Effects of Adding Antioxidants to CoQ-Deficient Yeast Cells Studied by in Vivo Confocal Raman Microspectroscopy
|關鍵字:||拉曼顯微光譜;抗氧化劑;裂殖酵母;Raman Microspectroscopy;antioxidant;fission yeast|
|摘要:||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.
|Appears in Collections:||Thesis|
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