利用共焦拉曼顯微光譜技術觀測在活體的缺乏輔酶Q酵母細胞添加抗氧化劑之影響

Abstract

我們利用共焦拉曼顯微技術去觀測缺乏輔酶Q10的活體酵母細胞在添加定量之抗氧化劑的影響。根據文獻，拉曼訊號1602 cm－1主要來自於酵母細胞中的麥角甾醇結構裡的共軛碳碳雙鍵。然而，生長環境中的活性氧會使得麥角甾醇轉換成過氧化麥角甾醇，因此，共軛碳碳雙鍵會被破壞，使得拉曼訊號1602 cm－1消失。缺乏輔酶Q10的酵母細胞本身的抗氧化能力相對弱於野生種，所以活性氧存在於環境中的濃度高於野生種，故缺乏輔酶Q10的活體酵母細胞的拉曼訊號1602 cm－1強度也弱於野生種。利用額外添加抗氧化劑去消除環境中的活性氧，使其訊號增加。我們分別測量添加各種抗氧化劑200隻細胞，其結果發現訊號增強大約2到3倍。經過時間推移實驗，發現添加抗氧化劑後10小時，訊號有顯著地增強。

In this study, we apply confocal Raman microspectroscopy to a strain of fission yeast that cannot produce CoQ10 due to a gene disruption (denoted ppt1) and quantitatively assess the effect of adding various antioxidants. According to the literature, the Raman band of yeast at 1602 cm−1 is contributed mainly from the conjugated C=C structure of ergosterol. The reactive oxygen species (ROS) present in yeast cells may oxidize ergosterol to form ergosterol peroxide, in which the conjugated C=C structure is destroyed, and consequently the 1602 cm−1 band may decrease. If exogenous antioxidative reagents are added to the ppt1 strain, we expect the Raman band at 1602 cm−1 to recover because ROS responsible for the loss of ergosterol can be detoxified by the antioxidant. We measured Raman spectra from 200 ppt1 cells with and without treatment of common antioxidants (lipoic acid, glutathione, ascorbic acid, and an inclusion complex of lipoic acid with -cyclodextrin) as well as 200 wild-type yeast cells. The band area ratio of the 1602 cm−1 band to the CH bending band at 1440 cm−1 was found to increase about 2–3 times when the antioxidant was added. Among the five antioxidants, glutathione showed the best performance of increasing the intensity of the 1602 cm−1 band. We also performed time-lapse experiments, in which the time dependence of the band intensity ratio (1602 vs. 1440) was monitored over 1 day. The results show that in all cases tested, the band intensity ratio markedly increases in 10 h after addition of the antioxidant. We demonstrate that Raman microspectroscopy in combination with the mutant yeast strains can be used as a novel quantitative tool for assessing the efficacy of various antioxidants in vivo.