Determination of urinary 8-hydroxy-2'-deoxyguanosine by an improved high performance liquid chromatography method

• Main Authors: Wei-Ling Lin, 林韋伶
• Other Authors: Hung-Yau Ho
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/11923004230294498588

碩士 === 長庚大學 === 醫學生物技術研究所 === 93 === 　　Urinary 8-OHdG(8-hydroxy-2’-deoxyguanosine), a DNA repair end product, is elevated in urine of various degenerative diseases and can be regarded as a marker of oxidative stress. Two major methods to determine urinary 8-OHdG are high performance liquid chromatography (HPLC) and enzyme-link immunosorbent assay (ELISA). In the present study, the current HPLC procedure was improved by incorporating a filter based immunoaffinity purification step. In comparison between conventional solid phase extraction (SPE) and filter based immunoaffinity purification method, the latter method greatly facilitated analysis, and enhanced the sensitivity of assay. Urinary 8-OHdG can be analyzed efficiently without resort to the current complicated procedure. To establish a more applicable method for urinary 8-OHdG measurement, a home-made competitive ELISA was developed. The detection limit of HPLC and home-made ELISA were 0.35 nM and 2.5 nM respectively. The between-run coefficient of variation of HPLC and home-made ELISA were 3.52% and 9.37 % respectively. These findings suggest that HPLC is superior to that of ELISA in terms of sensitivity and reproducibility. Urinary 8-OHdG levels determined by commercial ELISA kit was about thrice as much as values determined by HPLC, and was almost equal to the sum of 8-OHdG and 8-OHG determined by HPLC method, suggesting that the current commercial kit can detect both molecule species. The filter based HPLC method is adopted as standard method in our laboratory. We also tested its clinical applicability using samples from patients of degenerative diseases. As reported previously, diabetic patients had higher urinary 8-OHdG levels than controls (1.63 ± 0.66 vs. 1.13 ± 0.58 nmol/mmol creatinine respectively, p < 0.01). These findings suggest that with the filter based immunoaffinity purification pretreatment step, HPLC can accurate determine urinary 8-OHdG, which appears to be a good index for monitoring degenerative diseases such as diabetes.