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Free Radic Biol Med. 2014 May;70:78-85. doi: 10.1016/j.freeradbiomed.2014.02.008. Epub 2014 Feb 18.

Glutathionylated 4-hydroxy-2-(E)-alkenal enantiomers in rat organs and their contributions toward the disposal of 4-hydroxy-2-(E)-nonenal in rat liver.

Author information

1
Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
2
Department of Nutrition, Case Western Reserve University, Cleveland, OH 44106, USA.
3
Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address: tochtrop@case.edu.
4
Department of Nutrition, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address: gxz35@case.edu.

Abstract

The major route for elimination of 4-hydroxy-2-(E)-nonenal (4-HNE) has long been considered to be through glutathionylation and eventual excretion as a mercapturic acid conjugate. To better quantitate the glutathionylation process, we developed a sensitive LC-MS/MS method for the detection of glutathione (GSH) conjugates of 4-hydroxy-2-(E)-alkenal enantiomers having a carbon skeleton of C5 to C12. The newly developed method enabled us to quantify 4-hydroxy-2-(E)-alkenal-glutathione diastereomers in various organs, i.e., liver, heart, and brain. We identified the addition of iodoacetic acid as a critical step during sample preparation to avoid an overestimation of glutathione-alkenal conjugation. Specifically, we found that in the absence of a quenching step reduced GSH and 4-hydroxy-2-(E)-alkenals react very rapidly during the extraction and concentration steps of sample preparation. Rat liver perfused with d11-4-hydroxy-2-(E)-nonenal (d11-4-HNE) revealed enantioselective conjugation with GSH and transportation out of the liver. In the d11-4-HNE-perfused rat livers, the amount of d11-(S)-4-HNE-GSH released from the rat liver was higher than that of d11-(R)-4-HNE-GSH, and more d11-(R)-4-HNE-GSH than d11-(S)-4-HNE-GSH remained in the perfused liver tissues. Overall, the glutathionylation pathway was found to account for only 8.7% of the disposition of 4-HNE, whereas catabolism to acetyl-CoA, propionyl-CoA, and formate represented the major detoxification pathway.

KEYWORDS:

4-Hydroxy-2-(E)-alkenal; 4-hydroxy-2-(E)-nonenal; Free radicals; Glutathione conjugate; LC–MS/MS; Rat liver; Rat organs

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