JPS648790B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for accurately, rapidly and easily quantifying lipoprotein X in serum in routine clinical tests. It has long been known that phospholipids and free cholesterol levels increase in the serum of patients with obstructive jaundice, but this suggests that these are abnormal lipoproteins that have completely different lipid composition and properties from normal lipoproteins. was identified and named lipoprotein X. Quantification of lipoprotein Conventional methods for quantifying lipoprotein X include the following methods. (1) A method in which lipoprotein X migrated to the cathode side by agar gel electrophoresis is scraped off and its lipid components are chemically measured. (2) Rocket immunoelectrophoresis using anti-lipoprotein X serum. (3) A method in which lipoproteins other than lipoprotein X are removed by centrifugation using heparin and zinc, and then turbidity is measured by adding heparin and calcium. (4) Extract the phospholipids of lipoprotein X with ether,
A method of colorimetric quantitative determination by developing diazo color. (5) A method in which after dissolving the heparin/calcium precipitate, lipoprotein X is separated by hydroxyapatite chromatography and its lipid components are measured. However, all of the conventional quantitative methods require complicated operations and lack speed, making them unsuitable for routine clinical tests performed in general hospital clinics. As a result of repeated research to solve the above problems, the present inventors found that by sequentially using phosphotungstic acid + ethylenediaminetetraacetic acid and alkaline earth metal ions, lipoprotein By discovering that lipoprotein X can be isolated and further measuring the lipid components of the separated lipoprotein
We developed a quantitative method for That is, an acidic solution of phosphotungstic acid and ethylenediaminetetraacetic acid precipitates β-lipoprotein without precipitating lipoprotein X in serum under conditions in the absence of metal ions;
When a solution of alkaline earth metal ions is added to the centrifugation supernatant, lipoprotein . The reagent used in the present invention for separating lipoprotein
Acidic precipitation reagent with buffering capacity between 5.0 and 6.0 ()
and a precipitation reagent () whose main component is an alkaline earth metal ion.The method for quantifying lipoprotein X according to the present invention involves separating lipoprotein The amount of lipoprotein X is determined by measuring the lipid component of the separated lipoprotein X. The present inventors added a certain amount of phosphotungstic acid + ethylenediaminetetraacetic acid precipitation reagent () at various concentrations to a certain amount of normal serum and lipoprotein was measured by a known enzymatic method. The result is the first
As shown in the figure, it can be seen that for both normal serum and lipoprotein X-positive serum, the amount of cholesterol in the supernatant remains constant when the phosphotungstic acid concentration of the precipitation reagent ( ) is in the range of 0.6 to 1.0%. Separate studies using immunoelectrophoresis have confirmed that increasing the concentration of phosphotungstic acid causes precipitation of β-lipoprotein, and that this precipitation is completed within the range of 0.6 to 1.0%. From this, the concentration of phosphotungstic acid in the precipitation reagent () is 0.6~
A range of 1.0% has been found to be suitable. Next, the present inventors added precipitation reagent () to normal serum and lipoprotein X-positive serum and centrifuged the resulting supernatant. was added and centrifuged, and the amount of cholesterol in this supernatant was measured.
The results are shown in FIG. 2, and for lipoprotein X-positive serum, the amount of cholesterol in the supernatant becomes constant when the magnesium ion concentration of the precipitation reagent ( ) is 12.5 mM or higher. Although not shown, there was no change in the magnesium ion concentration up to 200 mM. In contrast, in normal serum, the amount of cholesterol in the supernatant remained constant regardless of changes in the magnesium ion concentration of the precipitation reagent (2). This shows that when a magnesium ion precipitation reagent () is added to the supernatant, only lipoprotein It has been confirmed. From the above, it was found that the magnesium ion concentration of the precipitation reagent (2) is preferably 15mM to 200mM. The first step precipitation reagent () in the present invention is an acidic solution containing phosphotungstic acid as a main component and ethylenediaminetetraacetic acid, and having a buffering capacity at a pH of 5.0 to 6.0. The buffer for the precipitation reagent (2) may be any acidic buffer that does not form a precipitate with alkaline earth metal ions, such as a 2(N-morpholino)ethanesulfonic acid buffer. The second step precipitation reagent () of the present invention is a buffer solution containing alkaline earth metal ions as a main component and having a buffering capacity at pH 6.0 to 10.0. Although calcium ions can also be used as alkaline earth metal ions, magnesium ions are more preferred. The buffer solution of the precipitation reagent () does not react with alkaline earth metal ions to form a precipitate;
Further, any buffer solution having a buffering capacity at pH 6.0 to 10.0 can be used. Examples of this buffer include Tris-HCl buffer, N-2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamidoiminodiacetic acid (ADA), NN-bis-(2
-hydroxyethyl)-2-aminoethanesulfonic acid (BES), NN-bis(hydroxyethyl)
Glycine (Bicine), Bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bis-tris), 1,3-bis[tris(hydroxymethyl)methylamino]propane (Bis-
trispropane), 3-cyclohexylaminopropanesulfonic acid (CAPS), cyclohexylaminoethanesulfonic acid (CHES), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2-(N-morpholino) Ethanesulfonic acid (MES), 3-(N-morpholine)propanesulfonic acid (MOPS), piperazine-N,N'-
Bis(2-ethanesulfonic acid) (PIPES), N-
Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tris(hydroxymethyl)-
A buffer solution such as methylglycine (Tricine),
Examples include glycylglycine, imidazole, and peronal. In order to reliably avoid precipitation of contaminant proteins in clinical tests, the pH is more preferably in the range of 8.0 to 9.5. The method of quantifying lipoprotein X in serum according to the present invention in a clinical test is as follows. First, an acidic precipitation reagent () containing phosphotungstic acid as a main component, ethylenediaminetetraacetic acid, and no metal ions is added to the test serum, and the reaction solution is centrifuged to remove the precipitate ().
A precipitation reagent (2) containing alkaline earth metal ions as a main component is added to the supernatant (2) obtained in the first stage reaction, and the reaction solution is centrifuged to remove the supernatant (2). The amount of lipoprotein X is determined by fractionating the precipitate obtained in the second stage reaction and measuring its lipid components. Furthermore, the amount of lipoprotein Test serum and precipitation reagent () are usually mixed in equal amounts. In this case, the pH of the precipitation reagent ( ) is 5.30,
The composition is, for example, 0.6 to 1.0% phosphotungstic acid,
Ethylenediaminetetraacetic acid is 10mM, and 2-(N-morpholino)ethanesulfonic acid is 0.2M. The mixing ratio of the test serum and precipitation reagent () is not limited to this, for example, the mixing ratio may be 1:20, but in this case, the pH of the precipitation reagent is 5.30 and the composition is, for example, phosphotungstic acid. is 0.012%, ethylenediaminetetraacetic acid is 1mM, and 2-(N-morpholino)ethanesulfonic acid is 0.2M. In this way, the composition of the precipitation reagent () is determined by considering the mixing ratio with the sample. The precipitation reagent () is used for β-
The composition is not limited to the above composition as long as it precipitates lipoproteins but does not precipitate lipoproteins. Next, add a precipitation reagent () containing alkaline earth metal ions as a main component to the supernatant () obtained from the first stage reaction.
is added to carry out the second stage reaction. A preferred mixing ratio of supernatant (2) and precipitation reagent (2) in the second stage reaction is 1:1. In this case, the composition of the precipitation reagent () is 0.05-0.5M Tris-HCl buffer (PH8.0-
9.5) is preferred. When determining the amount of lipoprotein
Considering the sensitivity of the method for measuring lipid components, the mixing ratio of supernatant (2) and precipitation reagent (2) is preferably 1:1. This mixing ratio is not limited to this, and the mixing ratio can be changed up to 1:20 even if the above composition of the precipitation reagent (2) remains unchanged. Also the second
In step reactions, precipitate reagent () to supernatant ()
The amount of lipoprotein X can also be determined by adding and reacting, and measuring the turbidity of the reaction solution. In this case the first
A suitable mixing ratio of the supernatant (2) obtained in the step reaction and the precipitation reagent (1) is 1:10 to 1:20. A preferred example of the method for quantifying lipoprotein X according to the present invention is systematically shown below.

[Table] Measurement

Claims (1)

[Scope of Claims] 1. Alkaline earth metal ions are added to the supernatant obtained by adding an acidic precipitation reagent () containing phosphotungstic acid as a main component, ethylenediaminetetraacetic acid, and no metal ions to the test serum. A method for quantifying lipoprotein X, which comprises adding a precipitation reagent () as a main component to separate lipoprotein X as a precipitate, and measuring the lipid component of the separated lipoprotein X. 2. The method for quantifying lipoprotein X according to claim 1, wherein the precipitation reagent () has a pH of 5.0 to 6.0. 3. The method for quantifying lipoprotein X according to claim 1, wherein the precipitation reagent () has a pH of 6.0 to 10.0. 4. The method for quantifying lipoprotein X according to claim 1, wherein the alkaline earth metal ion is a magnesium ion.