(1)Quantitative Analysis of 1,N2-Ethenoguanine in Human Urine by Isotope Dilution GC/NICI/MS and(2) Measurement of Apurinic Sites on DNA

• Main Authors: Hong-lun Lin, 林泓倫
• Other Authors: none
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/99004174383200610664

碩士 === 國立中正大學 === 化學所 === 93 === 1,N2-Ethenoguanine (1,N2-eGua) is promutagenic DNA lesions, known to derive from endogenous lipid peroxidation. Moreover, 1,N2-eGua is reported to be a primary substrate of human alkylpurine-DNA-N-glycosylase. Thus, this DNA lesion could be excised from tissue DNA and released into urine. In this study, a highly sensitive and quantitative assay based on isotope dilution gas chromatography/ negative ion chemical ionization/ mass spectrometry (GC/NICI/MS) was developed for analysis of 1,N2-eGua in human urine. The assay involved adduct enrichment by consecutive strong cation exchange and reversed phase solid phase extraction columns. The enriched sample was derivatized by pentafluorobenzyl bromide (PFB-Br) and PFB2-1,N2-eGua was analyzed by GC/NICI/MS after a Si SPE clean-up. The stable isotope of the analyte, [13C1,15N2] 1,N2-eGua, was used as internal standard for accurate quantification of urinary 1,N2-eGua in as little as 0.5 mL of human urine samples. This assay provides a valuable tool for measuring urinary 1,N2-eGua as a potential biomarker of DNA damage and repair derived form lipid peroxidation and oxidative stress in general. Apurinic (AP) sites are important DNA repair intermediates in mutagenesis and carcinogenesis. These lesions are generated by hydrolysis of the N-glycosidic bonds. We have described two methods for quantification of DNA apurinic sites, i.e. fluorescence and localized surface plasma resonance (LSPR). Based on the specific reaction of the amionooxy group and the aldehyde moiety of the open ring deoxyribose of DNA apurinic sites, a stable oxime ether is formed. A fluorescence probe is synthesized with an aminooxy group at one end to react with the apurinic sites of DNA. Another approach is to synthesize a nano-probe with an aminooxy group on one end and thiol group on the other. The thiol is attached to gold nanoparticles. When it reacts with the aldehydic group of DNA apurinic sites, formation of the oxime ether should lead to signal enhancement in LSPR.