High density lipoprotein cholesterin fraction esterification rate determination method

A technique for high-density lipoprotein and a determination method is applied in the field of determination of the fractional esterification rate of lipoprotein cholesterol, which can solve the problems of inconvenient research and application, difficult sample storage and the like, and achieve the effects of simple determination and low cost.

Inactive Publication Date: 2011-05-11
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This requires strict control over sample collection, handling and preparation, making it difficult to allow any form of sample storage
This sample request to FER HDL Research and application cause inconvenience, so there is an urgent need for simple, safe and low-cost determination of FER that can be applied to sample storage HDL Methods

Method used

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  • High density lipoprotein cholesterin fraction esterification rate determination method
  • High density lipoprotein cholesterin fraction esterification rate determination method
  • High density lipoprotein cholesterin fraction esterification rate determination method

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Experiment on changes in plasma high-density lipoprotein free cholesterol after incubation at 37°C for different times

[0037] FER HDL The determination of the method is similar to the enzymatic test, it is a determination of catalytic activity, and the determination result depends on the determination procedure. The content of this study is the effect of incubation time on plasma HDL free cholesterol. Take venous blood (15mL) from five healthy volunteers (A, B, C, D, E), put them into test tubes containing EDTA anticoagulant, mix them, put them in a water bath at 0°C immediately, and centrifuge after 30 minutes Plasma; Dextran Magnesium Sulfate Precipitant (MgCL 2 The concentration is 0.35mol / l, and the dextran sulfate is 10mg / ml, both of which are aqueous solutions. ), to prepare plasma HDL; divide the HDL into 6 tubes (500 μl each), keep the first tube at 0°C, and incubate the second, third, fourth, fifth, and sixth tubes at 37°C for 0.5, 1, 2, 4. Aft...

Embodiment 2

[0038] Embodiment 2: DTNB activity inhibition experiment to LCAT:

[0039] Adding a certain concentration of DTNB to whole blood samples can form disulfide bonds with two free sulfhydryl groups of LCAT, inhibit the activity of LCAT, and stabilize HDLFC at the initial level. Add 0.125, 0.25, 0.5, 1, 2, 4, and 8mmol / LDTNB solutions (prepared by adding Tris buffer solution to DTNB powder, the concentration of Tris solution should be 2 times the target concentration of DTNB) in whole blood samples, room temperature The serum was left for 1 hour to separate the serum, and HDL was prepared, and the HDL was incubated at 37° C. for 2 hours, and the HDLFC after incubation was determined by HPLC. HDLFC levels at different DTNB concentrations see figure 2 , the results showed that when the whole blood DTNB was low, the FC of HDL was also low after incubation, indicating that the activity of LCAT was not completely inhibited, and part of HDLFC was esterified; as the concentration of DTN...

Embodiment 3

[0040] Embodiment 3: the recovery experiment of the LCAT activity after inhibition by different concentrations of ME:

[0041] Volunteer blood was taken, DTNB was added to make the final concentration 1mmol / L, and HDL was prepared after the serum was separated. Divide the serum HDL into 8 tubes, place them in an ice-water bath, add ME aqueous solution to make the concentrations 0, 0.25, 0.5, 1, 2, 4, 8 and 16 mmol / L, Vortex (vortex mixing), and mix each tube at the same time Put it in a water bath at 37°C, take it out after 2 hours, put it in an ice-water bath, Vortex (vortex mixing), and measure HDLFC. The results of HDLFC at different ME concentrations are shown in image 3 . Depend on image 3 It can be seen that when there is no ME or the concentration of ME is low, HDLFC is high, and as the concentration of ME increases, the level of HDLFC decreases, and when ME>4mmol / L, HDLFC remains stable. The results indicated that ME could restore the activity of LCAT and decreas...

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Abstract

The invention discloses a measuring method of high density lipoprotein cholesterol fraction esterification velocity, which comprises: a) adding a lecithin cholesterol acyltransferase activity inhibitor in a fresh whole blood sample; b) adding a precipitator to obtain a sample only containing high density lipoprotein; c) dividing the sample into two parts with one part added with a lecithin cholesterol acyltransferase activity restorer and the other part added with nothing, and the two parts are cultivated at a temperature of 37 DEG C for one hour; and d) utilizing the high efficiency liquid phase chromatography to respectively measure high density lipoprotein free cholesterol of the two samples and calculating the difference between the two parts, and obtaining the high density lipoprotein cholesterol fraction esterification velocity by dividing the difference value by the detected percentage of high density lipoprotein free cholesterol of the sample without adding lecithin cholesterol acyltransferase activity restorer. The method of the invention has advantages of simplicity, safety, low cost and storability of samples.

Description

technical field [0001] The invention belongs to the detection field, and more specifically, the invention belongs to a method for measuring the esterification rate of lipoprotein cholesterol fraction. Background technique [0002] Lecithin cholesterol acyltransferase (LCAT) is the main enzyme of cholesterol esterification in the circulation. It transfers the fatty acyl group at the sn-2 position of lecithin to cholesterol to generate cholesterol and lysolecithin. LCAT binds to high-density lipoprotein (HDL) , so cholesterol esterification mainly occurs in HDL. LCAT plays an important role in HDL metabolism and cholesterol reverse transport (Glomset JA. The plasma lecithin: cholesterololacyltransferase reaction. J Lipid Res, 1968, 9: 155-167;, Sviridov D, Nestel PDynamics of reverse cholesterol transport: protection against atherosclerosis. Atherosclerosis, 2002, 161: 245-254.). There are many methods for studying LCAT activity, one of which is to measure the cholesterol es...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02C12Q1/60G01N33/00
Inventor 陈文祥董军彭涛王抒国汉邦李红霞满永
Owner BEIJING HOSPITAL
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