JPS587280B2 - Enzyme reagent system for total cholesterol analysis using oxygen consumption rate method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The invention of the present application relates to cholesterol analysis using an enzyme reagent containing cholesterol oxidase.

To measure cholesterol in a sample, cholesterol
This enzyme reagent, which uses oxidase, measures the amount of hydrogen peroxide produced by the reaction between oxygen and cholesterol and converts it into a measure of the amount of cholesterol in the sample.

Hydrogen peroxide measurements are generally based on the reaction of hydrogen peroxide with a dye (which results in a color change).

This color change is measured and converted into a measure of the amount of hydrogen peroxide present.

Reducing substances such as glutathione, ascobic acid and uric acid present in human body fluids react with hydrogen peroxide and prevent all hydrogen peroxide from reacting with the sting.

This results in a loss of accuracy in cholesterol analysis.

One solution to this problem is cholesterol-4
-en-one(cholest-4-en
This method measures the consumption rate of oxygen in the enzymatic conversion of hydrogen peroxide to hydrogen peroxide and converts it into the amount of cholesterol present.

An oxygen rate analyzer and method for making such measurements were developed by James C.
), U.S. Pat. Nos. 3,857,771 and 393
No. 3,593, which is hereby incorporated by reference.

Oxygen rate measurement systems, such as those described in the aforementioned Sternberg patents, can be effectively employed to measure the total amount of cholesterol in a sample; Substances that inhibit the reaction between enzymes or the possibility of the reaction between enzymes and cholesterol (hereinafter collectively referred to as inhibitors)
The presence of , makes the aforementioned measurements quite inaccurate.

In particular, inhibitors that may be present in cloudy serum containing above normal concentrations of triglycerides may reduce the efficacy of reactive cholesterol or the rate at which enzymes catalyze reactions resulting in oxygen consumption ( This is thought to reduce the oxygen consumption inhibitory effect (hereinafter collectively referred to as the oxygen consumption suppressing effect).

In either case, the result is lower than the normal oxygen consumption rate and therefore measurement of oxygen consumption rate in the presence of such inhibitors does not provide an accurate measurement of cholesterol concentration.

For example, when the oxygen rate measurement system is used to measure the total cholesterol value in a certain sample, the oxygen consumption rate is determined by the cholesterol content of the sample, the Abell
The actual cholesterol content of the samples determined by the total cholesterol analysis method by et al. was lower than expected.

The above-mentioned method by Abel et al. Biol Chem.
, 1.95:367 (1952), which is cited here.

Summary of the invention Cationic surfactant (Cholesterol oxydase and buffer solution)
It was found that by adding Cationic Surfactant) the rate of the oxygen consuming reaction was essentially unaffected by the presence of said inhibitor in the sample being analyzed.

As a result, the oxygen consumption rate measured with an oxygen rate analyzer such as that described in the Sternberg patent is a true measure of the cholesterol in the sample being analyzed as a secondary matter.

To avoid subjecting the sample to a separate saponification step, enzyme reagents for cholesterol analysis may be
Preferably it consists of an esterase.

Cholesterol esterase is believed to catalyze the reaction between water and cholesterol esters present in the sample to produce cholesterol and fatty acid byproducts.

Cholesterol produced by cholesterol esterase and any cholesterol initially present in the sample reacts with oxygen catalyzed by cholesterol oxidase to form cholest-4-en-3-one and hydrogen peroxide.

The rate of oxygen consumption in reaction with cholesterol can be converted to a measure of cholesterol concentration using well known techniques.

DESCRIPTION OF PREFERRED EXPERIMENTAL EMBODIMENTS The enzyme reagent for cholesterol analysis of the present invention comprises cholesterol oxidase, a buffer solution in an amount to produce a solution having a pH of about 5.5 to about 8, and an inhibitor of oxygen consumption present in the sample to be analyzed. It consists of an amount of cationic surfactant sufficient to neutralize essentially all of the effects.

Preferably, the reagent also comprises cholesterol esterase.

The cationic surfactant used in the present invention is used in an amount sufficient to neutralize the oxygen consumption inhibiting effects of triglycerides and other inhibitors.

One method for determining the minimum and maximum amounts, and therefore ranges, of suitable cationic surfactants for enzyme reagents for cholesterol analysis is as follows.

The amount of total cholesterol in a series of samples is first determined by the method of Abel et al., supra.

The total cholesterol values of said series of samples were then determined by the oxygen percentage method (e.g., as described in U.S. Pat. method).

The minimum amount of cationic surfactant required is that amount that gives total cholesterol values for all samples, which values are equal to those obtained with the cited method.

(As mentioned above, the total cholesterol value at the subminimum value of cationic surfactant appears to be lower than the cited method.

) If the amount of cationic surfactant is greater than the desired maximum, denaturation of the cholesterol oxidase and cholesterol esterase enzymes will occur.

Modification of the surfactant will inhibit the conversion of cholesterol esters to cholesterol and the conversion of cholesterol to cholest-4-en-3-one.

As the amount of cationic surfactant increases above this maximum value, the kinetics of oxygen consumption slows down progressively until it becomes impractical to measure.

Additionally, the enzyme reagent of the present invention desirably comprises about 0.01-0.4, preferably about 0.05-0.2% (by weight) cationic surfactant final solution.

Optimally, said reagent comprises 0.1% (by weight) of said cationic surfactant.

In fact, are any cationic surfactants originally developed? It can be used for.

A commercially available cationic activator that can be used in the present invention is "MCCutcheon ζ Detergent and Emulsifier" (Nrth American Edition).
tion. McCutcheon Division,
McPublishing Co. , Glen Ro
ck, N. J., 1975), the publication of which is hereby cited.

Quaternary imidazolium, pyridium, quinolinium, and quaternary ammonium cationic surfactants are all suitable for the present invention.

Representative cationic surfactants particularly useful in the present invention are β hydroxyethylbenzyl stearyl imidazolium chloride, laurylpyridium chloride, and laurylquinolinium bromide.

In addition, diisobutylphenoxyethoxyethyl dimethyl benzyl ammonium chloride, diisobutylcresoxyethoxyethyl dimethyl benzyl
Ammonium chloride and methyldotecylbenzyl trimethyl ammonium chloride? Cationic quaternary ammonium surfactants appear to be a desirable class of cationic surfactants.

Particular alkyldimethylbenzylammonium salts used in preferred embodiments of the invention include mono-iamine (
Hyamine) 3 5 0 0 CD-
Zephiran chloride (C), Winthrop (manufactured by 7 Star IJ Pharmaceutical Co., New York City);
Winthrop Institute).

Hyamine 3500 is the trade name for alkyldimethylbenzylammonium sodium chloride, the alkyl group of which is about 50% C, 4H29, about 40%
of C1H25, and a mixture of alkyl containing about 10% C,6H33.

Zephyrane chloride is the trade name for alkyldimethylbenzylammonium chloride, whose alkyl groups are a mixture of alkyls from C8H17 to C18H37.

Examples of buffer solutions that can be used include phosphates such as potassium orthophosphate, citrates such as sodium citrate, or acetates such as sodium acetate.

The buffer solution is present in an amount to produce a solution having a pH of about 5.5-8, preferably about 6.

Cholesterol oxidase of various types can also be used in the reagents of the present invention.

The amount of cholesterol oxidase used in the reagent does not need to be particularly precise.

About 0.2-5, preferably about 0.5-2 per ml of reagent
, more preferably about 1 to 1.5 international units of cholesterol oxidase is used in the present invention.

Approximately 1.2 international units of cholesterol per ml of reagent.
Optimally, oxidase is used.

Any cholesterol esterase can be used in the present invention.

The amount of cholesterol esterase used in the present invention does not need to be particularly precise; it is about 5 or less per 1 m of reagent, preferably about 0001 to 1, more preferably about 0.005 to 1.
0.05 international units of cholesterol esterase is used.

Approximately 0014 international units of cholesterol per ml of reagent
The use of esterases is optimal.

In a preferred embodiment, a nonionic surfactant is used to maintain serum cholesterol and cholesterol esters in solution.

When used in the reagents of the present invention, the amount of nonionic surfactant is not critical, but approximately 0.01 to 0.5 of the reagent;
Preferably, it is present in an amount of about 0.05% (by weight).

Virtually any nonionic surfactant can be used in the present invention.

Commercially available nonionic surfactants that can be used in the present invention are found in the aforementioned publication, ``Matuku Cation Detergents and Emulsifiers''.

Examples of nonionic surfactants that can be employed in the present invention include nonylphenoxypolyethyleneoxy ethanol and polyethoxylated sorbitol monolaurate.

about 0.005% (by weight) of the total solution nonionic surfactant;
Satisfactory results were obtained using Triton X-100.

Triton X-1 O O is the trade name of isooctylphenoxypolyethoxyethanol.

It is also desirable to add bile salts to the reagent system of the invention.

The amount of bile salts added is not critical, but it should be about 0.01 to 0.1, preferably about 0.08 (by weight) of the total solution.
% is desirable.

Bile salts render cholesterol esters and cholesterol in a soluble state, thus making them effective for reaction by enzymes.

Examples of bile salts suitable for the present reagents include sodium chlorate, taurochlorate, deoxychlorate and taurodeoxychlorate.

The bile salt in a preferred embodiment is sodium chlorate.

The following is the composition of a preferred reagent system of the present invention: Example I Cholesterol Ester 0. O O 7 I
U/ml Zecholesterol Oxider I Mixture of K2HPO4 and KH2PO4 to adjust pH of IU/Ebize solution to 6.0 1.0 molar Triton X-100 0.05% Sodium chlorate 0.08% Hyamine 3
500 0.1% Example■ Cholesterol esterase I JU/ml! Cholesterol oxidase 1 A mixture of K2HP04 and KH2PO4 that makes the pH of 1U/ml solution 6.0 1゛0ゝ''
Sodium chlorate O. OS% Hyamine 3500 0.08% Example■ Cholesterol esterase 1 ■TJ/m. l
Cholesterol oxidase 1 ■ Mixture of K2HPO4 and KH2P04 to adjust pH of U/ml solution to 6.0 1゜0 Mol Triton 0.1% Example■ Cholesterol esterase I IU/mlCholesterol oxidase I IU/mlpH
Citric acid and salt to make it 6.0 1. .

D. Sodium enoate mixture Triton X-100 0.5% Hyamine 3 5 0 0 0. 1% Sodium chlorate 0.08% Example V Cholesterol ester 0. O I. 4 I
TJ/ml zecholesterol oxidizer 1 1U/ml citric acid to bring the pH to 6.0 and i. Q mo/l/
Mixture of sodium citrate Laurylpyridinium chloride 0.1% Sodium chlorate 0.08% Example ■ Cholesterol esterase I IU/ml Cholesterol oxidase 1 1 TJ/ml pH
Citric acid and salt to make it 60 1. .

E/L/sodium enoate mixture Hyamine 3 5 0 0 0. 1%
Polyoxyethylated sorbitol 0.05% sodium monolaurate Example ■ Cholesterol esterase 1 ■ U/ml cholesterol oxitase 1 U/ml pH 6
．． Citric acid and chlorine to make it 0. .

E/L/sodium enoate mixture Hyamine 3500 0.08% Tori 1.
N
ml Zecholesterol Oxider 1.2 I[J/m
Mixing of K2HPO4 and KH2PO4 to adjust the pH of the lase solution to 60 1.0 molar Triton X-100 0.05% Sodium chlorate 0.08% Hyamine 3
500 0.1% Another desirable reagent system is shown in Example 2 - Iamine 3500 at 0.1% (by weight)
is substituted with any of the cationic and cationic quaternary ammonium surfactants.

The preferred reagent system described herein employs cholesterol esters to hydrolyze cholesterol esters to cholesterol.

However, the present invention is not limited to cholesterol esterase to effect this hydrolysis, but also includes reagents that employ other hydrolyzing agents, such as alkaline materials, in place of cholesterol esterase.

Examples of such alkaline materials are potassium hydroxide in ethanol and sodium hydroxide in ethanol.

If an alkaline material is used in place of cholesterol esterase to hydrolyze cholesterol esters, the serum will first react with the alkaline material and then a portion will react with the reagent system.

The reagent system of the invention described herein can be prepared as a single reagent containing all of the components described above, or as a kit of two or three reagents.

One reagent in the preparation of a two-reagent reagent kit is cholesterol esterase and cholesterol esterase.
The remaining reagents include the oxidase, its buffer solution and other components.

In the preparation of a three-reagent reagent kit, the first reagent consists of a solution of cholesterol oxydase, the second reagent consists of cholesterol esterase, and the third reagent consists of the buffers, surfactants, and others described above. It consists of an aqueous solution of the ingredients.

The preferred embodiment of the reagent system is such that keeping the enzyme and remaining components of the reagent separate from each other until use improves the shelf life of the reagent system (
The kit consists of two separate reagents because it is believed to improve shelf life.

This should prevent any reactions that might occur between the components of the reagent system if both reagents were mixed before use and left for an extended period of time.

The reagent system of the present invention can be stored and used in the form of an aqueous solution, or the solution can be freeze-dried in the conventional manner and reconstituted with deionized water at the time of use.

The reagent system can also be made into a powder and dissolved in water at the time of use.

The specific effects of adding a cationic surfactant to the enzyme reagent for cholesterol analysis according to the present invention will be explained below.

To examine this effect, approximately 500 random samples were collected from the Orange Country Hospital Pathologist Central Laboratory (
Hospital - Pathology stC
central Laboratory of Oran
ge Country, Inc., I 2 9 2
2 Sungrove, GardenGrove
, California, U. Liberman-Burchard (S, A)
-Burchard) endpoint method.

Based on the previous experience gained during the research and development of the present invention, two samples were selected from the 500 random samples: a normal serum sample and a serum sample containing an oxygen consumption inhibitor.

It was analyzed using the Liberman-Burchard method.

The total cholesterol content of these two samples is shown in Table 1 below.

Sample A is a normal serum sample. Sample B is a serum sample containing an oxygen consumption inhibitor.Next, an enzyme reagent for cholesterol analysis as shown in Table 3 was prepared.

Serum sample A and serum sample B were analyzed using the enzyme reagents ① to ① described above.

The data obtained from these analyzes are shown in Table ■ below.

Table III demonstrates that only cationic surfactants such as Hyamine 3500 can neutralize virtually all of the inhibitory effects of inhibitors present in samples to be analyzed for cholesterol by kinetic methods. There is.

Neither anionic nor nonionic surfactants have this unique property.

Furthermore, the data in Table 3 show that anionic surfactants interfere with oxygen analysis by kinetic methods.

Although specific embodiments of the invention have been described for purposes of disclosure, it should be understood that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (1)

[Scope of Claims] 1. Enzyme reagent for cholesterol analysis for rate-limiting cholesterol in an analyte sample, comprising cholesderol oxidase and an amount of buffer to produce a solution having a pH of about 5.5 to 8. , the reagent further comprises a cationic surfactant in an amount sufficient to neutralize essentially all of the enzyme consumption inhibiting effect of the inhibitor present in the sample to be analyzed, and the cationic surfactant is An enzyme reagent for cholesterol analysis that controls the rate of cholesterol in an analysis sample, characterized by being an alkyldimethylpendylammonium salt. 2. The enzyme reagent for cholesterol analysis according to claim 1, wherein the cholesterol analysis reagent is lyophilized. 3(a) with about 0.2 to 5 international units of cholesterol oxidase per ml of reagent; (b) with not more than about 0.5 international units of cholesterol esterase per ml of reagent; (C) with about 0.01 to 5 international units of cholesterol esterase per ml of reagent; 0.4% (by weight) of an alkyldimethyl ammonium salt; and (d) an amount of a buffer to produce a solution having a pH of about 5.5 to 8. Rate-limiting enzyme reagent for cholesterol analysis. 4 said alkyldimethylbenzylammonium salt is selected from the group consisting of alkyldimethylbenzylammonium chloride and mixtures thereof, and said alkyl moiety is
4. Reagent according to claim 3, characterized in that it contains 18 carbon atoms. 5 A mixture of the alkyldimethylbenzylammonium salts of about 50% tetradecyldimethylbenzylammonium sodium chloride, about 40% dodecyldimethylbenzylammonium sodium chloride, and about 10% hexadecyldimethylbenzylammonium sodium chloride. The reagent according to claim 3, characterized in that: 6 (a) buffer in an amount to produce a solution with a pH of about 6; (b) cholesterol oxidase at about 1.2 international units per ml of reagent; and (c) about 0.014 international units per ml of reagent. (d) about 0.08% (by weight) of the reagent bile sulfate; (e) about 0.05% (by weight) of the reagent inoctylphenoxypolyethoxyethanol; (f ) An enzyme reagent for cholesterol analysis that determines the rate of cholesterol in a sample to be analyzed, characterized in that the reagent comprises about 0.1% (by weight) of an alkyldimethylbenzyl ammonium salt. 7. The alkyldimethylbenzylammonium salt is a mixture of about 50% sodium tetradecyldimethylbenzylammonium chloride, about 40% sodium dodecyldimethylbenzylammonium chloride, and about 10% hexadecyldimethylbenzylammonium chloride. The reagent according to claim 6, characterized in that: 8 (a) a first reagent consisting of (i) cholesterol esterase and (11) cholesterol oxidase; (b) a second reagent consisting of a buffer solution having a pH of about 5-5 to 8; An enzyme reagent kit for cholesterol analysis used for the determination of cholesterol in a sample to be analyzed, in which the reagent and the second reagent are stored separately until used and are combined at the time of use, wherein the second reagent further comprises: , comprising an argyldimethylbenzyl ammonium salt in an amount sufficient to neutralize substantially all of the inhibitory effect on oxygen consumption of the inhibitor present in the sample to be analyzed. Enzyme reagent kit for cholesterol analysis used for rate-limiting purposes. 9 (a) Cholesterol. (b) a second reagent comprising cholesterol oxidase; and (b) a second reagent comprising cholesterol oxidase.
, a reagent; (c) a third reagent consisting of a buffer solution having a pH of about 5.5 to 8; each of the reagents being stored separately until used and combined at the time of use; In the enzyme reagent kit for cholesterol analysis used for rate-limiting cholesterol analysis, the third reagent further comprises a kit sufficient to neutralize substantially all of the enzyme consumption inhibitory effect of the inhibitor present in the sample to be analyzed. 1. An enzyme reagent kit for cholesterol analysis, which is used for rate-limiting cholesterol in a sample to be analyzed, and is characterized by comprising an alkyldimethylbenzyl ammonium salt in a sufficient amount. 10 A first reagent consisting of cholesterol esterase: a second reagent consisting of cholesterol oxidase; a buffer solution having a pH of about 6; a bile sulfate salt; a nonionic surfactant; and an alkyldimethylbenzylammonium salt. The main component is a mixed solution of a third reagent consisting of: (a) a solution having a pH of about 6; (b) about 0.08% (by weight) of said mixed solution of sodium chlorate; (e) about 0.05% (by weight) of said mixed solution of isooctylphenoloxy; (d) with polyethanol;
(e) about 1.2 international units of cholesterol oxidase per ml of the mixed solution; An enzyme reagent kit for cholesterol analysis used for rate-limiting cholesterol in a sample to be analyzed, characterized in that it consists of 0.014 international units of cholesterol esterase. 11 A mixture of the alkyldimethylbenzylammonium salts of about 50% tetradecyldimethylbenzylammonium sodium chloride, about 40% dodecyldimethylbenzylammonium sodium chloride, and about 10% hexadecyldimethylbenzylammonium sodium chloride. Claim 10 is characterized in that
Reagents listed in section. 12 (a) (!) Cholesterol oxidase, (11) Cholesterol esterase, (!!
i) the presence of a sensor serving to monitor the properties of oxygen with respect to oxygen concentration in an oxygen-saturated aqueous solution by means of an enzyme reagent for cholesterol analysis in the form of a buffer in an amount to produce a solution with a pH of from 5.5 to about 8; oxidizing cholesterol present in the sample in the oxygen-saturated aqueous solution; (b) generating a first electrical signal related to the oxygen concentration; and (c) generating the first electrical signal. (d) measuring said output signal to determine said cholesterol concentration. In the measurement method, a cationic interface consisting of an alkyldimethylbenzylammonium salt is added to the enzyme reagent for cholesterol analysis in an amount sufficient to neutralize substantially all of the inhibitory effect on oxygen consumption of the inhibitor present in the sample to be analyzed. A method for determining the reaction rate of cholesterol concentration in a cholesterol-containing sample, characterized in that substantially all of the oxygen consumption suppressing effect of the inhibitor in the sample to be analyzed is neutralized by containing an activator. 13 The reagent is (a) about 0.2 to 5 per ml of reagent.
International unit of cholesterol oxidase; (b)
Reagent Contains less than approximately 5 international units of cholesterol per ml.
esterase; (c) about 0.0% of said reagent; 0 1~0
．． 13. The method of claim 12, comprising a cationic surfactant comprising 4% (by weight) of said alkyldimethylbenzylammonium salt. 14. Claim 13, characterized in that said alkyldimethylbenzylammonium salt is selected from alkyldimethylbenzylammonium chlorides and mixtures thereof, and said alkyl moiety contains from 8 to 18 carbon atoms.
The method described in section. 15 A mixture of the alkyldimethylbenzylammonium salts of about 50% tetradecyldimethylbenzylammonium sodium chloride, about 40% dodecyldimethylbenzylammonium sodium chloride, and about 10% hexadecyldimethylbenzylammonium sodium chloride. Claim 13 characterized in that
The method described in section. 16 (a) (!) Cholesterol oxidase, (11) Cholesterol esterase, (ii
in an oxygen-saturated aqueous solution by an enzyme reagent for the analysis of cholesterol in the form of i) bile sulfate as a nonionic surfactant, and Ov) a buffer in an amount to produce a solution with a pH of from 5.5 to about 8. oxidizing cholesterol present in a sample in said oxygen-saturated aqueous solution in the presence of a sensor serving to monitor a characteristic of oxygen with respect to said oxygen concentration; and (b) a first electrical signal related to said oxygen concentration. (c) differentiating said first electrical signal to generate an output signal proportional to an instantaneous rate of change in oxygen concentration; (d) measuring said output signal to determine said cholesterol concentration. In the method for measuring the reaction rate of cholesterol concentration in a cholesterol-containing sample, the enzyme reagent for cholesterol analysis is added to substantially all of the oxygen consumption inhibiting effect of the inhibitor present in the sample to be analyzed. Substantially all of the oxygen consumption inhibitory effect of the inhibitor in the sample to be analyzed is neutralized by containing a cationic surfactant consisting of an alkyldimethylbenzylammonium salt in a sufficient amount to Kinetic method for determining cholesterol concentration in cholesterol-containing samples. 17 The reagent contains (a) about 0.2 to 5 per ml of reagent;
international units of cholesterol oxidase; (b) not more than about 5 international units of cholesterol esterase per ml of reagent; and (C) about 0.01% to 0.4% (by weight) of said alkyldimethylbenzylammonium salt of said reagent. a cationic surfactant consisting of: (d) about 0 of said reagent;
．． 0.01 to 0.1% (by weight) of said bile sulfate salt; and about 0.05 to 0.4% (by weight) of said nonionic surfactant of said reagent. Range 1
The method described in Section 6. 18 said reagent comprises: (a) said buffer in an amount to produce a solution having a pH of about 6; (b) about 1.2 ml of cholesterol oxidase per ml of reagent; (c) Reagent 1
Approximately 0.0% per ml. ．． 0 14 international units of cholesterol esterase; (d) about 0.08% (by weight) of said reagent bile sulfate; and (e) 0.05% (by weight) of said reagent isooctyloxy. 18. The method of claim 17, comprising: polyethoxyethanol and: (f) about 0.1% (by weight) of said reagent an alkyldimethylbenzylammonium salt. 19 A mixture of tetradecyldimethylbenzylammonium sodium chloride containing about 50% of the alkyldimethylbenzylammonium salt, about 40% dodecyldimethylbenzylammonium sodium chloride, and about 10% hexadecyldimethylbenzylammonium sodium chloride. Claim 17 characterized in that
The method described in section. 20 The cationic surfactant comprising the alkyldimethylbenzyl ammonium salt is about 0.01 to 0.20% of the reagent.
19. A method according to claim 18, characterized in that the method is present in an amount of 0.4% (by weight).