Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis

Krisztian Stadler*, Marcelo G. Bonini, Shannon Dallas, Danielle Duma, Ronald P. Mason, Maria B. Kadiiska

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

33 Scopus citations


Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the α-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression.

Original languageEnglish (US)
Pages (from-to)E456-E462
JournalAmerican Journal of Physiology - Endocrinology and Metabolism
Issue number2
StatePublished - Aug 2008
Externally publishedYes


  • Acetone
  • Electron paramagnetic resonance
  • Free radicals
  • Inducible nitric oxide synthase

ASJC Scopus subject areas

  • Physiology (medical)
  • Physiology
  • Endocrinology, Diabetes and Metabolism


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