Cholesterol-esterase colour substrate.

Cholesterol esterase (cholesterol ester hydrolase EC 3.1.1.13) hydrolyzes emulsified cholesterol esters of long-chain fatty acids at the interface between the lipophilic and aqueous phases. Cholesterol esterase occurs in the human organism, including in microorganisms. Cholesterol esterase is used particularly frequently as a research reagent and in biochemical analysis. In particular, it is used as an auxiliary enzyme in many (diagnostic) enzymatic tests. Cholesterol esterase determination is therefore of considerable importance for clinical chemistry, but also for biochemistry, microbiology and food chemistry. There is therefore a need for a specific, uncomplicated test for this and in the production and isolation of the enzyme.

A number of cholesterol esterase measurement methods are already known, which are based on the determination of the cleavage products formed during cholesterol ester cleavage. With radiolabelled cholesterol esters as substrates, the released radiolabelled acid can be determined (Biochim.Biophys.Acta<u style="single">617</u>, 446 (80)). The method is complex, prone to failure and cumbersome.

Photometric determination in UV is known. Released cholesterol is oxidized to cholestenone using cholesterol oxidase, which can be measured using the increase in absorption at λ = 240 nm (Bergmeyer, Methods of Enzymatic Analysis, 3th.Ed., Vol.II, p.168). Alternatively, the hydrogen peroxide formed during this oxidation is determined by a color reaction (Stähler, Med.Lab.<u style="single">30</u>, 29 (77)). These methods are cumbersome and also prone to failure.

Fluorescent substrates such as pyrenebutyric acid or pyrendecanoic acid esters of cholesterol are also used. After cleavage, a change in the fluorescence yield can be determined (Uster, Arch.Biochem.Biophys.<u style="single">209</u>, 385 (81)). However, this method is very susceptible to interference and most laboratories are not equipped with fluorescence measuring devices.

It is also known to use chromogenic substrates for the quantification of cholesterol esterase, for example fluorescein dilaurate or methylumbelliferylester (Meyer-Bertenrath, Enzyme Vol. <u style="single">28</u>, 336 (82); A. Negre, Biochim. Biophys. Acta<u style="single">794</u>, 89 (84)). However, these substrates are not specific and are also cleaved by esterases.

There is therefore a need for a color test that can be carried out with simple equipment and can be checked directly visually.

The object of the invention is to create a substrate and a color test for the determination of cholesterol esterase using this substrate, which does not have the disadvantages of the known color tests, provides accurate results, is easy to use, has high sensitivity and is only low lag phase, so that the adaptation to the various analyzer systems is not difficult.

According to the invention, this object is achieved by a cholesterol esterase substrate of the general formula I X - <img file="EP0328029A2_D0001.tif" /> - A - <img file="EP0328029A2_D0002.tif" /> - Y - Chol I wherein A is an alkylene or alkenylene group with 1 to 20 carbon atoms, chol is the 3-cholesterol residue, X is the residue of an aromatic hydroxy or thiol compound and Y is -S- or -O-.

In the context of the present invention, 3-cholesterol residue is understood to mean any unsubstituted or substituted steroid residue which is esterified via the 3-O group and which is hydrolyzed by the cholesterol esterase. Such steroid residues are, for example, cholesterol, cholestanol, dihydrocholesterol, β-sitosterol, stigmasterol or ergosterol.

Under the action of cholesterol esterase, the cholesterol ester according to the invention is cleaved to liberate the aromatic hydroxy or thiol compound corresponding to radical X, which is either determined optically directly or coupled with a suitable chromophore or fluorophore and the coupling product is measured. If the chain length of A 4 exceeds C atoms, an esterase is preferably added as an auxiliary enzyme to accelerate the reaction. Suitable esterases are, for example Carboxylesterase or aryl esterase.

The cholesterol esterase substrate according to the invention further contains the remainder of a dicarboxylic acid COOH-A-COOH, in which A preferably has 3 to 7 carbon atoms. Examples of acids from which A is derived are malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, nonandicarboxylic acid, decanedicarboxylic acid and undecanedicarboxylic acid. The acids from glutaric acid to azelaic acid, which correspond to A with 3 to 7 carbon atoms, are preferred as mentioned above.

X is the remainder of an aromatic hydroxy or thiol compound, which is a chromophore or can only be converted into a dye by a subsequent reaction. Typical examples are phenol, thiophenol, naphthol, thionaphthol and their derivatives and the chromogenic compounds such as the resorufin, chlorophenol red, indoxyl or thiofluorescein residue. An exhaustive list of the suitable hydroxy or thiol compounds is not possible due to their large number, but the directly chromophoric or convertible aromatic hydroxy or thiol compounds are known to the person skilled in the art.

The above-mentioned reaction to the dye can be carried out either by direct coupling (for example with a diazonium salt such as 4-chloro-2-methylbenzene diazonium salt (Fast Red), 4-benzamido-2-methoxy-5-methylbenzene diazonium salt (Fast Violet), diazotized sulfanilic acid, 2,4- and 2,5-substituted phenyldiazonium salts, for example 2,4-dichlorophenyldiazonium-1,5-naphthalenedisulfonic acid), or by an oxidative coupling, for example with 4-aminoantipyrine or other aminopyrazolones (such as trimethylaminopyrazolone, diaminoantipyrine) or MBTHS (3-methyl-2-benzothiazolinone hydrazone-6-sulfonic acid).

Chromophores which have a low polarity and are lipophilic are preferred. The lipophilic character of the above chromophores can be positively influenced by suitable substitution, such as with alkyl groups. The methyl, dimethyl and ethyl groups and bromine have proven to be suitable substituents, in particular for the resorufin radical.

The cholesterol esterase substrates according to the invention can be produced by methods known per se. For example, cholesterol and dicarboxylic acid anhydrides can be derived from dicarboxylic acids of the general formula HOOC - A - COOH derive, in which A has the meaning given above, synthesize the half esters in anhydrous medium such as chloroform / pyridine.

Suitable methods for the preparation of the dicarboxylic acid anhydrides are described in Houben-Weyl-Müller "Methods of Organic Chemistry", Volume IV / 4, page 786.

The esterification of the half ester thus obtained with the aromatic hydroxy or thiol compound from which the radical X is derived can be carried out, for example, by directly reacting the dicarboxylic acid monoester with the aromatic alcohol or thiol in the presence of a dehydrating agent such as dicyclohexylcarbodiimide. Alternatively, the dicarboxylic acid monoester is first converted to an activated ester, for example the hydroxysuccinimide ester or the imidazolide, and the activated ester is then reacted with the aromatic alcohol or thiol.

Likewise, it is also possible first to prepare a monoester of dicarboxylic acid with the aromatic alcohol or thiol, for example the mononitrophenyl adipate or monophenyl glutarate, and then to esterify it with the cholesterol, for example via the intermediate formation of an acid chloride, anhydride or activated ester. The dicarboxylic acid monoesters with the aromatic alcohol or thiol can be prepared, for example, from acid anhydride and aromatic compound in a molar ratio of 1: 1 or from activated dicarboxylic acid and aromatic compound or from a dicarboxylic acid monoester with an easily removable protective group and the aromatic compound. Suitable methods are described, for example, in Arch. Pharm.<u style="single">287</u>, 514 (1954).

The process according to the invention for the optical determination of cholesterol esterase is characterized in that a cholesterol esterase substrate according to the invention is subjected to the action of the sample containing cholesterol esterase and the amount of aromatic hydroxy or thiol compound released is determined optically or, after coupling with a suitable chromogen, the color formed therefrom.

A special feature of this process is that, in a preferred embodiment, it does not require any auxiliary enzymes or esterase inhibitors, as are frequently required in the known methods. Such additives are not only expensive, but also often not very stable.

Another object of the invention is a simple and long-lasting reagent for the optical determination of cholesterol esterase which, in addition to a cholesterol esterase substrate and buffer substance according to the invention, also contains a detergent, in particular a nonionic detergent, a chromogenic coupler and / or a salt such as sodium chloride. In addition, the reagent can suitably contain preservatives and / or activators.

In a preferred composition, this contains reagent 0.05 to 2 mg / ml substrate, 10 to 50 µl detergent, 10 to 50 µl dioxane, 50 to 500 mM buffer substance, each based on the ready-to-use solution in the test.

Polyethylene glycol monododecyl ester (Thesit®) is preferred as the detergent.

Suitable buffer substances are those which are able to set a pH between 5.5 and 10.5 in the context of the reagent according to the invention. The preferred pH range is between 6.5 and 7.8. Examples of suitable buffers are diethanolamine buffers, triethanolamine buffers, tris buffers and good buffers such as Hepes buffers (well suited for addition before lyophilization), taps buffers, CHES buffers (2- (cyclohexylamino) ethanesulfonic acid) and bicine. Potassium phosphate buffer is particularly preferred. The preferred amount of buffer substance is between 50 and 500 mM.

If the reagent according to the invention is in a dry or concentrated form, to be diluted to the final composition, it contains the substances mentioned in appropriate proportions, and preferably a protective colloid.

The protective colloid includes the substances known to those skilled in the art, such as polyhydroxy compounds, serum albumin, polyvinylpyrrolidone, solid polyethylene oxides and the like. Polyhydroxy compounds, in particular monomeric or polymeric pentose or hexose with 1 to 10 pentose or hexose units in the molecule and / or polyethylene glycol which is solid at room temperature, are preferred. Preferred examples of suitable polyhydroxy compounds are mannitol and similar sugar alcohols, oligosaccharide of glucose, mannose, maltoheptaose, polyethylene glycol with an average molecular weight between 3500 and 7000 u. Other protective colloids that can be used are e.g. B. amino acids, such as alanine, plant gums, such as gum arabic, etc. The preferred amount of protective colloid or a mixture of protective colloids is 20 to 90 wt .-%. A mixture of sugar alcohol and polyalkylene glycol proved to be particularly suitable.

The reagent according to the invention can also be impregnated on a suitable carrier material. Both an absorbent carrier material and a swellable, soluble film-forming carrier material can be considered. In this form, the reagent according to the invention enables the production of test strips which can be evaluated directly visually or by means of suitable measuring devices.

The color test according to the invention for determining the cholesterol esterase provides very precise results with high sensitivity. It is very easy to use and is even suitable for test strips. Since it has only a very small or no lag phase, it can be easily adapted to the various analyzer systems.

The determination itself can be carried out both as an end point determination and kinetically. Compared to many known processes, the kinetic feasibility has the advantage that the reaction product formed does not have to be stopped or shaken out.

The following examples and the figure further illustrate the invention.

FIG. 1 shows the comparison of the calibration curves (ΔE / min against substrate concentration mg / ml) for the cholesterol-oleate method and the method using a substrate according to the invention (cholesterol-3-glutaric acid resorufine ester).

<u style="single">example 1</u>

Cholesterol-glutaric acid resorufine ester

a) Cholesterol-glutaric acid monoester

19.5 g of cholesterol are dissolved in 150 ml of chloroform, 16 ml of pyridine, 11.5 g of glutaric anhydride and a spatula tip of dimethylaminopyridine are added. After heating at 65 ° C. for 8 hours, the mixture was cooled, then diluted with 200 ml of chloroform and washed twice with 2N hydrochloric acid and once with water. The organic phase is dried over sodium sulfate, concentrated and chromatographed on silica gel (mobile phase: ethyl acetate / petroleum ether 1: 1). Yield: 6 g Mp: 125 ° C 1 H-NMR (CDCl₃):. δ [ppm]: 0.68 (s, 3H); 0.83 (s, 3H); 0.90 (s, 3H); 1.02 (s, 3H); 0.95 - 1.95 (m, 31H); 2.0 (m, 2H) 2.4 (m, 4H); 4.6 (m, 1H); 5.37 (d, 1H).

b) cholesterol-glutaric acid chloride

2.55 g 1a) are dissolved in 20 ml of chloroform and 2.2 ml of oxalyl chloride are added while cooling with ice. Then 1 drop of dimethylformamide is added and the mixture is stirred at room temperature for 8 h. The solvent is drawn off and the residue is further processed raw.

c) cholesterol-glutaric acid resorufine ester

1.1 g of resorufin are slurried in 40 ml of chloroform, 0.75 ml of diazabicycloundecene and 0.1 mg of dimethylaminopyridine are added. With ice cooling, a solution of 1b) in 20 ml of chloroform is slowly added dropwise and the mixture is then stirred at room temperature for 8 h. Then it is diluted with 50 ml of chloroform and shaken with 50 ml of 2N hydrochloric acid. After drying over sodium sulfate, the organic phase is concentrated and the residue is chromatographed on silica gel (mobile phase: ethyl acetate / petroleum ether 1: 3). Yield: 200 mg DC: R_f = 0.6 (silica gel; ethyl acetate / petroleum ether 1: 2) 1 H-NMR (CDCl₃):. δ [ppm]: 0.68 (s, 3H); 0.83 (s, 3H); 0.90 (s, 3H); 1.02 (s, 3H); 0.95 - 2.6 (m, 35H); 2.70 (t, 2H); 4.62 (m, 1H); 5.37 (d, 1H); 6.33 (d. 1H); 6.86 (dd, 1H); 7.13 (dd, 1H); 7.17 (s. 1H); 7.43 (d. 1H); 7.80 (d, 1H).

<u style="single">Example 2</u>

2nd mg of cholesterol-glutaric acid re-ester are dissolved in 0.5 ml of dioxane and 0.5 ml of Thesit®.

For the measurement, 850 µl 0.1 M potassium phosphate buffer pH 6.8 and 50 µl of the substrate solution described above are mixed well in a cuvette. After adding 100 µl sample (enzyme solution), ΔE / min is determined photometrically at λ = 571 nm (temperature = 25 ° C).

When evaluated using a standard known cholesterol esterase activity, the cholesterol esterase activity of the sample is calculated as follows<maths id="math0001" num=""><img file="EP0328029A2_D0003.tif" /></maths> A calculation of the cholesterol esterase activity of the sample is also possible using the following formula:<maths id="math0002" num=""><img file="EP0328029A2_D0004.tif" /></maths> V_total : Total volume of the test batch [cm³] V_sample : Volume of sample [cm³] ε: extinction coefficient of the chromogen at 571 nm d: layer thickness of the cuvette [cm] ΔE / min: change in extinction per minute at 571 nm

Under the reaction conditions mentioned, the extinction coefficient is ε = 60.00 · cm² · µmol⁻¹.

<u style="single">Example 3</u>

For comparison, the cholesterol esterase test with cholesterol oleate as a substrate was carried out (Bergmeyer, Methods of Enzymatic Analysis, 3th.Ed., Vol.II, p. 168) and compared with the color test according to Example 2 and the correlation coefficient was determined. The results are shown in Table I and Figure 1.<tables id="tabl0001" num="0001"><table frame="all"><title>TABLE I</title><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="left">Cholesterol oleate</entry><entry namest="col3" nameend="col4" align="left">Cholesterol-glutaric acid resorufine ester</entry></row><row><entry namest="col1" nameend="col1" align="left">Conc. (Mg / ml)</entry><entry namest="col2" nameend="col2" align="center">ΔE / min</entry><entry namest="col3" nameend="col3" align="center">Conc. (Mg / ml)</entry><entry namest="col4" nameend="col4" align="center">ΔE / min</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">10,0</entry><entry namest="col2" nameend="col2" align="char" char=",">0,072</entry><entry namest="col3" nameend="col3" align="right">10,0</entry><entry namest="col4" nameend="col4" align="char" char=",">0,071</entry></row><row><entry namest="col1" nameend="col1" align="left">6,6</entry><entry namest="col2" nameend="col2" align="char" char=",">0,045</entry><entry namest="col3" nameend="col3" /><entry namest="col4" nameend="col4" /></row><row><entry namest="col1" nameend="col1" align="left">5,0</entry><entry namest="col2" nameend="col2" align="char" char=",">0,036</entry><entry namest="col3" nameend="col3" align="right">5,0</entry><entry namest="col4" nameend="col4" align="char" char=",">0,033</entry></row><row><entry namest="col1" nameend="col1" align="left">2,0</entry><entry namest="col2" nameend="col2" align="char" char=",">0,015</entry><entry namest="col3" nameend="col3" align="right">2,0</entry><entry namest="col4" nameend="col4" align="char" char=",">0,015</entry></row><row><entry namest="col1" nameend="col1" align="left">1,3</entry><entry namest="col2" nameend="col2" align="char" char=",">0,011</entry><entry namest="col3" nameend="col3" align="right">1,3</entry><entry namest="col4" nameend="col4" align="char" char=",">0,013</entry></row><row><entry namest="col1" nameend="col1" align="left">1,0</entry><entry namest="col2" nameend="col2" align="char" char=",">0,007</entry><entry namest="col3" nameend="col3" align="right">1,0</entry><entry namest="col4" nameend="col4" align="char" char=",">0,009</entry></row><row><entry namest="col1" nameend="col1" align="left">Blank value:</entry><entry namest="col2" nameend="col2" align="char" char=",">0,002</entry><entry namest="col3" nameend="col3" align="right">Blank value:</entry><entry namest="col4" nameend="col4" align="char" char=",">0,0027</entry></row><row rowsep="1"><entry namest="col1" nameend="col4" align="justify">Correlation coefficient: 0.998</entry></row></tbody></tgroup></table></tables>

<u style="single">Example 4</u>

Cholesterol azelaic acid resorufine ester

a) Cholesterol azelaic acid monoester

Preparation analogous to example 1a) from 5.68 g cholesterol, 7.5 g azelaic anhydride and 50 ml chloroform Yield: 2.8 g DC: R_f = 0.60 (silica gel; chloroform / methanol 9: 1)

b) cholesterol azelaic acid chloride

Preparation analogous to Example 1b) from 1.26 g 4a), and 1 ml oxalyl chloride.

c) cholesterol-azelaic acid resorufine ester

Preparation analogous to Example 1c) from 4b), 0.72 g resorufin and 0.52 ml diazabicycloundecene Yield: 210 mg DC: R_f = 0.41 (silica gel; ethyl acetate / petroleum ether 2: 3)

<u style="single">Example 5</u>

3β-cholestanyl glutaric acid resorufine ester

a) 3β-Cholestanyl glutaric acid monoester

Preparation analogous to Example 1a) from 19.4 g of 5 α-cholestan-3β-ol, 23 g of glutaric anhydride and 150 ml of chloroform. Yield: 22 g DC: R_f = 0.63 (silica gel; chloroform / methanol 8: 2)

b) 3β-cholestanyl glutaric acid chloride

Preparation analogous to Example 1b) from 5 g 5a) and 4.5 ml oxalyl chloride.

c) 3β-Cholestanyl glutaric acid resorufine ester

Preparation analogous to Example 1c) from 5b), 2.1 g resorufin and 1.5 ml diazabicycloundecene. Yield: 1.2 g DC: R_f = 0.29 (silica gel; ethyl acetate / petroleum ether 2: 3)