Method and reagent for the determination of .beta.-lipoprotein

The present invention provides a process for the direct turbidimetric determination of .beta.-lipoproteins (LDL) in body fluids by precipitation with polyanions and divalent cations, wherein the precipitation is carried out in the presence of a complex former at a pH value of from 7 to 9. The present invention also provides a reagent for the direct turbidimetric determination of .beta.-lipoproteins (LDL) in body fluids, comprising a polyanion, a salt of a divalent metal, a complex former and a substance buffering at pH 7 to 9.

This invention relates to a method and a reagent for the determination of 
the beta-lipoprotein fraction (low density lipoprotein (LDL)) in body 
fluids. 
Hypercholesterolaemia and hypertriglyceridaemia favor the development of 
atherosclerosis and of cardiac infarction. The determination of 
cholesterol and of triglycerides in serum belong, therefore, to the most 
frequently performed tests in routine clinical-chemical laboratories. 
Numerous investigations into fat metabolism have resulted in the conclusion 
that the individual coronary risk can be better assessed when there is 
determined not only the change of the triglyceride and cholesterol ledvel 
but also the fundamental pathological displacement in the lipoprotein 
picture (see Munch. med. Wschr., 121, 1639/1979). 
The known plasma lipoproteins contain a proportion of protein, 
phospholipids, cholesterol and triglycerides of varying magnitude. On the 
basis of their behavior (differing density), they can be subdivided in an 
analytical centrifuge into three different classes: 
pre-.beta.-lipoproteins=VLDL (very low density lipoprotein) 
.beta.-lipoprotein=LDL (low density lipoprotein) 
.alpha.-lipoprotein=HDL (high density lipoprotein). 
The investigation of the function of the lipoproteins showed that LDL, 
among the lipoproteins, represents the decisive atherogenic component, an 
increase of which in the blood results in an increased risk of coronary 
heart disease. The early recognition and combating of this condition is of 
great importance. Therefore, there is a need for a practical process for 
the quantitative determination of the LDL concentration in serum and 
plasma. 
Hitherto, for the determination of the LDL lipoprotein fraction, 
essentially four methods have been used which, however, suffer from 
certain disadvantages: 
1. Ultracentrifuging. 
This process is not suitable for use in a routine laboratory since special 
equipment is necessary and the carrying out of the process requires an 
extremely careful working procedure and a very large expenditure of time 
(2.times.20 hours at 105,000 g). Consequently, the use of this analytical 
process has hitherto been limited to the medical research laboratory. 
2. Precipitation reaction. 
The LDL content can also be determined by fractional precipitation with 
polyanions, for example heparin sodium or dextran sulphate, and divalent 
cations, for example calcium, manganese or magnesium cations. The 
lipoproteins can be precipitated out, with increasing concentration of the 
polyanions, in the following sequence: VLDL, LDL and HDL. However, this 
process requires two working steps and is thus not practical and cannot be 
automated: VLDL is separated off in a first precipitation step and 
subsequently, by increasing the concentration of the precipitation agent, 
the LDL lipoprotein fraction is precipitated and determined 
turbidimetrically (see H. Okabe, 10th Int. Congr. of Clin. Chem., Mexico, 
1978). 
3. Determination of the LDL concentration by means of the Friedewald 
formula. 
In the case of this process, the triglyceride, cholesterol and 
HDL-cholesterol content of the sample are determined and the content of 
LDL cholesterol calculated therefrom according to Friedewald's process 
(see Clin. Chem., 18, 499/1972). This laborious process is also not 
practical. 
4. Qualification by electrophoretic separation and polyanion precipitation. 
This process is also time-consuming and requires the use of an 
electrophoresis apparatus, as well as of a densitometer for the evaluation 
(see Lab. med., 1, 145/1977). 
Therefore, it is an object of the present invention to provide a practical 
process which can be automated and with which LDL can be determined 
directly in a routine laboratory. 
Thus, according to the present invention, there is provided a process for 
the direct turbidimetric determination of the .beta.-lipoproteins (LDL) in 
body fluids, such as serum or plasma, by precipitation with polyanions and 
divalent cations from an aqueous solution, wherein the precipitation is 
carried out in the presence of a complex former at a pH value of from 7 to 
9. 
The present invention is based upon the discovery of the surprising fact 
that LDL can be selectively and practically quantitatively precipitated in 
the presence of VLDL and HDL and can be determined turbidimetrically when 
the precipitation is carried out with the combination of complexing agent, 
polyanion and cation. This is surprising since, according to previously 
published investigations, when precipitating LDL, VLDL is always 
coprecipitated if it has not been previously separated off (see M. 
Burstein, H. R. Scholnick, "Protides of the biological fluids", ed. 
Peeters, pp. 21-28/1972; Arztl. Lab., 23, 101-110/1977). However, 
according to the present invention, it is, surprisingly, possible 
selectively to precipitate LDL in such a manner that a kinetic 
measurement, i.e. a measurement within a predetermined period of time, can 
be carried out. At the same time, the error in comparison with the known 
processes of joint precipitation of VLDL and LDL is substantially reduced. 
The complex formers used according to the present invention are to be 
understood to be compounds which are capable of forming complexes, i.e. 
compounds of a comparatively high order which are formed by combination of 
molecules. The polyligands have proved to be especially useful, for 
example the tri-, tetra- and hexaligands, the hexaligands being preferred, 
for example aminopolycarboxylic acids, such as ethylenediamine-tetraacetic 
acid (EDTA), nitrilotriacetic acid (NTA) and 
diethylenetriamine-pentaacetic acid. 
The complex former is generally used in an amount of from about 1 to 35 
g./liter and preferably of from 5 to 25 g./liter. 
The cations used according to the present invention are in the form of 
salts of divalent cations with an anion which does not disturb the 
reaction, divalent cations of the alkaline earth metal group being 
preferred. Examples of other divalent cations which can be used include 
manganese and cobalt. Calcium is especially preferred. 
According to a preferred embodiment of the process of the present 
invention, the cations and complex former are first reacted together to 
give a salt-like complex which, possibly after previous isolation, can be 
used for the process according to the present invention. If the complex is 
first separated out from the solution in which it is formed, it admittedly 
does not matter whether the components are used in stoichiometric amount 
since a component which is present in excess and does not participate in 
the complex formation is separated off with the solvent. Preferably, 
however, the cations and complex former are used in stoichiometric amount. 
This also applies when complex formation is carried out directly in the 
solution to be investigated. Since it is frequently desirable to have an 
excess of cation present during the precipitation, in the case of this 
embodiment it is preferable first to add the complex former and the 
cations in stoichiometric amount and then to introduce an excess of 
cations after complex formation has taken place. The concentration of 
cations in the solution is preferably from 0.01 to 0.3M and more 
preferably from 0.08 to 0.15M. The cations may be used, for example, in 
the form of nitrates, acetates, sulphates or halides, the chlorides being 
preferred. 
The third substance necessary for the process according to the present 
invention is a polyanion. Examples of suitable polyanions include 
sulphated polysaccharides, such as heparin, dextran sulphate and 
mepesulphate and phosphotungstates. Heparin is preferred, especially in 
the form of the sodium salt. Polysulphates can also be used, for example 
sodium polyethanolsulphonate and similar compounds. The preferred 
polyanion concentration is generally from 0.005 to 0.5%. 
According to a preferred embodiment of the process of the present 
invention, an ester-splitting enzyme is also added, examples of 
appropriate ester-splitting enzymes including the lipases and the 
esterases, a lipase from Rhizopus arrhizus or a cholesterol esterase being 
especially preferred. The addition of such an ester-splitting enzyme 
increases the exactitude of the process since errors due to coprecipitated 
VLDL can thereby be eliminated. Without the use of an ester-splitting 
enzyme, a lag phase frequently occurs so that measurement can only be 
commenced after 1 to 2 minutes. This lag phase disappears in the presence 
of an ester-splitting enzyme so that the period of determination can 
thereby also be shortened. Therefore, in the case of this preferred 
embodiment of the present invention, not only the practicability but also 
the sensitivity are increased. 
The turbidimetric measurement can be carried out preferably kinetically, 
the possibility of carrying out the measurement kinetically, i.e., within 
a predetermined time interval, thereby being especially advantageous. 
The adjustment of the pH to a value of from 7 to 9 can be carried out in 
known manner, preferably with the use of a buffer. In principle, all 
substances buffering in the given range can be used, examples of 
appropriate buffers including imidazole/HCl, as well as tra/HCl, tris/HCl 
being preferred. 
The present invention also provides a reagent for the direct turbidimetric 
determination of .beta.-lipoproteins (LDL) in body fluids, such as serum 
or plasma, said reagent comprising a polyanion, a salt of a divalent 
metal, a complex former and a substance buffering at pH 7 to 9. 
With regard to the individual components of the reagent and the amounts 
thereof, the statements made hereinbefore in connection with the process 
also apply. Therefore, the reagent according to the present invention 
preferably also contains an ester-splitting enzyme, especially a lipase 
and more especially a lipase from Rhizopus arrhizus. 
The reagent according to the present invention preferably contains the 
complex former already in a complexed state with a divalent metal, i.e. 
the cation. The preferred amount of complex salt is 2 to 25 g., referred 
to the preparation of a 1 liter amount of reagent. The polyanion is 
preferably present in an amount of from 0.02 to 0.5%. If the preferred 
polyanion heparin sodium is used, then this corresponds to 30,000 to 
750,000 USP, again referred to the amount required for the preparation of 
1 liter of liquid reagent. 
With regard to the buffer substance, there again apply the statements made 
above with regard to the process. The buffer substance is preferably 
present in an amount such that the concentration thereof in the liquid 
reagent is from 100 to 150 mMol/liter and more preferably from 120 to 140 
mMol/liter. This applies especially for the preferred tris/HCl buffer. 
If an ester-splitting enzyme is present, the amount thereof is preferably 
from 100 to 6000 U/liter of dissolved reagent and more preferably 500 to 
2000 U. 
Consequently, a preferred reagent according to the present invention has 
the following composition: 
0.02 to 0.5% by weight heparin sodium (30,000 to 750,000 USP/liter), 
dextran sulphate or polyphosphotungstic acid, 
2 to 25 g./liter of a complex salt of calcium and 
ethylenediamine-tetraacetic acid, 
15 to 50 mMol/liter calcium chloride, 
100 to 150 mMol/liter tris/HCl, pH 7 to 9 and optionally 
100 to 6000 and preferably 500 to 2000 U/liter of ester-splitting enzyme. 
Equally good results are obtained with a reagent of the above composition, 
regardless of whether use is made of heparin sodium or of an equivalent 
amount of dextran sulphate. 
According to the present invention, the VLDL precipitation can be neglected 
during the measuring interval (see the following Table 1).

It can be seen that completely different reaction rate constants are 
obtained. The result of this is that, according to the present invention, 
both courses of reaction can be differentiated. 
In the case of the known process in which LDL and VLDL are coprecipitated 
by means of the addition of heparin and calcium, the error, referred to 
the LDL fraction, is 30%. In contradistinction thereto, according to the 
present invention, even in the embodiment without the ester-splitting 
enzyme, this error is reduced to less than 8%. 
The process and reagent according to the present invention are thus 
characterized by the fact that, for the first time, LDL can be 
specifically determined by a single reaction which is easy to carry out. 
Slight disturbances by possible coprecipitated VLDL can be prevented by 
the addition of an esterase. By means of the turbidity measurement, which 
is preferably carried out according to the principle of the kinetic fixed 
time method, the following advantages are obtained: 
1. A blank value determination is unnecessary. 
2. Kinetic fixed time methods are especially suitable for carrying out the 
determination using modern automatic analyzers. This process is, 
therefore, readily adaptable to the analysis devices available in modern 
routine laboratories. 
The measurement value used is the increase in extinction due to the 
turbidity in the measurement time interval. This extinction increase can 
be measured preferably by the kinetic process. The test may be evaluated 
by means of a calibrated curve produced with standard samples. 
Without sample predilution, the present invention enables LDL values of up 
to about 600 mg./dl. to be determined. 
The following Examples are given for the purpose of illustrating the 
present invention. The salt complex used in all of the Examples was 
prepared by means of the following process: 
36 g. Ethylenediamine-tetraacetic acid are dissolved in 400 ml. double 
distilled water. After filtration, 16 g. calcium chloride are added to 
this solution. After stirring for 15 minutes at ambient temperature, the 
suspension is kept for one day at 4.degree. C. and subsequently suction 
filtered through a glass filter, washed free of chloride and dried. 
EXAMPLE 1 
The determination described in the following was carried out by the kinetic 
fixed time process, using the following reagents: 
Reagent 1: 
124 mMol/liter tris/HCl, pH 7.5 
151,000 USP/liter heparin 
20 g./liter Ca.sup.++ /EDTA (salt complex) 
Reagent 2: 
0.68 Mol/liter calcium chloride 
Test formulation: 
measurement wavelength: Hg 365 nm; layer thickness of 
the cuvette: 1 cm.; measurement temperature: 25.degree. C. 
2.5 ml. of Reagent 1 are pipetted into a cuvette, 0.05 ml. of sample is 
added thereto, mixed and the reaction is commenced with 0.1 ml. of Reagent 
2. E.sub.1 is read off 90 seconds after the start and E.sub.2 300 seconds 
after the start. The so obtained extinction difference .DELTA.E=E.sub.2 
-E.sub.1 is used for the evaluation. 
Evaluation: 
The measured extinction difference is referred to an LDL concentration 
reference curve produced with the use of one or more standard samples. 
If, in the case of this Example, there is used, on the one hand, a purified 
VLDL and, on the other hand, a VLDL-free serum and the extinctions 
measured at the different times are evaluated semilogarithmically, then 
there are obtained the different reaction kinetics for VLDL and LDL given 
in the accompanying drawing. The different slopes clearly show this. 
EXAMPLE 2 (WITH THE ADDITION OF LIPASE) 
Measurement took place by the fixed time process. 
Reagent 1: 
124 mMol/liter tris/HCl, pH 7.5 
151,000 USP/liter heparin 
20 g./liter Ca-EDTA 
1500 U/liter lipase 
Reagent 2: 
0.68 mol/liter calcium chloride 
The test formulation is analogous to that described in Example 1. 2.5 ml. 
of Reagent 1 are pipetted into the cuvette, 0.05 ml. of sample is added 
thereto, mixed and the reaction commenced with 0.1 ml. of Reagent 2. 
E.sub.1 is measured 30 to 90 seconds after the start of the reaction and 
E.sub.2 300 seconds after the start. The extinction difference 
.DELTA.E=E.sub.2 -E.sub.1 thus obtained is used for the evaluation. 
For the purpose of comparison, the determination was repeated according to 
the known method 2 described above. The results obtained are given in the 
following Table 1: 
TABLE 1 
______________________________________ 
Influence of different precipitation reagents on the 
precipitation of VLDL and LDL 
heparin/Ca.sup.++ / 
heparin/Ca.sup.++ / 
heparin/Ca.sup.++ 
EDTA, .DELTA.E 
EDTA/lipase 
.DELTA.E (90- 
(90-300 .DELTA.E (90- 
300 seconds) 
seconds) 300 seconds) 
______________________________________ 
VLDL 0.014 0.006 0.000 
fraction 
% error due 
30 7 0 
to VLDL, 
referred to 
LDL 
______________________________________ 
EXAMPLE 3 
The procedure described in Example 2 was repeated except that use was made 
of a reagent with the following composition: 
Reagent 1: 
124 mMol/liter tris/HCl, pH 7.5 
0.1% polyphosphotungstic acid 
24 g./liter Ca/EDTA 
1500 U/liter lipase 
Reagent 2: 
0.23 mol/liter calcium chloride. 
The results obtained correspond to those of Example 2. 
It will be understood that the specification and examples are illustrative, 
but not limitative, of the present invention and that other embodiments 
within the spirit and scope of the invention will suggest themselves to 
those skilled in the art.