Metabolomic and genetic analysis of biomarkers for peroxisome proliferator-activated receptor α expression and activation

Peroxisome proliferator-activated receptor alpha ( PPAR alpha) is a nuclear receptor with manifold effects on intermediary metabolism. To define a set of urinary biomarkers that could be used to determine the efficacy of PPAR alpha agonists, a metabolomic investigation was undertaken in wild-type and Ppar alpha- null mice fed for 2 wk either a regular diet or a diet containing the PPAR alpha ligand Wy- 14,643 ([ 4chloro- 6-( 2,3- xylidino)- 2- pyrimidinylthio] acetic acid), and their urine was analyzed by ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry. Principal components analysis of 6393 accurate mass positive ions revealed clustering as a single phenotype of the treated and untreated Ppar alpha (-/-) mice plus two additional discrete phenotypes for the treated and untreated Ppar alpha(+/+) mice. Biomarkers of PPAR alpha activation were identified from their accurate masses and confirmed by tandem mass spectrometry of authentic compounds. Biomarkers were quantitated from raw chromatographic data using appropriate calibration curves. PPAR alpha urinary biomarkers highly statistically significantly elevated by Wy- 14,643 treatment included 11 beta- hydroxy- 3,20dioxopregn-4-en-21-oic acid (> 3700- fold), 11 beta, 20-dihydroxy- 3- oxopregn-4-en-21-oic acid ( 50-fold), nicotinamide (> 2- fold), nicotinamide 1-oxide ( 5-fold), 1-methylnicotinamide ( 1.5-fold), hippuric acid ( 2- fold), and 2,8-dihydroxyquinoline-beta-D-glucuronide ( 3- fold). PPAR alpha urinary biomarkers highly statistically significantly attenuated by Wy- 14,643 treatment included xanthurenic acid ( 1.3-fold), hexanoylglycine ( 20-fold), phenylpropionylglycine ( 4-fold), and cinnamoylglycine ( 9-fold). These biomarkers arise from PPAR alpha effects on tryptophan, corticosterone, and fatty acid metabolism and on glucuronidation. This study underscores the power of mass spectrometry-based metabolomics combined with genetically modified mice in the definition of monogenic metabolic phenotypes.