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

Abstract

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-...

The metabolism of l-tryptophan by liver cells prepared from adrenalectomized and streptozotocin-diabetic rats

“…The incubation conditions for determining fluxes through tryptophan 2,3 -dioxygenase and kynureninase and to non-aromatic products of the glutarate pathway in isolated liver cell preparations by using L-[ring-2-'4C]-, L-[carboxyl-'4C1-and L-[benzene-ring-U-'4C1-tryptophan respectively were identical with those described previously (Smith & Pogson, 1980b;Smith et al, 1980).…”

“…Pharmaceuticals, Alderley Park, Cheshire, U.K.). The sources of all other materials have been detailed earlier (Smith & Pogson, 1980b;Smith et al, 1980).…”

A critical approach to the use of isolated liver cells for the study of metabolic events

“…These factors permit the experimenter to incubate more than IOO samples at a time, because processing, subsequent to quenching with acid, merely consists of absorption of 14C0, with phenylethylamine, adsorption of aromatic compounds on to Norit GSX, brief centrifugation and removal of supernatant fractions for counting (e.g. Smith & Pogson, 1980;Dickson et al 1981;Smith et al 1981).…”

Suppression and potentiation of 5‐hydroxytryptophan‐induced hypoglycaemia by α‐monofluoromethyldopa: correlation with the accumulation of 5‐hydroxytryptamine in the liver

“…Additionally, the agent has been shown not to be a specific inhibitor, because it also inhibits other enzymes in the tryptophan oxidative pathway at concentrations that are likely to be encountered in vivo after administration to experimental animals (Bender, 1980;Smith & Pogson, 1981). In fact, in spite of the protective effect of carbidopa on the tryptophan-induced hypoglycaemia, it has been suggested that 5-HT is unlikely to mediate the hypoglycaemic response to tryptophan (Smith et al, 1980;Lloyd et al, 1982a,b). Therefore, the possibility that metabolites of 5-HTP other than 5-HT may contribute to the development of 5-HTP-induced hypoglycaemia cannot be excluded.…”