Capillary zone electrophoresis separation of tryptophan and its metabolites, including quinolinic acid

Capillary zone electrophoresis with UV absorbance detection was used to separate tryptophan and ten its metabolites. Run buffers of pH 4.0–10.0 were evaluated for their effect on resolution; a pH 9.6 buffer was found to give optimum separation of all components. Ethylenediaminetetraacetic acid (EDTA), which prevents complexation of some analytes with polyvalent cations, was included in the run buffer to insure good peak shape and reproducible mobilities. The resulting method was used to detect the presence of quinolinic acid in a urine sample.     

Effects of transient, global, cerebral ischemia on striatal extracellular dopamine, serotonin and their metabolites

Rat striatal extracellular fluid levels of dopamine, serotonin, 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were measured before, during and after transient, global cerebral ischemia in awake rats using in vivo brain microdialysis. Before ischemia, extracellular levels of dopamine, DOPAC, HVA and 5-HIAA were detectable and consistent from sample to sample. During cerebral ischemia, there was a large increase in extracellular dopamine levels and a decrease in the extracellular levels of DOPAC, HVA, and 5-HIAA. During reperfusion, dopamine levels returned to normal as did those of DOPAC, HVA and 5-HIAA. Dialysate serotonin and 3-methoxytyramine concentrations were below detection limits except for samples collected during ischemia and early reperfusion.     

Effect of quinolinic acid on the distribution of hepatic metabolites

The intracellular distribution of hepatic metabolites in normal and quinolinic acid (QA)-treated rats has been calculated. QA, an inhibitor of gluconeogenesis, raises the total cell content of malate, aspartate, α-ketoglutarate and citrate. The calculated mitochondrial content of all four metabolites was raised, as was the mitochondrial/cytosolic gradient, and the cytosolic content of oxaloacetate and α-ketoglutarate decreased. The fall of cytosolic oxaloacetate in QA-treated rats suggests a control at PEPCK by substrate limitation. It is suggested that QA may have an action on the translocation of polycarboxylate anions across the mi tochondrial membrane.     

Effect of dietary tryptophan on plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid in rainbow trout

In order to determine the effect of dietary tryptophan level on plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid levels, juvenile rainbow trout (Salmo gairdneri) were raised for 16 weeks on semipurified diets containing 0.06%, 0.16%, 0.21%, 0.26%, 0.39%, or 0.59% tryptophan. After 14 weeks, feed intake was depressed in fish fed the diets containing 0.06% or 0.16% tryptophan. No further differences in feed intake were noted between the remaining treatments. In addition, body weight was lower in fish fed diets containing 0.06%, 0.16%, or 0.21% tryptophan compared with fish fed higher levels. After 16 weeks of feeding the test diets, plasma tryptophan levels were found to be directly related to dietary tryptophan levels. Similarly, increased dietary levels of tryptophan resulted in increased brain levels of tryptophan, serotonin, and 5-hydroxyindoleacetic acid. These results demonstrate that in rainbow trout, as in mammals, altered dietary levels of tryptophan result in alterations in plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid.     

Effects of two different loading doses of L-tryptophan on the urinary excretion of tryptophan metabolites in rats, mice and guinea pigs. Correlation with the enzyme activities

The effect of two different loading doses of L-tryptophan (0.5 and 1.0 g/Kg b.w.) on excretion of tryptophan metabolites and the relation to the enzyme activities were studied in rats, mice and guinea pigs. In rats there is no ratio between the dosage used and the levels of the metabolites excreted. Doubling the amount of tryptophan administered, a 5-fold increase in the elimination of the metabolites along the kynurenine pathway is obtained. The 1.0 g/Kg load provides a more complete pattern of the metabolites than with the 0.5 g/Kg b.w. load. Kynurenic acid, kynurenine and xanthurenic acid are the chief metabolites excreted. In mice, the urinary excretion of the metabolites is very low with both loads. In guinea pigs, xanthurenic acid is excreted in the highest amount and kynurenic acid and kynurenine also constitute the large fractions with both loadings. The load of 0.5 g/Kg b.w. is preferable to that of 1.0 g/Kg b.w. for not causing B6-deficiency. Liver tryptophan pyrrolase exists in two forms in rats, while in mice and in guinea pigs it is present only as holoenzyme. This enzyme is more active in rats than in the other two species of animals. Kynureninase activity is lower in guinea pigs, but it apparently correlated to the low levels of excretion of the metabolites following this step. Kynurenine aminotransferase is very active in rats and in mice, while it is apparently depressed in guinea pigs, in contrast with the high excretion of xanthurenic and kynurenic acids, that puts in evidence a B6-deficiency. The excretion of tryptophan metabolites and enzyme activities are better correlated in rats.     

The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid

Quinolinic acid (QUIN) is a glutamate agonist which markedly enhances the vulnerability of neural cells to excitotoxicity. QUIN is produced from the amino acid tryptophan through the kynurenine pathway (KP). Dysregulation of this pathway is associated with neurodegenerative conditions. In this study we treated striatal astrocytes in culture with QUIN and assayed the endogenous phosphorylating system associated with glial fibrillary acidic protein (GFAP) and vimentin as well as cytoskeletal remodeling. After 24 h incubation with 100 µM QUIN, cells were exposed to ³²P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca²⁺/calmodulin II (PKCaMII) or protein kinase C (PKC) inhibitors, H89 (20 μM), KN93 (10 μM) and staurosporin (10 nM), respectively. Results showed that hyperphosphorylation was abrogated by PKA and PKC inhibitors but not by the PKCaMII inhibitor. The specific antagonists to ionotropic NMDA and non-NMDA (50 µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50 µM MCPG), mGLUR1 (100 µM MPEP) and mGLUR5 (10 µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca²⁺ quelators (1 mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca²⁺ influx through voltage-dependent Ca²⁺ channel type L (L-VDCC) (blocker: 10 µM verapamil) is not implicated in this effect. Morphological analysis showed dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork, supporting misregulation of actin cytoskeleton. Both hyperphosphorylation and cytoskeletal remodeling were reversed 24 h after QUIN removal. Astrocytes are highly plastic cells and the vulnerability of astrocyte cytoskeleton may have important implications for understanding the neurotoxicity of QUIN in neurodegenerative disorders.     

The metabolism of L-tryptophan by isolated rat liver cells. Effect of albumin binding and amino acid competition on oxidatin of tryptophan by tryptophan 2,3-dioxygenase.

1. Novel methods, using L-[ring-2-14C]tryptophan, are described for the measurement of tryptophan 2,3-dioxygenase activity and tryptophan accumulation in isolated rat liver cells. 2. The effects of bovine serum albumin, non-esterified fatty acids and neutral amino acids on tryptophan oxidation by hepatocytes and on the partition of tryptophan between free and albumin-bound forms were investigated. 3. Oxidation of physiological concentrations (0.1 mM) of tryptophan was inhibited by approx. 50% in the presence of 2% (w/v) bovine serum albumin; no effects were found at tryptophan concentrations of 0.5 mM and above. 4. Increases in free tryptophan concentrations produced by displacement of 0.1 mM-tryptophan from albumin-binding sites by palmitate resulted in increased flux through tryptophan dioxygenase. 5. Addition of a mixture of neutral amino acids, at plasma concentrations, to hepatocyte incubations had no effect on the rate of tryptophan oxidation. 6. It is concluded that alterations in free tryptophan concentrations consequent to changes in albumin binding may be an important factor in regulating tryptophan uptake and catabolism by the liver. The results are briefly discussed with reference to possible consequences on brain tryptophan metabolism.     

Conversion of D- and L-tryptophan to brain serotonin and 5-hydroxyindoleacetic acid and to blood serotonin

Abstract— Intraperitoneal administration of both D- or L-tryptophan elevated the levels of serotonin and 5-hydroxyindoleacetic acid in the brains of hypophysectomized and intact rats. In intact rats, the increase in brain 5-hydroxyindoles was slower after D-tryptophan than after L-tryptophan. Similarly, brain tryptophan rose more slowly after administration of D-tryptophan. The uptake of L-tryptophan from blood into brain was at a rate about one-third that of ³H₂O. D-tryptophan uptake was at 1/25 that of ³H₂O. Brain and liver tryptophan aminotransferase activities were stereospecific for the L-isomer and no evidence could be found for a tryptophan racemase in brain. Evisceration prevented the increase in brain 5-hydroxyindoles following peripheral administration of D-tryptophan administration but not that after L-tryptophan. The serotonin ratios between the two brain regions examined remained constant following administration of either D- or L-tryptophan. On the basis of these results we concluded that the increase in brain 5-hydroxyindoles following administration of L-tryptophan was not dependent upon stress-induced changes in pituitary hormones and that the elevations after D-tryptophan were dependent upon its prior conversion to L-tryptophan via peripheral deamination and subsequent transamination.     

Rapid depletion of serum tryptophan, brain tryptophan, serotonin and 5-hydroxyindoleacetic acid by a tryptophan-free diet

Rats were trained for 20 days to eat their normal daily meal in a period of 2 hours. On the twentyfirst day they received a diet in which tryptophan was omitted instead of the usual balanced diet. The ingestion of the tryptophan-free diet produced a marked depletion of free serum tryptophan (90%), brain tryptophan (85%), brain 5-HT (58%) and brain 5-HIAA (76%). These changes were almost maximal within 2 hours after food presentation and persisted for more than 24 hours. The mechanism of these changes is discussed.     

Effect of kynurenine loading on quinolinic acid production in the rat: studies in vitro and in vivo

In vitro and in vivo techniques were used to examine the production and subsequent fate of the endogenous excitotoxin quinolinic acid (QUIN) following administration of its bioprecursor L-kynurenine (KYN). Incubation of liver slices in the presence of 10-1000 microM KYN resulted in a dose- and time-dependent release of QUIN into the incubation medium. Less than 15% of total QUIN produced was recovered from the tissue. In vivo experiments, performed with a microdialysis probe inserted in the jugular vein of anesthetized rats, showed that injection of KYN (20-600 mg/kg, i.v.) causes rapid and dose-dependent increases in the serum level of QUIN. Peak QUIN concentrations in serum dialysates were reached 75 minutes following KYN administration. Longer lasting increases were detected following the administration of pyrazinamide (20 mg/kg, i.p.), an indirectly acting stimulator of QUIN biosynthesis in the periphery. The data demonstrate the feasibility of assessing the mechanisms of QUIN production and disposition in experimental paradigms which can be expected to allow insights into the function and possible dysfunction of QUIN in the brain.     

Quantification of quinolinic acid in rat brain, whole blood, and plasma by gas chromatography and negative chemical ionization mass spectrometry: effects of systemic L-tryptophan administration on brain and blood quinolinic acid concentrations

A gas chromatography/mass spectrometry assay is described to quantify the endogenous neurotoxin quinolinic acid (QUIN) in brain, whole blood, and plasma. High specificity and high sensitivity were obtained by using negative chemical ionization and accuracy was achieved by using [18O]QUIN as internal standard. Neutralized perchloric acid extracts were washed with chloroform, applied to Dowex 1 x 8 (formate form), and eluted with 6 M formic acid. After lyophilization, QUIN and [18O]QUIN were esterified with hexafluoroisopropanol (to mass 467 and 471, respectively) using trifluoroacetylimidazole as catalyst. The esters were extracted into heptane and injected onto a gas chromatograph, DB-5 capillary column. QUIN and [18O]QUIN were quantified by selected ion monitoring of QUIN-specific anion currents from the molecular anions (m/z 467 and 471, respectively) and a specific anion fragment (m/z 316 from QUIN and m/z 320 from [18O]QUIN). Minimum sensitivity was 3 fmol, intraassay variability was 3.2%, and interassay variability was 8.1% QUIN concentrations in frontal cortex from over 200 rats ranged from 20 to 180 fmol/mg wet wt. Two hours after systemic L-tryptophan (L-Trp; 0.370 mmol/kg) administration, QUIN increased in whole blood 134.8-fold and in plasma, 74.3-fold. In frontal cortex, increases in QUIN (22.6-fold, corrected for QUIN in blood) exceeded increases in cortical L-Trp (2.54-fold), 5-HT (1.35-fold), and 5-HIAA (1.74-fold). These studies demonstrate that QUIN is present in brain and is sensitive to the availability of systemic L-Trp.     

The effect of quinolinic acid on the content and distribution of hepatic metabolites

The effect of quinolinic acid treatment on the hepatic metabolite profile and the flux of glucose through the alternative pathways of metabolism have been measured, and the distribution of metabolites between the cytosolic and mitochondrial compartments has been calculated. Marked increases of the total-cell polycarboxylic anions were found and these were, in order of magnitude: malate, citrate, isocitrate, aspartate, 2-oxoglutarate, and glutamate. Calculation of the compartmented values suggested that the major increase was in the mitochondrial compartment: cytosolic glutamate, 2-oxoglutarate, and oxaloacetate were decreased and only aspartate increased in this compartment.The changes of the mitochondrial/cytosolic anion ratio was most marked, 60-fold, in the case of 2-oxoglutarate. It is suggested that inhibition of transport of 2-oxoglutarate by quinolinic acid could, by blocking the operation of the aspartate shuttle, contribute to the inhibition of gluconeogenesis from lactate.Metabolite and flux data suggest an increase in the rate of lipogenesis in quinolinic acid-treated rats with the decrease of long-chain acyl CoAs, caused by this treatment, being the possible effector for this activation.     

Effect of tryptophan administration on serotonin and 5-hydroxyindoleacetic acid content in the brainstem of Lilly 110140-pretreated rats.

Adult male Sprague-Dawley rats were divided into 3 groups. One group was pretreated with Lilly 110140 (10 mg/kg) 27 hours and again 3 hours before sacrifice while a second group received Lilly 110140 only 3 hours before sacrifice. The third or control group received only equivalent volumes of saline. Animals from each group were administered 25 mg/kg L tryptophan intraperitoneally (i.p.) 0, 30, 60 or 90 minutes before sacrifice. Equivalent elevations in serum and also brainstem tryptophan content were observed in all three groups with the peak observed at 30 minutes. Brainstem serotonin content was significantly elevated in both groups of Lilly 110140-pretreated rats but not in the control group. Brainstem 5-hydroxyindoleacetic acid was significantly elevated after tryptophan administration in the control and the 3 hour and 27 hour, Lilly 110140-pretreated groups but not in the 3 hour Lilly 110140 pretreated group. The results indicate that neither 3 or 3 hours and 27 hours of Lilly 110140 pretreatment appreciably affects the increase in brainstem serotonin synthesis induced by the i.p. administration of 25 mg/kg of L tryptophan.     

Dose-response decrease in plasma tryptophan and in brain tryptophan and serotonin after tryptophan-free amino acid mixtures in rats

Rats fasted 15 hours were treated p.o. with increasing amounts (660 and 1320 mg/kg body weight) of a mixture containing a fixed proportion of seven essential amino acids (L-phenylalanine 13.6%, L-leucine 6.0%, L-isoleucine 12.1%, L-methionine 12.1%, L-lysine 30.3%, L-threonine 10.6%, L-valine 15.2%) and lacking tryptophan. The mixtures produced a dose-response decrease of free (by 34% after the lower dose and by 58% after the higher dose of the mixture) and total (by 10 and 31%) plasma tryptophan and of brain tryptophan (by 38 and 65%), serotonin (by 17 and 41%) and 5-hydroxyindole acetic acid (by 21 and 49%). The mechanisms of these changes are discussed.