METHOD FOR DETERMINATION OF FREE INTRACELLULAR AND EXTRACELLULAR METHYLGLYOXAL IN ANIMAL-CELLS GROWN IN CULTURE

Methylglyoxal is present at low levels in most cells as a by-product o f glycolysis and a product of lipid and amino acid catabolism, The mos t 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 resultin g 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 c ulture, Cell harvest and sample volume measurement techniques were dev eloped, Solid-phase extraction prior to methylglyoxal derivatization r educed interferences unique to cell culture, such as the phenol red in dicator dye used in most cell culture media, and extended the useful l ife of the HPLC column, In addition, this extraction step significantl y lessened the interference represented by oxidative degradation of nu cleic acids to methylglyoxal by perchloric acid under assay conditions , The concentration of free intracellular methylglyoxal in Chinese ham ster 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 t he cell, A possible explanation for the difference between measured fr ee intracellular and extracellular methylglyoxal levels is that the as say for free intracellular methylglyoxal also measures some reversibly bound methylglyoxal. (C) 1996 Academic Press, Inc.