Potential urinary and plasma biomarkers of peroxisome proliferation in the rat: identification of N-methylnicotinamide and N-methyl-4-pyridone-3-carboxamide by ¹H nuclear magnetic resonance and high performance liquid chromatography

Abstract

This study identified two potential novel biomarkers of peroxisome proliferation in the rat. Three peroxisome proliferator-activated receptor (PPAR) ligands, chosen for their high selectivity towards the PPARα, -δ and -γ subtypes, were given to rats twice daily for 7 days at doses known to cause a pharmacological effect or peroxisome proliferation. Fenofibrate was used as a positive control. Daily treatment with the PPARα and -δ agonists produced peroxisome proliferation and liver hypertrophy. ¹H nuclear magnetic resonance spectroscopy and multivariate statistical data analysis of urinary spectra from animals given the PPARα and -δ agonists identified two new potential biomarkers of peroxisome proliferation - N-methylnicotinamide (NMN) and N-methyl-4-pyridone-3-carboxamide (4PY) - both endproducts of the tryptophan-nicotinamide adenine dinucleotide (NAD⁺) pathway. After 7 days, excretion of NMN and 4PY increased 24- and three-fold, respectively, following high doses of fenofibrate. The correlation between total NMN excretion over 7 days and the peroxisome count was r=0.87 (r²=0.76). Plasma NMN, measured using a sensitive high performance liquid chromatography method, was increased up to 61-fold after 7 days' treatment with high doses of fenofibrate. Hepatic gene expression of aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) was downregulated following treatment with the PPARα and -δ agonists. The decrease was up to 11-fold compared with controls in the groups treated with high doses of fenofibrate. This supports the link between increased NMN and 4PY excretion and regulation of the tryptophan-NAD⁺ pathway in the liver. In conclusion, NMN, and possibly other metabolites in the pathway, are potential non-invasive surrogate biomarkers of peroxisome proliferation in the rat.

Keywords::

• Biomarkers
• N-methylnicotinamide
• N-methyl-4-pyridone-3-carboxamide
• Biofluid Nuclear Magnetic Resonance Spectroscopy
• Metabonomics
• Multivariate Data Analysis
• Rat Plasma
• Rat Urine
• Peroxisome Proliferator-activated Receptor
• Peroxisome Proliferation
• Aminocarboxymuconate-semialdehyde Decarboxylase
• Quinolinate Phosphoribosyltransferase
• Mrna Levels
• Tryptophan-nicotinamide Adenine Dinucleotide Pathway