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CN-121978245-A - Metabolic profile detection kit based on three different metabolites and application thereof

CN121978245ACN 121978245 ACN121978245 ACN 121978245ACN-121978245-A

Abstract

The invention discloses a metabolic profile detection kit based on three different metabolites and application thereof, belonging to biomedical detection technology. The invention provides a kit for detecting three metabolites of glycerol, 3-hydroxyisovalerate and methionine, which comprises a calibrator unit, a sample processing reagent unit, a derivatization reagent unit and a detection unit, wherein the calibrator unit comprises a glycerol standard, a 3-hydroxyisovalerate standard and a methionine standard with concentration gradients, the sample processing reagent unit comprises a protein precipitant, a metabolite extractant and a stabilizer, the derivatization reagent unit comprises a silanization reagent for gas chromatography-mass spectrometry combined analysis, and the detection unit comprises a specific ion pair and an internal standard for liquid chromatography-tandem mass spectrometry analysis. By utilizing the kit, comprehensive evaluation of mitochondrial function, amino acid metabolism and energy metabolism can be realized, and the kit can be applied to early screening of perinatal depression clinically.

Inventors

  • ZHENG XIAOMIN
  • WANG CHEN
  • Bu Chaozhi

Assignees

  • 无锡市妇幼保健院

Dates

Publication Date
20260505
Application Date
20260224

Claims (9)

  1. 1. A kit for detecting three metabolites of glycerol, 3-hydroxyisovalerate and methionine is characterized by comprising a calibrator unit, a sample processing reagent unit, a derivatization reagent unit and a detection unit, wherein the calibrator unit comprises a glycerol standard, a 3-hydroxyisovalerate standard and a methionine standard with concentration gradients, the sample processing reagent unit comprises a protein precipitant, a metabolite extractant and a stabilizer, the derivatization reagent unit comprises a silanization reagent for gas chromatography-mass spectrometry combined analysis, and the detection unit comprises a specific ion pair and an internal standard for liquid chromatography-tandem mass spectrometry analysis.
  2. 2. The kit of claim 1, wherein the glycerol standard has a concentration of 0.1-100 μg/mL, the 3-hydroxyisovaleric acid standard has a concentration of 0.05-50 μg/mL, and the methionine standard has a concentration of 0.05-50 μg/mL.
  3. 3. The kit of claim 1, wherein the volume ratio of the protein precipitant, the metabolite extract, and the stabilizer is 5:4:1, the protein precipitant is acetonitrile solution, the metabolite extract is methanol solution, and the stabilizer is ascorbic acid solution.
  4. 4. The kit according to claim 1, wherein the derivatizing reagent unit is a mixed solution of N-methyl-N-trimethylsilyl trifluoroacetamide and trimethylchlorosilane, and the volume ratio of the N-methyl-N-trimethylsilyl trifluoroacetamide to the trimethylchlorosilane is 9:1.
  5. 5. The kit of claim 1, wherein the detection unit comprises the following specific ion pairs: glycerin, m/z 116.9- > 75.0; 3-hydroxy isovaleric acid, m/z 131.0-87.0; Methionine with m/z 150.0- > 104.0; internal standard d 5-glycerol, 13C-3-hydroxyisovalerate and d 3-methionine.
  6. 6. Use of a kit according to any one of claims 1-5 for the preparation of a product for assessing mitochondrial dysfunction, abnormal amino acid metabolism and energy metabolism disorders.
  7. 7. Use of a kit according to any one of claims 1-5 for the preparation of a product for early screening of perinatal depression.
  8. 8. A method for detecting the concentration of glycerol, 3-hydroxyisovalerate and methionine in a biological sample, comprising the following steps using a kit according to any one of claims 1-5: (1) Sample pretreatment, namely taking a sample to be detected, adding the protein precipitant, vortex mixing, centrifuging and taking supernatant; (2) Metabolite extraction, namely adding the metabolite extractant and the stabilizer into the supernatant, fully mixing and centrifuging to obtain an extract; (3) Derivatization treatment, namely adding the derivatization reagent into the extracting solution to react; (4) The instrument analysis adopts gas chromatography-mass spectrometry or liquid chromatography-tandem mass spectrometry for analysis; (5) Data analysis, the concentration of the three metabolites was calculated according to the standard curve.
  9. 9. The method of claim 8, wherein in the step of (3) derivatizing, the temperature of the reaction is 70 ℃, and the time of the reaction is 30 minutes.

Description

Metabolic profile detection kit based on three different metabolites and application thereof Technical Field The invention relates to the technical field of biomedical detection, in particular to a metabolic profile detection kit based on three different metabolites and application thereof. Background Among the numerous metabolic pathways, lipid oxidation, branched Chain Amino Acid (BCAA) breakdown, and one-carbon unit cycling are recognized as "intersections" connecting the metabolism of three substances, sugar, lipid, and amino acid. Wherein: 1. glycerol (Glycerol) In obese, diabetic, nonalcoholic fatty liver disease (NAFLD) and long chain fatty acid oxidation deficiency (LC-FAOD) patients, fasting glycerol levels are often significantly elevated and positively correlated with insulin resistance index (HOMA-IR). 2.3-Hydroxyisovaleric acid (3-Hydroxyisovalerate, 3-HIV) Mitochondrial oxidative phosphorylation (OXPHOS) inhibition, hypoxia or high-fat diet can also lead to elevated 3-HIV. Recent researches show that 3-HIV can inhibit Histone Deacetylase (HDAC) and further regulate and control adipogenesis gene expression, and the 3-HIV has double identities of metabolic abnormality signals and epigenetic regulation molecules. 3. Methionine (Methionine, met) Methionine cycle is highly interleaved with folate cycle, choline pathway, nicotinamide N-methyltransferase (NNMT) pathway, and imbalance in homeostasis is seen in various pathological states such as hyperhomocysteinemia, alzheimer's disease, tumor, and gestational nerve tube defects. It is notable that Met itself is a zwitterionic, with large polarity, weak UV absorption, requiring pre-column derivatization for traditional chromatographic-UV/fluorescent detection, cumbersome steps and large fluctuations in recovery. Although the value of the three metabolites has been widely accepted in clinic and in scientific research, the prior art still has the following prominent bottlenecks: (1) Limitations of single index detection Currently, the clinical laboratory uses an enzymatic method (Glycerol), gas chromatography-mass spectrometry (GC-MS, 3-HIV) or an amino acid analyzer (Met) for individual measurement. All indexes are dispersed on different detection platforms, and the result is difficult to transversely compare due to the lack of a standardized calibrator and a unified quality control system, and more importantly, the concentration of a single metabolite is obviously influenced by eating, movement and circadian rhythm, so that two distinct biological scenes of pathway activation and substrate accumulation cannot be distinguished. For example, the measurement of elevated blood glycerol alone cannot be judged to be due to increased fat mobilization (e.g., starvation, exercise) or impaired hepatic gluconeogenesis (e.g., cortisol deficiency), and similarly, the measurement of elevated 3-HIV alone cannot exclude the secondary biotin deficiency rather than the hereditary MCC deficiency. (2) Complex sample pretreatment Glycerol and 3-HIV have strong polarity and volatility, wherein the glycerol has a boiling point of 290 ℃ but is easy to absorb moisture, 3-HIV can be lactonized into 3-methyl-gamma-butyrolactone under acidic conditions, so that the recovery rate in GC-MS analysis is reduced by 20-40%, and methionine is easily oxidized to form methionine sulfoxide at alkaline pH due to sulfur-containing groups, so that peak tailing or false positive is caused. Traditional liquid-liquid extraction (LLE) requires multiple lyophilization-reconstitution steps, which takes > 2h hours, and derivatizing reagents (e.g. bstfa+tmcs) are extremely moisture sensitive, and laboratory humidity differences can cause batch-to-batch CVs >15%. In addition, infant Dried Blood Spot (DBS) sample matrix interference is severe, and hemoglobin and phospholipid co-extracts inhibit electrospray ionization (ESI) efficiency, reducing detection sensitivity. (3) Insufficient technical integration Glycerol is a neutral small molecule, 3-HIV is an acidic organic acid, methionine is an amphoteric amino acid, the three are obviously different in polarity, pKa and protonation efficiency, the retention and peak shape are difficult to be compatible with conventional reversed-phase C18 chromatography, and if an HILIC mode is adopted, the risk of blocking a system due to high organic proportion and easy precipitation of buffer salt is encountered. In terms of mass spectrum parameters, glycerol has only one characteristic fragment (m/z 57) and is susceptible to background interference, 3-HIV needs to be monitored in negative ion mode, and methionine responds more in positive ion mode, resulting in an extended "positive-negative" switching period and reduced flux. Therefore, the prior literature/patent focuses on single or similar structural metabolites (such as CN119000968A only measures homocysteine; CN20171929132. X only measures ketone bodies such as hydroxybutyric acid, acetoacetic acid and the lik