1.Simultaneous Quantification of Etheno Adducts in human urine and DNA samples by Stable Isotope Dilution Capillary Liquid Chromatography/Nanospray Ionization-Tandem Mass Spectrometry 2.Post-translational modifications on human hemoglobin by glyoxal and

• Main Authors: Chia-yen Wu, 吳佳燕
• Other Authors: Hauh-Jyun Candy Chen
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/39716950631837908475

碩士 === 國立中正大學 === 化學所 === 96 === Etheno DNA adducts are promutagenic DNA lesions derived from exogenous as well as endogenous sources. The levels of etheno adducts in tissue DNA are elevated in cancer-prone tissues, and the urinary excretion of etheno adducts is associated with oxidative stress. In this report, we have developed an assay for the accurate quantification of etheno DNA adducts by isotope dilution capillary liquid chromatography/nanospray ionization-tandem mass spectrometry (capillary LC-NSI/MS/MS) in human urine and tissue samples. These promutagenic etheno DNA adducts are excised by base excision repair and possibly the nucleotide excision repair mechanisms and they are excreted into urine as adducted bases and the nucleosides. The etheno DNA adducts investigated in this study include 1,N6-ethenoadenosine(eAde),3,N4-ethenocytidine(eCyt),1,N6-etheno-2?-deoxyadenosine(edAdo),3,N4-etheno-2?-deoxycytosine(edCyt),and1,N2-etheno-2?-deoxyguanosine(edGuo). Sample purification before analysis by MS only requires a reversed phase solid phase extraction column. The detection limit (LOD) of eAde, eCyt, edAdo, edCyt and edGuo injected on-column using this capillary LC-NSI/MS/MS is 300 , 120, 1.8, 20, and 86 amol, respectively. Levels of eAde, eCyt and edAdo in 12 human urine are 88 ± 48,109 ± 96 and 4.4 ± 3.0 pg/mL. The origin of DNA are calf thymus, human placental and human white blood cell DNA. Levels of these etheno adducts in calf thymus DNA and human placental DNA were compared as nucleosides using six different enzyme hydrolysis procedures. In human placental DNA, the levels of edAdo, edCyd and edGuo was 6.45 ± 0.46, 27.8 ± 0.27 and 7.76 ± 0.64 adducts per 107 parent nucleoside, respectively. But in some calf thymus and WBC DNA samples, the adduct levels were below the detection limits. Larger quantities of DNA is needed for simultaneous quantification of low levels of these three etheno adducts. The non-enzymatic conjugated addition product of glucose or aldehyde derivatives and glycation reaction of protein are the main cause of vascular complications of diabetes. When the concentration of glucose remains at high level in the body, the amounts of ?dicarbonyl compounds, such as glyoxal and methylglyoxal, also increase. Glyoxal is derived from glucose and amino acid oxidation as well as lipid peroxidation; whereas methylglyoxal is mainly from self-decomposition of glyceraldehyde-3-phospate (G-3-P), a degradation intermediate product. This research makes use of liquid chromatography nanospray ionization tandem mass spectrometry to investigate sites of reaction sites on human hemoglobin with glyoxal and methylglyoxal. We found that there was a single glyoxal addition at Lys-11, Lys-139, Arg-92, His-20, His-45, His-50 on ?globin and at Lys-144, His-77, His-92, His-143, Cys-93 and Cys-112 on β-globin, when the concentration of glyoxal was 20 mM. When 0.5 mM of glyoxal and methylglyoxal react with hemoglobin under physiological conditions (pH7.4, 37℃) for 48 hr, we also found that there was a single glyoxal addition at Lys-11, His-20, His-45, His-50, Arg-92 and Lys-139 on ?globin and at His-77, His-92, Cys-93, Cys-112, His-143 and Lys-144 on β-globin. For methylglyoxal, a sigle addition was found at His-20 and Arg-92 on ?globin and at Cys-93, Lys-144 on β-globin. We have also confirmed that glyoxal and methylglyoxal form hydroimidazolone at Arg-92 of ?globin. Finally, we used 0.5 μM of glyoxal and methylglyoxal to react with hemoglobin under physiological conditions (pH7.4, 37℃) for 35 days, we also found a single glyoxal addition at Lys-11, His-20, His-45, His-50 on ?globin and at His-77, His-143, His-116, Cys-93 and Cys112on β-globin. For methylglyoxal addation, only addition product of Cys-93 on β-globin was found. In human blood hemoglobin, only glyoxal addation on β-globin was found.