| Summary: | 碩士 === 中山醫學大學 === 職業安全衛生學系碩士班 === 102 === A highly specific and sensitive liquid chromatography–tandem mass spectrometry method was first developed for simultaneously measuring 5-methyl-2’-deoxycytidine (5-mdC) and 5-hydroxymethyl-2’-deoxycytidine (5-hmdC) in DNA and urine. With the use of isotope internal standards (d3-5-mdC and d3-5-hmdC) and on-line solid-phase extraction, the detected limits of this method were 0.054 and 0.132 fmol on column for 5-mdC and 5-hmdC, respectively, and a total analysis time per sample as short as 15 min. Inter- and intraday imprecision was <10 % and the mean recovery of 5-mdC and 5-hmdC in DNA was 99-104 %.
This method was then applied to evaluate the global DNA methylation and hydroxymethylaion levels in calf thymus and rat liver DNA. The global DNA methylation levels, based on the mole ratio of 5-mdC/(5-mdC+5-hmdC+dC), were found to be 6.25 ± 0.11 (%) and 3.31 ± 0.01 (%) for the calf thymus and rat liver DNA, respectively. The global DNA hydroxymethylation levels, based on the mole ratio of 5-hmdC/(5-mdC+5-hmdC+dC), were found to be 0.0339 ± 0.0026 (%) and 0.1387 ± 0.0063 (%) for the calf thymus and rat liver DNA, respectively.
This method was further used to determine the levels of 5-mdC and 5-hmdC in human urine obtained from 30 healthy subjects and 6 lung cancer patients. The urinary levels of 5-mdC and 5-hmdC of healthy subjects were 6.89 ± 4.92 and 3.68 ± 2.60 ng/mg creatinine, respectively. The urinary levels of 5-mdC and 5-hmdC of lung cancer patients were 13.54 ± 14.91 and 3.06 ± 1.53 ng/mg creatinine, respectively. Overall, our newly developed on-line sample purification/enrichment system coupled with isotope-dilution LC-MS/MS method would be a useful tool for assessing global DNA methylation and hydroxymehtylation level in human.
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