IE44834B1

IE44834B1 - Method of determining lipase activity using a novel triglyceride reagent and method for preparing that reagent

Info

Publication number: IE44834B1
Application number: IE90777A
Authority: IE
Original assignee: Du Pont
Priority date: 1976-05-04
Filing date: 1977-05-04
Publication date: 1982-04-07
Also published as: DK194577A; IT1114613B; DE2719704A1; FR2350604A1; IE44834L; BE854219A; NL7704782A; CA1109373A; GB1530238A; JPS52134499A; LU77256A1; FR2350604B1; CH628929A5

Abstract

The reagent for the determination of lipase activity in a solution consists of a solid matrix of an inert carrier which is soluble in an aqueous reaction solution and is impregnated with a triglyceride. The carrier is preferably impregnated with triolein; the carrier is, for example, a sugar. To prepare the reagent, at least one triglyceride is dissolved in a solvent. The resulting solution is then mixed with an appropriate amount of the inert carrier which is soluble in an aqueous reaction solution. Finally, the solvent is evaporated off so that a solid matrix of the carrier impregnated with the triglyceride remains. The lipase activity in a sample is determined by reacting the sample with an emulsion of a triglyceride in an aqueous reaction solution and measuring the rate at which the triglyceride is hydrolysed to soluble fragments.

Description

PATENT APPLICATION BY (71) N.I. DU PONT DE NEMOURS AND COMPANY, A CORPORATION ORGANIZED AND EXISTING UNDER THE LAWS OF THE STATE OF DELAWARE, LOCATED AT WILMINGTON, DELAWARE, UNITED STATES OF AMERICA.

Pnct 12|p 4 8 3 4 / Background, of the Invention 1. Field of the Invention: The present invention relates to a method for determining the lipase activity of a sample hy reacting the sample with a triglyceride emulsion, and to a method of preparing the triglyceride reagent used to form the emulsion. .5 2, Discussion of the Prior Art: lipase is an enzyme produced hy the pancreas. lipase activity in hlood or blood serum is increased hy pancreatitis and other diseases of the pancreas.

A measure of lipase activity in hlood or hlood serum, is therefore, a useful diagnostic tool to physicians..

A traditional method of measuring lipase activity is the turbidimetric method described by Shihahi and Bishop in Clinical Chemistry, 17, pp. 1150 - 1155 (1S71), in which the decrease in absorbance of an olive oil/water emulsion is related to lipase activity. Olive oil is a mixture of substances called collectively triglyceride. Wh'en 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 emulsion. lipase hydrolyzes triglyceride into fragments which are soluble in the reaction mixture. As. the triglyceride is hydrolyzed, light scattering by the emulsion is decreased andr, transmission of the light through the medium is increased, - 2 *<834 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 in5 adequate. 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 the Invention The present invention provides a method for determining lhe lipase activity of an aqueous sample which comprises adding to an aqueous reaction solution containing the sample a solid excipient soluble in said solution, which has been uniformly impregnated with a triglyceride by uniformly wetting the excipient with a solution of the triglyceride in a solvent in which the excipient is insoluble and subsequently evaporating the solvent, so that the said excipient dissolves in. the said solution containing the sample and an emulsion is of the said triglyceride/formed, reacting the sample with the said emulsion, and measuring the rate at which the triglyceride is hydrolyzed to soluble fragments.

The invention includes within its scope a reagent for use in an aqueous reaction solution for determining lipase activity comprising - 3 4 4 8 3 4 a solid excipient uniformly impregnated with, a triglyceride by uniformly wetting the excipient with a solution of the triglyceride in a solvent in which the excipient is insoluble and subsequently evaporating the solvent, the said excipient being soluble in the aqueous reaction solution.

In a preferred embodiment of the invention, the excipient comprises polyethlyene glycol and a sugar or sugar alcohol such as mannitol, inositol, sorbitol, maltose, dextrose, lactose, dextran and mixtures thereof; and it is impregnated with triolein, one of the major trigLycerides.

The invention also provides 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 triglyceride (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 liquid solution being chosen i to substantially saturate the excipient; and 20 (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 - 4 44834 concentration of triglyceride per weight of solid matrix.

Description of the Preferred Embodiment The first 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 v/ill 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 triglyceride reagent of the present invention should satisfy four criteria. It must dissolve the particular triglyceride used. It must not dissolve the particulate 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, i.e., one that is not reactive with or harmful to the other reagent'used.

Solvents such as chloroform and ethanol will dissolve triolein. - 5 44834 It has teen found, that chloroform or ethanol or mixtures of chloroform, preferably mixtures containing up to about 50% ethanol, will produce a uniform solution of triolein.

The volume of solvent used to dissolve the triglyceride is chosen so' that when the liquid -triglyceride solution is applied to the excipient, the liquid will just saturate the solid material. The volume of solution must be sufficient so that it can be uniformly distributed throughout the excipient. Normally this volume is the maximum amount that the excipient can retain by capillary action after mixing, but less than this amount can be used provided the method of mixing used, ensures uniform distribution of the solution on the excipient. If too little solvent is used, a non-uniform distribution of the solvent, and hence the triglyceride, throughout the solid will occur.

When 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 nob, that portion of the triglyceride contained in the solution which does not impregnate the solid will coat the solid in a nonuniform manner. Depending upon the precision required, each of these conditions can be tolerated, but for best results, the amount of solvent used should be chosen so that the triglyceride solution substantially saturates the solid material. - 6 44834 Hie amount of triglyceride used in the solution is a matter of choice. Hie amount used should he 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, fhe excipient chosen to form 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 dissolution is required to release the triglyceride into the reaction solution to form the emulsion. Hie 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. Hiere 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 mannitoljinositol, sorbitol, maltose, dextrose, lactose, dextran and mixtures thereof.

A more uniform distribution of the triglyceride in the solid matrix will be formed if, in addition to the substances referred to above, polyethlyene glycol is used. Carbowax © 6000 is one suitable substance. Shis substance prevents agglomeration of the - 7 44834 triglyceride droplets in the solid matrix and aids in producing a uniform coating of ,-the triglyceride solution on the surface of the solid matrix. Ihe polyethylene glycol, however, 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 Hobart ©mechanical mixer can be used. Mixing continues until the excipient is just saturated. The saturated excipient is then dried to evaporate the solvent 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 parti20 cular, air drying' in a fume hood can be used. The impregnated solid matrix can be used as a powder, or it can be tableted using conventional tableting techniques.

As a particular example of the formation of the triglyceride impregnated matrix, 95 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 1 the excipient, a just saturated solid blend is formed. The - 8 4834 solvent mixture 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. lhis solid matrix is in powder form. The impregnated blend is then tableted to yield approximately 85 mg. tablets containing 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. lhis 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: Ca++, Lipase Triolein Emulsion -----Free Eatty Acids + (Turbid) pH 8.8 (25°C) Mixed Glycerides (Decreasing turbidity) As a particular example of a lipase assay, a test kit containing the following reagents is prepared: Tris Buffer, pH 8.8 ί 0.2; 50 μΜοΙ Deoxycholate 35.5 μΜοΙ Calcium chloride 0.7 μΜοΙ ‘ Triolein 0.98 μΜοΙ (in the tableted form described above) , These reagents are added to 4.8 ml. of water, and 0.20 ml. of sample containing a known 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. As 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 monitored at 340 nm. is found, to be proportional to the lipase activity. 1 The uSe tof a tablet described above yields highly reproducible emulsions and imparts long-term stability to the substrate (at,least 12 months).

Claims

1. CLAIMS:1. A method for determining the lipase activity of an aqueous sample which comprises adding to an aqueous reaction solution containing the sample a solid excipient soluble in said solution which has been uniformly impregnated with a triglyceride by 5' uniformly wetting the excipient with a solution of the triglyceride in a solvent in which the excipient is insoluble and subsequently evaporating the solvent, so that the said excipient dissolves in the said solution containing the sample and an emulsion of the said triglyceride is formed, reacting the sample with the 10 said emulsion, and measuring the rate at which the triglyceride is hydrolysed to soluble fragments.

2. A method according to Claim 1 wherein the said triglyceride comprises triolein.

3. A method according to Claim 1 or 2 wherein the said excipient 15 comprises a sugar or sugar alcohol.

4. A method according to Claim 5 wherein the said excipient comprises mannitol, inositol, sorbitol, maltose, dextrose, lactose, dextran, ^or a mixture thereof. 20 5. A method according to any of Claims 1 to 4 wherein the said excipient further comprises polyethylene glycol. - .n ^4834

5. A method according to any of Claims 1 to 4 wherein the said excipient further comprises polyethylene glycol.

6. A reagent for use in an aqueous reaction solution for determining lipase activity comprising a solid excipient uniformly impregnated with a triglyceride hy uniformly wetting the excipient with a solution of the triglyceride in a solvent in which the excipient is insoluble and subsequently evaporating the solvent, the said excipient being soluble in the aqueous reaction solution.

7. A reagent according to Claim 6 wherein the said triglyceride comprises triolein.

8. A reagent according to Claim 6 or 7 wherein the said excipient comprises sugar or sugar alcohol.

9. A reagent according to Claim 8 wherein the said excipient comprises mannitol, inositol, sorbitol, maltose, dextrose, lactose, dextran or a mixture thereof.

10. A reagent according to any of Claims 6 to 9 wherein the excipient further comprises polyethlene glygol.

11. A process for preparing a reagent for use in. an aqueous reaction solution for determining lipase activity, comprising: (a) forming a liquid solution of at least one triglyceride in a solvent for that triglyceride; (b) mixing the said, liquid, solution with a predetermined, quantity of an excipient which is soluble in an aqueous reaction solution, the volume of solvent used to form said liquid solution being chosen to substantially saturate said excipient; and 5 (o) evaporating the said solvent to leave the solid excipient uniformly impregnated with the triglyceride, the amount of triglyceride used to form said liquid solution being chosen to give the desired weight concentration of the triglyceride in the solid excipient. 10

12. A process according to Claim 11 wherein the said triglyceride comprises triolein.

13. A process according to Claim 11 or 12 wherein the said excipient comprises sugar or sugar alcohol.

14. 15 14. A process according to Claim 13 wherein the said sugar or sugar alcohol is mannitol,.inositol, sorbitol, maltose, dextrose, lactose, dextran or a mixture thereof. i 15. A process according to any of Claims 11 to 14 wherein the said excipient fufther comprises polyethylene glycol. 20

15. 16. A prooesp according to any of Claims 11 to 15 wherein the said solvent comprises chloroform.

16. 17. A process according to any of Claims 11 fco 15 wherein the ..448 3 4 said solvent comprises a mixture of chloroform and ethanol.

17. 18. A process according to any of Claims 11 to 17 further comprising the step of' tahleting the impregnated excipient.

18. 19. A method according to Claim 1 substantially as hereinbefore 5 described.

19. 20. A reagent according to any of Claims 6 to 10 in the forms of a tablet.

20. 21. A process according to Claim 11 substantially as hereinbefore 10 described.

21.

22. A reagent according to Claim 6 when prepared by a process as claimed in any of ClaimB 11 to 18 or 21.