TitleMethod for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture.
Publication TypeJournal Article
Year of Publication1996
AuthorsChaplen, FW, Fahl, WE, Cameron, DC
JournalAnal Biochem
Volume238
Issue2
Pagination171-8
Date Published1996 Jul 1
ISSN0003-2697
KeywordsAnimals, Cells, Cultured, CHO Cells, Chromatography, High Pressure Liquid, Cricetinae, Culture Media, Pyruvaldehyde
Abstract

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.

DOI10.1006/abio.1996.0271
Alternate JournalAnal. Biochem.
PubMed ID8660607
Grant ListP01-CA-22484 / CA / NCI NIH HHS / United States
T32 GM08349-03 / GM / NIGMS NIH HHS / United States