CA1104041A - Hydrolysis of protein-bound cholesterol esters - Google Patents

Abstract

Abstract of the Disclosure A novel process is described for hydrolyzing protein-bound cholesterol esters such as are found in blood serum.
The method comprises contacting sample containing protein-bound cholesterol esters with a compatible mixture of an enzyme preparation which demonstrates cholesterol ester hydrolase activity and, as an effector, a surfactant which is an alkyl phenoxy polyethoxy ethanol comprising a polyoxyethylene chain of less than about 20 oxyethylene units.

Hydrolysis compositions comprising compatible mixtures of an enzyme preparation which demonstrates cholesterol ester hydrolase activity and an effector which is a surfactant as described are also disclosed, as are analytical elements com-prising at least one layer which includes such a hydrolysis composition.

Description

Field of the Invention The present invention relates to methods for the hydrolysis of cholesterol esters and more particularly to methods and compositions ror the hydrolysis of protein-bound cholesterol esters such as serum cholesterol esters.
Back~round of the Invention 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 (KOH, NaOH, etc.), 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 îneffective or ver~ slow when used to treat protein-bound cholesterol esters. The i 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.

U.S. Patent No. 3~869,349 issued March 4, 1975 to .~.
Goodhue et al describes an improved technique for hydrolyzing serum cholesterol esters which involves the use of compositions comprising a lipase preparation which demonstrates cholesterol esterase activity and a protease. This patent contains no suggestion that a surfactant can replace the protease.

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"'' , U.S. Patent No. 3,703,591 to Bucolo et al describes the use of a combination of a lipase and a protease to achieve serum (i.e. 5 protein-bound) triglyceride hydrolysis. No sug-gestion is made to use a surfactant either in combination with or as a substitute for the protease in the hydrolysis of cholesterol esters.

U.S. Patent No. 3,759,793 to Stork et al describes ~ the hydrolysis of serum triglycerides using a lipase from - Rhizopus arrhizus which is apparently identical to that sug-gested by Bucolo et al, however7 with no requirement for a protease. The reasons for this apparent anomaly are not clear, however~ it is noted in British Patent No. 1~395,126 of the same assignee that the Stork et al hydrolysis technique ~; is very slow. This British Patent describes an improved method for hydrolyzing triglycerides with the aforementioned Rhizopus arrhi~us lipase comprising contacting the triglyceride with the lipase in a buffer and in the presence of carboxyl-esterase and an alkali metal or alkaline earth metal alkyl sulfate, the alkyl radical of which contains 10 to 15 carbon atoms. The pre~erred alkyl sulfate is sodium dodecyl sulfate.
There is no suggestion that the use of surfactant alone in the absence of carboxylesterase stimulates hydrolase activity of kriglycerides.
Helenius, Ari and Simons, Kai, Biochemistry, Vol. 10, No. 13 (1971) describe a method for removing all major lipids from human plasma low-density lipoprotein comprising treatment :

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of the human plasma with high concentrations of ~atural and synthetic surfactants. Lipid removal is applied for purposes of characterizing the lipid free protein moiety of human plasma low-density lipoprotein. There is no suggestion in this publication that the combination o~ a surfactant and a lipase would yield a use~ul analytical tool which would simplify the assay of serum for cholesterol content by pro-viding a fast and accurate hydrolysis method and a ~table assay composition.
U.S. Patent No. 3,689,364 issued September 5, 1972 describes an assay for lipase contained in body fluids such as blood serum using a "free" triglyceride emulsion as sub-strate for the lipase. It is suggested that the bile salts which stabilize the substrate emulsion of "free" triglyceride (i.e., triglycerides not bound to protein) also concurrently e~ert an "activating effect" on the lipase lmder assay when it is a pancreatic lipase. The activating e~fect apparently results in an increase in the hydrolytic activity of the lipase on the free triglycerides of the substrate emulsion.
There is no teaching or suggestion in this patent that such bile salts exert any effect on lipase preparations when con-tacted with lipids bound to proteins as are found in blood serum. In particular there is no suggestion that such lipase can hydrolyze protein-bound cholesterol esters.
U.S. Patent No. 3,898,130 to Konatsu issued August 5 1975 describes a method for hydrolyzing triglycerides com-; prising contacting triglyceride with a composition comprising a mixture of a microbial lipase, particularly Candida lipase(sic), a pancreatic lipase and a bile salt selected from sodium tauro-

3~ deoxycholate, taurocholate, taurocheno~eoxycholate and tauro-dehydrocholate. Both the microbial and the pancreatic lipase enzymes are critical components o~ the hydrolysis composition.

German Offenlegungsschrift No. 2,522,432 published December 4, 1975 describes an enzymatic method for hydrolyzing cholesterol esters using a cholesterol esterase from Pseudomonas fluorescens. There is no suggestion of the use of or a need for a surfactant to achieve protein-bownd cholesterol ester hydrolysis.
French Patent No. 2,2239696 and U.S. Patent No.
3,925,164 issued December ~ 1975 describe an assay for total cholesterol in blood serum wherein cholesterol esters ~re ~ 10 hydrolyzed with an ~nzyme preparation from Candida ru~_a, E~ or Aspergillus in the presence of a surfactant.
The only suggested surfactant is hydroxypolyethoxy dodecane.
; As will be shown in the examples below, such surfactants are not as effective as the materials described herein, possibly - because of incompatibility wlth the cholesterol ester hydrolase.

German Offenlegu~gsschrift No. 2,509,156 published September 23, 1975 describes an enzymatic method for the assay of total cholesterol using as the cholestervl ester hydrolyzing medium cholesterol esterase and a gallic acid or a salt of a gallic acid. The cholesterol esterase is identified as ~C 3.1.1.13 which is derlved from Nocardia restrictus. There is no suggestion in the foregoing publica-tion that the synthetic surfactants described herein are useful effectors for cholesterol esterase.
Related A~plications Commonly-owned U. S. Patent No. 4~179,3349 issued on December 18, 1979, entitled "Hydrolysis of Protein-Bound Triglycerides" discloses a process and a composition for hydrolyzing protein-bound triglycerides, featuring a com-patible mixture of an enzyme preparation and a surfactant.

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Summary of the Invention It has now been discovered that protein-bound cholesterol esters can be hydrolyzed in relatively short periods of time on the order of less than about 10 minutes (preferably in about 5 minutes) by contacting the protein-bound cholesterol esters with a compatible mixture of a lipase preparation which demonstrates cholesterol ester hydrolase activity and an alkyl phenoxy polyethoxy ethanol comprising a polyoxyethylene chain of less than about 20 oxyethylene units.
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 pos- ¦
sible 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.
Description of the Preferred mbodiments As discussed hereinabove, there are enzyme preparations 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 effe~t of the protein-lipid complex which prevents the enzyme ~rom catalyzing the hydrolysis in the usual fashion. The prior art has suggested the use of what might be termed effectors3 namely, agents which increase the rate at which lipase materials can hydrolyze protein-bound cholesterol esters. Although the mechanism by which - such agents act is not known, it is theorized that they disrupt - 30 the ester-protein complex in some way to "free" the ester for hydrolysis in a conventional mode. Protease enzymes, for example, have been suggested ~or this purpose.

We have found that certain 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 multilaye~ elements for ;~
the detection of anaIyte as described in Belgian Patent No. 801,742, the compositions described herein are particu~
larly useful in such elements.
The novel hydrolysis 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 alkyl phenoxy , polyethoxy ethanol comprising a polyoxyethylene chain of less than about 20 oxyethylene units.

Enzyme preparations potentially useful in the methods and compositions described herein are those demonstrating free- `
(l.e., not protein-bound) cholesterol ester hydrolase activity. ;
~ipase preparations which demonstrate such activity are 20 specifically preferred.
; A useful screening technique for determining the cholesterol ester hydrolase (esterase) activity of enzyme, and particularly lipase, preparations comprises adding a flxed amount of the enzyme preparation to a standard cholesteryl linoleate solution at pH 7.0, incubating at 37C under N2 for 2 hours and determining the amount of ester left in the solu-tion by the hydroxylamine method of J. Vonhoeffmyr and R. Fried, r 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 about 25 mg/dl cholesterol in the-screening procedure should be considered a useful candidate in the practice of the present invention.
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Useful enzyme preparations for cholesterol ester hydrolysis may be derived from plant or animal sources but we prefer preparations from microbial sources such as from Candida rugosa, Chromobacterium viscosum, variant ~
lipolyticum, crude or purified. Other useful enzyme preparations and methods for their preparation are described in the following U.S. Patents: 25888,385 to Grandel issued May 26, 1959; 3,168,448 to Melcer et al issued February 2, 1965; 3,189,529 to Yamada et al issued June 15, 1965;
3,262,863 to Fukumoto et al issued July 26, 1966; and 3,513,073 to Mauvernay et al issued May 19, 1970.
Preferred commercial enzyme prepara-tions include wheat germ Iipase from Miles Laboratorles of Elkhart, Indiana, Lipase 3000 from Wilson Laboratories, Steapsin from Sig~a Chemical Company (both of the latter are pancreatic enzymes), and Lipase M (from Candida rugosa) from Enzyme Development Company.
Certain surfactants inhibit the cholesterol esterase activlty of certain enzyme preparations. Consequently, it is important that before any attempt is made to combine an enzyme preparakion 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 descrlbed below. An enzyme preparatlon and sur~actant mixture 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 compositio~s 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 . ~ ~

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added and the mixture incubates at 37C for a period of about 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 o~ 1.2 ml by the well known cholesterol oxidase system described below. A slmilar "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 but 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% Or 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 ab~ut 90% of the available cholesterol esters in less than about 10 minutes.
From testing of this type9 it has been discovered that phenox~ polyethoxy ethanols are highly superior effectors, Specifically preferred are materials available commercially from Rohm and Haas Company under the trade~ s Triton X-114, 100, 102 and Triton n-101. Preferred alkyl phenoxy polyethoxy ethanols comprise a polyoxyethylene chain of less than about 3 20 oxyethylene units, As will be shown in the following éxamples, similar materials outside Qf these broad limits do 9 _ 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 below3 certain prior art surfactants discussed above do not produce acceptably high dissociation when combined with the enzyme preparations evaluated. This includes the hydroxypolyethoxy dodecanes o~ French Patent No. 2,223,696 and U.S. Patent No.
3,925,164. (Exemplary useful hydrolysis compositions are shown in Table I.) ~ he concentration of enzyme preparation and sur-factant 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, the particular ~` surfactant used, etc. Generally, however, surfactant concen-trations of from about 0.25 to about 10% by weight of the analytical solution have been found useful wlth concentrations .
of between about 0.5 and 5% bv weight of surfactant providing optimum results. The useful range of concentrations of enzyme preparation will vary similarly3 but concentrations of between about 10 and 80 mg/ml of the total analytical solution have been found quite useful when commercial preparations are used.
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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 ~; 30 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 composi~ions described herein are incorporated into one or more layers of multilayer analytical elements of the type described, or example, in Belgian Patent No. 801,742.
; Such 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 com-ponents 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 as described in detail hereinafter. 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 contiyuous : ! :

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or effectively so for purposes of fluid passage. Alternatively, it may be appropriate to prepare elements that have layers initially non-contiguous, and which ~ur~her can be spaced apart, such as by the use of interleaves as descrlbed, 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, i~ the element has initially non-contiguous layers, it may be necessary to apply compressive force or otherwise provlde 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 compo-sition sensitive to hydrogen peroxide for cholesterol detection, ~`
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The following description of standardized procedures and examples are presented to further illustrate the useful scope of the present invention.
~ Standard Procedures:
`~ Quantification of Total Serum Cholesterol - Choles--- terol esters must first be hydrolyzed to free cholesterol.
Incubation mixtures contained in a total volume of 8 ml:2.4 units cholesterol oxidase (N. cholesterolicum), 0.768 mg 4-amino-__ antipyrene HCl, 0.256 mg 1,7-dihydroxynaphthalene, 0.22 mg peroxidase (125 purpurogallin units/mg), 6.4 mg crude lipase preparation~ and either o.48 mg protease (B subt11is (Sigma Corporation Type VII)) or 160 mg octyl pheno~y polyethoxy ethanol (Triton X-100). Incubation mixtures were equilibrated at 37C for five minutes and the reaction was initiated by .

addition of 20 ~1 of human serum. A~ter 10 minutes, the absorbance at 490 nm was measured. Blank tubes contained all components except serum. Tot~] cholesterol concentra-tions were obtained from a standard curve which was con-structed~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, (1965~ pp. 1052-1064. This method involves extrac~ion of the cholesterol and cholesterol esters from serum prior to quantitation.

Example 1 ~ Enzyme Catalyzed Hydrolysis of Serum Cholesterol Esters in the Presence of Alkyl Phenoxy Pol ethox Ethanol Surfactant Y Y - :.
Human serum (20 ~1) was added to 8 ml of buffer ~, reagent (equilibrated---at--37) which contained---either enzyme ~' preparation and a protease or the enzyme preparation and surfactant :::
~, to-~effect ~ydro-lysis of the cholestero~ esters. After 10 ; 20 mlnutes, 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 con-.i, .
'~, comitant production of H202. The H2O2 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 ,, ,~, 3O mixture. The data in Table I show that in the presence of ~' S-l (an octylphenoxy polyethoxy ethanol having about lO ethoxy ~ units and an HL3 number of 13.5) complete hydrolysis of ,- -13-- : .,.:: . .

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cholesterol esters was observed. The surfactant efficiently replaced the protease and thus eliminated the need for this ex~raneous protein which may undesirably (1) hydroly~e protein components o~ the cholesterol detection system or (2) alter the pH of the system. Quantitation of serum cholesterol with a lipase preparation and S-1 as the hydrolytic system gave results (see Table I~ ~hich compared very favorably with the reference method.
TABLE I
Total Serum Cholesterol Concentration Lipase Protease Lipase Referenae Sample (Gontrol) 2% S-l Method 2 150 1~5 190 .

aThe reference method was a semi-automated :` Liebermann-Burchard method , .
~ 20 An S-l concentration of 2~ gave complete hydrolysis -. and the surfactant produced no harmful effects at concentra-:
tions as high as 4~. Although final color densities were measured after 10 minutes7 reactions were essentially complete in as little as five minutes at 37C.
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Comparative Example I
Repetition of the test described in Example I above using alkylphenoxy polyethoxy ethanols having polyoxyethylene chains above about 20 yields results which indicate that such ` 30 surfactants somehow inhibit the cholesterol esterase activity of the enzyme~

. , . -14-Comparative Example II
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 surf'actants described herein.
Incubation mixtures were prepared containing in a total volume of` o.6 ml:

O,5 ml normal human serum mg cholesterol esterase (Lipase M commercial preparation f'rom Candida rugosa) ~ moles potassium phosphate buff'er (pH 7.0) mg ef'fector 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.

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From the foregoing, it is apparent that the poly-oxyethylene 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.
The invention has been described in detail with particular reference to certain pre~erred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the inventionO

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Claims (30)

What is claimed is:

1. A process for hydrolyzing protein-bound cholesterol esters comprising contacting protein-bound choles-terol esters in an aqueous medium with a compatible mixture of an enzyme preparation having cholesterol ester hydrolase activity and an alkyl phenoxy polyethoxy ethanol comprising a polyoxyethylene chain of less than about 20 oxyethylene units.

2. A process for hydrolyzing blood serum cholesterol esters comprising contacting serum containing cholesterol esters with a compatible mixture of an enzyme preparation having cholesterol ester hydrolase activity and an alkyl phenoxy polyethoxy ethanol comprising a polyoxyethylene chain of less than about 20 oxyethylene units.

3. The process of claim 2 wherein the enzyme preparation is an esters preparation derived from a micro-bial or an animal source.

4. The process of claim 3 wherein the esters preparation is derived from a microbial source selected from the group consisting of Candida rugosa, Chromobacterium viscosum, variant paralipolyticum, and Rhizopus arrhizus.

5. The process of claim 4 wherein the esterase preparation is derived from Candida rugosa.

6. The process of claim 3 wherein the esterase preparation comprises a pancreatic esterase.

7. The process of claim 2 wherein the surfactant is an alkyl phenoxy polyethoxy ethanol of from about 7 to about 13 ethoxy units.

8. The process of claim 7 wherein the surfactant is an octyl phenoxy polyethoxy ethanol.

9. A process for hydrolyzing protein-bound cholesterol esters comprising contacting protein-bound cholesterol esters in an aqueous medium with a compatible mixture of a microbially derived enzyme preparation having cholesterol ester hydrolase activity and an effector which is an alkyl phenoxy polyethoxy ethanol comprising a polyoxyethylene chain of less than about 20 oxyethylene units.

10. In a process for assaying blood serum for total serum cholesterol content in which cholesterol esters are first hydrolyzed, and subsequently an assay for total cholesterol is performed, the improvement comprising hydrolyzing the cholesterol esters in an aqueous medium with a compatible mixture of an enzyme preparation having cholesterol ester hydrolase activity and an alkyl phenoxy polyethoxy ethanol comprising a poly-oxythylene chain of less than about 20 oxyethylene units.

11. The process of claim 10 wherein the enzyme preparation is an enzyme preparation derived from a microbial or an animal source.

12. The process of claim 11 wherein the microbial source is selected from the group consisting of Candida rugosa, Chromobacterium viscosum, variant paralipolyticum, and Rhizopus arrhizus.

13. The process of claim 12 wherein the enzyme preparation is derived from Candida rugosa.

14. The process of claim 10 wherein the esterase preparation comprises a pancreatic esterase,

15. The process of claim 10 wherein the surfactant is an alkyl phenoxy polyethoxy ethanol of from about 7 to about 13 ethoxy units.

16. The process of claim 15 wherein the surfactant is an octyl phenoxy polyethoxy ethanol.

17. A composition for hydrolyzing protein-bound cholesterol esters comprising a compatible mixture of an enzyme preparation having cholesterol ester hydrolase activity and an alkyl phenoxy polyethoxy ethanol comprising a poly-oxyethylene chain of less than about 20 oxyethylene units.

18. The composition of claim 17 wherein the enzyme preparation is an esterase preparation derived from a microbial or an animal source.

19. The composition of claim 18 wherein the esterase preparation is derived from a microbial source selected from the group consisting of Candida rugosa, Chromobacterium viscosum, variant paralipolyticum, and Rhizopus arrhizus.

20. The composition of claim 19 wherein the esterase preparation is derived from Candida rugosa.

21. The composition of claim 18 wherein the esterase preparation comprises a pancreatic esterase.

22. The composition of claim 18 wherein the surfactant is an alkyl phenoxy polyethoxy ethanol of from about 7 to about 13 ethoxy units.

23. An analytical element for the detection of cholesterol esters in an aqueous medium, said element comprising, in at least one layer, a hydrolysis composition for protein-bound cholesterol esters comprising an enzyme preparation having cholesterol ester hydrolase activity and an alkyl phenoxy polyethoxy ethanol comprising a polyoxyethylene chain of less than about 20 oxyethylene units.

24. The element of claim 23 wherein the element comprises a spreading layer and a reagent layer.

25. The element of claim 24 wherein the enzyme preparation is derived from a microbial or an animal source.

26. The element of claim 25 wherein the microbial source is selected from the group consisting of Candida rugosa, Chromobacterium viscosum, variant paralipolyticum, and Rhizopus arrhizus.

27. The element of claim 26 wherein the enzyme preparation is derived from Candida rugosa.

28. The element of claim 25 wherein the enzyme preparation comprises a pancreatic esterase.

29. The element of claim 23 wherein the surfactant is an alkyl phenoxy polyethoxy ethanol of from about 7 to about 13 ethoxy units.

30. The element of claim 29 wherein the surfactant is an octyl phenoxy polyethoxy ethanol.