SE462047B - SET TO ANALYZE BLOOD SERIES WITH REGARD TO TOTAL QUANTITY OF SERUM CHOLESTEROL AND A MULTI-LAYER ANALYSIS ELEMENT ADAPTED FOR ANALYSIS - Google Patents

Description

462 047 Aspergillus in the presence of a surfactant. The only proposed surfactant is hydroxypolyethoxydodecane.

U.S. Pat. No. 3,925,164 discloses a method for hydrolyzing cholesterol esters using cholesterol esterase, but has not realized the problem of protein-bound cholesterol esters, much less its solution. U.S. Pat. Nos. 3,884,764 and 3,983,005 disclose a method for hydrolyzing protein-bound cholesterol esters I using a protease, a lipase having cholesterol esterase activity and, optionally, a surfactant. Among the listed surfactants is TRITON X-100, which is an alkylphenoxy polyethoxyethanol. When a surfactant is used, this is done in relatively small concentrations.

The present invention provides a method for hydrolyzing protein-bound cholesterol esters without the use of a protease and capable of higher efficiency than the prior art methods. The present invention relates to a process for the hydrolysis of protein-bound cholesterol esters, which process comprises treating the esters in an aqueous medium with a compatible mixture, as defined below, of an enzyme preparation having cholesterol ester hydrolase activity and an alkylphenoxy polyethoxyethanol with a polyoxyethylene chain with less than 20 oxyethylene units.

Protein-bound cholesterol esters can be hydrolyzed within a relatively short period of time in the order of less than 10 minutes (preferably 5 minutes) according to the invention.

The methods and compositions described in connection with the present invention allow the use of a much larger number of enzyme preparations such as cholesterol ester hydrolyzing agents than was possible with prior art methods. Thus, cheaper materials can be used to achieve reaction times and states of completed reaction which are at least equal to and often superior to the reaction times and states of completed reaction which can be “achieved by known methods and materials.

Enzyme formulations which catalyze the hydrolysis of free cholesterol esters are known in the art. However, such materials catalyze the hydrolysis of cholesterol esters bound to protein, which are found in blood serum, only at very low rates 466,047 or incompletely. This is obviously due to some effect of the protein-lipid complex, which prevents the enzyme from catalyzing the hydrolysis in the usual way. It has been proposed to use something which can be termed as effectors, namely agents which increase the rate at which the lipase materials can hydrolyze protein bound cholesterol esters. Although the mechanism by which such agents act is not known, it is believed that they break the ester-protein complex in some way to "free" the ester for hydrolysis in a conventional manner. Protease enzymes, for example, have been proposed for this purpose. It has now been found that certain surfactants are effectors and can be used as substitutes for protease to make enzyme preparations which are not normally capable of catalyzing the hydrolysis of protein-bound cholesterol esters or which catalyze such hydrolysis only at unacceptably low rates, useful as hydroly protein-binding cholesterol esters. Furthermore, since the protease tends to degrade the proteinaceous binders, such as gelatin, used in multilayer analyte detection elements as described in GB PS 1440 464, the compositions described herein are particularly suitable in such elements.

The hydrolysis compositions according to the invention thus comprise a compatible mixture of an enzyme preparation which exhibits cholesterol esterase activity and as effector a surfactant which consists of an alkylphenoxypolyethoxyethanol which comprises a polyoxyethylene chain with less than 20 oxyethylene units.

Enzyme formulations which can be used in the methods and compositions of the invention are those which exhibit free (ie not protein bound) cholesterol ester hydrolase activity. Lipase formulations having such activity are especially preferred.

A suitable selection technique for determining the cholesterol ester hydrolase (esterase) activity of enzyme, and especially lipase, formulations involves adding a certain amount of the enzyme preparation to a standard cholesterol ylin lineate solution at pH 7.0, incubating at 37 ° C. in a nitrogen atmosphere for 2 hours and determines the amount of ester remaining in the solution by the hydroxylamine method of J. Vonhoeffmyr and R. Fried, Z. Klin. Chem. U. Klin. Biochem., §, 134 (1970). Any preparation which exhibits a cholesterol esterase activity which releases more than 25 mg / dl of cholesterol in the selection process of this method should be considered suitable in the practice of the present invention. Suitable enzyme preparations for cholesterol-cluster hydrolysis may be derived from vegetable or animal sources, but preparations from microbial sources, such as Candida rugosa, Chromobacterium viscosum, variant paralipolyticum, which may be crude or purified are preferred. Other suitable enzyme preparations and methods for their preparation are described in the following U.S. Pat. No. 2,888,385; 3,168,448; 3 189 529; 3,262,863 and 3,513,073. P fl Suitable commercial enzyme preparations include lipase from wheat germ from Miles Laboratories, Elkhart, Indiana, Lipase 3000 from Wilson Laboratories, Steapsin from Sigma Chemical Company (the latter two being pancreatic enzymes), and Lipase M (from Candida rugosa) from Enzyme Development Company.

Certain surfactants inhibit the cholesteryl activity of certain enzyme preparations. Accordingly, before attempting to combine an enzyme preparation and a surfactant for use as described, it is important to determine the compatibility of the two components of the composition. Such a determination is conveniently performed by the test described below. A mixture of a single formulation and a surfactant which successfully passes this test is referred to herein as a compatible mixture and each component of the mixture is said to be compatible with the other.

The hydrolysis compositions of the invention are characterized by the test used in Comparative Example 2 below. The proposed surfactant to be tested is added to normal human serum. A sample of a proposed enzyme preparation is added and the mixture is incubated at 37 ° C 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. The cholesterol is quantified to a total volume of 1.2 ml with the well-known cholesterol oxide system, which is described below. A similar "control" test is performed simultaneously with only the enzyme preparation without surfactant.

When performing the indicated test, it is advisable to run a blank, which contains all the components of the mixture except the enzyme preparation, so that a reaction which may be due to free cholesterol or other components in the serum can be subtracted. The preferred compositions provide hydrolysis to at least 70% of available cholesterol esters in less than 10 minutes and the most suitable compositions are those which provide substantially complete hydrolysis, i.e. hydrolysis to at least 90% of available cholesterol esters in less than 10 minutes. 5,462,047 It has been discovered that phenoxy polyethoxyethanols are very superior to effectors. Particularly suitable are the materials which can be obtained from Rohm and Haas Company under the trademarks Triton X-114, 100, 102 and Triton n-101. Preferred alkylphenoxy polyethoxyethanols include a polyoxyethylene chain of less than 20 oxyethylene units.

The most suitable alkylphenoxy polyethoxyethanols include polyoxyethylene chains having 7 to 13 oxyethylene units. As will be shown in the following examples, similar materials outside these wide limits do not provide the improved hydrolysis described. Particularly suitable are those materials in which the alkyl group has either 8 or 9 carbon atoms.

As will be shown in the following examples, the hydroxypolyethoxydodecanes of French Pat. No. 2,223,696 and U.S. Pat. No. 3,925,164 are ineffective when used in place of the surfactants of the invention (suitable useful hydrolysis compositions are shown in Table I). The concentration of the enzyme preparation and the surfactant in the compatible mixtures which can be used for hydrolysis according to the invention can vary greatly depending on, for example, such factors as the enzyme preparation purity, the enzyme preparation activity, the nature of the bound cholesterol ester and the particular surfactants. was used. As a rule, however, concentrations of the surfactant from 0.25 to 10% by weight of the analytical solution have proved suitable and concentrations from 0.5 to 5% by weight of surfactant give optimum results. A suitable concentration range for the enzyme preparation varies similarly, but concentrations from 10 to 80 mg / ml of the total assay solution have been found to be quite useful when commercial formulations are used. Optimization of such compositions is of course within the scope of professional measures.

It is further understood that hydrolysis compositions of the type described may be included in any of the monolayer or multilayer absorbents or other assay elements (eg, test paper) described early and the use of compositions and methods of the invention in such elements. for the detection or determination of protein-bound cholesterol esters falls within the scope of the invention.

According to a preferred embodiment, the described hydrolysis compositions are incorporated in one or more layers of the multilayer analysis elements described in, for example, GB PS 1,440,464 and US PS 3,992,158. Such analysis elements are intended for analysis of liquids with respect to the presence of a given analyte and they comprise a suitably non-fibrous spreading layer which gives a uniform apparent concentration of analyte components in a sample applied to the reagent layer, which layer contains at least some of the materials which are interactive in the presence of the analyte for formation of a detectable product or detectable change. Such layers @ are in fluid contact under the conditions of use.

The term "fluid contact" between the layers of an assay element refers to the ability of the fluid, whether this fluid is liquid or gaseous, to pass into such an element between superimposed areas of the spreading layer and the reagent layer. In other words, the term fluid contact refers to the ability of the components of a fluid to pass between the layers of fluid contact.

Although such layers in fluid contact may be adjacent to each other, they may also be separated by intermediate layers. However, layers in the element that physically separate a spreading layer and a reagent layer in mutual fluid contact do not prevent the passage of fluidity between the spreading layers and reagent layers that are in fluid contact.

Fluid contact between the layers can be achieved by producing elements with layers which initially adjoin each other or act in this way to provide fluid passage. Alternatively, it may be convenient to produce elements having layers which are initially not adjacent to each other and which can be further separated by, for example, the use of intermediate layers as described in US PS 3,511,608 or the use of an elastic absorbent material or deformable carriers as described in U.S. Pat. No. 3,917,453 and U.S. Pat. No. 3,933,594. It will be appreciated that if the element initially has non-adjacent layers, it may be necessary to apply compressive forces or otherwise bring the layers of the element into fluid contact at the opportunity to achieve an analysis result.

According to a highly preferred embodiment of such an element, the hydrolysis composition described is incorporated in the dispersion layer and a detection system, eg cholesterol oxidase, and an indicator composition, which is sensitive to hydrogen peroxide, for cholesterol detection is incorporated in the reagent layer. The following description of standardization methods and examples is provided to further illustrate the utility of the invention. 462 047 STANDARD METHODS: Determination of the total amount of serum cholesterol ~ The cholesterol esters must first be hydrolyzed to free cholesterol. Incubation mixtures contained a total volume of 8 ml 72.4 units of cholesterol oxidase (N. cholesterolicum), 0.768 mg of 4-amino-antipyrene.HCl, 0.256 mg of 1.7 dihydroxynaphthalene, 0.22 mg of 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 octylphenoxy polyethoxyethanol (Triton X-100). The incubation mixtures were equilibrated at 37 ° C for 5 minutes and the reaction was initiated by the addition of 20 μl of human serum. After 10 minutes, the absorbance was measured at 490 nm. Test tubes with blank samples contained all components. except serum. The total cholesterol concentration was obtained from a. standard curve, obtained by replacing the serum substrate with aliquots of Fermco Test Aqueous Cholesterol Standard v (from Fermco Laboratories, Chicago, Illinois).

The reference method was the Liebermann-Burchard method described. in Hawk's Physiological Chemistry, B.L. Oser, 4th Edition, I McGraw-Hill Book Company, New York, pp. 1062-1064. This method involves extracting cholesterol and cholesterol esters from serum before quantification.

EXAMPLE 1 - Enzyme catalyzed hydrolysis of serum cholesterol esters in the presence of alkylphenoxy polyethoxyethanol surfactant.

Human serum (20 μl) was added to a buffer buffer reagent (equilibrated at 37 ° C), which contained either enzyme preparation and a protease or enzyme preparation and a surfactant to effect hydrolysis of the cholesterol esters. After 10 minutes, the absorbance was measured at 490 nm and the total serum cholesterol amount was calculated as described above.

Serum cholesterol levels were determined using the cholesterol oxidase peroxidase system. In doing so, the cholesterol esters were first hydrolyzed to free cholesterol, which was then oxidized to cholesterol with concomitant hydrogen peroxide formation. The hydrogen peroxide was then coupled to dye via peroxidase reaction. It has been reported that crude lipase preparations catalyze the hydrolysis of serum cholesterol esters if protease is added to the incubation mixture. The results in Table I show that in the presence of S-1 (an octylphenoxy polyethoxyethanol with 10 ethoxy units and a hydrophilic-lipophilic balance number (HLB) of 13.5) complete hydrolysis of cholesterol esters is observed. The surfactant successfully replaced the protease and thus eliminated the need for this foreign protein, which can undesirably (1) hydrolyze the protein components of the cholesterol detection system or (2) change the pH of the system.

Determination of the amount of serum cholesterol with a lipase preparation and S-1 as the hydrolytic system gave results (see Table I) which performed well in a comparison with the reference method.

TABLE I Total concentration of serum cholesterol (mg / ai) 'Sample Lipase protease (control) Lipase 2% S-1 Reference method a 1 225 225 232 2 150 185 190 3 150 180 176 4 240 230 220 a The reference method was a semi-automated Liebermann-Burchard method.

An S-1 concentration of 2% gave incomplete hydrolysis and the surfactant gave no adverse effects at concentrations as high as 4%. Although the final dye densities were measured after 10 minutes, the reactions were essentially completed via 37 ° C. In as short a time as Repetition of the indicated test with alkylphenoxypolyethoxyethanols with polyoxyethylene chains over about 20 gives results indicating that such surfactants in some way inhibit the cholesterol esterase activity of the enzyme and are ineffective in the process of the invention.

Direct comparative tests were performed with a cholesterol esterase from Candida rugosa and (a) a surfactant according to French Patent Specification 2,223,695 and (b) a representative number of surfactants used in the process of the invention.

The incubation mixtures were prepared so that the volume of 0.6 ml contained: that in a total of 20 ml of normal human serum 20 mg of cholesterol esterase (a commercial Lipase M preparation from Candida rugosa) .4 Pmol of potassium phosphate buffer ( pH 7.0) 10 mg effects The reactions were allowed to proceed for 10 minutes via 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 The cholesterol was then quantified with cholesterol oxidase the peroxidase system with aqueous cholesterol standard for the determination of a standard curve, the results of these tests are shown in Table II below.

From the above it appears that polyoxyethylene lauryl ethers (ie dodecane material according to French patent specification 2,223,696) are not suitable as effectors according to the invention. 10 462 047 O ß ~ mm N_N Oø fi m.H ^ .HmuwnamcøHäum fl xo uw flfl un fl Human mmunwumm Eöc fi mcnouww fl m. mod «.m ßm fl«. ~ H flflfiflflflfi UÜCC5> # 5 W HÜUMO fl0 GC5> UD ßm fl ßm fl ßm fi ßm fl ßm fi. Houwumw fi ox xm fl uxmm Hw \ ms HH Anmmêâ. nwuw fi æusm fl Am fl vcw fl æum fl xoæ fl om Hwum fl æusm fl Ammvcw fi hum fl xoæ fl om uwum fi æuøm fi ^ w.cwHauw fl xoæHom Hnamnwwwouwh fi om fl xocwmn ux hux.

Claims (5)

ll PATENT REQUIREMENTS 1. A method of analyzing blood serum for total serum cholesterol, characterized in that the cholesterol esters are first hydrolysed by treatment in the absence of protease with a compatible mixture of an enzyme preparation having cholesterol ester hydrolase activity and an alkylphenoxy polyethoxyethanol with a polyoxyethylene chain than 20 oxyethylene units, the mixture containing the alkylphenoxy polyethoxyethanol in an amount effective to hydrolyze at least 70% of the available cholesterol esters in the serum, and that the amount of cholesterol formed is subsequently analyzed. 2. A method according to claim 1, characterized in that the hydrolysis and analysis takes place in a multilayer analysis element with a layer containing the hydrolysis mixture and a reagent layer which gives a detectable color change in response to cholesterol. A multilayer assay element for analyzing blood serum for total serum cholesterol according to claim 1 or 2, characterized in that the assay element comprises at least one layer containing a protein-bound cholesterol esterase composition for hydrolysis of protein-bound cholesterol esters in the absence of protease. which composition comprises an enzyme preparation having cholesterol ester hydrolase activity and an alkylphenoxypolyethoxyethanol having a polyoxyethylene chain of less than 20 oxyethylene units, the mixture containing the alkylphenoxypolyethoxyethanol in an amount effective to hydrolyze at least 70% of the available cholesterol esters in the serum. A multilayer analysis element according to claim 3, characterized in that the element has a spreading layer and a reagent layer. Multilayer assay element according to claim 3 or 4, characterized in that it contains one or more layers, which give a detectable color change in response to the presence of cholesterol.