Method and composition for the determination of cholesterol

The present invention provides a process for the determination of cholesterol in free or bound form by means of cholesterol oxidase and optionally of cholesterol esterase by measurement of the oxygen consumption, of the hydrogen peroxide formed or of the cholestenone formed, wherein the determination is carried out kinetically with the use of a cholesterol oxidase which has been obtained from a micro-organism of the genus Streptomyces and with the addition of 3,4-dichlorophenol. The present invention also provides a reagent for the kinetic determination of cholesterol, comprising cholesterol oxidase and a system for the determination of hydrogen peroxide or of cholestenone and optionally cholesterol esterase, wherein it additionally contains 3,4-dichlorophenol and the cholesterol oxidase used originates from a micro-organism of the genus Streptomyces.

This invention relates to a method and a reagent for the determination of 
cholesterol, using cholesterol oxidase. 
It is known to react cholesterol, possibly after previous liberation from 
its esters by chemical or enzymatic saponification, by means of 
cholesterol oxidase in the presence of molecular oxygen to give 
cholestenone and hydrogen peroxide and to utilise these quantitatively 
proceeding reactions for the determination of the cholesterol, either the 
cholestenone or hydrogen peroxide formed or the consumed oxygen thereby 
being measured. This enzymatic cholesterol determination process has 
admittedly brought a considerable advance due to its specificity in 
comparison with the previously used chemical methods of determination but 
hitherto the process has not been suitable for a rapid and practical 
kinetic carrying out. Because of the low K.sub.M value of cholesterol 
oxidase, which catalyses the most specific part of the reaction sequence, 
the reaction in the concentration range of up to 13 mMol/l., which is of 
interest for the determination of cholesterol, does not proceed according 
to the first or pseudo-first order. However, such a course of reaction is 
a prerequisite for a rapid and practical kinetic method of determination 
which does not require a sample blank value, which would make possible a 
substantial shortening of the time requirement per individual analysis in 
comparison with the previously necessary end point or kinetic methods. The 
time requirement for the previous photometric processes was from 10 to 6 
minutes and, in the case of kinetic methods, analysis times of 1 to 3 
minutes are desired. The most modern automatic analysers are intended for 
a high sample throughput and permit only short incubation times which 
could not be achieved with the previous processes used for the detection 
of cholesterol. Consequently, the automatic analysers with high analysis 
frequency which are today usual cannot be fully utilised. 
Therefore, it is an object of the present invention to provide a kinetic 
process of determination for the cholesterol oxidase reaction in which 
this proceeds according to the pseudo-first order. 
It is known that, in many cases, it is possible to achieve such a course of 
reaction at too low K.sub.M values of the enzymes involved by an 
artificial increase of the K.sub.M value. From the theory of Michaelis and 
Menton, it follows that enzyme-catalysed single substrate reactions then 
proceed over a wide concentration range according to the first order when 
the Michaelis constant of the enzyme is very much greater than the maximum 
substrate concentration. Since the previously known cholesterol oxidases 
from different microorganism genuses, such as Nocardia, Brevibacterium or 
Streptomycetes, have K.sub.M values of the order of 10.sup.-4 to 10.sup.-5 
mol/l., only low cholesterol concentrations can here be measured. Attempts 
artificially to increase the K.sub.M value of the cholesterol oxidase in 
known manner by the addition of a competitive inhibitor proved to be 
unsuccessful. 
Surprisingly, however, we have now found that it is possible, in the case 
of the selection of a cholesterol oxidase of particular origin and of a 
particular phenol derivative, to achieve a course of reaction according to 
the pseudo-first order. 
Thus, according to the present invention, there is provided a process for 
the determination of cholesterol in free or bound form by means of 
cholesterol oxidase and optionally of cholesterol esterase by measurement 
of the oxygen consumption, of the hydrogen peroxide formed or of the 
cholestenone formed, wherein the determination is carried out kinetically 
with the use of a cholesterol oxidase which has been obtained from a 
micro-organism of the genus Streptomyces and with the addition of 
3,4-dichlorophenol. 
Neither with the cholesterol oxidases from Nocardia or Brevibacterium 
previously used for the determination of cholesterol nor with the steroids 
or bile acids similar to cholesterol, which are primarily considered as 
competitive inhibiting materials, could such a course of reaction be 
achieved which, for the first time, makes possible a kinetic 
determination. The other isomers of dichlorophenol also have no or only an 
insufficient influence on the course of the reaction. Therefore, it is 
assumed that a special exchange action occurs between the 
3,4-dichlorophenol and the enzyme from the micro-organisms of the genus 
Streptomyces. 
The obtaining of cholesterol oxidase from Streptomycetes is described in 
Federal Republic of Germany Patent Specification No. 2,924,875. Examples 
of suitable strains include Streptomyces griseofuscus DSM 40191, 
Streptomyces hygroscopicus DSM 40771 and Streptomyces acidomyceticus DSM 
40798. 
The aimed for course of reaction of the first order can be achieved with 
widely varying concentrations of 3,4-dichlorophenol and cholesterol 
oxidase. However, it is preferable to use 0.5 to 10 mmol/liter 
3,4-dichlorophenol and 0.1 to 20.times.10.sup.3 U/liter cholesterol 
oxidase from Streptomyces. 
The process of the present invention is otherwise carried out in the usual 
manner for kinetic determinations. Preferably, at least two measurements 
are carried out in a previously determined time interval. The 
determination can be carried out in the whole pH value range in which the 
cholesterol oxidase from Streptomyces is active, with the proviso that any 
adjuvant enzymes possibly used are also active in this pH range. The 
determination is preferably carried out in a buffered solution at a pH 
value of from 6.5 to 8. 
As already mentioned, in principle all known variants of the cholesterol 
determination with the use of cholesterol oxidase can be used in the scope 
of the present invention. Only the measurement of the cholestenone 
formation at 230 to 250 nm is less suitable in the case of comparatively 
high 3,4-dichlorophenol concentrations since 3,4-dichlorophenol then 
absorbs at the measurement wavelength of the cholestenone. Therefore, in 
the case of this embodiment of the process according to the present 
invention, a concentration of 1.5 mmol/liter of 3,4-dichlorophenol should 
not be exceeded. 
One preferred embodiment of the process according to the present invention 
consists in the determination of the hydrogen peroxide formed by the 
addition of 4-aminoantipyrine, phenol, peroxidase and a buffer substance 
appropriate for this system, preferred buffers including phosphate buffer, 
hepes buffer (hepes: 4-(2-hydroxyethyl)-1-piperazinoethane-sulphonic acid) 
and tris buffer. 
In the case of this embodiment of the present invention, the best results 
are obtained when, in addition, a non-ionic detergent and optionally also 
a detergent of the cholic acid group is added. 
The present invention also provides a reagent for the kinetic determination 
of cholesterol, comprising cholesterol oxidase and a system for the 
determination of hydrogen peroxide or of cholestenone and optionally 
cholesterol esterase, wherein it additionally contains 3,4-dichlorophenol 
and the cholesterol oxidase originates from a micro-organism of the genus 
Streptomyces. 
A preferred system for the determination of hydrogen peroxide is a 
combination of 4-aminoantipyrine or of a derivative thereof, phenol or a 
phenol derivative, buffer and detergent, this system frequently being 
referred to as the PAP system. Therefore, the PAP system and the 
modifications thereof are especially advantageous because the coloured 
material formed absorbs in ultra-violet light at wavelengths other than 
that of 3,4-dichlorophenol so that no impairment of the sensitivity can 
arise due to overlapping of the extinctions. The presence of a non-ionic 
detergent has proved to be especially appropriate, which may be used alone 
or optionally together with a detergent of the cholic acid group. 
The reagent according to the present invention preferably contains a buffer 
substance; all buffers can be used which are able to buffer in the 
activity range of the participating enzymes and preferably in the pH range 
of from 6.5 to 8. Phosphate buffer, hepes buffer and tris buffer have 
proved to be especially useful. 
A reagent according to the present invention which has proved to be 
especially preferred is one which, as the system for determining hydrogen 
peroxide, contains the PAP system and has the following quantitative 
composition: 
0.1 to 10.times.10.sup.3 U/l. cholesterol oxidase, 
0.1 to 20.times.10.sup.3 U/l. cholesterol esterase, 
0.1 to 5.times.10.sup.3 U/l. peroxidase, 
1 to 10 mmol/l. 3,4-dichlorophenol, 0.5 to 10 mmol/l. 4-aminoantipyrine, 
5 to 20 mmol/l. phenol or phenol derivative, 
1 to 10 g./l. non-ionic detergent, 
0 to 15 mmol/l. detergent of the cholic acid group and 
50 to 200 mmol/l. buffer, pH 6.5 to 8. 
In addition, the reagent according to the present invention can also 
contain additional materials usual for enzymatic reagents, such as 
stabilising agents, for example mannitol, bactericides, such as azides, 
and inorganic salts. 
In the above-mentioned PAP system according to Trinder, instead of phenol, 
there can be used phenol derivatives, aniline derivatives, naphthol, 
naphthol derivatives, naphthylamine, naphthylamine derivatives, 
aminoquinolines, hydroxyquinolines, dihydroxyphenylacetic acid and 
similarly reacting substances. The 4-aminoantipyrine can be replaced, for 
example, by phenylenediamine-sulphonic acid, 
methylbenzothiazolone-hydrazone, sulphonated 
methylbenzothiazolone-hydrazone derivatives and similarly constituted 
compounds. 
However, instead of the PAP system, according to the present invention 
other known hydrogen peroxide determination systems can also be employed. 
These include the luminescent methods in which the fluorescence or 
chemi-luminescence is measured, preferably, for example, the 
peroxidase/luminol system. Other appropriate systems comprise catalase, a 
.beta.-diketone, for example acetylacetone, and an alcohol, for example 
methanol, ethanol or methylene glycol. Another appropriate system 
comprises peroxidase and a chromophore, such as 
2,3'-aminobenzthiazoline-sulphonic acid. 
When the reagent according to the present invention contains a system for 
the determination of cholestenone, then the known detection systems for 
this purpose can be used. If desired, as already mentioned above, the 
cholestenone formation can also be measured directly at 240 nm if a 
certain loss of sensitivity is not important since, at this wavelength, 
the 3,4-dichlorophenol also absorbs. 
The reagent according to the present invention can also be present 
impregnated on to a solid carrier. In this case, the determination can be 
carried out either 
(a) by dipping the carrier impregnated with the reagent into the sample, or 
(b) by dropping a definite amount of sample on to the reagent carrier, or 
(c) by eluting the reagent from the carrier with a definite volume of 
sample. 
If the rate of reaction is measured directly on the carrier, then this is 
possible, for example, reflectometrically or by the measurement of the 
luminescence. If the determination takes place after elution of the 
reagent, then the measurement can be carried out as in the case of the use 
of a carrier-free reagent. 
The carrier materials for the analytical detection reagents are the 
conventional carriers, such as paper, cellulose, fibre fleece, porous 
synthetic resin membranes and the like. They can be produced by immersion 
into or spraying with the reagent according to the present invention. 
The process according to the present invention permits a substantially 
quicker ascertainment of the amount of cholesterol in a sample and leads 
to a great saving of time, in comparison with conventional analysis 
processes. For automated analysis systems, in many cases the enzymatic 
determination of cholesterol hereby becomes a practicable possibility for 
the first time. 
The following Examples are given for the purpose of illustrating the 
present invention: