人類富含血小板血漿中抗壞血酸自由基生成機轉探討

Mechanisms of Ascorbyl Free Radical Formation in Human Platelet-Rich Plasma

葉宇捷(Yu-Chieh Yeh)

指導教授 ： 周敦穗

共同指導教授 ： 許準榕

臺北醫學大學/醫學院/醫學科學研究所/碩士(2013年)

摘要

抗壞血酸是體內還原力最強的抗氧化物，存在生物體內的抗壞血酸會去還原大部分的活性分子，進而轉變成抗壞血酸自由基(ascorbyl free radical；AFR)，由於抗壞血酸自由基十分穩定且對於氧化物有高度敏感性，因此常被當作生物內生性總氧化程度的指標。在先前研究指出，電子自旋共振儀(electron spin resonance；ESR)可以直接且即時的去偵測血漿中抗壞血酸自由基的濃度，藉此來評估血液中的氧化壓力程度。近來有許多臨床的文獻指出，在人類血液中抗壞血酸自由基可當作是一個非侵入性，可靠且即時的氧化壓力指標，但目前對於人類血液中抗壞血酸自由基生成機轉探討的文獻卻很少。
本研究主題即運用電子自旋共振儀直接偵測自由基的能力，並分別透過加入上游來源物質，酵素抑制劑及自由基清除劑等方式，藉此推論在人類血液中抗壞血酸自由基的生成路徑。由實驗結果得知，在人類富含血小板血漿(human platelet-rich plasma；PRP)的ESR圖譜中，偵測到一個明顯的雙峰自由基訊號，此訊號再經由WIN-EPR分析軟體進行超精細分裂分析。由以上結果得知，此自由基訊號的氫原子分裂係數(aH )為1.88 gauss，絕對位置為g-factor : 2.00627，此訊號會隨著外加抗壞血酸的濃度升高而增大。因此，証實所偵測到的自由基訊號為抗壞血酸自由基。在加入ROS清除劑的實驗中，發現不論是將superoxide或者是nitric oxide清除，都會使抗壞血酸自由基的訊號降低，因此初步判定，超氧自由基和一氧化氮可能是形成抗壞血酸自由基的上游來源。在外加花生四烯酸(arachidonic acid；AA)以及phospholipase A2 (PLA2) 抑制劑的實驗當中，也發現外加AA會使抗壞血酸的訊號上升，而在加入PLA2抑制劑後會使抗壞血酸的訊號下降，由結果顯示AA可能是抗壞血酸自由基生成的上游物質之一。在酵素抑制劑的實驗中，發現在各別加入NADPH oxidase (NOX)，cyclooxygenase (COX)，lipoxygenase (LOX)，cytochrome P450 (CYP450)，mitochondria complex III，nitric oxide synthase (NOS)的抑制劑後，也都會降低抗壞血酸自由基的生成，只有xanthine oxidase (XO)抑制劑不影響抗壞血酸的訊號。最後，在Fenton-reaction試驗中，證實全部的酵素抑制劑都沒有明顯的抗氧化能力。綜合上訴實驗結果，証實在human PRP中抗壞血酸自由基的生成路徑中，A A可能是產生抗壞血酸自由基的上游物質，並且抗壞血酸自由基可能是透過NOX，LOX，COX，CYP450和mitochondria產生的superoxide以及NOS產生的nitric oxide反應生成的。

關鍵字

富含血小板血漿,抗壞血酸自由基

並列摘要

Ascorbic acid is a strong reducing agent in vivo and ascorbyl free radical is produced via ascorbate oxidation which may be performed by almost all reactive species intrinsic to the biological milieu. This makes the level of ascorbyl free radical highly sensitive to oxidative status. In addition, ascorbyl free radical is relatively stable and these characteristics make it the most exploited endogenous marker of oxidative status. In previous study, electron spin resonance (ESR) spectrometer was used to detect the concentration of ascorbyl free radical in plasma directly and immediately, thereby to assess the level of oxidative stress in the blood. Recently, many clinical articles report that ascorbyl free radical can be treated as a noninvasive, reliable and real-time maker of oxidative stress, but the generate mechanisms of ascorbyl free radical in human blood has rarely been discussed. In this study, we used upstream substances, enzyme inhibitors and free radical scavengers to check the mechanisms of ascorbyl free radical formation in human platelet-rich plasma. Our results show that the doublet signal was detected in PRP samples by ESR and the hyperfine splitting of doublet signal was analyzed by WIN-EPR analysis software (aH =1.88 gauss and g-factor = 2.00627). This doublet signal intensity increases as the concentration of exogenous ascorbate increases confirming that the doublet signal is ascorbate free radical. The ascorbyl free radical intensity increased in the presence of ROS scavenger such as superoxide and nitric oxide scavengers. This indicated that superoxide and nitric oxide maybe an upstream radical in ascorbyl free radical formation in the PRP. In the experiments of adding exogenous AA and PLA2 inhibitor, the ascorbyl free radical signal intensity increased when the concentration of AA increased and the signal intensity decreased when the concentration of PLA2 decreased. This indicated that AA maybe an upstream substance in ascorbyl free radical formation in the PRP. In the studies of inhibition formation of ascorbyl free radical, the ascorbyl free radical signal intensity decreased as the concentration of NADPH oxidase (NOX), cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 (CYP450), mitochondria complex III, nitric oxide synthase (NOS) but not xanthine oxidase (XO) inhibitors increased. Finally, all enzyme inhibitors showed no obvious antioxidant activity during Fenton-reaction assay. To sum up, the present data suggest that ascorbyl free radical formation is associated with AA, NOX, LOX, COX, CYP450 and mitochondria in human PRP.

並列關鍵字

platelet-rich plasma, ascorbyl free radical

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