Gas Chromatography of the Metabolites in the Tryptophan-Niacin Cycle

Altschuler, Henry; Gold, Martin

doi:10.1093/chromsci/9.6.377

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“…Subsequently, quinolinate may be quantified colorimetrically (McDaniel et al, 1972b) or by g.l.c. (Toseland, 1969;Altschuler & Gold, 1971). For isolation of quinolinic acid from hepatocyte incubations, we used a combination of ion-exchange and charcoal-adsorption methods; the latter step was included to avoid practical difficulties encountered in the freeze-drying of strongly acidic solutions.…”

Section: Quinolinic Acidformation By Liver Cellsmentioning

confidence: 99%

The metabolism of l-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism

Smith¹,

Carr²,

Pogson³

1980

Biochemical Journal

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1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-14C], L-[carboxy-'4C1-and L-[benzene-ring-U-'4C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10%o of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5mm-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5-6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-"4C]-and [benzene-ring-U-'4C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.In the mammal, four separate pathways exist for the metabolism of L-tryptophan (see Scheme 1). These are: (i) oxidation by tryptophan 2,3-dioxygenase [L-tryptophan-02 2,3-oxidoreductase (decyclizing), EC 1.13.11.111 to kynurenine and subsequently to acetyl-CoA (the 'glutarate' pathway) or nicotinamide nucleotides (the 'NAD' pathway); (ii) transamination to yield indol-3-ylpyruvic acid, which may then be oxidized to indol-3-ylacetic acid; (iii) decarboxylation to yield the neurotransmitter tryptamine (Saavedra & Axelrod, 1973;Young et al., 1980) (Young et al., 1978). Several methods have been used to investigate the control of tryptophan oxidation by the kynurenine pathway in intact isolated liver preparations. These include measurements of rates of appearance of specific metabolites such as kynurenine (Green et al., 1976) or of removal of added tryptophan (Kim & Miller, 1969;Ng et al., 1970). The disadvantages of these methods have been discussed elsewhere (Smith & Pogson, 1980). S. A. Smith, F. P. A. Carr and C. I. Pogson kynurenine pathway has been derived from experiments with the use of specifically radiolabelled tryptophan (Altman & Gerber, 1967;Ng et al., 1970). However, interpretation of these radioisotope data is complicated because, in most instances, a racemic mixture of D-...

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Section: Quinolinic Acidformation By Liver Cellsmentioning

confidence: 99%