JPS5991897A - Method for measuring concentration of phospholipid fraction in lipoprotein - Google Patents

Abstract

PURPOSE:To obtain a sharp and clear fraction pattern, by subjecting a specimen to the electrophoresis, fractionating a phospholipid in a lipoprotein, and reacting a novel dyeing reagent with the fraction to develop a color. CONSTITUTION:A specimen is subjected to the electrophoresis to fractionate a phospholipid in a lipoprotein. The fractionated phospholipid in the lipoprotein is reacted with a dyeing reagent containing phospholipase D, choline oxidase, ethanol, catalase, acetaldehyde dehydrogenase, nicotinamide adenine dinucleoside phosphate (NADP), diaphorase and NTB to develop a color. After stopping the color developing reaction with a color developing stopping solution, the specimen is washed with distilled water and dried. The fraction % of the phospholipid in the alpha, pre-beta and beta fractions is measured by a densitometer, etc., and the concentration is determined by multiplying the total phospholipid determined separately by the conventional method by the respective factor.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring the fractional strength of phospholipids in riboproteins.

In general, measurement of the phospholipid concentration present in riboproteins in a dish is useful in determining the etiology and diagnosis of diseases such as coronary artery sclerosis.

Conventionally, electrophoretic staining of phospholipids in lipoprotein fractions has been carried out using, for example, H2O2,4-aminoantipyrine and 2,4-dichlorophenol produced by phosphorase D and choline oxidase in the presence of peroxidase. The method used is to react and form a water-bathable red quinone dye. However, this dyeing power method has two problems: (1) Since the quinone dye is water bathable, the dyed pattern tends to diffuse over time and become unclear; (2)
) There were problems in use, such as the low color development sensitivity of the quinone dye, and it was not fully satisfactory in clinical civil engineering.

Therefore, the inventor of the present invention has carried out various studies in order to overcome these conventional difficulties and provide a highly reliable and clinically effective measurement method. The present invention was completed by developing a new staining reagent that can be used as a staining reagent.

That is, the present invention subjects a sample to electrophoresis to fractionate phospholipids in riboproteins, and then injects phospholipase D (hereinafter abbreviated as PLD) and choline oxidase (hereinafter abbreviated as COD) into the phospholipids in riboproteins. ), ethanol, catalase, acetaldehyde dehydrogenase (hereinafter At
This study provides a method for measuring the concentration of phospholipid fraction in ribo-1 protein by applying a staining reagent containing DH, NADP, diaphorase P (hereinafter referred to as DIP), and NTB to develop color. .

To carry out the present invention, first, body fluids such as blood and semen are subjected to electrophoresis to fractionate riboproteins and phospholipids. Therefore, as long as electrophoresis can fractionate phospholipids in riboproteins, it does not matter what the specific electrophoresis method is. Gallose film is preferably used with barbital slow release as the support buffer, Tricine-L+ buffer as the migration buffer, and the migration conditions are Corning Acarose Film System at 90V for 60 to 70 minutes. You can achieve your goals through electrophoresis.

Next, a staining reagent containing PLDlCOD, ethanol, catalase, AtDH%NADP, DIP, and NTB is applied to the protein-negative medium phospholipid fraction separated by electrophoresis.

Here, the staining reagent is, for example, 0.3 M MOPS-Li
(pH 7.0-8.5) 3-medium, PLDIO-30
u.

C0D15-50u% DIP15-45m, catalase 2,600-30,000u, AIDH5-20u
, NADP 2-7 mM, NTB Q, 5-1.
0 mM, j-tanol 0.2-1.0M.

The reagent of the present invention contains the above-mentioned essential component #Kani, one or more selected from the group consisting of MfCl, CaC4, and KCl as a reaction activator, and a clear staining reagent. If a surfactant such as Triton x-ioo is blended for this purpose, 4- can be obtained.

Therefore, as a method for causing such a staining reagent to act on the phospholipid fraction in riboproteins, the immersion method and the sandwich method can also be used, and usually at 35 to 40°C for 25 to 30 minutes. By incubating for about 45 minutes, the phospholipid fraction in riboproteins can be colored.

The principle of this color development is as follows.

OD Choline → 2H20, 10 Betaine AtDM DIP Next, the coloring reaction was stopped using a coloring reaction stop solution (10% acetic acid), and then washed with purified water and dried.
α, p by denmltometer
The % phospholipid fraction in the re-β and β fractions is measured, and the concentration is determined by multiplying the total phospholipids separately determined by a conventional method by each ratio.

As the present invention is constructed as described above, by using the method of the present invention, a sharp and clear fractionation pattern can be obtained through a rapid staining reaction, and an extremely accurate determination of the fractional concentration of phospholipids in lipoproteins can be made. This measurement method is clinically extremely advantageous.

The present invention will be further explained below with reference to Examples.

Example (1) Electrophoresis conditions Support for electrophoresis: Thin layer agarose film Buffer in support: 60 mM barbital buffer Buffer for electrophoresis: C
aC4 at 10 mM% M? Lysine-Ll buffer (pH 8,6) containing 5mM C4 (59mM) Electrophoresis conditions: Corning agarose film system,
9 (1', 60 minutes electrophoresis (2) Reagent preparation ■ Staining reagent composition As the composition in 3 m of staining reagent, PLD 20
uCOD 40m DIP 30 ucatalase
26000uAtDH10u・l+NADP
5mMNTB
lmM ethanol
0.5MMtC1 snow
10mMCaC1 mushroom
10mM KCt
0,3y MOP8-Ll containing 120mM Triton X-1000,3% (PH7,5
) buffer was used.

■ Staining reaction stop solution: 10% acetic acid ■ Washing solution: Purified water (3) Measurement example After electrophoresing 3 μt of serum as a sample under the above electrophoresis conditions, the agarose film was removed from the electrophoresis cell, and the stain reagent 3- with the above composition was added to the electrophoresis cell. 8- When spread evenly on the surface of the cell and incubated at 37℃ for 30 minutes, a sharp reddish-purple phospholipid fraction appeared.
, pre-β and β fractions.

After the reaction, it was immersed in 10% acetic acid for about 1 hour and then in purified water for about 1 hour. Soaked in 3% acetic acid containing ot7't glycerol for approximately 2 minutes.

After that, after drying in a dryer at 70°C for 20 minutes, the phospholipids in the fractionated riboprotein bond were analyzed by densitometry.
itometry) L, phospholipid fraction in serum: α fraction 40%, prs-β fraction 10%, β fraction 50%
I got it. Total lentil 9125 obtained separately from this using a conventional method
0111/at multiplied by α fraction phospholipid fraction 100
q/” 5pre-β fraction phospholipid concentration 25η/dl
, a β-fractionation lipid score of 125 W/dz was obtained.

that's all Applicant: Nippon Chemifa Co., Ltd. 11- 612-

Claims (1)

[Claims] After subjecting the specimen to electrophoresis to fractionate the phospholipids in riboproteins, the phospholipids in riboproteins are allowed to react with a staining reagent containing Phosphoriber Zero 1 choline oxidizer and NTB to develop color. A method for measuring the degree of phospholipid fraction in riboproteins, characterized by: