Method for the enzymatic hydrolysis of cholesterol esters

Abstract

A totally enzymatic method for the hydrolysis of cholesterol esters using a lipase having cholesterol esterase activity and a protease and a method for quantitative determination of total cholesterol in compositions containing both free and esterified cholesterol comprising enzymatically hydrolyzing the cholesterol esters with a lipase having cholesterol esterase activity and a protease and determining total cholesterol by gas-liquid chromatography or some other suitable technique are described.

Description

FIELD OF THE INVENTION

This invention relates to an enzymatic method for hydrolyzing cholesterol esters in complex aqueous solutions which may contain both free and esterified cholesterol, for example, blood serum and in particular to a process comprising enzymatically hydrolyzing cholesterol esters using a lipase having cholesterol esterase activity and a protease.

BACKGROUND OF THE INVENTION

The most common clinical estimations of cholesterol in blood serum are for "total cholesterol." This value is a measure of cholesterol and cholesterol esters present in the serum and anything else such as cholesterol precursors that respond indiscriminately to the usual tests which are based on reactions involving "free" cholesterol and require prior conversion of cholesterol esters to "free" cholesterol.

In a well-known conventional procedure, serum is extracted with an organic solvent, the extract is saponified with alcoholic KOH and the liberated cholesterol is isolated and assayed. These methods require the handling of corrosive chemicals and are tedious, time-consuming, and not readily automated.

German, Offenlegungsschrift No. 2,246,695 published Mar. 29, 1973, describes an enzymatic assay for free cholesterol using a cholesterol oxidase, however, this technique requires hydrolysis of the blood serum cholesterol esters using the cumbersome techniques of the prior art prior to application of the enzymatic assay.

G. Bucolo, and H. David, Clin. Chem., 19 476 (1973) describe a lipase-protease system for hydrolyzing serum triglycerides; however, it is specifically stated that cholesterol esters are not hydrolyzed in this system.

OBJECTS OF THE INVENTION

It is therefore an object of the present invention to provide a simplified and improved technique for hydrolyzing cholesterol esters.

It is another object of the present invention to provide a reproducible enzymatic process for the quantitative hydrolysis of cholesterol esters in blood serum, useful as an initial step in the quantitative determination of total cholesterol in blood serum.

SUMMARY OF THE INVENTION

The foregoing objects are accomplished by an improved process for hydrolyzing cholesterol esters which comprises treating a sample of an aqueous solution containing cholesterol esters, for example blood serum, with a mixture of a lipase having esterase activity and a protease to release free cholesterol. Subsequently, the free cholesterol can be assayed using any suitable technique including gas-liquid chromatography, cholesterol oxidase assay, or any of a number of other well known techniques for the assay of "free" cholesterol.

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred embodiment of the present invention, hydrolysis of cholesterol esters in complex aqueous solutions (referred to hereinafter generally as blood serum) is achieved by treating the blood serum with a mixture comprising per ml of serum from about 20 to about 50 mg of a lipase having cholesterol esterase activity and from about 5 to about 50 mg of a protease at a temperature of from about 25.degree. to about 55.degree.C and a pH of from about 6.5 to about 9.5 for about 5 to about 15 minutes, preferably with agitation and in an inert atmosphere. When the foregoing concentrations of lipase and protease are utilized, the lipase should contain at least about 30 international units per mg and the protease at least about 10 units per mg. One unit of lipase is defined as the amount of the enzyme which will liberate 1 micro mole of fatty acid in a given time at a given pH and temperature using a substrate containing esterified fatty acid. For the preferred lipase materials described hereinbelow the conditions are 1 minute at pH 7 and 37.degree.C with olive oil as substrate. One unit of protease will hydrolyze casein to produce color equivalent to 1 micro mole (181 .mu.g) tyrosine per minute at pH 7.5 and a temperature of 37.degree.C. (Color per Folin-Ciocalteu Reagent). It should, of course, be clear that as the level of enzyme activity per unit by weight of preparation increases or decreases, so also will the quantity of enzyme preparation added vary. Most preferably, the ratio of lipase to protease on an activity basis should range from about 3 to about 10 and at least about 1000 units of lipase should be used per ml of serum. Relative lipase activity to esterase activity is usually about 10 to about 50.

Generally speaking and in accordance with illustrative embodiments of our invention, we contact an aqueous medium containing the cholesterol ester, preferably blood serum, which contains both esterified and free cholesterol, with a mixture of a lipase which demonstrates cholesterol esterase activity as defined in Example 3 and a protease. The lipase may be of plant or animal origin, but we prefer and find best a microbial lipase such as the lipase from Candida cylindracca. Lipases from Chromobacterium viscosum, variant paralipolyticum, crude or purified, the lipase from Rhizopus delemar, purified, for example as noted in Fukumoto et al, J. Gen. Appli. Microbiol, 10, 257-265 (1964), and lipases having similar activity, which are those described in the aforementioned Bucolo and Davis publication, do not demonstrate the required cholesterol esterase activity. Specifically preferred commercial lipase preparations include wheat germ lipase supplied by Miles Laboratories of Elkhart, Ind., Lipase 3000 supplied by Wilson Laboratories, Chicago, Ill., Steapsin supplied by Sigma Chemical Co., St. Louis, Mo., (both of the last two enzymes are pancreatic enzymes) and Lipase M (from Candida cylindracca) supplied by Enzyme Development Corporation, New York, N.Y. Screening of lipases for this purpose to determine their cholesterol esterase activity may be accomplished using the technique described in Example (3) below. Using this technique, any lipase which demonstrates a cholesterol esterase activity which releases above about 25 mg% cholesterol in the screening procedure of Example 3 should be considered useful in the successful practice of the present invention.

Proteases in general may be used in the successful practice of this invention. These include by way of example, chymotrypsin, Streptomyces griseus protease (commercially available under the registered trademark "Pronase"), Aspergillus oryzae protease, Bacillus subtilis protease, elastas, papain and bromelain. Mixtures of such enzymes, of course, may also be employed, at times, with advantageous results as demonstrated in the examples below.

The protease utilized as described above, is only necessary where the cholesterol ester is present in a protein containing solution the most notable and important of which is, of course, blood serum. In the case where a simple non protenaceous solution is being assayed, it is possible, as demonstrated in Example 3 below, to obtain ester hydrolysis using only the lipase. As also demonstrated by the Examples, however, in a protein containing solution such as serum, the presence of the protease is essential to useful results.

As will be further elaborated below, the free cholesterol liberated by the action of the foregoing enzyme mixture may be assayed in a number of ways. According to a preferred embodiment of the instant invention, this assay is performed using gas-liquid chromatography.

According to a preferred embodiment of this "free" cholesterol assay technique, an aliquot generally from about 0.5 to about 5 ml of the hydrolyzed aqueous composition to be tested, hereinafter blood serum, is mixed with from about 0.5 to about 2 ml of heptane or some other suitable organic solvent, for example, isooctane, containing from about 25 mg to about 50 mg weight percent octacosane or some other organic suitable for use as an internal standard. The heptane may, of course, be replaced with any other solvents suitable for gas-liquid chromatography, for example, isooctane. The solvent mixture is then extracted with water according to conventional techniques, preferably using from about 3 to about 10 ml of water per ml of solvent solution. The water extracted solvent solution is then reacted with a silylating agent, for example, (N,O-bis(trimethylsilyl) trifluoroacetanide with 1% trimethylchlorosilane or a mixture of equal volumes of trimethylchlorosilane and 1,1,1,3,3,3,-hexamethyldisilazane for a period of from about 2 to about 15 minutes. The silylated solvent solution is then passed through a conventional gas-liquid chromatograph to determine the total cholesterol in the sample under examination. This method for determining cholesterol concentration is an adaptation of the technique described in detail in J. L. Driscoll, D. Aubuchon, M. Descoteaux and R. F. Martin, Anal. Chem., 43, 1196 (1971).

One of the most significant advantages of the instant enzymatic hydrolysis technique involves the requirement for dilution of blood serum for hydrolysis and assay. Surprisingly, using the techniques described herein, undiluted serum is hydrolyzed as rapidly and readily as diluted serum. This is quite surprising in view of the uniform requirements for serum dilution described in the prior art.

The following examples serve to illustrate particular embodiments of the present invention.

"Validate," a reconstituted serum standard produced by the Warner-Lambert Company, was used in the examples below. The total cholesterol content of "Validate" (lot 2560121) was checked by saponifying an aliquot according to the method of Driscoll et al, and analyzing the heptane extract by both glc and the Liebermann-Burchard method. Values of 160 and 162 mg% respectively were obtained. These are well within the range of values quoted by the supplier (148-192 mg%).

EXAMPLE 1 HYDROLYSIS OF CHOLESTEROL ESTER IN SERUM

A mixture of 1 ml "Validate" (a serum cholesterol standard sold by Warner-Lambert and containing 148-192 mg% cholesterol), 40 mg Lipase M, 40 mg papain, 8 mg .alpha.-chymotrypsin, and 0.1 M tris buffer to 3 ml total volume (pH 7.2) is incubated in a 25 ml flask under nitrogen at 50.degree.C and 250 rpm for 10 min.

The hydrolysis is performed in an atmosphere of nitrogen in order to minimize artifacts introduced by autooxidation of cholesterol and its esters; of course, proper correction for such autooxidation factors will permit hydrolysis to be performed in a normal atmosphere.

EXAMPLE 2

QUANTITATIVE ESTIMATION OF TOTAL CHOLESTEROL BY GAS-LIQUID CHROMATOGRAPHY (GLC)

One ml samples of serum or reconstituted serum standards ("Validate," Warner-Lambert) containing up to 150 mg% cholesterol are mixed with 5 ml ethanol and shaken 3 minutes with 1 ml heptane containing 50 mg% octacosane. Five ml of water is added and the mixture is shaken again for 3 minutes. When the layers separate, equal portions of the heptane layer and N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane are mixed. After 5 minutes reaction 1 .mu.l samples are injected into a Hewlett Packard F and M 810 chromatograph with a single stainless steel column (1/8 inch .times. 4 feet) packed with 3% SE30 on ov 1. Gas flow rate 20 ml/min, oven temperature 250.degree.C, injection port 260.degree.C, flame detector 265.degree.C, range 10.sup.2, attenuation .times. 1, chart speed 1/2 inch/min. Octacosane retention time is about 1 min. Cholesterol retention time is about 21/2 min. Runs are complete in about 4 min. Under these conditions the amount of cholesterol in the sample is proportional to the peak height ratios of cholesterol to octacosane. This method is adapted from Driscoll et al referred to above.

EXAMPLE 3

SCREENING OF LIPASES FOR CHOLESTEROL ESTERASE ACTIVITY

Tests were conducted with cholesteryl linoleate as the substrate because it is the major ester component of human serum and because it gives relatively stable emulsions compared to saturated esters such as the palmitate.

A solution of 200 mg redistilled cholesteryl linoleate in 5 ml ethyl ether was mixed with rapid stirring into 100 ml boiling water containing 430 mg sodium cholate. Five ml of this suspension was added to a solution of 50 mg of lipase preparation in 5 ml 0.1 M phosphate, pH 7.0. This mixture was incubated 2 hours at 37.degree.C, 400 rpm under N.sub.2. Cholesterol esters remaining after this treatment were determined by the hydroxylamine method of J. Vonhoeffmayr and R. Fried, Z. Klin. Chem. u Klin., Biochem., 8, 134 (1970) which involves quantitative conversion of esters to hydroxamic acids. The results are shown in Table 1.

Table 1 ______________________________________ Hydroxylamine Assay with Cholesteryl Linoleate Suspension ______________________________________ Cholesterol Enzyme released (mg%) ______________________________________ Lipase (Miles) 30 Lipase 3000 (Wilson) 42 Wheat Germ Lipase (Miles) 59 Steapsin (Sigma) 59 Lipase M (Enzyme Development Corp.) 68 ______________________________________

All of the enzymes show esterase activity. However, Lipase M is preferred because of its significantly greater esterase activity and also because it is a relatively inexpensive commercial enzyme. As purchased, the preparation is about 80% lactose, so on a protein basis its activity is about five times its activity on a weight basis.

EXAMPLE 4

ACTIVITY OF LIPASE WITH SERUM CHOLESTEROL ESTERS

Mixtures containing 40 mg. of lipase in 1 ml "Validate" were incubated 10 min. at 50.degree.C under nitrogen in 25 ml flasks at 250 rpm. The mixture was extracted and cholesterol was estimated as in Example 2. The enzymes tested and the results are given in Table II.

Table II ______________________________________ Esterase Activity with Serum as Substrate ______________________________________ Enzyme Cholesterol (mg%) ______________________________________ None 23 Lipase 3000 26 Lipase M 36 ______________________________________

Lipase M and lipase 3000 while exhibiting considerable esterase activity on cholesteryl linoleate emulsions show very little activity with serum esters.

EXAMPLE 5

ESTERASE ACTIVITY OF LIPASE-PROTEASE COMBINATIONS ON SERUM CHOLESTEROL ESTERS

Combinations of Lipase M and various proteases were tested in the same manner as in Example 4. Proteases, except for .alpha.-chymotrypsin, were added directly to serum at 40 mg per ml; .alpha.-chymotrypsin was added at 8 mg per ml. The amount of cholesterol (mg%) released by each combination is shown in Table III.

Table III ______________________________________ Enzymes Cholesterol (mg%) ______________________________________ None 23 Lipase M 36 .alpha.-Chymotrypsin (Sigma, Type 11 3X crystallized) 37 Chymotrypsinalpha. 129 Papain (Sigma, Grade-Crude Type 11) 29 Lipase M + papain 115 chymotrypsin + papain 114 ______________________________________

It is seen that in the presence of proteases such as .alpha.-chymotrypsin or papain the cholesterol esterase activity of Lipase M was enhanced nearly four fold. The proteases themselves may have a slight esterase activity but their major effect probably is to increase the availability of cholesterol esters to the lipase by breaking up ester-lipoprotein complexes in serum. Most cholesterol esters in serum are bound to lipoproteins.

Thus, for Lipase M to be optimally effective on serum cholesterol esters, a protease must also be present.

EXAMPLE 6

A series of commercially prepared proteases were tested for their ability to enhance esterase activity of Lipase M in serum. Protease was added directly to serum in the amounts shown in Table IV. Lipase M concentration was 40 mg per ml serum. Otherwise assay and conditions are the same as in Example 4. The results are contained in Table IV.

Table IV ______________________________________ Amount added Cholesterol Protease (mg/ml) (mg%) ______________________________________ 1. Aspergillus oryzae 20 118 (Sigma Type II) 40 123 80 145 2. Streptomyces griseus 5 147 (Sigma Type VI) 10 144 20 140 3. Bacillus subtilis 5 140 (Sigma Type VIII) 10 118 20 135 4. Bromelain 5 114 (Sigma Grade II) 10 127 20 132 5. Protease 30 20 80 (Rohm and Haas) 40 127 80 161 6. Pronase (Calbiochem, Grade A) 5 133 7. Subtilisin BPN (Sigma Type VII) 5 87 8. Protease, bacterial (Calbiochem, Grade B) 10 142 9. Lipase M 36 ______________________________________

All of the proteases tested appear to enhance the activity of Lipase M somewhat more than .alpha.-chymotrypsin and papain. However, it is difficult to compare the activities of these proteases on the basis of units given by suppliers since several different assays are used. In general, the enzymes judged less pure were used in the higher amounts.

The effect of pH value on the esterase activity was tested with different buffers and enzymes as described in Examples 7 and 8 below.

EXAMPLE 7

Four different buffers were tested in a system containing three enzymes. Each sample consists of 1 ml "Validate," 40 mg Lipase M, 40 mg papain, 8 mg .alpha.-chymotrypsin and 0.1 M buffer to 3 ml total volume. The mixtures were incubated and tested as in Example 4. The results are depicted in FIG. 1.

The measurement of pH optimum in this assay with serum as a substrate may be somewhat ambiguous because two enzymes (lipase and protease) are necessary. It is possible that the pH optimum of each enzyme may not coincide. In this study tris buffer (tris(hydroxymethyl)aminomethane) at pH 7.2 was superior.

EXAMPLE 8

Studies with tris buffer were conducted on combinations of Lipase M with the series of proteases described in Example 6. The amount of each protease used was that which gave the best result in the experiment shown in Table IV. Three pH values between 7 and 9 were tested. The assay was the same as described in Example 6 except that time was reduced to 5 minutes so that it would be expected to find cholesterol values well below 160 mg%, the maximum available in the "Validate" substrate.

Each incubation mixture contained 0.5 ml "Validate," 0.5 ml 0.2M tris buffer at pH shown, 40 mg lipase M and the protease in the amounts shown. Incubation was for 5 minutes under N.sub.2 at 50.degree.C, 250 rpm. Samples were analyzed by glc as in Example 2. The results are indicated in Table V.

Table V ______________________________________ Protease Amt Cholesterol mg% (mg) pH pH pH 7.2 8.1 9.0 ______________________________________ 1. A. oryzae 80 66 86 72 2. Thermolysin (Calbiochem, Grade A) 10 58 74 52 3. Bromelain 20 66 100 72 4. B. subtilis 5 54 65 70 5. S. griseus 5 56 58 30 6. Pronase 5 67 74 40 7. Subtilisin 5 56 66 56 8. .alpha.-chymotrypsin 8 84 56 22 9. Protease, bacterial (Calbiochem) 10 86 100 102 ______________________________________

Most of these proteases show an optimum within the three values tested. The best results were obtained with bromelain at pH 8.1, Calbiochem bacterial protease at pH values from 8 to 9, A. oryzae protease at pH 8.1 and .alpha.-chymotrypsin at pH 7.2.

EXAMPLE 9

Undiluted serum (1 ml "Validate" ) was incubated with 40 mg Lipase M, protease in amounts indicated in Table VI, and 12.1 mg of "Trisma Base" or Tris buffer at pH 7.2. The incubations were run at 50.degree.C for 10 min. under nitrogen and at 250 rpm. Cholesterol was analyzed as before by glc. The results are shown in Table VI.

Table VI __________________________________________________________________________ Hydrolysis of Cholesterol Esters in Undiluted Serum with Lipase M and Various Proteases __________________________________________________________________________ No Additions "Trizma Base".sup.a Tris Buffer pH 7.2.sup.b Amt Cholesterol Cholesterol Cholesterol Protease (mg) pH Range (mg%) pH Range (mg%) pH Range (mg%) __________________________________________________________________________ 1. B. subtilis 5 7.10-6.89.sup.c 104 8.61-7.85 84 6.98-6.64 208.sup.d 2. Protease (Calbiochem) 5 7.03-6.52 116 8.52-7.71 100 7.01-6.82 206 3. A. oryzae 80 6.90-6.27 90 7.60-7.25 103 6.72-6.70 174 4. Bromelain 20 6.78-6.56 119 8.25-7.93 52 6.82-6.45 165 5. .alpha.-Chymotrypsin 8 7.08-6.93 91 8.65-8.19 51 7.00-6.94 162 6. Pronase 5 6.82-6.66 134 8.72-7.99 64 7.01-6.89 160 7. S. griseus 5 6.88-6.48 96 8.60-7.94 63 6.89-6.68 119 __________________________________________________________________________ .sup.a "Trizma Base" (Sigma) powder was added directly to serum. .sup.b Tris-HCl buffer, pH 7.2 freeze dried, then added as powder directl to serum. .sup.c pH value at start of incubation and pH value at end. .sup.d Some values higher than 160 mg% occasionally obtained from same lo of "Validate" calibrated at 160 mg%.

Although according to a preferred embodiment of the present invention quantitative estimation of total cholesterol is achieved using gas-liquid chromatography, any of the well known conventional techniques for the analysis of total "free" cholesterol (after cholesterol ester hydrolysis has been achieved) may be used. These include the Pearson, Stern and McGarack, Carr and Drecker, and Zak methods described at pages 355-361 of Fundamentals of Clinical Chemistry, TIETZ, N. W., W. B. SAUNDERS CO. (1970) as well as the well known Lieberman-Burchard technique and the cholesterol oxidase method described in German Offenlegungschrifft No. 2,246,695 referred to hereinabove which is incorporated herein by reference insofar as it describes a technique for quantitatively determining total free cholesterol by treating a cholesterol solution with cholesterol oxidase and measuring the quantity of one or more of the products of this oxidation. Furthermore, hydrolysis as described herein may be used as an integral part of a single solution assay using cholesterol oxidase as is described in concurrently filed U.S. Pat. application Ser. No. 454,622 filed Mar. 25, 1974, in the names of Goodhue, Risley and Snoke entitled "Method and Composition for Blood Serum Cholesterol Analysis," which is incorporated herein by reference insofar as it describes another useful application of the novel hydrolysis described herein.

While the invention has been described in detail with particular reference to preferred embodiments thereof, it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

Claims

What we claim is:

1. In a process of assaying an aqueous proteinaceous liquid containing cholesterol esters for total cholesterol content in which said ester is hydrolyzed to liberate all of said cholesterol followed by determining the amount of cholesterol, the improvement comprising effecting said hydrolysis by treating said aqueous proteinaceous liquid with both a lipase having cholesterol esterase activity and a protease.

2. The improved method of claim 1 wherein said lipase having cholesterol esterase activity releases at least 25 mg% cholesterol (in 2 hours at 37.degree.C under nitrogen) when 50 mg of a preparation of said lipase in 5 ml 0.1 M phosphate buffer, pH 7.0, is used to treat a dispersion of cholesteryl linoleate prepared by dispersing 200 mg cholesteryl linoleate in 5 ml of ethyl ether and 100 ml boiling water containing 430 mg of sodium cholate.

3. The improved method of claim 1 wherein said aqueous liquid is serum and said treating step is accomplished with a mixture comprising from about 600 to about 1500 units of said lipase having cholesterol esterase activity and from about 50 to about 500 units of said protease per ml of serum.

4. The method of clain 1 wherein said treatment is carried out at a temperature of between about 25.degree. and 55.degree.C and at a pH of between about 6.5 and about 9.5.

5. The improved method of claim 1 wherein said lipase having esterase activity is a microbial lipase.

6. The improved method of claim 5 wherein said microbial lipase is the lipase from Candida cylindracca.

7. The improved method of claim 1 wherein said lipase is selected from the group consisting of wheat germ lipase, pancreatic lipases and the lipase from Candida cylindracca.

8. The improved method of claim 1 wherein said protease is selected from the group consisting of .alpha.-chymotrypsin, papain, bromelain, Bacillus subtilis protease, Aspergillus oryzae protease, Streptomyces griseus protease and mixtures thereof.

9. The improved method of claim 8 wherein said lipase is selected from the group consisting of wheat germ lipase, pancreatic lipases and the lipase from Candida cylindracca.

10. The method of claim 9 wherein said treatment is carried out at a temperature of between about 25 and 55.degree.C and at a pH of between about 6.5 and about 9.5.