John Knight1, Ross P Holmes. 1. Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. jknight@wfubmc.edu
Abstract
BACKGROUND/AIMS: Primary hyperoxaluria results from an alteration in enzymes that metabolize glyoxylate. The metabolism that leads to glyoxylate synthesis is not well defined. The aim of this study was to investigate the production of glyoxylate in liver mitochondria when they metabolize hydroxyproline. METHODS: Mitochondria were isolated from mouse liver using Percoll gradient centrifugation. The metabolism of hydroxyproline was examined by a combination of HPLC and ion chromatography/mass spectrometry techniques. RESULTS: Glyoxylate production was substantially greater when mitochondria were incubated with hydroxyproline in comparison with proline. Inclusion of malate and glutamate with hydroxyproline resulted in a drop in glyoxylate and an increase in glycolate in the incubation mixture. This suggests an increased NAD(P)+ reduction which occurred with the inclusion of glutamate/malate and that the NAD(P)H production was required to stimulate the glyoxylate reductase-catalyzed conversion of glyoxylate to glycolate. The presence of glyoxylate reductase in these mitochondria was confirmed by measuring enzymatic activity and by Western blotting. CONCLUSION: These results indicate that studies on isolated mitochondria have the potential to help unravel the metabolism associated with glyoxylate and oxalate production and understand the metabolic function of glyoxylate reductase. 2005 S. Karger AG, Basel
BACKGROUND/AIMS: Primary hyperoxaluria results from an alteration in enzymes that metabolize glyoxylate. The metabolism that leads to glyoxylate synthesis is not well defined. The aim of this study was to investigate the production of glyoxylate in liver mitochondria when they metabolize hydroxyproline. METHODS: Mitochondria were isolated from mouse liver using Percoll gradient centrifugation. The metabolism of hydroxyproline was examined by a combination of HPLC and ion chromatography/mass spectrometry techniques. RESULTS:Glyoxylate production was substantially greater when mitochondria were incubated with hydroxyproline in comparison with proline. Inclusion of malate and glutamate with hydroxyproline resulted in a drop in glyoxylate and an increase in glycolate in the incubation mixture. This suggests an increased NAD(P)+ reduction which occurred with the inclusion of glutamate/malate and that the NAD(P)H production was required to stimulate the glyoxylate reductase-catalyzed conversion of glyoxylate to glycolate. The presence of glyoxylate reductase in these mitochondria was confirmed by measuring enzymatic activity and by Western blotting. CONCLUSION: These results indicate that studies on isolated mitochondria have the potential to help unravel the metabolism associated with glyoxylate and oxalate production and understand the metabolic function of glyoxylate reductase. 2005 S. Karger AG, Basel
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