第一部份: 本實驗主要是研究臺灣地區G6PD缺乏的病人紅血球內carbonic anhydrase isoenzyme變化的情形。carbonic anhydrase(CA)是一種含鋅離子的金屬酵素，為紅血球中除了血紅素外主要的蛋白，在紅血球中扮演著Cl-/HCO3-轉換代謝的重要角色，已證實會與band 3蛋白結合。我們使用12.5 % SDS PAGE (0.8 % bisacrylamide)電泳分析，並以專一的carbonic anhydrase II抗體進行西方墨點法分析正常個人(n=30)及G6PD缺乏病人(n=30)紅血球內CAI及CAII的蛋白變化。結果顯示G6PD缺乏病人(n=30)紅血球中CA II明顯上升。所得數值以student’s test及stepwise regression進行分析後發現在G6PD缺乏病人組別中CA I / CA II的平均比值為2.3，明顯低於正常個體的平均比值5.2 (p<0.05)。正常個體樣本中band 3的蛋白表現明顯高於G6PD缺乏病人。由結果我們得知當G6PD缺乏可能會影響CA II及 band 3等蛋白的基因表現或蛋白穩定度，當有氧化性傷害出現更加強改變spectrin與cytoskeleton的結合，使cytoskeleton自spectrin蛋白中流失，導致band 3蛋白減少及CA II蛋白合成也會跟著上升，造成溶血之臨床症狀，而其詳細機轉則需深入探討。 第二部份: 本研究使用商業化試劑及Beckman Synchrom CX-5自動化分析系統，提供快速準確的分析方法評估血液中superoxide dismutase (SOD)、 total antioxidant status (TAS)和glutathione peroxidase (GSHPx)等活性分析。以本方法分析SOD、TAS和GSHPx所得再現性非常好，CVs小於6。我們收集了188個健康個體的樣本(90個女性及98個男性)進行分析。發現女性的SOD與GSHPx (SOD:1082±261 unit/g Hb; GSH-Px: 91±16 unit/g Hb)較男性高(SOD: 989±196 unit/g Hb; GSH-Px: 79±11unit/gHb)。令人感興趣的是在以上樣本中具有飲酒及機車作為交通工具者，其血中GSHPx的活性較對照組低(P < 0.05)。而在分析抽煙及不抽煙樣本中， SOD、TAS和GSHPx的數據並沒有發現明顯的差異。 Part 1: The aim of the present study was to determine the level of eryth-rocyte carbonic anhydrase isoenzyme of G6PD deficiency patient in Taiwan . Carbonic anhydrase (CA) is a zinc-containing enzyme, it reversibly catalyzes the hydration of carbon dioxide to bicarbonate and hydrogen ions. CA II is believed to be a chloride/bicarbonate exchanger in red blood cells, and also directly coupled to band 3 protein. Human erythrocyte CA I and CAII were measured in normal subjects (n=30) and G6PD deficiency patients (n=30), by using Western blot assay (with 12.5 % SDS-PAGE- 0.8 % bisacrylamide). In the groups of G6PD deficiency patients, there was a tendency for CA I / CA II ratio (mean= 2.3) to be lower than normal subjects (mean= 5.2)(P<0.001). The band 3 protein were resol-ved by 8 % SDS-PAGE, normal subjects was also significantly higher than the G6PD deficiency patients. We may speculate that in G6PD deficiency might be to a change in CA II and band 3 protein expression and stability. The cytoskeletal proteins which bind spectrin, and that this change makes the affected protein more susceptible to oxidative damage, band 3 protein loss and increased levels of CAII in G6PD deficiency patients may lead to hemolytic anemia. Detailed mechanism is unclear. Part 2: A rapid automated system for assessing the activities of superoxide dismutase (SOD), total antioxidant status (TAS), and glutathione peroxidase (GSHPx) in blood was developed using a set of commercial kits and the Beckman Synchrom CX-5 automatic analyser. Reproducibility data for assaying SOD, GSHPx, and TAS by our proposed procedure were excellent (CVs < 6.0 %). We then recruited a total of 188 apparently healthy individuals and their antioxidative status were assessed by our proposed method. We found that female (n=90) had a significant higher SOD (1082±261 unit/g Hb) and GSH-Px (91±16 unit/g Hb) than its male counterparts (n=98)[ SOD: 989±196 unit/g Hb; GSH-Px: 79±11 unit/g Hb] (P < 0.01). GSH-Px (R = 0.26) and TAS (R = -0.38) was correlated with age. Interestingly, we also found that alcoholics and motor cyclists seemed to have lower GSH-Px (P < 0.05). In contrast, we did not find any significant discrepancy in SOD, GSH-Px and TAS between smokers and non- smokers.
