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Method for detecting enzymatic activity of PON-1 in blood sample and application thereof

A technology of PON-1 and enzyme activity, applied in the medical field, can solve the problems of low comparability, large inter-batch and intra-batch coefficient of variation, large difference in enzyme activity measurement level, etc., and achieve the effect of small variation coefficient and stable detection

Inactive Publication Date: 2011-10-26
WUHAN UNIV
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Problems solved by technology

[0011] The above two types of enzyme activity measurement methods respectively reflect the esterase catalytic activity of PON-1. The purpose is to reflect the anti-oxidation function of HDL in blood in different studies. The experimental results have intuitive advantages in various research reports, but in the operation method Each sample in the sample is measured respectively with cuvettes, and the coefficient of variation between batches and within batches is large (the paraoxonase activity assay method is respectively 5% and 8%, and the aromatic esterase activity assay method is respectively 13.3% and 10.9%) , and in different laboratories with different operating methods and experimental standard conditions, the PON-1 enzyme activity measurement level varies greatly, and the comparability is low. At present, there is an urgent need for a unified standardized analysis and measurement method and reference value, so as to facilitate the comparability of research and use it as Effective Clinical Diagnostic Auxiliary Criteria

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  • Method for detecting enzymatic activity of PON-1 in blood sample and application thereof
  • Method for detecting enzymatic activity of PON-1 in blood sample and application thereof
  • Method for detecting enzymatic activity of PON-1 in blood sample and application thereof

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Embodiment 1

[0044] The present invention reforms the experimental operation mode and conditions on the basis of the commonly used PON-1 paraoxonase activity determination principle and method, and uses the ratio (P / A value) of PON-1 activity and apo AI level as the HDL particle Evaluation of antioxidant function. A method for detecting PON-1 enzyme activity in a blood sample, the specific steps of which are:

[0045] (1) Measurement: The enzymatic activity of PON-1 from serum or plasma samples was measured by using the principle of enzymatically hydrolyzing the specific substrate paraoxon into p-nitrophenol. in Ca 2+ In the Tris-Cl buffer (pH8.5) analysis system, the generation rate of the enzymatic reaction product within 4min was continuously scanned and recorded at 405nm on a microplate reader, and the enzymatic activity was expressed as p-nitrophenol per milliliter (ml) serum per minute expressed in nanomoles (nmol).

[0046] The specific testing conditions and methods are as follo...

Embodiment 2

[0060] Measurement of PON-1 enzyme activity levels in transgenic mouse models of different hyperlipidemia:

[0061] The apo E gene knockout (apo E - / - ) mouse, scavenger receptor BI gene knockout (SRBI - / - ) mice, apo E / SRBI double gene knockout (apo E - / - SRBI - / - ) mouse, lipoprotein lipase gene knockout (LPL - / - ) mice were fasted for 12 hours, blood was collected from the retro-orbital venous plexus, and serum was prepared by centrifugation (10min×4000rpm), and the following indicators were directly tested on the serum samples of each group: total cholesterol (TG), triglyceride (TC), PON -1 enzyme activity level, or freeze blood samples at -80°C and then detect PON-1 activity. N: Number of individual subjects.

[0062] Table 1. Serum PON-1 enzyme activity levels of different hyperlipidemia model mice

[0063]

[0064] Since about 30% of PON-1 in mouse serum can be freed from HDL, but this lipid-free (not bound to HDL) PON-1 also has antioxidant effects, therefore,...

Embodiment 3

[0072] Measurement of PON-1 activity and apo AI level in patients with coronary heart disease of different etiologies:

[0073] A total of 165 patients (92 males, 73 females, average age 61 years old) in Hubei with diagnostic indicators of coronary heart disease or myocardial infarction (22 patients with coronary heart disease were re-visited after more than half a month of probucol treatment). 65 normal persons (35 males, 30 females, average age 58 years old) and 19 seemingly normal subjects with low levels of HDL-C were detected in the relevant research. Subjects were asked to undergo the following tests: ①Coronary angiography for the diagnosis of coronary heart disease; ②Echocardiography, for evaluating information such as myocardial ischemia and myocardial infarction in high-risk groups of coronary heart disease; ③A full set of blood lipid testing: including plasma or Serum TC, TG, LDL-C, HDL-C, lipoprotein (a), apo AI, apo B100 levels. ④ Detection of PON-1 enzyme activit...

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Abstract

The invention discloses a method for detecting enzymatic activity of PON-1 in a blood sample and application thereof. The method comprises the following steps: (a) taking paraoxon as a substrate, and detecting the activity of paraoxonase-1, PON-1 in a blood serum or blood plasma sample; (b) detecting the apolipoprotein AI (apo AI) level in the sample by immunity transmission turbidity; (c) calculating the ratio of the activity of the PON-1 to the apo AI level; and (d) comparing the calculated ratio with at least one known index indicating existence or inexistence of coronary heart disease. The method specifically and sensitively evaluates the existence of fatalness of the coronary heart disease by reflecting the relative activity of the PON-1 in HDL particles. The detection of the enzymatic activity is applied in preparing medicaments for treating or preventing cardiovascular and cerebrovascular diseases such as the coronary heart disease, miocardial infarction and the like. The PON-1is a characteristic antioxidant enzyme having main antioxidant function in the HDL, and the activity thereof can reflect the antioxidant function of the HDL, so activity detection of the PON-1 is taken as a method and a tool for evaluating the fatalness of the coronary heart disease.

Description

technical field [0001] The invention relates to the field of medical technology, more specifically to a method for detecting the enzyme activity of paraoxonase 1 (PON-1) in plasma or serum, and also relates to a method for detecting the enzyme activity of PON-1 in coronary heart disease and myocardial infarction. Applications in laboratory-assisted diagnosis, early detection, risk assessment and treatment monitoring of cardiovascular and cerebrovascular diseases. Background technique [0002] Cardiovascular and cerebrovascular diseases such as coronary heart disease and stroke caused by atherosclerosis (As) are the "number one killer" that seriously endanger human health. The occurrence of As is closely related to the disorder of blood lipid metabolism in the body. A large number of epidemiological investigations have confirmed that the level of plasma high-density lipoprotein cholesterol (HDL-C) is negatively correlated with the occurrence of coronary heart disease, so it i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/44G01N21/00
Inventor 喻红郑伟郑悦玲李小明胡汉宁沈丹周赤燕
Owner WUHAN UNIV
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