Speaker: Sandford P. Markey, Laboratory of Neurotoxicology, National Institute of Mental Health, Bethesda, MD

Topic: Tryptophan Metabolism: New Insights from Mass Spectrometric Studies

Place: Building 426, Conference Room, NCI-Frederick, Frederick, MD

Time: Thursday, September 16, 1999, at 1:30 PM

Abstract: Tryptophan is a well studied essential amino acid whose metabolism is significantly altered by immune mediated events. Mass spectrometric studies were directed first to understanding the neurotransmitter metabolites (quinolinic and kynurenic acids) in studies by Dr. M. P. Heyes in this laboratory. The development of quantitative mass spectrometric assays for these metabolites presented interesting challenges in separation and derivatization chemistry, and the opportunity to learn about fundamental pathophysiological processes in inflammatory diseases in the central nervous system. Quinolinate, an excitotoxic amino acid, is elevated markedly in the CNS following trauma or inflammation producing infiltration of peripheral blood macrophages or activation of resident microglia. Stable isotope labelling and GC/MS studies demonstrate that human macrophages and microglia metabolize tryptophan preferentially through the kynurenine pathway. Kinetic studies of quinolinate distribution show that elevated levels of CNS quinolinate result from quinolinate formed in the CNS, not from peripheral sources. Labeling studies are also in progress to define the contribution of peripherally formed kynurenine to CNS metabolism.

Tryptophan can be a metabolic precursor of NAD (nicotinamide adenine dinucleotide) via the kynurenine pathway. Studies to trace ¹³C or ²H-labelled tryptophan to NAD require LC/MS methods for the measurement of labelled NAD. Reverse phase HPLC conditions were not successful in analyzing NAD using ESI/MS in perchlorate extracts of tissue. Hydrophilic interaction chromatography is shown to provide useful separation of perchloric acid extracts of cultured cells, permitting quantification of labeled NAD (¹³C₅) formed from ¹³C₆-tryptophan. Yeast grown on ¹³C, ¹⁵N enriched culture medium provided an NAD internal standard containing ¹³C₂₁, ¹⁵N₇. Hydrophilic interaction chromatography is a useful separation tool enabling mass spectrometric analyses of polar organic compounds in solvents compatible with ESI.