CN-121992066-A - Quantitative detection method for nitrate medicine metabolizing enzyme activity

Abstract

The scheme discloses a nitrate medicament metabolizing enzyme activity quantitative detection method in the technical field of medicament metabolism and individual medication, the scheme method incubates a reaction system containing theophylline acetaldehyde or hydrate thereof with whole blood of an individual to be detected, the amount of theophylline acetic acid produced by the specific metabolism of theophylline acetaldehyde by ALDH1A1 and ALDH2 in an LC-MS/MS detection system is adopted, and the metabolic clearance is estimated based on the produced amount. The invention uses whole blood as an enzyme source, does not need to separate blood cells, has simple and convenient flow, can directly and quantitatively reflect the actual activity of relevant enzymes in vivo, overcomes the limitation of traditional genotype detection, can accurately guide the clinical medication of nitrate drugs, and predicts the failure and tolerance risk of the drugs.

Inventors

• YANG LING
• LIU YU
• HU CONG
• WANG XIAOJING
• ZOU LIWEI

Assignees

• 遵义医科大学

Dates

Publication Date

20260508

Application Date

20260211

Claims (8)

1. 1. A nitrate medicament metabolizing enzyme activity quantitative detection method is characterized by comprising the following steps: incubating a reaction system containing theophylline acetaldehyde or a hydrate thereof with whole blood of an individual to be tested at 36.5-37.5 ℃; Step two, detecting the amount of theophylline acetic acid generated by the specific metabolism of the theophylline acetaldehyde by nitrate medicine metabolizing enzyme in a reaction system; And thirdly, evaluating the metabolic clearance capacity of the individual to be tested on the nitrate medicine based on the generation rate of theophylline acetic acid.
2. 2. The method for quantitatively detecting the activity of nitrate medicine metabolizing enzyme according to claim 1, wherein the theophylline acetaldehyde or the hydrate thereof is used as a specific probe substrate of the nitrate medicine metabolizing enzyme.
3. 3. The method for quantitatively detecting the activity of nitrate medicine metabolizing enzyme according to claim 2, wherein the nitrate medicine metabolizing enzyme is aldehyde dehydrogenase.
4. 4. The method for quantitatively detecting the activity of nitrate ester drug metabolizing enzyme according to claim 3, wherein the incubation condition is that the reaction is carried out for 3-10 minutes at 37 ℃.
5. 5. The method for quantitatively detecting the activity of nitrate-based drug metabolizing enzyme according to claim 4, wherein the whole blood is peripheral venous blood subjected to anticoagulation treatment and is not subjected to cell separation.
6. 6. The method for quantitatively detecting the activity of nitrate medicine metabolizing enzyme according to claim 5, wherein the nitrate medicine comprises any one of isosorbide dinitrate, isosorbide mononitrate and nicorandil.
7. 7. The method for quantitatively detecting the activity of nitrate medicine metabolizing enzyme according to claim 6, wherein the aldehyde dehydrogenase is one or both of ALDH1A1 and ALDH 2.
8. 8. The quantitative detection method for nitrate medicine metabolizing enzyme activity according to claim 7, wherein the production rate of theophylline acetic acid in whole blood of the individual is measured after the third step, and the risk of the individual developing drug resistance to the nitrate medicine is estimated based on the comparison of the production rate and the in vitro nitrate medicine metabolizing activity.

Description

Quantitative detection method for nitrate medicine metabolizing enzyme activity Technical Field The invention relates to the technical field of drug metabolism and personalized medicine application, in particular to a nitrate drug metabolizing enzyme activity quantitative detection method. Background Nitrate drugs, such as nitroglycerin, isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN), etc., are classical first-line drugs for the clinical treatment of cardiovascular diseases such as coronary heart disease, angina pectoris, heart failure, etc. The core action mechanism is that the biological conversion is completed in vivo through specific enzymatic reaction, and finally Nitric Oxide (NO) is released. NO as a key effector can activate guanylate cyclase in vascular smooth muscle cells, raise cyclic guanosine monophosphate (cGMP) level, and further dilate coronary artery and peripheral blood vessels, thereby relieving myocardial ischemia and playing an irreplaceable role in preventing and treating cardiovascular diseases. However, the treatment effect of nitrate drugs has obvious individual differences, and is mainly represented in the aspects of treatment failure, drug resistance, adverse reaction and the like of partial patients. Part of patients can have the phenomenon that symptom relief is not obvious or completely ineffective after administration, namely, the drug resistance of nitroglycerin. In addition, continuous or frequent use is susceptible to inducing the development of drug resistance, leading to reduced efficacy and even increased risk of adverse cardiovascular events. The key mechanism behind these individual differences is closely related to the in vivo bioconversion efficiency of nitrate drugs. Studies indicate that mitochondrial acetaldehyde dehydrogenase 2 (ALDH 2) is a key enzyme that catalyzes the activation of nitroglycerin, whose activity directly affects the efficiency of nitroglycerin conversion to Nitric Oxide (NO), while also being closely related to the tolerance of nitroglycerin. In addition, other subtypes such as mitochondrial acetaldehyde dehydrogenase 1 (ALDH 1 A1) have been demonstrated to be involved in the metabolic activation of long-acting nitrate drugs such as isosorbide dinitrate in vitro. Notably, ISMN metabolism is not dependent on ALDH2, but rather is more dependent on ALDH1A1. Therefore, the enzyme activities of the ALDH1A1 and the ALDH2 are evaluated simultaneously, and the method has important clinical significance for comprehensively predicting the curative effects of different nitrate drugs and evaluating the failure and drug resistance risks. The current and clinically usual assessment method is to detect the ALDH2 genotype (e.g. Glu504Lys mutation), and indirectly infer the enzymatic activity by genotype, thereby judging whether the patient is likely to be unresponsive to nitroglycerin treatment. However, genotypes can only reflect genetic background, and cannot accurately quantify the actual activity level of enzymes, and cannot comprehensively reflect the influence of other factors (such as drug interactions, coexisting diseases, environmental factors, and the like) on enzyme functions. Thus, there are still limitations to relying on genotype detection alone, which may result in partial patient classification errors or inaccurate guidance. In order to more accurately guide clinical medication, predict drug failure or tolerance risk, it is necessary to establish a functional assay for directly and quantitatively assessing enzymatic activity of ALDH1A1 and ALDH 2. Disclosure of Invention The invention aims to provide a quantitative detection method for nitrate medicine metabolizing enzyme activity, which aims to solve the problem of insufficient genotype detection in the prior art. The method for quantitatively detecting the activity of nitrate medicament metabolizing enzyme in the scheme, Incubating a reaction system containing Theophylline Acetaldehyde (TA) or a hydrate thereof with whole blood of an individual to be tested at 36.5-37.5 ℃; step two, detecting the quantity of Theophylline Acetic Acid (TAA) generated by the specific metabolism of the theophylline acetaldehyde by nitrate medicine metabolizing enzyme in a reaction system by adopting LC-MS/MS; Step three, based on the generation rate of theophylline acetic acid, evaluating the metabolic clearance capacity of an individual to be tested on nitrate drugs; The structural formula of theophylline acetaldehyde and its hydrate is shown as follows: 。 The working principle of the scheme and the beneficial effects thereof are that: And (3) establishing a nitrate medicament metabolizing enzyme activity quantitative detection method by using theophylline acetaldehyde as a probe through a detection technology (LC-MS/MS). According to the invention, whole blood is used as an enzyme source, no additional blood cells are needed to be separated, the detection flow is simplified, the obtained result dire