Cellular oxidant stress and advanced glycation endproducts of albumin: Caveats of the dichlorofluorescein assay

Ram Subramaniam, Xingjun Fan, Vincenzo Scivittaro, Jianqi Yang, Chung Eun Ha, Charles E. Petersen, Witold K. Surewicz, Nadhipuram V. Bhagavan, Miriam F. Weiss, Vincent M. Monnier

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

In order to understand the mechanism by which advanced glycation endproducts (AGEs) elicit oxidative stress, macrophage-like RAW264.7 cells were exposed to various AGE-albumins, and oxidant stress was estimated from the fluorescence of oxidized dichlorofluorescein using the microtiter plate assay. Strongest fluorescence was observed with methylglyoxal modified albumin (MGO-BSA) compared with native albumin. Similar effects that were prevented by arginine coincubation were seen with phenylglyoxal-BSA. MGO-BSA had increased affinity for Cu2+ and Ca2+, but was conformationally similar to native albumin. Surprisingly, the mere addition of unmodified albumin to cells suppressed the fluorescence of oxidized DCF. While, several site-directed mutants of human serum albumin (BSA), including C34S and recombinant domains II and III retained fluorescence suppressing activity, proteolytic digests, recombinant domain I, and several nonalbumin proteins failed to suppress. Kinetic and ANS binding studies suggested albumin quenches DCF fluorescence by binding to hydrophobic pockets in domains II and III and that MGO-BSA is less hydrophobic than BSA. Finally, BSA also prevented H2O2 catalyzed DCF fluorescence more potently than MGO-BSA. These studies reveal important caveats of the widely used dichlorofluorescein assay and suggest methods other than the microtiter plate assay are needed to accurately assess cellular oxidant stress in presence of native or modified albumin.

Original languageEnglish (US)
Article number92776
Pages (from-to)15-25
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume400
Issue number1
DOIs
StatePublished - Apr 1 2002

Keywords

  • Albumin
  • Dichlorofluorescein
  • Glycation
  • Methylglyoxal
  • Oxidative stress

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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    Subramaniam, R., Fan, X., Scivittaro, V., Yang, J., Ha, C. E., Petersen, C. E., Surewicz, W. K., Bhagavan, N. V., Weiss, M. F., & Monnier, V. M. (2002). Cellular oxidant stress and advanced glycation endproducts of albumin: Caveats of the dichlorofluorescein assay. Archives of Biochemistry and Biophysics, 400(1), 15-25. [92776]. https://doi.org/10.1006/abbi.2002.2776