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大学・研究所にある論文を検索できる 「Comparative evaluation of the extraction and analysis of urinary phospholipids and lysophospholipids using MALDI-TOF/MS」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。
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Comparative evaluation of the extraction and analysis of urinary phospholipids and lysophospholipids using MALDI-TOF/MS

Li, Xin 京都大学 DOI:10.14989/doctor.k23410

2021.07.26

概要

1. Background
　Urine is a unique, easily accessible, and noninvasive sample widely used for clinical diagnostics, especially in urology.
　Lipids, particularly phospholipids (PLs) and lysophospholipids (LPLs), are attracting increasing scientific interest for their potentials as disease biomarkers. Due to their low concentrations in urine, efficient extraction and sensitive detection are required for analyzing urinary PLs and LPLs.
　Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) has been used for lipid analysis. However, the poor quantification ability limits its application in clinical biomarker screenings.

2. Objectives
　To improve the clinical usage of MALDI-TOF/MS, especially for detection of urinary PLs and LPLs, three main strategies include selecting a proper matrix, optimizing lipid extraction protocol, and improving quantitative measurement were performed.

3. Methods & Results
3.1. MALDI matrix
　Two most commonly used matrices for the MALDI lipid analysis, 9- aminoacridine (9-AA) and 2,5-dihydroxybenzoic acid (DHB), were compared for analyzing PLs and LPLs. Results showed that 9-AA was superior to DHB. Meanwhile, 5mM of 9-AA dissolved in a mixture of isopropanol and acetonitrile (60:40, v/v) could lower the matrix background while remaining sensitive detection of urinary PLs and LPLs.

3.2. Urinary lipids extraction
　Six lipid extraction protocols, including Folch method, Bligh and Dyer (B&D) method, acidified B&D method, methyl-tert-butyl ether method, butanol/methanol method, and hexane/isopropanol method were compared. The acidified B&D method showed the highest recovery rates in 9 of the 12 subcategories of urinary PLs and LPLs and the second-best in the other three remaining subcategories. Meanwhile, anti-oxidative additives such as Irgafos® 168 could be released from the disposable polypropylene (PP) plastic tubes by organic solutions and dramatically impact the detection of urinary PLs and LPLs by MALDI. Thus, glass vials that are resistant to organic solutions were used in this study.

3.3. Quantification of urinary PLs and LPLs
　Before the MALDI analysis, a drop of dissolved matrix and analyte was applied onto a target plate where the solvent was left to evaporate. This “drop-dry” method introduced a heterogeneous distribution of matrix and analyte on the target plate which caused a poor reproducibility among different analyses. To overcome this problem, a specially designed target plate which could limit the migration of the matrix and analyte molecules was used to form precise and concentrated distribution of the sample. Meanwhile, an ionization standard (including two commercially available PL standards with known concentrations) was spiked into the matrix and analyte solution to improve the well-to-well and day-to-day reproducibility. By the combination of these two methods, a reliable quantification of urinary PLs and LPLs was achieved.

3.4. Screening for PLs and LPLs in human urine samples
　Using this analytical system, a total of 40 PLs and LPLs were detected in the urine samples from individuals.

4. Conclusion
　A high-throughput screening system of the urinary PLs and LPLs was established. It offers a quick, qualitative, and quantitative detection of urinary PLs and LPLs, which has a promising clinical application prospect. The changed compositions of urinary PLs and LPLs might be used as non-invasive biomarkers for various human diseases like urologic tumors

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