Kriska Tamas, Girotti Albert W
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Nov 15;827(1):58-64. doi: 10.1016/j.jchromb.2005.03.045.
Thiol-dependent peroxidases catalyzing the reductive detoxification of lipid hydroperoxides (LOOHs) are crucial antioxidant components of mammalian cells. There is a growing interest in manipulating expression of such enzymes to better understand their biological roles. A new approach for determining their cellular activity is described, whereby LOOH reduction kinetics are tracked by high performance thin layer chromatography with peroxide-sensitive tetramethyl-p-phenylenediamine detection (HPTLC-TPD). The approach was tested on a tumor cell transfectant clone (7G4) over-expressing selenoperoxidase GP x 4. Timed incubation of Triton-solubilized 7G4 cells with GSH and peroxidized phosphatidylcholine (PCOOH), followed by lipid extraction, HPTLC-TPD and densitometry revealed an exponential decay of PCOOH at a rate approximately 80-times greater than that for GP x 4-deficient controls (VC). A TPD-detectable cholesterol hydroperoxide (7alpha-OOH) was also reduced much faster by 7G4 than VC extracts. Spraying with H(2)SO(4) after TPD revealed both 7alpha-OOH loss and resolved diol product (7alpha-OH) accumulation, the kinetics of which were identical. The approach described is relatively convenient, highly specific, and much more sensitive than conventional assays for cellular LOOH reducing enzymes.
催化脂质氢过氧化物（LOOHs）还原解毒的硫醇依赖性过氧化物酶是哺乳动物细胞重要的抗氧化成分。人们对操纵这类酶的表达以更好地理解其生物学作用的兴趣日益浓厚。本文描述了一种测定其细胞活性的新方法，即通过对过氧化物敏感的四甲基对苯二胺检测的高效薄层色谱法（HPTLC - TPD）跟踪LOOH的还原动力学。该方法在过表达硒过氧化物酶GP x 4的肿瘤细胞转染克隆（7G4）上进行了测试。将用Triton溶解的7G4细胞与谷胱甘肽（GSH）和过氧化磷脂酰胆碱（PCOOH）进行定时孵育，随后进行脂质提取、HPTLC - TPD和光密度测定，结果显示PCOOH呈指数衰减，其速率比缺乏GP x 4的对照（VC）快约80倍。7G4对TPD可检测的胆固醇氢过氧化物（7α - OOH）的还原速度也比VC提取物快得多。TPD后用H₂SO₄喷雾显示7α - OOH损失和分解的二醇产物（7α - OH）积累，其动力学相同。所描述的方法相对简便、特异性高，并且比传统的细胞LOOH还原酶检测方法灵敏得多。
