Method and kit for rapidly quantifying amino acids and derivatives thereof in saliva

A technology of amino acids and derivatives, which is applied in the field of rapid quantification of amino acids and their derivatives in saliva, can solve the problems of large sample consumption, complicated processing, long operation and detection time, and achieve high reliability and simple detection technology Effect

Pending Publication Date: 2022-07-15
SHANGHAI SIXTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The present invention requires invasive acquisition of biological samples for the measurement of blood biomarkers of metabolic diseases such as obesity and diabetes, and the current quantitative detection method of saliva amino acid has a complicated pretreatment process and requires a large amount of samples, and the detection and quantification process requires professional mass spectrometer technicians to operate , The problem of long detection time is improved, and a method and kit for rapid quantification of amino acids and their derivatives in saliva are provided

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  • Method and kit for rapidly quantifying amino acids and derivatives thereof in saliva
  • Method and kit for rapidly quantifying amino acids and derivatives thereof in saliva
  • Method and kit for rapidly quantifying amino acids and derivatives thereof in saliva

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Embodiment 1

[0039] The present embodiment provides a simple and rapid quantitative method for amino acids and derivatives thereof in saliva, comprising the following steps:

[0040]Step 1, obtain 20 μL of saliva sample, add 60 μL of -20°C pre-cooled methanol solution to remove proteins, centrifuge at 13200 rpm for 10 minutes at 4°C, take 20 μL of supernatant, and add 70 μL of boric acid buffer solution with pH=9.18;

[0041] In step 2, 20 μL of 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC) derivatization reagent prepared with acetonitrile to prepare 10 mmol / L was added, and the reaction solution was heated at 50° C. and shaken for 10 minutes. Place at -20°C for 20 minutes, and centrifuge at 13200 rpm for 10 minutes at 4°C;

[0042] Step 3, take the supernatant after centrifugation and use the AcquityUPLC-QDa chromatography-mass spectrometry detector of Waters to quantify amino acids, specifically adopting a reversed-phase chromatographic column with a particle size of 1.6 μm and a...

Embodiment 2

[0044] The present embodiment verifies the quantitative method provided in Example 1, and the specific experimental steps and conclusions are as follows:

[0045] The 28 kinds of amino acids were prepared with deionized water into a mother solution with a concentration of 20 mmol / L each, and diluted with deionized water to contain 3 groups of standards with different concentrations in the mixed standard standard solution, 1 group: Asp, EA, Sar, Cit, β-Ala, GABA, 2-AAA, BAIBA, AABA, Met 10, the concentration is 100μmol / L; 2 groups: Asn, Arg, Glu, Orn, Tyr, Ile, Leu, Phe, Trp 9, the concentration All were 400 μmol / L; 3 groups: 9 Ser, Gln, Tau, Gly, Thr, Ala, Pro, Lys, Val, and the concentration was 400 μmol / L. Accurately take 20 μL of the mixed standard solution, add 70 μL of boric acid buffer solution with pH=9.18, and then add 20 μL of AQC derivatization reagent prepared with acetonitrile to prepare 10 mmol / L. Place at °C for 20 minutes, centrifuge at 13200 rpm for 10 minutes...

Embodiment 3

[0052] The present embodiment adopts the method provided in Example 1 to explore the correlation between salivary branched-chain amino acids and blood glucose levels and serum branched-chain amino acids. The specific operations and results are as follows:

[0053] According to literature research reports, serum branched-chain amino acids are biomarkers for the diagnosis of diabetes, and the level of serum branched-chain amino acids in diabetic patients is significantly higher than that in normal people. Age (42 ± 12.2 and 44.3 ± 12.1), sex (1:1) and BMI (23.7 ± 2.6 and 24.8 ± 2.7) matched normal subjects were used (fasting blood glucose 3.5 ± 1.5, 2h postprandial blood glucose 7.7 ± 1.4, glycation Hemoglobin 6.2±0.9) and diabetics (fasting blood glucose 4.5±1.5, 2h postprandial blood glucose 12.4±2.5, glycated hemoglobin 14.6±3.9) saliva and serum, take 20μL saliva or serum, add 60μL-20℃ pre-cooled methanol Remove the protein from the solution, centrifuge at 13200rpm for 10 mi...

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Abstract

The invention provides a method and a kit for rapidly quantifying amino acids and derivatives thereof in saliva. The amino acid and the derivative thereof comprise asparagine, arginine, serine, aspartic acid, taurine, glycine, ethanolamine, guanidine, sarcosine, glutamic acid, glutamine, threonine, beta-alanine, alanine, ornithine, lysine, tyrosine, proline, methionine, valine, isoleucine, leucine, phenylalanine, tryptophan and gamma-aminobutyric acid. Amino adipic acid, beta-aminoisobutyric acid and alpha-amino acid butyric acid; the method comprises the following steps: carrying out saliva derivatization pretreatment on a saliva sample without protein by using an AQC derivatization reagent, and carrying out quantitative determination on the treated saliva sample by adopting ultra-high performance liquid chromatography-mass spectrometry detection. The method is simple and rapid, does not need to be operated by mass spectrum professionals, is high in reliability, and provides a practical and large-scale popularization means for rapid diagnosis of metabolic diseases such as obesity, diabetes mellitus and cardiovascular diseases.

Description

technical field [0001] The invention relates to the technical field of biological sample detection, in particular to a method and a kit for rapidly quantifying amino acids and derivatives thereof in saliva. Background technique [0002] More than a decade ago, Christopher B. Newgard of Duke University in the United States found that branched-chain amino acids are closely related to metabolic diseases including diabetes and obesity. Plasma branched-chain amino acids can predict the occurrence of obesity, diabetes, and the results of therapeutic interventions. At the same time, it has also been reported that the level of aromatic amino acids changes in the blood of obese and diabetic patients. [0003] According to the 2021 International Diabetes Federation IDF "Diabetes Atlas 10th Edition" report, there will be 537 million adults with diabetes worldwide in 2021, and it is expected to increase to 643 million in 2030 and 784 million in 2045. According to the analysis of the "G...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCG01N30/02G01N30/06G01N30/72
Inventor 贾伟赵爱华瞿纯包玉倩
Owner SHANGHAI SIXTH PEOPLES HOSPITAL
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