CA1174149A - Process for the enzymatic determination of free and esterified chloresterol - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Cholesterol is determined by incubating a sample suspec-tod of containing cholesterol in an aqueous medium with cholester-ol oxidase and determining either the oxygen consumption, the hydrogen peroxide formed, or cholestenone, as a measure of the initial cholesterol content; reagent compositions comprising choloaterol oxidase and a system for the determination of H2O2 or for the determination of cholestenone are provided.

Description

i~'f~

The pre~ent invention i8 concerned w~th a procos~
for the enzymatic determination of free and esterified cholesterol and ~ith a reagent ~or the carrying out of this process.
The determination of choleQterol ~8 o~ considerable importancs for medical diagno3i~. An increased cholesterol level ln the blood i8 an important risk factor of arterlo-sclerosis. In the case of high cholesterol level~, l.e.
hypercholesterolaemia, coronarg insu~iciency and cardiac infarct occur more ~requently than in the case of low -cholesterol levels. EypercholesterolaeJnia ~avours the occurrence o~ arteriosclerosis and o~ coronary di~eases and must, therefore, be recognised in good time in order that treatment can be commenced as 800n aB possible.
Increased cholesterol levels are frequentlg also found in the case of diabetes mellitus, nephrotic syndrome, hypothyreosis and liver diseases, such as biliary cirrhosis. There~ore, a rapid and dependable method rOr the determination o* cholesterol is of great lmportance.
The known and used methods for the determlnation o~
cholesterol a~e based on the ~iebermann-Burchardt test.
Accordlng to this test, ~ree and esterified cholesterol ~orms, with acetic anhydride and concentrated sulphuric acid, blue-green coloured compound~, the colour intensity of which is proportional to the choleeterol concentration and is measured spectrophotometrically.
However, this known test su~fers from several dls-adva~tages~ the chie~ o~ which is its non-specificlty.
The ~iebermann-Burchardt test is a relative non-~peciflc steroid reaction which, apart from cholesterol, also

-2-145~

involves other steroids. Since, for example, plasma also con-tains 1 to 3% dihydrocholesterol and 0.5 to 1.4% ~ -cholesterol as well as other steroids, an undesirably large error is obtain-ed. Furthermore, this test reaction is disturbed by the pre-sence of bilirubin and haemoglobin, which simulate increased and thus falsely positive cholesterol levels. A further disadvantage is that it is necessary to use aggressive and corrosive reagents.
It is, therefore, an object of the present invention to provide a new process and an agent for the determination of cholesterol which does not suffer from the above-mentioned dis-advantages and, in particular, is absolutely specific for cholesterol and does not require the use of aggressive reagents.
According to the present invention there is provided a process for the determination of cholesterol, wherein cholesterol in an aqueous medium is incubated in the presence of oxygen with cholesterol oxidase which converts cholesterol in the presence of oxygen into cholestenone and hydrogen peroxide and thereafter there is determined either the amount of oxygen used up or the amount of hydrogen peroxide or of cholestenone formed.
Cholesterol oxidase is a new enzyme which catalyses the followîng reaction:

cholesterol + 0 cholesterol oxidase~ cholestenone + H 0 The above reaction takes place quantitatively and thus enables an absolutely specific and precisely quantit-ative determination of cholesterol to be carried out.
Cholesterol oxidase, the starting materials for the production thereof, the purification thereof and the properties thereof are described in German Patent Speci-fications Nos. 2,224,133; 2,224,131 and 2,307,518.

~ccordlng to the disclosures in the~e ~erm~n Patent Specificatlon~, cholesterol oxida~e i8 obtalned by dlgest-ing and extracting a cholesterol-reactlng micro-organismJ
optionally after washing with a butanol_~ater mixture, with a bu~er solution containing a non-ionlc, surface-active agent and ~solating the cholesterol oxida6e rrom the extract, ~or example, by adsorption on an anlon exchanger and elution with a buffer solution containing a surface-actl-ve agent. As starting material for produ¢-ing cholesterol oxida~e, there can bo used, in partlcular, the micro-organisms Proactinomycetes~~rvthro~oli~
~TCC 1789~, ATCC 4277 and Nocardia formica ATCC 14811.
There i~ pre~erably used a micro-organlsm which has been cultured on cholesterol as the 801e source of carbo~ ln that, to the micro-organism cultured on a mineral ~alt medium, which contains peptone or an acetate as the~ 801e source o~ carbon, there is added an aqueous emulsion o~
cholesterol as soon as the logarlthmic growth pha~e le reached and therea~ter the micro-organism is ~urther ¢ultured with cholesterol as the 801e ~ource o~ carbon.
The process according to the present in~entlon can be used ~or the determination o~ cholesterol in agueous media of all lsinds, ~or example, in ~oodstu~ extracts, body ~luids and, in particular, in serum. Be¢ause of the great speci~icit~ of the new process according to the pre~ent invention, only ~ree cholesterol i~ determined.
Bound cholesterol, which, a i8 known, i~ present in esterified form, can, ho~e~er, also be determined after chem~ cal or enzymatic saponification. Chemical saponiflc-ation ca~ be carried out, for example, wlth an alcoholic ll f '~l4~

potassium hydroxide solution and enzymatic saponificatioll with an esterase, preferably with cholesterol esterase from the liver or pancreas.
The measurement of the oxygen utilisation, the hydrogen peroxide formation or the cholestenone formation according to the above-given equation can be carried out by the known and conventional methods for this purpose.
Appropriate methods for the determination of oxygen utilisation include, for example, gas chromatography and depolarisation processes. Polarometric determination by means of oxygen electrodes is preferably used since this process is particularly suitable for carrying out auto-matic cholesterol determinations~ Determination methods of this kind are known, the methods described in Canadian Patent Applications, S.N. 144,991 and 144,994, both of Alexander Hagen et al both filed June 16, 1972, being especially appropriate for the polarometric measurement of oxygen utilisation in aqueous media.
Hydrogen peroxide formed can be determined not only potentiometrically, polarographically and colorimetrically but also enzymatically. Preferably, there are used the enzymatic methods employing catalase or peroxidase since these methods are not only extremely specific and depend-able but can also be combined, in a very simple manner, with the main reaction i.e. the formation of hydrogen peroxide. The determination by means of catalase in the presence of a ~-diketone, for example of acetylacetone, and of a lower aliphatic mono- or polyhydroxy alcohol, for example methanol, ethanol or methylene glycol, as well as the determination by means of peroxidase in the presence of a chromogen, have proved to be especially useful. In the case of a determination by means of catalase, acetyl-acetone and methanol, the latter is oxidised to formalde-hyde which undergoes a colour reaction with acetylacetone, which can be measured. In the case of the determination by means of peroxidase, as chromophor there is added a compound which can be determined photometrically after the reaction.
An example o~ a chromophor which can be used for this purpose is 2,2'-aminobenzthiazoline-sulphonic acid.
The determination of cholestenone can be carried out with the use of a keto reagent, preferably of a hydrazine derivative which reacts with keto groups with hydrazone formation, for example, 2,4-dinitrophenyl-hydrazine. However, cholestenone can also be determined directly by measurement of the absorption at 240 nm.
The present invention also provides a reagent for the determination of cholesterol, comprising cholesterol oxidase in an amount effective to catalyse reaction between cholesterol and oxygen to produce hydrogen peroxide and cholestenone and a component for the determination of hydrogen peroxide or for the determination of cholestenone.
An especially preferred embodiment of this reagent comprises cholesterol oxidase, catalase, acetylacetone, methanol and an ammonium ion-containing buffer, separately or mixed.
Another preferred embodiment of the reagent comprises cholesterol oxidase, peroxidase, a chromogen and a buffer, separately or mixed. The preferred chromogen is 2,2'-amino-benzthiazoline-sulphonic acid.
A fuxther preferred embodiment of the reagent according to the present invention comprises cholesterol oxidase and a h~drazine deri~atlYe which react8 ~ith k~to groups ~ith the formation of a hydrazone, and optionally also a buffer. A prererred hydrazine derivative for this purpose is 2,4-dinitrophenylhydrazine.
T~e aboYe-mentioned preferred reagent combinatlons preferably also contain, in addition to~the mentioned es~ential components, conventional solYents, stabilisers and surface-active substances. All the~e additional materials are kno~n and are conventional in determinatio~
~ystems for hydrogen peroxide or cholestenone.
The above-mentioned reagent combinations prererably contain the essential components in the following amounts:
1. 13 - 150 U cholesterol oxidase, 2 x 104 to 5 x 105 U
commercially available catalase, 0.05 to 0.2 ml.
acetylacetone a~d 2 to 10 ml. metha~ol in 100 ml.
ammonium ion-containing buffer (pH 5 to 7), as well as ~.02 to 0.3 ml. o~ a surface-act~e agent (prefer-ably hydro~ypolyethoxydodecane).
2. 3 to 40 U cholesterol oxidase, 2 2 102 to 1 x 104 U
commercially available peroxidase, 50 to 200 mg.
2,2'-aminobenzthiazoline-sulphonic acld and 0.05 to 0.5 ml. surrace-active agent (preferably hydroxypoly-ethoxydodecane) in 100 ml. bu~fer (pH 6 to 8).

3. 0.1 to 1 U chole~terol oxidase, 1 to 5 ml. of a 1 mM
solution o~ 2,4-dinitrophenylhydrazlne and optlonallg 0.005 to 0.1 ml. of a surface-actl~e agent.

4. 2 to 100 U cholesterol oxidase and 0.1 to 2.0 ml. of a ~urface-acti~e agent (preferably hydroxypolyetho~y-dodecane) in 50 ml. o~ a bu~fer (p~ 5 to 9), prefer-ably 0.5 M 30dium phosphate buffer (pH 7.5).

i~ ~ 4149 In another embodiment of the invention there is pro-vided a test composition for determining total cholesterol in a biological fluid sample comprising (1) a mixture of a chemical system having cholesterol ester hydrolase activity and a chemical system having cholesterol oxidase activity and (2) reagent means for determining hydrogen peroxide or ~4-cholesten-3-one.

The invention is further described by reference to the accompanying drawings in which -s 10 FIGURE 1 is a cross-section of an apparatus for the ' enzymatic determination of total cholesterol in body fluids by measuring the oxygen consumption, FIGURE 2 is a diagram of the dependence of the cholesterol concentration upon oxygen consumption (polarometrically measured), FIGURE 3 is a diagram of the dependence of the cholesterol concentration upon the colour , intensity of the formed Lutidin derivative at 405 nm (catalase-reaction), and FIGURE 4 is a diagram of the dependence of the cholesterol concentration upon the colour intensity of the formed dyestuff, at 436 nm (peroxidase reacti.on).

1 ~

The ~ollo~ing ~amples are gl~on ~or tho purpo80 0 illustrating the present invention:-_~ample 1.
Polarometric d ~
There is uæed the deYice illustrated in Fig.l of the accompanying drawings. The device comprises a reaction vessel 1 in the form o~ a cylindrioal chamber of tran3parent synthetic resin ~ith an internal diameter of 143 mm. and an internal height o~ 220 mm. The chamber contains 1.8 ml.
of a solution of 18 mM potassium iodide, 7.5 mM ammonium heptamolybdate, 800 m~q sodium chloride and 2 U cholesterol oxidase in 0.2 M potassium phosphate bu~fer (pH 6.0).J Into this reactor there pro~ects a detector 3, which comprises an oY.ygen-sensitive electrode (WTW: OXI-electrode E 016).
The detector i8 connected with an analy~er 4 (WTW:
digital meter DIGI 610 with plugged in OXI 610 D). On the bottom o~ the reaction ~essel 1, th~re i8 provided a magnetic stlrrer 5. Through a filling opening 6, there is introduced a 20 ~1. ~ample o~ an aqueous cholesterol-containing solution. During ~igorous stirring with the magnetic stirrer, the decrease o~ the o~ygen concentration o~ the solution is mea6ured. The oxygen utili6ation observed after 2.5 minutes reaction is plotted against the cholesterol concentration. The measurement resultR
for di~erent concentrations of the cholesterol-containing solution i~ shown in ~ig.2 o~ the accompanying drawings.
It shows a linear dependence between the measurement signal6 and the chole6terol concentrations.
The process was repeated but with the uee o~ a serum 6ample which had been saponi~ied by means of alcoholic i~414~

potassium hydro~ide solution. The determination gavo a concentratlon of 193 mg. cholesterol/100 ml. A compara-tive value obtained by the ~iebermann-Burchardt method gave a value of 182 mg./100 ml.
~xample 2.

~0 g. ammonlum hydrogen phosphate were dissolved in 100 ml. water and ad~usted to pH 7.0 with 85% phosphorlc acid. 105 U catalase were then added thereto. Th~ 801-ution so obtained was used to make up to 100 ml. a mixture o~ 0.2 ml. acetylacetone and 10 ml. methanol.
7.5 ml. of the solution thus obtained were mixed ~ith 0.5 ml. serum or with 0.5 ml. of a cholesterol standard solution containin~ 200 mg.% cholesterol.
Aliquots`o~ the serum-containing sample, as well as Or the cholesterol standard sample, were each mi~ed with

5 U cholesterol o~idase and 0.02 ml. of a 10~ hydroxy-polyethoxydodecane solution and incubated for 70 minutes at 37C. The coloured material formed iB then measured photometrically at 405 nm, having regard to the blank value Or the reagent. With the cholesterol standard solutions, there was prepared a calibratlon curve, whlch i8 illu~trated in Fig.3 of the accompanying drawi~gs. In this calibration curve, the cholesterol concentration 18 plotted against the extinctlon at 405 nm.
The cholesterol content of the serum-containing sample Y~a~ determined ~ith the use o~ a standard as re~erence. Control determinations by the ~ibermann-Burchardt method gave deviations of about 5%.

_9_ 1 ~ ~ 4 1 4 9 Example 3.
Determination o~ hydrogen peroxide To 3 ml. potassium phosphate bufrer (pH 7), satur-ated with oxygen, which contained 100 mg.% 2,2'-amino-benzthia~oline-sulphonic acid, were added 0.05 ml. Or a 20% solu~ion o~ hydro~ypolyethoxydoaecane and 0.02 ml.
(36 U) commercially-available peroxidase solutlon, as well as 0.02 ml. hydrogen peroxide (0.02 ml. 30~ v/v hydrogen peroxide in 100 ml. water) for the removal Or reduclng substances. The mixture was monitored in a photometer at 405 nm or at 436 nm. As soon as the reaction comes to a stop, it i3 started again with 0.~1 ml.
serum (diluted 1:15 with water). A~ter 10 minute~, 0.15 U
cholesterol oxida~e were added thereto and after a further 10 minutes, the extinctlon difference wa~ read of r.
In the ~ame manner but with the use of a cholesterol standard~solution with a conte~t of 200 mg.g cholesterol, there ~as plotted the calibration curve shown in Fig.4 o~
the accompanying drawings. With the help o~ this cali-bratlon curve, the cholesterol content o~ the serum ~amplo was determined. The measurement gave a content Or 180 mg.%
cholesterol. A control measurement using ~iebermann-Burchardt method gave 185 mg.% cholesterol.
E~amPle 4.
Determination o~ cholestenone 0.2 ml. ~erum (dilute 1:10 with water) were mlxod with 0.05 ml. 20% hydroxypolyetho~ydodecane solution and 0.15 U cholesterol oxidase. After 15 minutes, 2.0 ml. Or a ~olution containing 1 mM 2,4-dinitrophenylhydrazine in 1N hydrochloric acid were added. Arter a rurther 30 1~ ~ 41~9 minutes, 4.0 ml. water were added and the hydrazone formed was mea~ured in a photometer at 405 nm. The evaluatlon was carried out with reference to a standard curve, having regard t'o the blank value. The measure~ent can also take place at 546 nm a~ter the addition o~ an alkali.
Thè mea~urement of a typical samp~e gave 60 mg.%
~ree cholesterol a~d 154 mg.~ total cho~esterol (sapon-i~ication with alcoholic potas~ium hydroxide).
A comparative determination carried out by the ~iebermann-Burchardt method gave a total cholesterol content of 170 mg.%.
For the saponification of esterlfied chole8terol (mea3urement of total cholesterol in serum), 1 ml. serum, 1 ml. 20% hydro~ypolyethoxyd~decane solutlon and 5 ml.
0.5~ potassium hydroxide solution ln 90~ ethanol are mixed and heated to 70C. for 30 minutes. The solutlon is then allowed to cool, mixed with 10 ml. 0.1~ magnesium sulphate 601ution and centrifuged of~. The supernatant (13 ml.) contains the total cholesterol in ~ree form. ^
The saponi~ication of e3teri~ied cholesterol with liver esterase or with cholesterol esterase 18 carried out by incubation o~ the serum ~or 30 minutes at 37C.
and a pH o~ 6 to 8.
Example 5.
0.2 ml. serum were added to 2.5 ml. 0.5~ sodlum phosphate bu~fer (pX 7.5) containlng 0.4% hydroxypoly-ethoxy dodecane. The extinction (E1) was read off at 240 nm in an appropriate spectrophotometer and the reaction initiated with 0.02 ml. (1.5 U) cholesterol oxidase. After 2 minutes, the new extlhction (E2) was _1 1 -l49 read of~. The concentration of the choleetenone ~ormed and thus of the cholesterol was given by the di~ference between E1 and ~2' having regard to the molar extlnctlon coe~ficients for cholestenone at 240 nm The measurement of a typical sample gave 58 mg.%
~ree cholesterol and 163 mg.% total cholesterol (a~ter saponi~ication).
A comparative determination by the Liebermann-Burchardt method gave a total cholesterol content o~
173 mg-%-

Claims (32)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Process for the determination of cholesterol, wherein cholesterol in an aqueous medium is incubated in the presence of oxygen with cholesterol oxidase which converts cholesterol in the presence of oxygen into cholestenone and hydrogen peroxide and thereafter there is determined either the amount of oxygen used up or the amount of hydrogen peroxide or of cholestenone formed.

2. A process according to claim 1 comprising incubating cholesterol in an aqueous medium in the presence of cholesterol oxidase to react said cholesterol with oxygen to produce hydrogen peroxide and cholestenone and thereafter determining the amount of oxygen used up.

3. Process according to claim 2 wherein the amount of oxygen used up is measured polarometrically.

4. A process according to claim 1 comprising incubating cholesterol in an aqueous medium in the presence of cholesterol oxidase to react said cholesterol with oxygen to produce hydrogen peroxide and cholestenone and thereafter determining the amount of hydrogen peroxide formed.

5. Process according to claim 4 wherein the amount of hydrogen peroxide formed is determined enzymatically with catalase or peroxidase.

6. A process according to claim 1 comprising incubating cholesterol in an aqueous medium in the presence of cholesterol oxidase to react said cholesterol with oxygen to produce hydrogen peroxide and cholestenone and thereafter determining the amount of cholestenone formed.

7. Process according to claim 6 wherein the amount of cholestenone formed is measured directly at 240 nm or with the use of a keto reagent.

8 Process according to claim 1 wherein free and total cholesterol are determined separately by first testing an unsaponified sample to give the free cholesterol content and then testing a saponified sample to give the total cholesterol content.

9. Process according to claim 8 wherein the saponification is carried out with alcoholic potassium hydroxide solution.

10. Process according to claim 1 wherein the free cholesterol is first determined and then, after saponification, the bound cholesterol is determined.

11. Process according to claim 10 wherein the saponification is carried out with an esterase.

12. Process according to claim 11 wherein the saponification is carried out with cholesterol esterase of non-microbial origin.

13. Reagent for the determination of cholesterol, comprising cholesterol oxidase in an amount effective to catalyse reaction between cholesterol and oxygen to produce hydrogen peroxide and cholestenone and a component for the determination of hydrogen peroxide or for the determination of cholestenone.

14. A reagent according to claim 13 further including an agent for saponifying esterified cholesterol.

15. A reagent according to claim 13 comprising said cholesterol oxidase and a component for the determination of hydrogen peroxide which comprises catalase, a .beta.-diketone, a lower aliphatic alcohol and a buffer.

16. A reagent according to claim 15 wherein the .beta.-diketone is acetylacetone.

17. A reagent according to claim 15 or 16 wherein the lower aliphatic alcohol is methanol.

18. A reagent according to claim 13 comprising said cholesterol oxidase and a component for the determination of hydrogen peroxide which comprises peroxidase, a chromogen and a buffer.

19. A reagent according to claim 18 wherein the chromogen is 2,2'-aminobenzthiazoline-sulphonic acid.

20. A reagent according to claim 13 comprising said cholesterol oxidase and a component for the determination of cholestenone which comprises a keto group-reacting derivative.

21. A reagent according to claim 18 wherein the keto group-reacting derivative i9 2,4-dinitrophenylhydrazine.

22. A reagent according to claim 15 which comprises 13 to 150 U cholesterol oxidase, 2 x 104 to 5 x 105 U catalase, 0.05 to 0.2 ml. acetylacetone, 2 to 10 ml. methanol and 0.02 to 0.3 ml. of a surface-active agent in 100 ml. ammonium ion-containing buffer with a pH of 5 to 7.

23. A reagent according to claim 18 which comprises 3 to 40 U cholesterol oxidase, 2 x 102 to 1 x 104 U peroxidase, 50 to 200 mg. 2,2'-aminobenzthiazoline-sulphonic acid and 0.05 to 0.5 ml. of a surface-active agent in 100 ml. buffer with a pH of 6 to 8.

24. A reagent according to claim 20 which comprises 0.1 to 1 U cholesterol oxidase and 1 to 5 ml. of a 1 mM
solution of 2,4-dinitrophenylhydrazine.

25. A reagent according to claim 24 which further contains 0.005 to 0.1 m1. of a surface-active agent.

26. A reagent according to claim 22, 23 or 25 wherein the surface-active agent is hydroxypolyethoxydodecane.

27. A reagent according to claim 13, 14 or 15 including a surface-active agent.

28. A reagent according to claim 14, wherein said agent is a cholesterol esterase of non-microbial origin.

29. A test composition for determining total cholesterol in a biological fluid sample comprising (1) a mixture of a chemical system having cholesterol ester hydrolase activity and a chemical system having cholesterol oxidase activity and (2) reagent means for determining hydrogen peroxide or .DELTA.4-cholesten-3-one.

30. A test composition as claimed in claim 29, in which the mixture (1) additionally comprises a buffering material.

31. , A test composition as claimed in claim 30, wherein the said buffering material is capable of maintaining a pH of between 5 and 8 when contacted with the fluid sample.

32. A reagent according to claim 13, further including an agent for saponifying esterified cholesterol, said agent being alcoholic potassium hydroxide.