阅读说明：本技术 一种新型氘代试剂的合成和应用 (Synthesis and application of novel deuterated reagent ) 是由 叶永蓁 陈龙 朱海岸 徐小为 于 2021-05-25 设计创作，主要内容包括：本发明公开了一种之前已经授权的氘代试剂在乳腺癌患者中甘油单酯的测定。该化合物与单甘油酯进行衍生化反应后,可增加单甘油酯的质谱响应。通过与轻标试剂联合使用,可用于乳腺癌患者癌旁组织和癌组织中单甘油酯的检测和相对定量分析。(The present invention discloses the determination of monoglycerides in breast cancer patients of a previously authorised deuterated agent. After the compound and the monoglyceride are subjected to derivatization reaction, the mass spectrum response of the monoglyceride can be increased. The reagent can be used for detecting and relatively quantitatively analyzing the monoglyceride in the para-cancer tissues and the cancer tissues of the breast cancer patients by combining with a light standard reagent.)

1. The synthesis and application of a novel deuterated reagent are characterized in that: the single-chain fatty acid is respectively marked with the same amount of 3-nitro-phenylboronic acid in cancer tissues and tissues beside the cancer in a breast cancer patient, and the content difference of the monoglyceride in the two tissues can be distinguished through relative quantitative analysis, so that the single-chain fatty acid can be used as an early diagnosis method of breast cancer.

2. The synthesis and application of a novel deuterated reagent are characterized in that: deuterated 3-nitrophenylboronic acid (d)₄NPB) can further modify a fluorescent group to realize clinical fluorescent detection of the monoglyceride.

Technical Field

The invention relates to application of a reported deuterated reagent in early diagnosis of breast cancer, belonging to the field of clinical medicine.

Background

Monoglycerides are a class of active substances that participate in a variety of physiological processes in the body. Studies have shown that monoglycerides are closely related to various diseases, such as diabetes, obesity, cancer, etc. At present, an indirect detection method is mostly adopted to identify a monoglyceride mixture in a complex sample, namely, monoglyceride is degraded into glycerol and corresponding free fatty acid, the free fatty acid is separated by thin-layer chromatography, and the obtained free fatty acid mixture is separated and identified by a gas chromatography-mass spectrometry combined method. The method is time-consuming and complex in pretreatment process, and is easy to cause sample loss. In addition, the mass spectrum detection of the whole molecules of the monoglyceride is limited by the ionization property of hydroxyl groups in the monoglyceride structure, and the detection can be carried out only in a negative ion mode, so that the detection sensitivity is reduced, and meanwhile, partial structural information is easy to lose in a secondary mass spectrum, and the difficulty in detection, identification and quantification is improved.

Therefore, in the present invention, the previously reported deuterated 3-nitrophenylboronic acid (d) is utilized₄NPB), the light standard derivatization reagent and the heavy standard derivatization reagent are respectively used for monoglyceride derivatization in breast cancer and para-cancer tissues, and relative quantitative analysis of monoglyceride under different physiological states can be realized through measuring the mass spectrum response ratio of the light standard product and the heavy standard product.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of the deuterated 3-nitrophenylboronic acid in early diagnosis of the breast cancer.

The principle of derivatization reaction:

after the 3-nitrophenylboronic acid is combined with hydroxyl in the solution to form a negative ion form, the negative ion form is combined with dihydroxy groups in MAG, and two molecules of H are removed₂And O, a stable five-membered ring is formed, so that the original structure of the monoglyceride molecule is successfully fixed and is not easily converted into an isomer thereof. The derivative product is in the form of negative ions and has the characteristic isotope distribution characteristic of boron atoms, so that the derivative product can be more easily detected in a negative ion mode, and the response of mass spectrometry is improved. 3-NPB dissolved in acetonitrile: water 1:1,10mM, pH 9 was adjusted by addition of ammonia. For derivatization, 100uLMAG standard solution was mixed with 10uL of 10mM 3-NPB, and reacted at 37 ℃ and 400rpm for 2 hours.

Mainly takes palmitic acid monoglyceride (PG), oleic acid monoglyceride (OG), arachidonic acid monoglyceride (AG), linoleic acid monoglyceride (LG) and stearic acid monoglyceride (SG) as examples, and firstly, the separation result of the liquid phase shows that the monoglyceride passes through d₄-NPB derivatization does not affect its liquid chromatography behavior, and d₀-elution of the NPB-labelled derivatised product takes place simultaneously. Secondly, the mass number difference of each monoglyceride detected from the mass spectrum is 4DaThe double peaks of (1) and the specific isotope distribution of the B element are simultaneously provided, which is beneficial to quickly identifying the monoglyceride in a complex sample. By d₀/d₄And (3) carrying out derivatization after mixing the-NPB 1:1 ratio, wherein the fluctuation of mass spectrum signals of the obtained product is within 10%, and the synthesized deuterated reagent has no significant difference from the efficiency of a light standard reagent. FIG. one A is the sn-2 isomer of MAG18:2 chromatographically separated after derivatization of 3-NPB, corresponding to 4.39 and 4.58 min; panel B shows the sn-1 isomer, corresponding to an elution time of 4.58 min. Comparison shows that the peak time of sn-2 isomer is 4.39min, indicating that 3-NPB derivatization can be used for capturing relatively unstable isomers. Panels C and D are mass spectra of corresponding isomers, which are expected to give m/z 502.299 equivalent to that expected, and show a characteristic isotopic distribution of boron, which can be used to further confirm the presence of the derivatized product. Detection of MAG based on 3-NPB derivatization is applied to characterization of MAG as a potential cancer marker.

Secondly, the effect of the deuterated reagent is verified by introducing samples after the derivatization of breast cancer tissues and tissues beside the breast cancer.

Selecting 38 pairs of breast cancer tissues and tissues beside the breast cancer, performing 3-NPB derivatization, performing chromatographic separation, and performing mass spectrometry for detection. The MAG to be detected is quantitatively found through establishing a standard curve, and the MAG in 5 shown in the figure II has significant difference in the distribution of cancer tissues and tissues beside the cancer and can be used as a potential biomarker. 60mg of tissue was ground with 4 degrees pre-cooled methanol followed by 400uL of methanol at-80 degrees pre-cooled to precipitate the protein. MAG was extracted by shaking at-20 ℃ and centrifuged at 18000rpm at 4 ℃ for 10 min. The supernatant was transferred to a new EP tube and redissolved in methanol after nitrogen blowing. 3-NPB was added at 37 ℃ and 400rpm for 2 hours.