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. 2015:4:136-48.
doi: 10.1016/j.redox.2014.12.016. Epub 2014 Dec 24.

"Twin peaks": searching for 4-hydroxynonenal urinary metabolites after oral administration in rats

Julia Keller  1 , Maryse Baradat  1 , Isabelle Jouanin  2 , Laurent Debrauwer  2 , Françoise Guéraud  3
Affiliations

"Twin peaks": searching for 4-hydroxynonenal urinary metabolites after oral administration in rats

Julia Keller et al. Redox Biol. 2015.

Abstract

4-Hydroxynonenal (HNE) is a cytotoxic and genotoxic lipid oxidation secondary product which is formed endogenously upon peroxidation of cellular n-6 fatty acids. However, it can also be formed in food or during digestion, upon peroxidation of dietary lipids. Several studies have evidenced that we are exposed through food to significant concentrations of HNE that could pose a toxicological concern. It is then of importance to known how HNE is metabolized after oral administration. Although its metabolism has been studied after intravenous administration in order to mimick endogenous formation, its in vivo fate after oral administration had never been studied. In order to identify and quantify urinary HNE metabolites after oral administration in rats, radioactive and stable isotopes of HNE were used and urine was analyzed by radio-chromatography (radio-HPLC) and chromatography coupled with High Resolution Mass Spectrometry (HPLC-HRMS). Radioactivity distribution revealed that 48% of the administered radioactivity was excreted into urine and 15% into feces after 24h, while 3% were measured in intestinal contents and 2% in major organs, mostly in the liver. Urinary radio-HPLC profiles revealed 22 major peaks accounting for 88% of the urinary radioactivity. For identification purpose, HNE and its stable isotope [1,2-(13)C]-HNE were given at equimolar dose to be able to univocally identify HNE metabolites by tracking twin peaks on HPLC-HRMS spectra. The major peak was identified as 9-hydroxy-nonenoic acid (27% of the urinary radioactivity) followed by classical HNE mercapturic acid derivatives (the mercapturic acid conjugate of di-hydroxynonane (DHN-MA), the mercapturic acid conjugate of 4-hydroxynonenoic acid (HNA-MA) in its opened and lactone form) and by metabolites that are oxidized in the terminal position. New urinary metabolites as thiomethyl and glucuronide conjugates were also evidenced. Some analyses were also performed on feces and gastro-intestinal contents, revealing the presence of tritiated water that could originate from beta-oxidation reactions.

Keywords: 4-Hydroxynonenal; HRMS; Isotope tracking; Lipoperoxidation; Metabolism.

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Figures

None
Graphical abstract
Fig. 1
Fig. 1
Labeling position of the HNE isotopes used for metabolite tracking and quantification.
Fig. 2
Fig. 2
Workflow for metabolites identification and quantification.
Fig. 3
Fig. 3
Work flow of the 2nd set of experiments. Metabolites were identified using radio-HPLC separation, ‘twin peaks’ tracking by High Resolution Mass Spectrometry and further ion fragmentation by MS/MS.
Fig. 4
Fig. 4
Radioactivity distribution after 3H-HNE oral administration in rats (n=3). Percentage of the given radioactivity found in 0–24 h urine and feces (A), intestinal contents and tissues after 24 h (B) (volume or weight related).
Fig. 5
Fig. 5
Radio-HPLC profiles of 3H-HNE urinary metabolites after oral administration in rats. A: 0–24 h urine. B: 0–6 h urine. C: 6–24 h urine.
Fig. 6
Fig. 6
General fragmentation pattern and fragment ion notation of mercapturic acid derivatives.
Fig. 7
Fig. 7
Fragmentation mass spectra and interpretation for ions 187.0977 (12C-HNE metabolite) and 189.1044 (13C-HNE metabolite).
Fig. 8
Fig. 8
Fragmentation mass spectra and interpretation for ions 189.1132 (12C-HNE metabolite) and 191.1198 (13C-HNE metabolite).
Fig. 9
Fig. 9
Cecal radio-HPLC profile at 24 h (A) and fecal 0–24 h radio-HPLC profile (B) of rats treated with HNE.
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