Process and reagent for the specific determination of HDL cholesterol in serum or plasma

The present invention provides a process for the specific determination of HDL cholesterol in serum or plasma by incubation with a cholesterol detection system, containing cholesterol oxidase and cholesterol esterase in a buffered aqueous medium, and measurement of a product of the cholesterol oxidase reaction or of the oxygen consumption, wherein a sample to be tested is incubated in the presence of a salt of a bile acid or of a bile acid derivative or of dioctylsulphosuccinate, a first measurement is then carried out, subsequently a non-ionic, polyethylene oxide group-containing detergent or a secondary alkane sulphonate is added, again incubated and a second measurement is carried out, the amount of HDL cholesterol being determined from the difference between the first and second measurement. The present invention also provides a reagent for the specific determination of HDL cholesterol in serum or plasma containing cholesterol oxidase, buffer and cholesterol esterase, wherein, referred to the final solution, it contains PA0 0.1 to 10 U/ml. cholesterol esterase PA0 0.05 to 10 U/ml. cholesterol oxidase PA0 20 to 500 mmole/liter buffer substance (pH 6.0 to 8.0) PA0 0.2 to 20 mmole/liter of a salt of a bile acid or of a bile acid derivative or of dioctylsulphosuccinate and, separately therefrom, PA0 0.02 to 2% non-ionic polyethylene oxide group-containing detergent or secondary alkane sulphonate, and optionally 0.05 to 2% of an alcohol containing up to 3 carbon atoms.

The present invention is concerned with a process and reagent for the 
specific determination of HDL cholesterol in serum or plasma. 
Besides the determination of the total cholesterol in serum (or plasma), 
which has long since been firmly established in the clinical laboratory, 
in recent years the additional analysis of the cholesterol bound to the 
"high density lipoprotein" fraction (HDL cholesterol) has achieved a 
considerable importance for the improved diagnosis of atherosclerosis or 
myocardial infarction risk. 
Extensive clinical studies have shown that an increased level of total 
serum cholesterol (sum of the free and fatty acid esterified cholesterol 
bound to the lipoprotein fractions chylomicrons, VLDL ("very low density 
lipoproteins"), LDL ("low density lipoproteins"), as well as HDL) leads to 
an increased myocardial infarction risk, whereas, on the other hand, there 
is an inverse relationship between the serum HDL-cholesterol content and 
the danger of atherosclerotic changes of blood vessels (see e.g. G. 
Assmann, Lipidstoffwechsel und Atherosklerose, pub. Schattauer-Verlag, 
Stuttgart, 1982). 
For the determination of the serum total cholesterol, for about 15 years 
there have been available simple but, nevertheless, very exact completely 
enzymatic analysis processes which can be carried out not only manually 
but also equally well on automated analysis systems without any sample 
pretreatment step and which usually proceed according to the reaction 
scheme: 
##STR1## 
The cholesterol detection takes place either via the hydrogen peroxide 
formed, preferably colorimetrically and especially by the 
peroxidase-catalysed oxidative coupling of phenol or of phenol or aniline 
derivatives with 4-aminoantipyrine to give a red to violet coloured 
quinone-imine material ("Trinder reaction") or via the cholestenone formed 
or via the oxygen consumed according to equation 2). These methods of 
determination are known, for example, from Federal Republic of Germany 
Patent Specifications Nos. 22 24 132 and 25 06 712. 
An important characteristic of the appropriate reaction systems or analysis 
reagents for the determination of total cholesterol is the presence of 
detergents, especially of non-ionic alkyl or alkylaryl polyethylene oxide 
ethers, possibly together with bile acid salts, such as sodium cholate. 
The detergents additionally serve as solubilising agents, without which 
the cholesterol esterase would not manifest any hydrolytic 
As cholesterol esterases, there are thereby preferably used enzymes of 
microbial origin. 
The determination of the serum HDL cholesterol takes place according to the 
same principle but, for that purpose, the other cholesterol-containing 
lipoprotein particles (chylomicrons, VLDL and LDL) must be previously 
separated from the serum in one or more process steps. 
For the separation of these lipoproteins, there are used, in particular, 
ultracentrifugation electrophoresis or diverse precipitation methods, such 
as immune precipitation, precipitation with phosphotungstic 
acid/Mg.sup.2+, dextran sulphate/MG.sup.2+ or heparin/Mn.sup.2+ or with 
polyethylene glycol (see e.g. Arztl. Lab., 29, 107-114/1983; Clin. Chem., 
31, 252-256/1985). 
Besides the additional apparatus necessary herefor, these separation 
processes also require a considerable time and personnel expense, which 
makes a routine HDL-cholesterol determination in the clinical laboratory 
much more difficult and thus also more expensive. 
Because of the equally high diagnostic value of the total cholesterol 
content as well as of the HDL-cholesterol content in the serum, it is, 
therefore, all the more important to have available a process and reagent 
which makes possible the analysis of HDL-cholesterol and serum total 
cholesterol in a reaction batch, without the necessity of a special sample 
preparation. 
Therefore, it is an object of the present invention to provide a process 
for the direct carrying out of HDL-cholesterol determinations in serum or 
plasma without previous separation of LDL-cholesterol, VLDL-cholesterol 
and chylomicron cholesterol from the sample. It is a further object of the 
present invention to carry out such a process in combination with a total 
cholesterol determination in serum or plasma. 
Thus, according to the present invention, there is provided a process for 
the specific determination of HDL-cholesterol in serum or plasma by 
incubation with a cholesterol detection system, containing cholesterol 
oxidase and cholesterol esterase in a buffered aqueous medium, and 
measurement of a product of the cholesterol oxidase reaction or of the 
oxygen consumption, wherein a sample to be tested is incubated in the 
presence of a salt of a bile acid or of a bile acid derivative or of 
dioctylsulphosuccinate (Aerosol.RTM. OT), a first measurement is then 
carried out, subsequently a non-ionic, polyethylene oxide group-containing 
detergent or a secondary alkane sulphonate is added, again incubated and a 
second measurement is carried out, the amount of HDL cholesterol being 
determined from the difference between the first and second measurement. 
The present invention is based upon the surprising ascertainment that 
certain detergents only make available to the reaction with cholesterol 
esterase and cholesterol oxidase the cholesterol contained in the 
chylomicrons, VLDL and LDL so that it is possible to allow these 
cholesterol fractions to react of in a first incubation step with the said 
enzymes and only then, in a second incubation step, by the addition of a 
non-ionic, polyethylene oxide group-containing detergent, or of a 
secondary alkane sulphonate, to introduce the HDL cholesterol to the 
reaction. This makes possible, in a single reaction batch, either first to 
determine the cholesterol contained in the chylomicrons, VLDL and LDL and 
thereafter separately the HDL cholesterol, so that there is obtained not 
only total cholesterol but also HDL cholesterol in a single batch, or the 
chylomicron, VLDL and LDL cholesterol is first allowed to react off 
without determination and then only the amount of HDL cholesterol is 
measured. In the first case, the amount of HDL cholesterol is determined 
from the difference between the measurements after the first incubation 
and the second incubation and the total amount of cholesterol from the 
measurement after the second incubation alone. In the second case, for the 
sole determination of HDL cholesterol, during the first incubation, a 
phenol or a phenol or aniline derivative, as well as peroxidase, are 
present but no aminoantipyrine, 3-methyl-2-benzothiazolinone hydrazone 
(MBTH) or its sulphonated derivative (MBTH-S). The hydrogen peroxide 
formed according to equation,2) thereby reacts off without colour 
formation. Colour is only formed when carrying out the second incubation 
in the presence of 4-aminoantipyrine (4-AAP), MBTH or MBTH-S which is a 
direct measure for the HDL concentration. Consequently, in the case of 
this process modification, only one measurement is necessary for the HDL 
determination. 
Therefore, the process according to the present invention is preferably 
carried out in such a manner that a cholesterol detection system is used 
which measures hydrogen peroxide as a product of the cholesterol oxidase 
reaction (2) by colour formation. Appropriate chromogenic systems which 
can be used for the hydrogen peroxide detection are well known. The 
so-called Trinder system is preferred which consists of phenol or a phenol 
derivative or a substituted aniline, 4-aminoantipyrine and peroxidase. 
Amongst the phenol compounds which can be used, phenol itself, as well as 
chlorophenol, triiodo- and tribromohydroxybenzoic acid and 
dichlorophenolsulphonic acid have achieved especial importance. Instead of 
4-aminoantipyrine, there can also be used, for example, 
3-methyl-2-benzothiazolinone hydrazone (MBTH) and its sulphonated 
derivative (MBTHS), preferably in combination with a substituted aniline, 
such as dimethylaniline, dimethylaminobenzoic acid, 
N-ethyl-N-.beta.-sulphoethyl-m-toluidine or 
N-ethyl-N-.beta.-hydroxyethyl-m-toluidine, the last two compounds also 
having proved to be useful in combination with 4-aminoantipyrine. 
As cholesterol esterase, there can be used a microbial cholesterol 
esterase, for example from Candida cylindracea, or the pancreatic enzyme, 
which is preferred. 
As bile acids or bile acid derivatives are to be mentioned the alkali metal 
and magnesium salts of cholic acid, desoxycholic acid, lithocholic acid, 
taurocholic acid, glycocholic acid, desoxyglycocholic acid and 
desoxytaurocholic acid and choleic acids, sodium cholate being preferred. 
Alternatively, there can also be used sodium dioctylsulphosuccinate 
(Aerosol.RTM. OT). We have found that these detergents only digest the 
lipid-rich lipoproteins provided with a relatively small protein part, 
i.e. chylomicrons, VLDL and LDL, and make available for the enzymatic 
reaction the cholesterol contained in them. 
The cholesterol of the HDL fraction is made available to the enzymatic 
determination by non-ionic, polyethylene oxide group-containing 
detergents, such as the non-ionic alkyl or alkylaryl polyethylene oxide 
ethers, for example n-dodecyl polyethylene glycol ether (Thesit.RTM.), 
isotridecanol polyethylene oxide ether (Genapol.RTM. x-80) and 
isooctylphenyl polyethylene oxide (Triton.RTM. X-lOO). Anionic secondary 
alkane sulphonates, such as are, for example, commercially available under 
the name Hostapur.RTM., can also be used. These HDL-digesting detergents 
are preferably employed together with methanol, ethanol, n-propanol or 
isopropanol, propanol preferably being used. The amount of alcohol used is 
preferably from 0.05 to 2%. These and all other statements of 
concentration given herein refer to the end concentration in the reaction 
mixture. 
The salt of the bile acid or of the bile acid derivative is preferably 
added in a concentration of from 0.2 to 20 mmole/liter. However, these 
amounts can be gone below or exceeded, depending upon the sample to be 
investigated. The same applies to the dioctylsulphosuccinate. 
With regard to the second detergent, i.e. the non-ionic polyethylene oxide 
group-containing detergent or the secondary alkane sulphonate, the 
preferred concentration is from 0.02 to 2%. However, depending upon the 
sample, these concentrations can also be gone below or exceeded. 
For the rest, when carrying out the process according to the present 
invention, as in the case of previously known cholesterol determinations, 
the pH value and buffer substances used are those appropriate for the 
amounts of enzyme used. 
The present invention also provides a reagent for the specific 
determination of HDL cholesterol in serum, containing cholesterol 
esterase, cholesterol oxidase and buffer, wherein, referred to the final 
aqueous solution, it contains 
0.1 to 10 U/ml. cholesterol esterase, 
0.05 to 10 U/ml. cholesterol oxidase 
20 to 500 mmole/liter of buffer substance (pH 6.0 to 8.0), 
0.2 to 20 mmole/liter of a salt of a bile acid or bile acid derivative or 
dioctylsulphosuccinate and, separately therefrom, 
0.02 to 2% non-ionic polyethylene oxide group-containing detergent or 
secondary alkane sulphonate, as well as optionally 
0.05 to 2% of an alcohol containing up to 3 carbon atoms. 
According to a first embodiment, the reagent according to the present 
invention contains 0.05 to 20 U/ml. peroxidase, 0.2 to 20 mmole/liter of a 
phenol or aniline and optionally 0.02 to 5% polyethylene glycol and/or 
0.05 to 2% n-propanol. 
A preferred reagent according to the present invention contains 0.2 to 2 
U/ml. cholesterol esterase, 0.05 to 0.5 U/ml. cholesterol oxidase, 1 to 5 
U/ml. peroxidase, 50 to 200 mmole/liter potassium phosphate buffer (pH 6.5 
to 7.0), 1.4 to 10 mmole/liter phenol or phenol derivative, 1 to 5 
mmole/liter of a salt of a bile acid or bile acid derivative or 
dioctylsulphosuccinate, optionally separately therefrom 0.2 to 2 
mmole/liter 4-aminoantipyrine and separately from the other components, 
except possibly from the 4-aminoantipyrine, 0.5 to 5.0% non-ionic, 
polyethylene group-containing detergent or secondary alkane sulphonate and 
optionally 0.1 to 0.5% n-propanol and 0.5 to 5.0% polyethylene glycol. 
In a further embodiment, the reaction components can be impregnated on or 
in a carrier material. The carrier material used can be an absorbent or 
swellable or film-forming carrier material, such as paper or a similar 
fleece material, for example tea bag paper. The reaction components can 
thereby be distributed on several carriers which are in contact with one 
another. 
By means of the present invention, the cholesterol contained in the HDL can 
be determined directly without previous separation from other 
cholesterol-containing lipoprotein fractions. This means a substantial 
easing and simplification of the determination methods. 
The following Examples are given for the purpose of explaining the present 
invention, the following abbreviations thereby being used: 
CHOD=cholesterol oxidase 
CHOL=esterase cholesterol esterase 
POD=peroxidase 
4=AAP 4-aminoantipyrine 
PEG 6000 polyethylene glycol 6000 
TRA triethanolamine

EXAMPLE 1 
(a) Basic reagent: potassium phosphate 0.1 mole/liter (pH 6.7) 
3 mM sodium cholate 
0.8 mM 4-AAP 
1.2 U/ml. CHOD 
0.5 U/ml. CHOL esterase 
1.4 mM phenol 
0.1% PEG 6000 
3 U/ml. POD 
(b) Start reagent for the TRA buffer 0.05M, pH 7.1 
second step : 10% Thesit 
10% propanol 
(c) Performance of the test: 
wavelength: 546 nm 
temperature: 25.degree. C. 
measurement at 25.degree. C. against reagent blank 
Into cuvettes with an optical pathlength of 10 mm., pipette: 
______________________________________ 
sample reagent blank (RB) 
______________________________________ 
basic reagent 2.00 ml. 2.00 ml. 
sample 0.02 ml. -- 
water -- 0.02 ml. 
incubate 10 (-30) minutes, subsequently measure E.sub.1 
(sample) against E.sub.1 (RB) 
start 0.02 ml. 0.02 ml. 
reagent 
incubate at least a further 10 minutes, subsequently 
measure E.sub.2 (sample) against E.sub.2 (RB) (= E.sub.2) 
______________________________________ 
(d) Calculation 
The calculation takes place by simultaneously using a serum standard with a 
known total and HDL cholesterol concentration, the serum standard thereby 
being used in the test batch in the same way as the sample 
##EQU1## 
The time course of the stepwise colour formation taking place corresponding 
to the test batch according to the Example is reproduced in FIG. 1 of the 
accompanying drawings. 
EXAMPLE 2 
(a) Basic reagent: as in Example 1 but without 4-AAP 
(b) Start reagent: as in Example 1 but additionally with 80 mM 4-AAP 
(c) Performance of the test: 
wavelength: 546 nm 
temperature: 25.degree. C. 
measurement at 25.degree. C. against reagent blank 
Into cuvettes with an optical pathlength of 10 mm., pipette: 
______________________________________ 
sample reagent blank (RB) 
______________________________________ 
basic reagent 2.00 ml. 2.00 ml. 
sample 0.02 ml. -- 
water -- 0.02 ml. 
incubate 10 (-30) minutes 
start 0.02 ml. 0.02 ml. 
reagent 
incubate at least for further 10 minutes, subsequently 
measure E (sample) against E (RB) (= .DELTA.E) 
______________________________________ 
(d) Calculation: 
The calculation takes place by simultaneously using a serum standard with a 
known total and HDL cholesterol concentration, the serum standard thereby 
being used in the test batch in the same way as the sample 
##EQU2## 
A comparison of the HDL cholesterol determination according to Example 2 in 
various human sera using the process according to the present invention 
with the method using a phosphotungstic acid (PTA)/Mg sulphate 
precipitation is illustrated in FIG. 2 of the accompanying drawings. The 
method comparison gives a regression of Y=-0.907+1.019 X with a 
correlation coefficient of 0.814. 
Identical results for HDL cholesterol are obtained when working according 
to Example 1.