride in a suitable solvent. The triglyceride is a substrate for the lipase.
In the past, purified olive oil or triolein, one of the major triglycerides, have been used. For convenience, the discussion which follows will be limited to a discussion of triolein, but lipase will react with any triglyceride, so any -triglyceride or combination of triglycerides, can be used in the present invention.
The solvent used to dissolve the triglyceride will, of course, depend upon the triglyceride chosen.
Numerous solvents for triglyceride are known to those skilled in the art. The solvent used to form the trigly-ceride reagent of the present invention should satisfy four criteria. It must dissolve the particular trigly-ceride used. It must not dissolve the particular excipient chosen. It must evaporate under conditions which will not harm the triglyceride or the excipient. Finally, it must leave no residue or, at least, only an inert residue,
produced by the pancreas. Lipase activity in blood or blood serum is increased by pancreatitis and other diseases of the pancreas. A measure of lipase activity in blood or blood serum is, therefore, a useful diagnostic tool to physicians.
A traditional method of measurinq lipase activity is the turbidimetri.c method described ~y Shihabi and Bishop in Clinical Chemistry, 17, pp. 1150-1153 (1971), in which the decrease in absorbance oE an olive oil/water emulsion is related to lipase activity. Olive oil is a mixture of substances called collectively triglyceride. When mixed in aqueous solution, triglyceride forms an emulsion. Light incident on this emulsion is scattered by the oil droplets so that very little light is transmitted through the emul~
sion. Lipase hydrolyzes triglyceride into fragments which are soluble in the reaction mixture. As the triglyceride is hydrolyzed, light scattering by the emulsion is decreased and transmission of the light through the medium is increased.
It is currently believed that the lipase attacks the triglyceride at the liquid-oil interface so that the emulsified state of the triglyceride is important to the reaction. Important as it is, current techniques of forming the triglyceride emulsion are inadequate. Triglyceride emulsions formed in a homogenizer, such as a blender,or by a solvent/aqueous mixture, exhibit limited stability ~four to eight weeks). In addition, they are difficult to make and in general lack reproducibility. Both deficiencies limit the usefulness of the assay in automated analyzers.
SUMMARY OF T~IE INVENTION
The present invention provides a method for deter-mining the lipase activity of a sample by reacting the sample with an emulsion of a triglyceride in an aqueous reaction solution, and measuring the rate at which the triglyceride is hydrolyzed to soluble fragments. The emulsion is formed by providing as the triglyceride reagent, a solid matrix of an excipient uniformly impregnated with the triglyceride, the excipient being soluble in the aqueous reaction solution, and dissolving the solid matrix to release the triglyceride, as an emulsion, into thé aqueous reaction solution.
In the preferred embodiment, the excipient com-prises polyethylene glycol and a sugar or sugar alcohol such as mannitol, inositol, sorbitol, maltose, dextrose, lactose, dextran and mixtures thereof; and it is impreg-nated with triolein, one of the major triglycerides.
The invention also provides a process for pre-paring a reagent for use in conjunction with an aqueous reaction solution to determine the lipase activity of a sample, comprising the steps of:
(a) ~orming a liquid solution of at least one trigly-ceride (e.g., triolein) with a solvent for that triglyceride;
(b) mixing the liquid solution with a predetermined quantity of an excipient which is soluble in the aqueous reaction solution, the volume of solvent used to form the ~ ~ ~3~3~
liquid solution being chosen to substantially saturate the excipient; and (c) evaporating the solvent to leave a solid matrix of the excipient impregnated with the triglyceride, the amount of triglyceride used to form the liquid solution being chosen to give the desired concentration of trigly-ceride per weight of solid matrix.
DESCRIPTION OF THE PREFERRF,D EMBODIMEN~
The Eirst step in the process of preparing the triglyceride reagent for use in determining the lipase activity of a sample, particularly a sample of blood or blood serum, is to dissolve the triglyceride in a suitable solvent. The triglyceride is a substrate for the lipase.
In the past, purified olive oil or triolein, one of the major triglycerides, have been used. For convenience, the discussion which follows will be limited to a discussion of triolein, but lipase will react with any triglyceride, so any -triglyceride or combination of triglycerides, can be used in the present invention.
The solvent used to dissolve the triglyceride will, of course, depend upon the triglyceride chosen.
Numerous solvents for triglyceride are known to those skilled in the art. The solvent used to form the trigly-ceride reagent of the present invention should satisfy four criteria. It must dissolve the particular trigly-ceride used. It must not dissolve the particular excipient chosen. It must evaporate under conditions which will not harm the triglyceride or the excipient. Finally, it must leave no residue or, at least, only an inert residue,
3,~ 3 i.e., one that is not reactive with or harmEul to the other reagent used.
~ olvents such as chloroform and ethanol will dissolve triolein. It has been found that chloroform or ethanol or mixtures of chloroform, preferably mixtures con-taining up to about 50~ ethanol,will produce a uniform solution of triolein.
The volume of solvent usecl to dissolve the tri-glyceride is chosen so that when the liquid triglyceride solution is applied to the excipient, the liquid will just saturate the solid material. If too little solvent is used, a non-uniform distribution of the solvent, and hence the triglyceride, throughout the solid will occur. I~hen the powder is divided, this will produce reagent portions with variable concentrations of triglyceride. If too much solvent is used, not all of the solution, and in particular, the triglyceride which it contains, will impregnate the solid. If the solid is drained before the drying step, this will lead to a loss of some of the triglyceride; if not, that portion of the triglyceride contained in the solution which does not impregnate the solid will coat the solid in a non-uniform manner. Depending upon the precision required, each of these conditions can be toler-ated, but for best results, the amount of solvent used should be chosen so that the triglyceride solution sub-stantially saturates the solid material.
The amount of triglyceride used in the solution is a matter of choice. The amount used should be chosen to yield the desired concentration of triglyceride per given volume of the solid matrix formed when the solvent is evaporated.
. .
._ ., By definition, an excipient is an inert substance that forms a vehicle for a particular reagent, in this case, the triglyceride. The excipient chosen to ~orm the solid matrix of the present invention should satisfy three criteria.
It must not dissolve in the solvent chosen to dissolve the triglyceride. It must dissolve in the medium in which the lipase assay takes place. Such dissolu-tion is required to release ~he triglyceride into the reaction solution to form the emulsion. The reaction solution is normally an aqueous solution which is maintained at a pH of between about 8.5 and about 9.5. Finally, the excipient should be inert and nondetectable in the assay. There are many materials known to those skilled in the art which qualify for use as excipients in the present invention. Common among them are sugars and sugar alcohols, such as mannitol, inositol~ sorbitol, maltose, dextrose, lactose, dextran and mixtures thereoE.
A more uniform distribution of the triglyceride in the solid matrix will be formed if, in addition to the 2d substances referred to above, polyethylene glycol is used. Carbowax~ 6000 is one suitable substance. This substance prevents agglomeration of the triglyceride droplets in the solid matrix and aids in producing a uniform coating of the triglyceride solution on the sur-face of the solid matrix; The polyethylene glycol, how-ever, is not absolutely necessary.
Once the triglyceride and the solvent for the triglyceride have been chosen and mixed to form the desired solution, the triglyceride solution and the excipient are blended together. This can be done in a number of ways known to those skilled in the art. In particular, a ~lobart~
mechanical mixer can be used. ~ixing continues until the excipient is just saturated. The saturated excipient is then dried to evaporate the solve~nt used to dissolve the triglyceride, and a solid matrix uniformly impregnated with the triglyceride is formed. Drying can be accomplished in any number of ways well known to those skilled in the art.
In particular, air drying in a fume hood can be used. The impregnated solid matrix can be used as a powder, or it 1~ can be tableted using conventional tableting techniques.
As a particular example of the formation oE the triglyceride impregnated matrix, 94 parts of mannitol and 5 parts of Carbowax~ 6000 are blended together to form an excipient. One part of triolein is dissolved in a 50~
chloroform, 50~ ethanol mixture, the amount of solvent being chosen such that when blended with the excipient, a just saturated solid blend is formed. The solvent mix-ture containing the triolein is then added to the mannitol-Carbowax~ 6000 blend in a Hobart~ mechanical mixer and blended for 30 minutes. The saturated excipient so formed is then air dried for 60 minutes, or until the solvent has completely evaporated, in a fume hood. A solid matrix uniformly impregnated with triolein is formed. This solid matrix is in powder form. The impregnated blend is then tableted to yield approximately 85 mg. tablets contain-ing about 0.87 mg. of triolein for each tablet.
An assay to determine lipase activity in human serum is then performed by a kinetic turbidimetric approach in which the rate of "clearing" of the triolein emulsion is monitored at 340 nm. This rate is proportional to the lipase activity of the sample. Calcium chloride and bile salts are used as activators for the lipase. The system is also buffered using tris(hydroxylmethyl) aminomethane ("Tris") buffer which serves as a pH control (pH of 8.6 to 9.0) for the aqueous reaction solution. The reaction proceeds as follows:
Triolein Emulsion ~ ~ Free Fatty ~cids pH 8.8 (25C) Mixed Glycerides (Turbid) (Decreasing turbidity) As a particular example of a lipase assay, a test kit containing the following reagents is prepared:
Tris Buffer, pH 8.8 -~ 0.2; 50 ~Mol Deoxycholate 35.5 ~Mol Calcium chloride 0.7 ~Mol Triolein 0.98 ~Mol (in the tableted form described above) These reagents are added to 4.8 ml. of water, and 0.20 ml.
of sample containing an unknown amount of lipase is then added to the solution. With a small amount of mixing, the excipient in the tablet dissolves and a triolein emulsion is formed in the aqueous reaction solution. ~s the lipase enzyme contained in the sample reacts with the triolein emulsion, free fatty acids and mixed glycerides are formed and the mixture becomes increasing optically transparent.
The rate of "clearing" of the triolein emulsion as moni-tored at 340 nm. is found to be proportional to the lipase activity.
The use of a tablet described above yields highly reproducible emulsions and imparts long-term stability to the substrate (at least 12 months).
The above description is intended to teach those skilled in the art how to make and use the triglyceride 3~33~
reagent of the pre.sent invention. It is not intendcd to limit the scope of the invention as set forth in the appended claims.
~ olvents such as chloroform and ethanol will dissolve triolein. It has been found that chloroform or ethanol or mixtures of chloroform, preferably mixtures con-taining up to about 50~ ethanol,will produce a uniform solution of triolein.
The volume of solvent usecl to dissolve the tri-glyceride is chosen so that when the liquid triglyceride solution is applied to the excipient, the liquid will just saturate the solid material. If too little solvent is used, a non-uniform distribution of the solvent, and hence the triglyceride, throughout the solid will occur. I~hen the powder is divided, this will produce reagent portions with variable concentrations of triglyceride. If too much solvent is used, not all of the solution, and in particular, the triglyceride which it contains, will impregnate the solid. If the solid is drained before the drying step, this will lead to a loss of some of the triglyceride; if not, that portion of the triglyceride contained in the solution which does not impregnate the solid will coat the solid in a non-uniform manner. Depending upon the precision required, each of these conditions can be toler-ated, but for best results, the amount of solvent used should be chosen so that the triglyceride solution sub-stantially saturates the solid material.
The amount of triglyceride used in the solution is a matter of choice. The amount used should be chosen to yield the desired concentration of triglyceride per given volume of the solid matrix formed when the solvent is evaporated.
. .
._ ., By definition, an excipient is an inert substance that forms a vehicle for a particular reagent, in this case, the triglyceride. The excipient chosen to ~orm the solid matrix of the present invention should satisfy three criteria.
It must not dissolve in the solvent chosen to dissolve the triglyceride. It must dissolve in the medium in which the lipase assay takes place. Such dissolu-tion is required to release ~he triglyceride into the reaction solution to form the emulsion. The reaction solution is normally an aqueous solution which is maintained at a pH of between about 8.5 and about 9.5. Finally, the excipient should be inert and nondetectable in the assay. There are many materials known to those skilled in the art which qualify for use as excipients in the present invention. Common among them are sugars and sugar alcohols, such as mannitol, inositol~ sorbitol, maltose, dextrose, lactose, dextran and mixtures thereoE.
A more uniform distribution of the triglyceride in the solid matrix will be formed if, in addition to the 2d substances referred to above, polyethylene glycol is used. Carbowax~ 6000 is one suitable substance. This substance prevents agglomeration of the triglyceride droplets in the solid matrix and aids in producing a uniform coating of the triglyceride solution on the sur-face of the solid matrix; The polyethylene glycol, how-ever, is not absolutely necessary.
Once the triglyceride and the solvent for the triglyceride have been chosen and mixed to form the desired solution, the triglyceride solution and the excipient are blended together. This can be done in a number of ways known to those skilled in the art. In particular, a ~lobart~
mechanical mixer can be used. ~ixing continues until the excipient is just saturated. The saturated excipient is then dried to evaporate the solve~nt used to dissolve the triglyceride, and a solid matrix uniformly impregnated with the triglyceride is formed. Drying can be accomplished in any number of ways well known to those skilled in the art.
In particular, air drying in a fume hood can be used. The impregnated solid matrix can be used as a powder, or it 1~ can be tableted using conventional tableting techniques.
As a particular example of the formation oE the triglyceride impregnated matrix, 94 parts of mannitol and 5 parts of Carbowax~ 6000 are blended together to form an excipient. One part of triolein is dissolved in a 50~
chloroform, 50~ ethanol mixture, the amount of solvent being chosen such that when blended with the excipient, a just saturated solid blend is formed. The solvent mix-ture containing the triolein is then added to the mannitol-Carbowax~ 6000 blend in a Hobart~ mechanical mixer and blended for 30 minutes. The saturated excipient so formed is then air dried for 60 minutes, or until the solvent has completely evaporated, in a fume hood. A solid matrix uniformly impregnated with triolein is formed. This solid matrix is in powder form. The impregnated blend is then tableted to yield approximately 85 mg. tablets contain-ing about 0.87 mg. of triolein for each tablet.
An assay to determine lipase activity in human serum is then performed by a kinetic turbidimetric approach in which the rate of "clearing" of the triolein emulsion is monitored at 340 nm. This rate is proportional to the lipase activity of the sample. Calcium chloride and bile salts are used as activators for the lipase. The system is also buffered using tris(hydroxylmethyl) aminomethane ("Tris") buffer which serves as a pH control (pH of 8.6 to 9.0) for the aqueous reaction solution. The reaction proceeds as follows:
Triolein Emulsion ~ ~ Free Fatty ~cids pH 8.8 (25C) Mixed Glycerides (Turbid) (Decreasing turbidity) As a particular example of a lipase assay, a test kit containing the following reagents is prepared:
Tris Buffer, pH 8.8 -~ 0.2; 50 ~Mol Deoxycholate 35.5 ~Mol Calcium chloride 0.7 ~Mol Triolein 0.98 ~Mol (in the tableted form described above) These reagents are added to 4.8 ml. of water, and 0.20 ml.
of sample containing an unknown amount of lipase is then added to the solution. With a small amount of mixing, the excipient in the tablet dissolves and a triolein emulsion is formed in the aqueous reaction solution. ~s the lipase enzyme contained in the sample reacts with the triolein emulsion, free fatty acids and mixed glycerides are formed and the mixture becomes increasing optically transparent.
The rate of "clearing" of the triolein emulsion as moni-tored at 340 nm. is found to be proportional to the lipase activity.
The use of a tablet described above yields highly reproducible emulsions and imparts long-term stability to the substrate (at least 12 months).
The above description is intended to teach those skilled in the art how to make and use the triglyceride 3~33~
reagent of the pre.sent invention. It is not intendcd to limit the scope of the invention as set forth in the appended claims.
Claims (18)
1. In a method for determining the lipase activity of a sample by reacting the sample with an emulsion of a triglyceride in an aqueous reaction solution and measuring the rate at which the triglyceride is hydrolyzed to soluble fragments, the improvement wherein said emulsion is formed by providing a solid matrix of an excipient uniformly impregnated with said triglyceride, said excipient being soluble in said aqueous reaction solution, and dissolving said solid matrix to release said triglyceride into said aqueous reaction solution.
2. The method of Claim 1 wherein said triglyceride comprises triolein.
3. The method of Claim 2 wherein said excipient comprises a sugar or a sugar alcohol.
4. The method of Claim 2 wherein said excipient comprises a sugar or sugar alcohol selected from the group consisting of mannitol, inositol, sorbitol, maltose, dextrose, lactose, dextran and mixtures thereof.
5. The method of Claim 2 wherein said excipient further comprises polyethylene glycol.
6. A reagent for use in an aqueous reaction solution to determine the lipase activity of a sample comprising a solid matrix of a water soluble excipient uniformly impregnated with a triglyceride by dissolving the triglyceride in a solvent that will not dissolve the excipient, mixing the excipient with a volume of triglyceride solution which substantially saturates the excipient and evaporating the solvent from the saturated excipient.
7. The reagent of Claim 6 wherein the triglyceride is triolein.
8. The reagent of Claim 6 wherein the excipient is selected from the group consisting of mannitol, inositol, sorbitol, maltose, dextrose, loctose, dextran and mixtures thereof.
9. The reagent of Claim 8 wherein the excipient contains polyethylene glycol and wherein the triglyceride is triolein.
10. A process for preparing a reagent for use in conjunction with an aqueous reaction solution to determine the lipase activity of a sample, comprising the steps of:
(a) forming a liquid solution of at least one trigly-ceride with a solvent for that triglyceride;
(b) mixing said liquid solution with a predetermined quantity of an excipient which is soluble in the aqueous reaction solution, the volume of solvent used to form said liquid solution being chosen to substantially saturate said excipient; and (c) evaporating said solvent to leave a solid matrix of the excipient impregnated with the triglyceride, the amount of triglyceride used to form said liquid solution being chosen to give the desired concentration of triglyceride per weight of solid matrix.
(a) forming a liquid solution of at least one trigly-ceride with a solvent for that triglyceride;
(b) mixing said liquid solution with a predetermined quantity of an excipient which is soluble in the aqueous reaction solution, the volume of solvent used to form said liquid solution being chosen to substantially saturate said excipient; and (c) evaporating said solvent to leave a solid matrix of the excipient impregnated with the triglyceride, the amount of triglyceride used to form said liquid solution being chosen to give the desired concentration of triglyceride per weight of solid matrix.
11. The process of Claim 10 wherein said trigly-ceride comprises triolein.
12. The process of Claim 11 wherein said solvent comprises chloroform.
13. The process of Claim 11 wherein said solvent comprises a mixture of chloroform and ethanol.
14. The process of Claim 10 wherein said excipient comprises sugar or sugar alcohol.
15. The process of Claim 10 wherein said exci-pient comprises a sugar or sugar alcohol selected from the group consisting of mannitol, inositol, sorbitol, maltose, dextrose, lactose, dextran and mixtures thereof.
16. The process of Claim 15 wherein said exci-pient further comprises polyethylene glycol.
17. A process for preparing a reagent for use in conjunction with an aqueous reaction solution to deter-mine the lipase activity of a sample comprising the steps of:
(a) forming a liquid solution comprising triolein and chloroform;
(b) mixing said liquid solution with a predetermined quantity of an excipient comprising polyethylene glycol and a sugar or sugar alcohol selected from the group con-sisting of mannitol, inositol, sorbitol, maltose, dextrose, lactose, dextran and mixtures thereof, the volume of solvent used to form said liquid solution being chosen to substan-tially saturate the excipient; and (c) evaporating said solvent to leave a solid matrix of said excipient impregnated with triolein, the amount of triolein used to form said liquid solution being chosen to give the desired concentration of triolein per weight of excipient.
(a) forming a liquid solution comprising triolein and chloroform;
(b) mixing said liquid solution with a predetermined quantity of an excipient comprising polyethylene glycol and a sugar or sugar alcohol selected from the group con-sisting of mannitol, inositol, sorbitol, maltose, dextrose, lactose, dextran and mixtures thereof, the volume of solvent used to form said liquid solution being chosen to substan-tially saturate the excipient; and (c) evaporating said solvent to leave a solid matrix of said excipient impregnated with triolein, the amount of triolein used to form said liquid solution being chosen to give the desired concentration of triolein per weight of excipient.
18. The process of Claim 17 further comprising the step of tableting said solid matrix of triolein impreg-nated excipient.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US68297776A | 1976-05-04 | 1976-05-04 | |
| US682,977 | 1976-05-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1109373A true CA1109373A (en) | 1981-09-22 |
Family
ID=24742046
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA277,628A Expired CA1109373A (en) | 1976-05-04 | 1977-05-02 | Method of determining lipase activity using a novel triglyceride reagent and method for preparing that reagent |
Country Status (12)
| Country | Link |
|---|---|
| JP (1) | JPS52134499A (en) |
| BE (1) | BE854219A (en) |
| CA (1) | CA1109373A (en) |
| CH (1) | CH628929A5 (en) |
| DE (1) | DE2719704A1 (en) |
| DK (1) | DK194577A (en) |
| FR (1) | FR2350604A1 (en) |
| GB (1) | GB1530238A (en) |
| IE (1) | IE44834B1 (en) |
| IT (1) | IT1114613B (en) |
| LU (1) | LU77256A1 (en) |
| NL (1) | NL7704782A (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5510447A (en) * | 1978-07-07 | 1980-01-24 | Nippon Electric Co | Oxide permittivity material |
| DE2904305C2 (en) * | 1979-02-05 | 1981-07-02 | Boehringer Mannheim Gmbh, 6800 Mannheim | Lipase determination reagent and process for its preparation |
| DE2905531A1 (en) * | 1979-02-14 | 1981-01-08 | Boehringer Mannheim Gmbh | DIAGNOSTIC AGENT FOR DETECTING LEUCOCYTES IN BODY LIQUIDS |
| EP0021572A1 (en) * | 1979-06-04 | 1981-01-07 | American Hospital Supply Corporation | A substrate for use in a turbidimetric assay for lipase and a method for making this substrate |
| US4555483A (en) * | 1982-08-11 | 1985-11-26 | Eastman Kodak Company | Methods, compositions and elements for the determination of lipase |
| US4820627A (en) * | 1986-03-24 | 1989-04-11 | Em Diagnostic Systems, Inc. | Method of preparing particles suitable for tabletting into diagnostic reagents |
| US5009994A (en) * | 1986-03-24 | 1991-04-23 | Em Diagnostic Systems, Inc. | Particles containing mannitol suitable for tabletting into diagnostic reagents |
| FR2619219A1 (en) * | 1987-08-07 | 1989-02-10 | Strasbourg I Louis Pasteur Uni | Dry reagent for the determination of pancreatic lipase, process for preparing the said dry reagent and dry reagent and reagent in emulsion form obtained by reconstituting the dry reagent |
| JP2711332B2 (en) * | 1988-04-15 | 1998-02-10 | 株式会社ヤトロン | Method for producing freeze-dried product from which transparent aqueous solution of water-insoluble substance is obtained |
| FR2824334B1 (en) | 2001-05-03 | 2003-10-10 | Coletica | METHOD FOR TESTING A SUBSTANCE POSSIBLY ACTIVE IN THE FIELD OF LIPOLYSIS AND ITS MAINLY COSMETIC USE |
| US20090093007A1 (en) * | 2007-05-16 | 2009-04-09 | Shigeki Kageyama | Method for producing dry analytical element for pancreatic lipase measurement |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3539450A (en) * | 1966-06-30 | 1970-11-10 | Calbiochem | Stabilization of enzymes |
| ZA707714B (en) * | 1969-12-10 | 1971-09-29 | Boehringer Mannheim Gmbh | Reagent for the determination of lipase activity |
| US3917515A (en) * | 1974-03-13 | 1975-11-04 | Jack M Goldberg | Serum lipase method and medium |
-
1977
- 1977-04-15 GB GB1585977A patent/GB1530238A/en not_active Expired
- 1977-05-02 CA CA277,628A patent/CA1109373A/en not_active Expired
- 1977-05-02 NL NL7704782A patent/NL7704782A/en not_active Application Discontinuation
- 1977-05-03 BE BE177233A patent/BE854219A/en not_active IP Right Cessation
- 1977-05-03 DE DE19772719704 patent/DE2719704A1/en not_active Withdrawn
- 1977-05-03 FR FR7713341A patent/FR2350604A1/en active Granted
- 1977-05-03 IT IT2313477A patent/IT1114613B/en active
- 1977-05-03 DK DK194577A patent/DK194577A/en unknown
- 1977-05-03 CH CH554077A patent/CH628929A5/en not_active IP Right Cessation
- 1977-05-04 LU LU77256A patent/LU77256A1/xx unknown
- 1977-05-04 JP JP5194277A patent/JPS52134499A/en active Pending
- 1977-05-04 IE IE90777A patent/IE44834B1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| LU77256A1 (en) | 1977-12-13 |
| IE44834L (en) | 1977-11-04 |
| IT1114613B (en) | 1986-01-27 |
| DK194577A (en) | 1977-11-05 |
| BE854219A (en) | 1977-11-03 |
| DE2719704A1 (en) | 1977-11-24 |
| NL7704782A (en) | 1977-11-08 |
| JPS52134499A (en) | 1977-11-10 |
| FR2350604B1 (en) | 1983-08-05 |
| GB1530238A (en) | 1978-10-25 |
| CH628929A5 (en) | 1982-03-31 |
| IE44834B1 (en) | 1982-04-07 |
| FR2350604A1 (en) | 1977-12-02 |
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