GB1592632A

GB1592632A - Hydrolysis process for protein-bound cholesterol esters

Info

Publication number: GB1592632A
Application number: GB4302/78A
Authority: GB
Original assignee: Eastman Kodak Co
Current assignee: Eastman Kodak Co
Priority date: 1977-02-03
Filing date: 1978-02-02
Publication date: 1981-07-08
Also published as: DE2804356A1; JPS5396378A; SE462047B; FR2379815A1; SE468093B; SE8303292D0; SE7801248L; DE2804356C2; CA1104041A; SE8303292L; FR2379815B1; JPS6237957B2; BE863657A

Description

(54) HYDROLYSIS PROCESS FOR PROTEIN BOUND CHOLESTEROL ESTERS (71) We, EASTMAN KODAK COMPANY, a Company organized under the Laws of the State of New Jersey, United States of America of 343 State Street, Rochester, New York 14650, United States of America do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to methods for the hydrolysis of cholesterol esters, together with compositions and analytical elements for performing the process.

In the assay of body fluids, especially blood serum, for cholesterol concentration, the initial step requires hydrolysis of cholesterol esters to free cholesterol.

Conventional procedures for cholesterol ester hydrolysis use a strong base, or for reasons of simplicity and selectivity, a hydrolase enzyme (i.e., a cholesterol esterase). Handling of caustic materials may be inconvenient or undesirable and, as discussed in relation to prior publications below, while enzymatic techniques can be useful for the hydrolysis of "free" cholesterol esters, i.e., those not bound to protein, they are either ineffective or very slow when used to treat protein-bound cholesterol esters. The binding of the ester to protein apparently inhibits the action of the esterase and thus requires some means for breaking the protein-ester complex before the enzyme can act on the ester.

French Patent No. 2,223,696 and U.S. Patent No. 3,925,164 describe an assay for total cholesterol in blood serum wherein cholesterol esters are hydrolyzed with an enzyme preparation from Candida rugosa, Rhizopus or Aspergillus in the presence of a surfactant. The only suggested surfactant is hydroxypolyethoxy dodecane. The present invention provides a process for hydrolyzing protein-bound cholesterol esters using an enzyme and a surfactant which has a much greater efficiency.

According to the present invention there is provided a process for hydrolyzing protein-bound cholesterol esters consisting of treating the esters in an aqueous medium with a compatible mixture, as herein defined, of an enzyme preparation having cholesterol ester hydrolase activity and an alkylphenoxypolyethoxyethanol having a polyoxyethylene chain of less than 20 oxyethylene units.

Protein-bound cholesterol esters can be hydrolyzed in relatively short periods of time of the order of less than 10 minutes (preferably in 5 minutes) by the process of the invention. Protein-bound cholesterol esters are contained in blood serum and may be hydrolyzed by the process of the invention.

The techniques and compositions described herein permit the use of a much broader range of enzyme preparations as cholesterol ester hydrolyzing agents than has been possible with prior art methods. Thus, lower cost materials can be used to attain reaction times and states of reaction completeness at least equal to and often superior to those attainable with the prior art methods and materials.

Enzyme preparations are known which catalyze the hydrolysis of free cholesterol esters. Such materials, however, catalyze the hydrolysis of cholesterol esters bound to protein, as found in blood serum, only at very slow rates or in an incomplete fashion. This result is apparently due to some effect of the protein-lipid complex which prevents the enzyme from catalyzing the hydrolysis in the usual fashion. The use has been suggested of what might be termed effectors, namely, agents which increase the rate at which lipase materials can hydrolyze proteinbound cholesterol esters. Although the mechanism by which such agents act is not known, it is theorized that they disrupt the ester-protein complex in some way to "free" the ester for hydrolysis in a conventional mode. Protease enzymes, for example, have been suggested for this purpose.

It has now been found that some surfactants are effectors and may be used as substitutes for protease to render useful, as hydrolyzers of protein-bound cholesterol esters, enzyme preparations which are normally incapable of catalyzing the hydrolysis of protein-bound cholesterol esters or which catalyze such hydrolysis only at undesirably slow rates. Furthermore, since protease tends to degrade proteinaceous binders, such as gelatin, used in multilayer analytical elements for the detection of analytes as described in U.K. Patent No. 1,440,464, the compositions described herein are particularly useful in such elements.

The compositions of the present invention thus comprise a compatible mixture of an enzyme preparation which demonstrates cholesterol esterase activity and as an effector a surfactant which is an alkylphenoxypolyethoxyethanol comprising a polyoxyethylene chain of less than 20 oxyethylene units.

Enzyme preparations potentially useful in the methods and compositions described herein are those demonstrating free (i.e., not protein-bound) cholesterol ester hydrolase activity. Lipase preparations which demonstrate such activity are specifically preferred.

A useful screening technique for determining the cholesterol ester hydrolase (esterase) activity of enzyme, and particularly lipase, preparations comprises adding a fixed amount of the enzyme preparation to a standard cholesteryl linoleate solution at pH 7.0, incubating at 37"C under nitrogen for 2 hours and determining the amount of ester left in the solution by the hydroxylamine method of J. Vonhoeffmyr and R. Fried, Z. Klin. Chem. U. Klin. Biochem., 8, 134 (1970).

By means of this technique, any preparation which demonstrates a cholesterol esterase activity which releases above 25 mg/dl cholesterol in the screening procedure should be considered useful in the practice of the present invention.

The enzyme preparations for cholesterol ester hydrolysis may be obtained from plant or animal sources but preparations from microbial sources are preferred such as from Candida rugosa, Chromobacterium viscosum variant paralipolyticum, or Rhizopus arrhizus, crude or purified. Other useful enzyme preparations and methods for their preparation are described in the following U.S. Patents: 2,888,385; 3,168,448; 3,189,529; 3,262,863 and 3,513,073.

Preferred commercial enzyme preparations include wheat germ lipase from Miles Laboratories of Elkhart, Indiana, Lipase 3000 from Wilson Laboratories, Steapsin from Sigma Chemical Company (both of the latter are pancreatic esterases), and Lipase M (from Candida rugosa) from Enzyme Development Company.

Certain surfactants inhibit the cholesterol esterase activity of certain enzyme preparations. Consequently, it is important that before any attempt is made to combine an enzyme preparation and a surfactant for use as described herein some determination of the compatibility of the two members of the composition be made. Such a determination is preferably made by using the test described below.

A mixture of enzyme preparation and surfactant which successfully meets this test is referred to herein as a compatible mixture and each member thereof is said to be compatible with the other.

Hydrolysis compositions of the present invention are characterized by the test used in comparative Example II below. The proposed surfactant under evaluation is added to normal human serum. A sample of a proposed enzyme preparation is added and the mixture incubated at 370C for a period of 10 minutes. Aliquots (0.1 ml) of this solution are then diluted to 1.9 ml with water and placed in a boiling water bath for 10 minutes. Cholesterol is quantified in a total volume of 1.2 ml by the well known cholesterol oxidase system described below. A similar "control" test is performed concurrently using only the enzyme preparation without the surfactant. When performing the foregoing test it is most desirable to run a blank which contains all of the components of the mixture except the enzyme preparation so that any reaction which may be due to free cholesterol or other components of the serum can be subtracted. The preferred compositions accomplish hydrolysis of at least 70% of the available cholesterol esters in less than 10 minutes and most preferred are those which achieve substantially complete hydrolysis, i.e., hydrolysis of at least 90 /O of the available cholesterol esters in less than 10 minutes.

It has been discovered that alkylphenoxypolyethoxyethanols comprising a polyoxyethylene chain of less than 20 oxyethylene units are highly superior effectors. Specifically preferred are materials available commercially from Rohm and Haas Company under the Trade Marks 'Triton' X-114, 100, 102 and 'Triton' n101. The most preferred alkylphenoxypolyethoxyethanols comprise polyoxyethylene chains having from 7 to 13 oxyethylene units. As will be shown in the following examples, similar materials outside of these broad limits do not provide the improved hydrolysis described herein. Most preferred are those materials wherein the alkyl is either 8 or 9 carbon atoms.

As will be demonstrated in the examples below, the hydroxypolyethoxy dodecanes of French Patent No. 2,223,696 and U.S. Patent No. 3,925,164 are not effective when used in place of the surfactants of the process of the present invention. (Exemplary useful hydrolysis compositions are shown in Table I.) The concentration of enzyme preparation and surfactant in the compatible mixtures useful for hydrolysis according to the methods described herein can vary greatly depending, for example, on such factors as the purity of the enzyme preparation, the activity of the enzyme preparation, the nature of the bound cholesterol ester and the particular surfactant used. Preferably, however, surfactant concentrations of from 0.25 to 10% by weight of the analytical solution are used with concentrations of from 0.5 to 5% by weight of surfactant providing optimum results. The useful range of concentrations of enzyme preparation will vary similarly, but concentrations of from 10 to 80 mg/ml of the total analytical solution are preferred when commercial preparations are used. Optimization of any such composition is, of course,within the skill of the art.

It should be apparent that hydrolysis compositions of the type described herein can be incorporated into any of the single or multiple layer absorbent or other analytical elements (for example, test papers) described in the prior art and that the use of the compositions and methods described herein in such elements for the detection or determination of protein-bound cholesterol esters is within the scope of the invention.

In accordance with one preferred embodiment, the hydrolysis compositions described herein are incorporated into one or more layers of multilayer analytical elements of the type described, for example, in U.K. Patent No. 1,440,464 and U.S.

Patent 3,992,158. Such analytical elements are intended to analyze liquids for the presence of a predetermined analyte and they include a preferably non-fibrous spreading layer, which delivers a uniform apparent concentration of analysis-active components in an applied sample to a reagent layer which contains at least some of the materials interactive in the presence of analyte to produce a detectable product or detectable change. Such layers are in fluid contact under conditions of use.

Reference herein to fluid contact between layers in an analytical element identifies the ability of a fluid, whether liquid or gaseous, to pass in such element between superposed regions of the spreading layer and the reagent layer. Stated in another manner, fluid contact refers to the ability of components of a fluid to pass between the layers in fluid contact, although such layers in fluid contact can be contiguous, they may also be separated by intervening layers. However, layers in the element that physically intervene a spreading layer and reagent layer in mutual fluid contact will not prevent the passage of fluid between the fluid contacting spreading and reagent layers.

Fluid contact between layers can be achieved by preparing elements having layers that are initially contiguous or effectively so for purposes of fluid passage.

Alternatively, it may be appropriate to prepare elements that have layers initially non-contiguous, and which further can be spaced apart, such as by the use of interleaves as described, for example, in U.S. Patent 3,511,608 or by the use of a resilient absorbent material or deformable supports as described in U.S. Patent 3,917,453 and U.S. Patent 3,933,594. As will be appreciated, if the element has initially non-contiguous layers, it may be necessary to apply compressive force or otherwise provide means to bring layers of the element into fluid contact at the time of its use to provide an analytical result.

According to a highly preferred embodiment of such an element, the hydrolysis composition described herein is incorporated into the spreading layer and a detection system, for example, a cholesterol oxidase and an indicator composition sensitive to hydrogen peroxide for cholesterol detection, is included in the reagent layer.

The following description of standardized procedures and examples are presented to further illustrate the useful scope of the present invention.

Standard Procedures: Quantiftcation of Total Serum Cholesterol-Cholesterol esters must first by hydrolyzed to free cholesterol. Incubation mixtures contained in a total volume of 8 ml:2.4 units cholesterol oxidase (N. cholesterolicum), 0.768 mg 4aminoantipyrene MCI, 0.256 mg 1,7-dihydroxynaphthalene, 0.22 mg peroxidase (125 purpurogallin units/mg), 6.4 mg crude lipase preparation, and either 0.48 mg protease (B. subtilis (Sigma Corporation Type VII)) or 160 mg octyl phenoxy polyethoxy ethanol (Triton X-100). Incubation mixtures were equilibrated at 370C for five minutes and the reaction was initiated by addition of 20 ul of human serum.

After 10 minutes, the absorbance at 490 nm was measured. Blank tubes contained all components except serum. Total cholesterol concentrations were obtained from a standard curve which was constructed by substituting aliquots of Fermco Test Aqueous Cholesterol Standard (available from Fermco Laboratories, Chicago, Illinois) for the serum substrate.

The reference method was the Liebermann-Burchard method as described in "Hawk's Physiological Chemistry", B. L. Oser (editor), 14th Edition, McGraw-Hill Book Company, New York, pp 1062-1064. This method involves extraction of the cholesterol and cholesterol esters from serum prior to quantitation.

Example 1 Enzyme Catalyzed Hydrolysis of Serum Cholesterol Esters in the Presence of Alkylphenoxypolyethoxyethanol Surfactant Human serum (20 ,ul) was added to 8 ml of buffer reagent (equilibrated at 37"C) which contained either enzyme preparation and a protease or the enzyme preparation and surfactant to effect hydrolysis of the cholesterol esters. After 10 minutes, the absorbance at 490 nm was measured, and total serum cholesterol calculated as described above.

Serum cholesterol was quantified using the cholesterol oxidase, peroxidase system. In this system cholesterol esters are first hydrolyzed to free cholesterol which is subsequently oxidized to cholestenone with concomitant production of hydrogen peroxide. The hydrogen peroxide is then coupled to dye formation via a peroxidase reaction. It has been reported that a crude lipase preparation catalyzes hydrolysis of serum cholesterol esters if a protease is added to the incubation mixture. The data in Table I show that in the presence of S-l (an octylphenoxy polyethoxy ethanol having about 10 ethoxy units and an hydrophile-lipophile balance (HLB) number of 13.5) complete hydrolysis of cholesterol esters was observed. The surfactant efficiently replaced the protease and thus eliminated the need for this extraneous protein which may undesirably (1) hydrolyze protein components of the cholesterol detection system or (2) alter the pH -of the system.

Quantitation of serum cholesterol with a lipase preparation and S-l as the hydrolytic system gave results (see Table I) which compared very favourably with the reference method.

TABLE I Total Serum Cholesterol Concentration (mg/dl) Lipase Protease Lipase Reference Sample (Control) 2% S-l Methoda 1 225 225 232 2 150 185 190 3 150 180 176 4 240 230 220 aThe reference method was a semi-automated Liebermann-Burchard method An S-l concentration of 2% by weight of the reaction mixture gave complete hydrolysis and the surfactant produced no harmful effects at concentrations as high as 4% by weight of the reaction mixture. Although final colour densities were measured after 10 minutes, reactions were essentially complete in as little as five minutes at 370C.

Repetition of the above test using alkylphenoxypolyethoxyethanols having polyoxyethylene chains above 20 yields results which indicate that such surfactants somehow inhibit the cholesterol esterase activity of the enzyme and are ineffective in the process according to the invention.

Direct comparative tests were conducted with a cholesterol esterase from Candida rugosa and (a) surfactant as described in French Patent No. 2,223,696 and (b) representative of the surfactants used in the process of the present invention.

Incubation mixtures were prepared containing in a total volume of 0.6 ml: 0.5 ml normal human serum 20 mg cholesterol esterase (Lipase M commercial preparation from Candida rugosa) 5 ,u moles potassium phosphate buffer (pH 7.0) 10 mg effector Reactions were allowed to proceed for 10 minutes at 37"C and then 0.1 ml aliquots were added to 1.9 ml of water and placed in a boiling water bath for 10 minutes. Cholesterol was then quantitated via the cholesterol oxidase-peroxidase system using aqueous cholesterol standards to prepare a standard curve. The results of these tests are shown in Table II below.

TABLE II Cholesterol Ester Actual Recovered V Effector mg/dl mg/dl Recovery None 157 2.4 1.5 Octylphenoxypolyethoxyethanol 157 157 100 Polyoxyethylene (4) lauryl ether 157 3.4 2.2 Polyoxyethylene (23) lauryl ether 157 103 65.7 Polyoxyethylene (12) lauryl ether 157 0 0 (The numbers in parenthesis indicate the number of oxyethylene units.) From the foregoing, it is apparent that the polyoxyethylene lauryl ethers (i.e., the dodecane materials of French Patent No. 2,223,696) are not useful as effectors in accordance with the invention described herein.

WHAT WE CLAIM IS: 1. A process for hydrolyzing protein-bound cholesterol esters consisting of treating the esters in an aqueous medium with a compatible mixture, as herein defined, of an enzyme preparation having cholesterol ester hydrolase activity and an alkylphenoxypolyethoxyethanol having a polyoxyethylene chain of less than 20 oxyethylene units.

2. The process as claimed in claim 1 wherein the polyoxyethylene chain of the alkylphenoxypolyethoxyethanol has from 7 to 13 oxyethylene units.

3. The process as claimed in claim 1 or 2 wherein the alkyl group of the alkylphenoxypolyethoxyethanol has 8 or 9 carbon atoms.

4. The process as claimed in any of the preceding claims wherein the proteinbound cholesterol esters are contained in blood serum.

5. The process as claimed in any of the preceding claims wherein the enzyme preparation is obtained from a microbial or an animal source.

6. The process as claimed in claim 5 wherein the enzyme preparation is obtained from Candida rugosa, Chomobacterium viscosum v. paralipolyticum or Rhizopus arrhizus.

7. The process as claimed in claim 5 wherein the enzyme preparation is a pancreatic esterase.

8. The process for assaying blood serum for total serum cholesterol content wherein the cholesterol esters are first hydrolyzed by the method of any of the claims 1 to 7 and the amount of cholesterol produced is assayed.

9. The process as claimed in claim 8 wherein the hydrolysis and assay take place in a multilayer analytical element having a layer containing the hydrolysis mixture and a reagent layer for forming a detectable colour change in response to cholesterol.

10. Processes for hydrolyzing protein-bound cholesterol esters as claimed in claim 1 and as herein described.

11. A composition comprising a compatible mixture, as herein defined, of an enzyme preparation having cholesterol ester hydrolase activity and an

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. somehow inhibit the cholesterol esterase activity of the enzyme and are ineffective in the process according to the invention. Direct comparative tests were conducted with a cholesterol esterase from Candida rugosa and (a) surfactant as described in French Patent No. 2,223,696 and (b) representative of the surfactants used in the process of the present invention. Incubation mixtures were prepared containing in a total volume of 0.6 ml: 0.5 ml normal human serum 20 mg cholesterol esterase (Lipase M commercial preparation from Candida rugosa) 5 ,u moles potassium phosphate buffer (pH 7.0) 10 mg effector Reactions were allowed to proceed for 10 minutes at 37"C and then 0.1 ml aliquots were added to 1.9 ml of water and placed in a boiling water bath for 10 minutes. Cholesterol was then quantitated via the cholesterol oxidase-peroxidase system using aqueous cholesterol standards to prepare a standard curve. The results of these tests are shown in Table II below. TABLE II Cholesterol Ester Actual Recovered V Effector mg/dl mg/dl Recovery None 157 2.4 1.5 Octylphenoxypolyethoxyethanol 157 157 100 Polyoxyethylene (4) lauryl ether 157 3.4 2.2 Polyoxyethylene (23) lauryl ether 157 103 65.7 Polyoxyethylene (12) lauryl ether 157 0 0 (The numbers in parenthesis indicate the number of oxyethylene units.) From the foregoing, it is apparent that the polyoxyethylene lauryl ethers (i.e., the dodecane materials of French Patent No. 2,223,696) are not useful as effectors in accordance with the invention described herein. WHAT WE CLAIM IS:

1. A process for hydrolyzing protein-bound cholesterol esters consisting of treating the esters in an aqueous medium with a compatible mixture, as herein defined, of an enzyme preparation having cholesterol ester hydrolase activity and an alkylphenoxypolyethoxyethanol having a polyoxyethylene chain of less than 20 oxyethylene units.

alkylphenoxypolyethoxyethanol having a polyoxyethylene chain of less than 20 oxyethylene units.

12. The composition as claimed in claim 11 in which the polyoxyethylene chain of the alkylphenoxypolyethoxyethanol has from 7 to 13 oxyethylene units.

13. The composition as claimed in claim 11 or 12 in which the alkyl group of the alkylphenoxypolyethoxyethanol has 8 or 9 carbon atoms.

14. The composition as claimed in claims 11, 12 or 13 in which the enzyme preparation is obtained from a microbial or animal source.

15. The composition as claimed in claim 14 in which the enzyme preparation is obtained from Candida rugosa, Chromobacterium viscosum v. parallpolvticum or Rhizopus arrhizus.

16. The composition as claimed in claim 14 in which the enzyme preparation is a pancreatic esterase.

17. Compositions as claimed in claim 11 and as herein described.

18. A multilayer analytical element comprising at least one. layer containing a composition as claimed in any of the claims 11 to 17.

19. A multilayer analytical element as claimed in claim 18 in which the element has a spreading layer and a reagent layer containing a composition as claimed in any of the claims 11 to 17.

20. A multilayer analytical element as claimed in claim 18 or 19 which contains a layer or layers which provide a detectable colour change in response to the presence of cholesterol.

21. Multilayer analytical elements as claimed in claim 18 and as herein described.