Chaplen F W, Fahl W E, Cameron D C
Department of Chemical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, 53706-1691, USA.
Anal Biochem. 1996 Jul 1;238(2):171-8. doi: 10.1006/abio.1996.0271.
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 high-performance 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 microM (mean +/- 2 standard deviations; n = 4) to 1.2 +/- 0.3 microM (mean +/- 2 standard deviations; n = 7). The concentration of free extracellular methylglyoxal in the growth medium was 0.07 +/- 0.02 microM (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.
甲基乙二醛在大多数细胞中以糖酵解副产物以及脂质和氨基酸分解代谢产物的形式少量存在。测量甲基乙二醛最广泛接受的方法涉及用1,2 - 二氨基苯衍生物（如邻苯二胺）对甲基乙二醛进行衍生化，然后用高效液相色谱法（HPLC）对生成的喹喔啉进行定量。在此，我们描述了对该方法的改进，用于测量培养的动物细胞中细胞内和细胞外游离甲基乙二醛。开发了细胞收获和样品体积测量技术。在甲基乙二醛衍生化之前进行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。此外，该萃取步骤显著减少了在测定条件下高氯酸将核酸氧化降解为甲基乙二醛所代表的干扰。培养的中国仓鼠卵巢（CHO）细胞中细胞内游离甲基乙二醛的浓度范围为0.7±0.3微摩尔（平均值±2个标准差；n = 4）至1.2±0.3微摩尔（平均值±2个标准差；n = 7）。生长培养基中细胞外游离甲基乙二醛的浓度为0.07±0.02微摩尔（平均值±2个标准差；n = 4），比细胞内发现的浓度低几倍。细胞内和细胞外游离甲基乙二醛测量水平差异的一个可能解释是，细胞内游离甲基乙二醛的测定还测量了一些可逆结合的甲基乙二醛。
