Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture
Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture
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DOI：
10.1006/abio.1996.0271
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发表时间：
1996-07-01
影响因子：
2.9
通讯作者：
Cameron, DC
中科院分区：
文献类型：
--
作者：
Chaplen, FWR;Fahl, WE;Cameron, DC
甲基乙二醛是糖酵解的副产物，也是脂质和氨基酸分解代谢的产物，在大多数细胞中含量很低。测量甲基乙二醛的最广泛接受的方法是将甲基乙二醛与1,2-二氨基苯衍生物（如邻苯二胺）衍生化，然后用高效液相色谱（HPLC）定量得到喹诺啉。在这里，我们描述了在培养的动物细胞中测量游离的细胞内和细胞外甲基乙二醛的程序的修改，开发了细胞收获和样品体积测量技术，甲基乙二醛衍生化之前的固相萃取减少了细胞培养特有的干扰，例如大多数细胞培养基中使用的酚红指示染料，并延长了HPLC柱的使用寿命。实验条件下，该提取步骤显著降低了高氯酸氧化降解核酸对甲基乙二醛的干扰，培养的中国仓鼠卵巢（CHO）细胞内游离甲基乙二醛浓度范围为0.7 +/- 0.3 μ M（平均+/- 2个标准差，n = 4）至1.2 +/- 0.3 μ M（平均+/- 2个标准差，n = 4）；n = 7)，生长培养基中游离细胞外甲基乙二醛的浓度为0.07 +/- 0.02 μ M（平均+/- 2个标准差；n = 4），比细胞内的浓度低几倍。细胞内游离甲基乙二醛水平与细胞外游离甲基乙二醛水平差异的可能解释是，细胞内游离甲基乙二醛的测定也测量了一些可逆结合的甲基乙二醛。(C) 1996学术出版社，Inc.
Methylglyoxal is present at low levels in most cells as a by-product of glycolysis and a product of lipid and amino acid catabolism, The most widely accepted method for measurement of methylglyoxal involves the derivatization of methylglyoxal with 1,2-diaminobenzene derivatives, such as o-phenylenediamine, followed by quantification of the resulting quinoxaline with highperformance liquid chromatography (HPLC), Here we describe the modification of this procedure for the measurement of free intra- and extracellular methylglyoxal in animal cells grown in culture, Cell harvest and sample volume measurement techniques were developed, Solid-phase extraction prior to methylglyoxal derivatization reduced interferences unique to cell culture, such as the phenol red indicator dye used in most cell culture media, and extended the useful life of the HPLC column, In addition, this extraction step significantly lessened the interference represented by oxidative degradation of nucleic acids to methylglyoxal by perchloric acid under assay conditions, The concentration of free intracellular methylglyoxal in Chinese hamster ovary (CHO) cells grown in culture ranged from 0.7 +/- 0.3 mu M (mean +/- 2 standard deviations; n = 4) to 1.2 +/- 0.3 mu M (mean +/- 2 standard deviations; n = 7), The concentration of free extracellular methylglyoxal in the growth medium was 0.07 +/- 0.02 mu M (mean +/- 2 standard deviations; n = 4), severalfold less than that found inside the cell, A possible explanation for the difference between measured free intracellular and extracellular methylglyoxal levels is that the assay for free intracellular methylglyoxal also measures some reversibly bound methylglyoxal. (C) 1996 Academic Press, Inc.