Amino Acid Metabolism is Significantly Altered at the Time of Admission in Hospital for Severe COVID-19 Patients: Findings from Longitudinal Targeted Metabolomics Analysis.

The heterogeneity in severity and outcome of COVID-19 cases points out the urgent need for early molecular characterization of patients followed by risk-stratified care. The main objective of this study was to evaluate the fluctuations of serum metabolomic profiles of COVID-19 patients with severe illness during the different disease stages in a longitudinal manner. We demonstrate a distinct metabolomic signature in serum samples of 32 hospitalized patients at the acute phase compared to the recovery period, suggesting the tryptophan (tryptophan, kynurenine, and 3-hydroxy-DL-kynurenine) and arginine (citrulline and ornithine) metabolism as contributing pathways in the immune response to SARS-CoV-2 with a potential link to the clinical severity of the disease. In addition, we suggest that glutamine deprivation may further result in inhibited M2 macrophage polarization as a complementary process, and highlight the contribution of phenylalanine and tyrosine in the molecular mechanisms underlying the severe course of the infection. In conclusion, our results provide several functional metabolic markers for disease progression and severe outcome with potential clinical application. IMPORTANCE Although the host defense mechanisms against SARS-CoV-2 infection are still poorly described, they are of central importance in shaping the course of the disease and the possible outcome. Metabolomic profiling may complement the lacking knowledge of the molecular mechanisms underlying clinical manifestations and pathogenesis of COVID-19. Moreover, early identification of metabolomics-based biomarker signatures is proved to serve as an effective approach for the prediction of disease outcome. Here we provide the list of metabolites describing the severe, acute phase of the infection and bring the evidence of crucial metabolic pathways linked to aggressive immune responses. Finally, we suggest metabolomic phenotyping as a promising method for developing personalized care strategies in COVID-19 patients.

Kynurenine derivative 3-HAA is an agonist ligand for transcription factor YY1.

The 3-hydroxyanthranilic acid (3-HAA), a derivative of kynurenine, was reported to suppress tumor growth. However, the function of 3-HAA largely remains unclear. Here, we report that 3-hydroxyanthranilic acid (3-HAA) is lower in tumor cells, while adding exogenous 3-HAA induces apoptosis in hepatocellular carcinoma by binding YY1. This 3-HAA binding of YY1 leads to phosphorylation of YY1 at the Thr 398 by PKCζ, concomitantly enhances YY1 chromatin binding activity to increase expression of target genes. These findings demonstrate that 3-HAA is a ligand of YY1, suggesting it is a promising therapeutic candidate for HCC.

Kynurenine, 3-OH-kynurenine, and anthranilate are nutrient metabolites that alter H3K4 trimethylation and H2AS40 O-GlcNAcylation at hypothalamus-related loci.

Epigenetic mechanisms can establish and maintain mitotically stable patterns of gene expression while retaining the DNA sequence. These mechanisms can be affected by environmental factors such as nutrients. The importance of intracellular dosages of nutrient metabolites such as acetyl coenzyme A and S-adenosylmethionine, which are utilized as donors for post-translational modifications, is well-known in epigenetic regulation; however, the significance of indirect metabolites in epigenetic regulation is not clear. In this study, we screened for metabolites that function as epigenetic modulators. Because the expression of genes related to hypothalamic function is reportedly affected by nutritional conditions, we used a neural cell culture system and evaluated hypothalamic-linked loci. We supplemented the culture medium with 129 metabolites separately during induction of human-iPS-derived neural cells and used high-throughput ChIP-qPCR to determine the epigenetic status at 37 hypothalamus-linked loci. We found three metabolites (kynurenine, 3-OH-kynurenine, and anthranilate) from tryptophan pathways that increased H3K4 trimethylation and H2AS40 O-GlcNAcylation, resulting in upregulated gene expression at most loci, except those encoding pan-neural markers. Dietary supplementation of these three metabolites and the resulting epigenetic modification were important for stability in gene expression. In conclusion, our findings provide a better understanding of how nutrients play a role in epigenetic mechanisms.

Protective Effects Induced by Microwave-Assisted Aqueous Harpagophytum Extract on Rat Cortex Synaptosomes Challenged with Amyloid ß-Peptide.

Harpagophytum procumbens is a plant species that displays anti-inflammatory properties in multiple tissues. The iridoid glycosides arpagoside, harpagide, and procumbide appear to be the most therapeutically important constituents. In addition, harpagoside treatment exerted neuroprotective effects both in vitro and in vivo. Considering these findings, the aim of the present work is to explore the possible protective role of the previously described microwave-assisted aqueous extract of H. procumbens on rat hypothalamic (Hypo-E22) cells, and in rat cortex challenged with amyloid ß-peptide (1-40). In this context, we assayed the protective effects induced by H. procumbens by measuring the levels of malondialdehyde, 3-hydroxykynurenine (3-HK), brain-derived neurotrophic factor, and tumor necrosis factor-α, 3-HK. Finally, we evaluated the effects of H. procumbens treatment on cortex levels of dopamine, norepinephrine, and serotonin. H. procumbens extract was well tolerated by Hypo-E22 cells and upregulated brain-derived neurotrophic factor gene expression but down-regulated tumor necrosis factor-α gene expression. In addition, the extract reduced amyloid ß-peptide stimulation of malondialdehyde and 3-HK and blunted the decrease of dopamine, norepinephrine, and serotonin, in the cortex. In this context, our work supports further studies for the evaluation and confirmation of Harpagophytum in the management of the clinical symptoms related to Alzheimer's disease. Copyright © 2017 John Wiley & Sons, Ltd.

Systemic administration of l-kynurenine sulfate induces cerebral hypoperfusion transients in adult C57Bl/6 mice.

The kynurenine pathway is a cascade of enzymatic steps generating biologically active compounds. l-kynurenine (l-KYN) is a central metabolite of tryptophan degradation. In the mammalian brain, l-KYN is partly converted to kynurenic acid (KYNA), which exerts multiple effects on neurotransmission. Recently, l-KYN or one of its derivatives were attributed a direct role in the regulation of the systemic circulation. l-KYN dilates arterial blood vessels during sepsis in rats, while it increases cerebral blood flow (CBF) in awake rabbits. Therefore, we hypothesized that acute elevation of systemic l-KYN concentration may exert potential effects on mean arterial blood pressure (MABP) and on resting CBF in the mouse brain. C57Bl/6 male mice were anesthetized with isoflurane, and MABP was monitored in the femoral artery, while CBF was assessed through the intact parietal bone with the aid of laser speckle contrast imaging. l-KYN sulfate (l-KYNs) (300mg/kg, i.p.) or vehicle was administered intraperitoneally. Subsequently, MABP and CBF were continuously monitored for 2.5h. In the control group, MABP and CBF were stable (69±4mmHg and 100±5%, respectively) throughout the entire data acquisition period. In the l-KYNs-treated group, MABP was similar to that, of control group (73±6mmHg), while hypoperfusion transients of 22±6%, lasting 7±3min occurred in the cerebral cortex over the first 60-120min following drug administration. In conclusion, the systemic high-dose of l-KYNs treatment destabilizes resting CBF by inducing a number of transient hypoperfusion events. This observation indicates the careful consideration of the dose of l-KYN administration by interpreting the effect of kynurenergic manipulation on brain function. By planning clinical trials basing on kynurenergic manipulation possible vascular side effects should also be considered.

Associations between intake of fish and n-3 long-chain polyunsaturated fatty acids and plasma metabolites related to the kynurenine pathway in patients with coronary artery disease.

PURPOSE: Enhanced tryptophan degradation via the kynurenine pathway has been related to several pathological conditions. However, little is known about the effect of diet on individual metabolites of this pathway. We investigated cross-sectional associations between reported intake of fish and omega-3 (n-3) long-chain PUFA (LC-PUFA) and plasma metabolites related to the kynurenine pathway. METHODS: Participants were 2324 individuals with coronary artery disease from the Western Norway B Vitamin Intervention Trial. Fish and n-3 LC-PUFA intakes were assessed using a food frequency questionnaire. Plasma concentrations of tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid, neopterin, and kynurenine-to-tryptophan ratio (KTR) were analyzed. Associations were investigated using partial Spearman's rank correlations and multiple linear regressions. RESULTS: Median age at inclusion was 62 years (80 % males), and 84 % had stable angina pectoris. Intake of fatty fish and n-3 LC-PUFA was inversely associated with plasma 3-hydroxykynurenine. Consumption of total fish, lean fish, and n-3 LC-PUFA was inversely associated with plasma neopterin. Intake of total fish, fatty fish, and n-3 LC-PUFA was inversely associated with KTR. All these correlations were weak (ρ between -0.12 and -0.06, P < 0.01). In 306 patients with diabetes, lean fish intake was positively associated with plasma 3-hydroxyanthranilic acid (ρ = 0.22, P < 0.001, P for interaction = 0.01), and total fish intake was inversely associated with KTR (ρ = -0.17, P < 0.01, P for interaction = 0.02). CONCLUSION: Fish intake was not an important determinant of individual metabolites in the kynurenine pathway. However, some correlations were stronger in patients with diabetes. The inverse associations of fish or n-3 LC-PUFA with neopterin and KTR may suggest a slightly lower IFN-γ-mediated immune activation with a higher intake.

The effect of conjugated linoleic acid supplements on oxidative and antioxidative status of dairy cows.

Dairy cows develop frequently negative energy balance around parturition and in early lactation, resulting in excessive mobilization of body fat and subsequently in increased risk of ketosis and other diseases. Dietary conjugated linoleic acid (CLA) supplements are used in dairy cows mainly for their depressing effect on milk fat content, but are also proposed to have antioxidative properties. As negative energy balance is associated with oxidative stress, which is also assumed to contribute to disease development, the present study was conducted to examine effects of CLA on oxidative and antioxidative status of lactating dairy cows. German Holstein cows (primiparous n=13, multiparous n=32) were divided into 3 dietary treatment groups receiving 100g/d of control fat supplement, containing 87% stearic acid (CON; n=14), 50g/d of control fat supplement and 50g/d of CLA supplement (CLA 50; n=15), or 100g/d of CLA supplement (CLA 100; n=16). The CLA supplement was lipid-encapsulated and contained 12% of trans-10,cis-12 CLA and cis-9,trans-11 CLA each. Supplementation took place between d1 and 182 postpartum; d 182 until 252 postpartum served as a depletion period. Blood was sampled at d -21, 1, 21, 70, 105, 140, 182, 224, and 252 relative to calving. The antioxidative status was determined using the ferric-reducing ability of plasma, α-tocopherol, α-tocopherol-to-cholesterol mass ratio, and retinol. For determination of oxidative status concentrations of hydroperoxides, thiobarbituric acid-reactive substances (TBARS), N'-formylkynurenine, and bityrosine were measured. Mixed models of fixed and random effects with repeated measures were used to evaluate period 1 (d -21 to 140) and 2 (d182-252) separately. Cows showed increased oxidative stress and lipid peroxidation during the periparturient period in terms of increased serum concentrations of hydroperoxides and TBARS, which decreased throughout lactation. During period 1, the supplemented cows had lower TBARS concentrations, which was not detectable in period 2. The other determined parameters were not affected by CLA supplementation. The obtained results show that dietary CLA supplementation in the chosen dosage, formulation, and application period had a marginal antioxidative effect in terms of lipid peroxidation in lactating dairy cows.

Pyridoxine supplementation does not alter in vivo kinetics of one-carbon metabolism but modifies patterns of one-carbon and tryptophan metabolites in vitamin B-6-insufficient oral contraceptive users.

BACKGROUND: Low chronic vitamin B-6 status can occur in a subset of women who use oral contraceptives (OCs) with uncertain metabolic consequences. An insufficiency of cellular pyridoxal 5'-phosphate (PLP), which is the coenzyme form of vitamin B-6, may impair many metabolic processes including one-carbon and tryptophan metabolism. OBJECTIVE: We investigated the effects of vitamin B-6 supplementation on the in vivo kinetics of one-carbon metabolism and the concentration of one-carbon and tryptophan metabolites in vitamin B-6-deficient OC users. DESIGN: A primed, constant infusion of [(13)C5]methionine, [3-(13)C]serine, and [(2)H3]leucine was performed on 10 OC users (20-40 y old; plasma PLP concentrations <30 nmol/L) before and after 28 d of supplementation with 10 mg pyridoxine hydrochloric acid/d. In vivo fluxes of total homocysteine remethylation, the remethylation of homocysteine from serine, and rates of homocysteine and cystathionine production were assessed. Targeted metabolite profiling was performed, and data were analyzed by using orthogonal partial least-squares-discriminant analysis and paired t tests adjusted for multiple testing. RESULTS: Pyridoxine supplementation increased the mean ± SD plasma PLP concentration from 25.8 ± 3.6 to 143 ± 58 nmol/L (P < 0.001) and decreased the leucine concentration from 103 ± 17 to 90 ± 20 nmol/L (P = 0.007) and glycine concentration from 317 ± 63 to 267 ± 58 nmol/L (P = 0.03). Supplementation did not affect in vivo rates of homocysteine remethylation or the appearance of homocysteine and cystathionine. A multivariate analysis showed a clear overall effect on metabolite profiles resulting from supplementation. Leucine, glycine, choline, cysteine, glutathione, trimethylamine N-oxide, and the ratios glycine:serine, 3-hydroxykynurenine:kynurenine, 3-hydroxykynurenine:3-hydroxyanthranilic acid, and 3-hydroxykynurenine:anthranilic acid were significant discriminating variables. CONCLUSIONS: Consistent with previous vitamin B-6-restriction studies, fluxes of one-carbon metabolic processes exhibited little or no change after supplementation in low-vitamin B-6 subjects. In contrast, changes in the metabolic profiles after supplementation indicated perturbations in metabolism, suggesting functional vitamin B-6 deficiency. This study was registered at clinicaltrials.gov as NCT01128244.

Tryptophan catabolism and vitamin B-6 status are affected by gender and lifestyle factors in healthy young adults.

BACKGROUND: Abnormalities of tryptophan (Trp) metabolism through the kynurenine (Kyn) pathway have been reported in various diseases; however, nutritional and lifestyle factors that affect this pathway in healthy individuals are not well documented. OBJECTIVE: Our aim was to examine the effect of vitamin B-6 status and lifestyle factors including the use of vitamin B-6 supplements, alcohol, smoking, and oral contraceptives on Trp and its Kyn metabolites in a cohort of 2436 healthy young adults aged 18-28 y. METHODS: Anthropometric and lifestyle data were collected by questionnaire. Participants provided blood samples for analysis of Trp, Kyn, anthranilic acid, kynurenic acid (KA), 3-hydroxykynurenine (HK), 3-hydroxyanthranilic acid (HAA), and xanthurenic acid (XA). Vitamin B-6 species were also measured. RESULTS: Serum Trp metabolites were 10-15% higher among men (n = 993) compared with women (n = 1443; P < 0.0001), except for HK and XA. In all participants, serum Trp was positively associated with plasma pyridoxal 5'-phosphate (PLP; r = 0.28, P < 0.0001), reaching a plateau at PLP concentrations of ∼83 nmol/L. HK was inversely associated with PLP (r = -0.14, P < 0.01). Users of vitamin B-6 supplements (n = 671) had 6% lower concentrations of HK than nonusers (n = 1765; P = 0.0006). Oral contraceptive users (n = 385) had lower concentrations of KA (20.7%) but higher XA (24.1%) and HAA (9.0%) than did nonusers (n = 1058; P < 0.0001). After adjustment for gender and other lifestyle variables, XA concentrations were 16% higher in heavy drinkers (n = 713) than in never or occasional drinkers (n = 975; P = 0.0007). Concentrations of 2 other essential amino acids, methionine and arginine, also were positively associated with serum Trp (r = 0.65 and 0.33, respectively; P < 0.0001). CONCLUSIONS: In this population of healthy young adults, gender has the largest influence on serum Kyn metabolite concentrations. The significant covariance of Trp with unrelated amino acids suggests that protein intake may be an important consideration in evaluating Kyn metabolism.

Supplementing healthy women with up to 5.0 g/d of L-tryptophan has no adverse effects.

Because of the frequent use of L-tryptophan (L-Trp) in dietary supplements, determination of the no-observed-adverse-effect-level is desirable for public health purposes. We therefore assessed the no-observed-adverse-effect-level for L-Trp and attempted to identify a surrogate biomarker for excess L-Trp in healthy humans. A randomized, double-blind, placebo-controlled, crossover intervention study was performed in 17 apparently healthy Japanese women aged 18-26 y with a BMI of ≈ 20 kg/m(2). The participants were randomly assigned to receive placebo (0 g/d) or 1.0, 2.0, 3.0, 4.0, or 5.0 g/d of L-Trp for 21 d each with a 5-wk washout period between trials. Food intake, body weight, general biomarkers in blood and urine, and amino acid composition in blood and urine were not affected by any dose of L-Trp. Administration of up to 5.0 g/d L-Trp had no effect on a profile of mood states category measurement. The urinary excretion of nicotinamide and its catabolites increased in proportion to the ingested amounts of L-Trp, indicating that participants could normally metabolize this amino acid. The urinary excretion of L-tryptophan metabolites, including kynurenine (Kyn), anthranilic acid, kynurenic acid, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and quinolinic acid (QA), all of which are intermediates of the L-TRP→Kyn→QA pathway, was in proportion to L-Trp loading. The response of 3-HK was the most characteristic of these L-Trp metabolites. This finding suggests that the urinary excretion of 3-HK is a good surrogate biomarker for excess L-Trp ingestion.

Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme whose expression by a broad range of clinical tumors is associated with immunosuppression and poor patient outcome. Here we describe a new fluorescence assay for measuring IDO1 activity suitable for high-throughput screening of compound libraries for novel IDO1 inhibitors. This assay is easy to perform, requiring the addition of only one reagent prior to readout. In place of measuring kynurenine, it uses the in situ formation of an N-formylkynurenine-derived fluorophore (NFKPIP) measured at an excitation wavelength of 400 nm and an emission wavelength of 500 nm. The fluorescence intensity of the NFKPIP formed is directly related to the amount of enzyme activity, and the signal is stable over 8 h. This assay has a lower limit of detection, equating to 153 nM N-formylkynurenine, which is over 30-fold lower than the limits of detection of existing assays for IDO1 activity. When we compared the performance of the new assay with that of the published colorimetric absorbance assay in screening the National Cancer Institute Diversity Set III of 1,597 compounds for IDO1 inhibitors, we obtained an identical list of the 25 most active compounds in the two assays. Although 93 compounds (aldehydes, ketones, and aromatic amines) in the library interfered with the absorbance readout, only 18 compounds (conjugated systems and fused cycles) interfered with the readout of the new fluorescence assay. IC(50) values determined using the new assay for three known IDO1 inhibitors-1,4-naphthoquinone, 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide and 4-phenyl-1H-imidazole-were consistent with their literature values, further validating the new assay for measuring IDO1 activity.

Low plasma vitamin B-6 status affects metabolism through the kynurenine pathway in cardiovascular patients with systemic inflammation.

It is unclear whether reduced plasma pyridoxal 5'-phosphate (PLP) during inflammation reflects an altered distribution or increased requirement of vitamin B-6 that may impair overall vitamin B-6 status in tissues. In plasma from 3035 patients undergoing coronary angiography for suspected coronary heart disease, we investigated if plasma concentrations of any metabolites in the kynurenine pathway, which depend on PLP as cofactor, may serve as metabolic marker(s) of vitamin B-6 status. We also examined the association of vitamin B-6 status with serum or plasma concentrations of several inflammatory markers. Among the kynurenines, only 3-hydroxykynurenine (HK) was inversely related to PLP and showed a positive relation to 4 investigated inflammatory markers. A segmented relationship was observed between PLP and HK, with a steep slope at PLP concentrations < 18.4 nmol/L, corresponding to the 5th percentile, and an almost zero slope at higher PLP concentrations. Low PLP and the steep PLP-HK slope were essentially confined to participants with 1 or more inflammatory markers in the upper tertile. Oral supplementation with pyridoxine hydrochloride (40 mg/d) for 1 mo increased plasma PLP 8-fold, reduced the geometric mean (95% CI) of HK from 29.5 to 20.2 nmol/L (P < 0.001), and abolished the steep segment of the PLP-HK curve. The steep inverse relationship of plasma PLP with HK at low plasma PLP and the lowering of HK by pyridoxine suggest plasma HK as a metabolic marker of vitamin B-6 status. Thus, low plasma PLP during inflammation may reflect impaired cellular vitamin B-6 status, as indicated by the concurrent increase in plasma HK.

Cellular mechanisms involved in the melatonin inhibition of HT-29 human colon cancer cell proliferation in culture.

The antiproliferative and proapoptotic properties of melatonin in human colon cancer cells in culture were recently reported. To address the mechanisms involved in these actions, HT-29 human colon cancer cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum at 37 degrees C. Cell proliferation was assessed by the incorporation of [(3)H]-thymidine into DNA. Cyclic nucleotide levels, nitrite concentration, glutathione peroxidase and reductase activities, and glutathione levels were assessed after the incubation of these cells with the following drugs: melatonin membrane receptor agonists 2-iodo-melatonin, 2-iodo-N-butanoyl-5-methoxytryptamine, 5-methoxycarbonylamino-N-acetyltryptamine (GR-135,531), and the antagonists luzindole, 4-phenyl-2-propionamidotetralin, and prazosin; the melatonin nuclear receptor agonist CGP 52608, and four synthetic kynurenines analogs to melatonin 2-acetamide-4-(3-methoxyphenyl)-4-oxobutyric acid, 2-acetamide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid, 2-butyramide-4-(3-methoxyphenyl)-4-oxobutyric acid and 2-butyramide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid. The results show that the membrane receptors are not necessary for the antiproliferative effect of melatonin and the participation of the nuclear receptor in this effect is suggested. Moreover, the antioxidative and anti-inflammatory actions of melatonin, counteracting the oxidative status and reducing the production of nitric oxide by cultured HT-29 cells seem to be directly involved in the oncostatic properties of melatonin. Some of the synthetic kynurenines exert higher antiproliferative effects than melatonin. The results reinforce the clinical interest of melatonin due to the different mechanisms involved in its oncostatic role, and suggest a new synthetic pathway to obtain melatonin agonists with clinical applications to oncology.

Protein oxidation in plant mitochondria detected as oxidized tryptophan.

The formation of N-formylkynurenine by dioxygenation of tryptophan was detected in peptides from rice leaf and potato tuber mitochondria. Proteins in matrix and membrane fractions were separated by two-dimensional gel electrophoresis and identified using a Q-TOF mass spectrometer. N-Formylkynurenine was detected in 29 peptides representing 17 different proteins. With one exception, the oxidation-sensitive aconitase, all of these proteins were either redox active themselves or subunits in redox-active enzyme complexes. The same site was modified in (i) several adjacent spots containing the P protein of the glycine decarboxylase complex, (ii) two different isoforms of the mitochondrial processing peptidase in complex III, and (iii) the same tryptophan residues in Mn-superoxide dismutase in both rice and potato mitochondria. This indicates that Trp oxidation is a selective process.

3-Hydroxykynurenine and quinolinate: pathogenic synergism in early grade Huntington's disease?

Huntington's Disease (HD), an inherited neurodegenerative disorder, is caused by an abnormal polyglutamine extension of a protein named huntingtin. This genetic defect is believed to result in heightened neuronal susceptibility to excitotoxic injury, a likely mechanism of neurodegeneration in HD. Two neuroactive kynurenine pathway metabolites, quinolinate (QUIN) and kynurenate (KYNA), have been proposed to play critical roles in the precipitation and prevention, respectively, of excitotoxic neuron death in HD. We now provide evidence that a third kynurenine pathway metabolite, 3-hydroxykynurenine (3-HK), should also be considered a pathogen in HD. The brain levels of this free radical generator are increased 5-10-fold in early stage (Grade 1) HD patients. In the same brains, QUIN levels are also significantly elevated in the cortex and in the neostriatum, but not in the cerebellum. In contrast, brain 3-HK and QUIN levels are either unchanged or reduced in Grade 2 and end stage (Grade 3-4) HD patients. Brain KYNA levels are moderately increased during the early disease stages and decrease as the illness progresses. In rats, 3-HK potentiates striatal QUIN toxicity, and this pro-excitotoxic effect can be prevented by free radical scavengers. Taken together, these studies provide further evidence for an involvement of kynurenine pathway metabolites in the early phases of HD neuropathology and suggest novel therapeutic strategies for the disease.

The presence of a human UV filter within the lens represents an oxidative stress.

It has recently been demonstrated that, with age, UV filters such as 3-hydroxykynurenine glucoside, bind to proteins in the human lens. This covalent interaction leads to colouration of the normal lens, and results from the instability of the kynurenine side chain. Other primate UV filters, in addition to containing the same side chain, can also be readily oxidized. One such compound is 3-hydroxykynurenine (3OHKyn). It has been proposed that oxidation of bound and/or free UV filters, such as 3OHKyn may give rise to the lens colouration associated with age-related nuclear cataract. Therefore it has become important to understand the oxidation of 3OHKyn within the lens. In this study, intact bovine lenses (which lack UV filters) were incubated with 3OHKyn and various lens parameters monitored. The effect of exposure to hyperbaric oxygen (HBO) was also assessed, both alone, and in combination with prior 3OHKyn incubation. Glutathione (GSH), protein sulfhydryl and protein-bound sulfhydryl levels, as well as soluble protein content and gel filtration profiles, were obtained for cortical and nuclear regions after defined periods of incubation. The presence of the primate UV filter, 3OHKyn, at concentrations similar to those present in the human lens, was shown to produce considerable oxidative stress within the lens, as judged by its effect on GSH. This effect was noted under normobaric conditions, but was exacerbated by increased oxygen. Exposure of lenses to HBO caused a marked fall in GSH in cortical and nuclear regions. This effect was exaggerated in the presence of 3OHKyn. HBO treatment also lead to a fall in protein sulfhydryl content, however, this was only partial (approximately 1 mol SH per mol protein) and changed only slowly, even with extended periods of exposure to HBO, suggesting that most crystallin sulfhydryl groups may be buried. 3OHKyn did not appreciably affect this oxidation although it did cause an increase in the level of protein-bound sulfhydryl. HBO treatment produced a more than two-fold increase in protein-bound sulfhydryl content in the cortex. There was little influence of 3OHKyn alone on protein solubility, even with extended periods of incubation, however, incubation for 72 hr in the presence of HBO caused a significant increase in insoluble protein particularly in the nucleus. This insolubilization was further increased in the presence of 3OHKyn. FPLC profiles showed that the proportion of gamma and beta crystallins in the soluble fraction decreased following HBO, suggesting that these may be involved in disulfide bond formation. This study demonstrates that a readily oxidized compound, such as the primate UV filter 3OHKyn, represents an oxidative stress within the lens and that such oxidative processes can be exacerbated if the concentration of oxygen within the lens is increased. We speculate that this factor may account for the evolution of unusually high levels of glutathione reductase in human lenses.

Systemic administration of 4-chlorokynurenine prevents quinolinate neurotoxicity in the rat hippocampus.

The synthetic compound 4-chlorokynurenine has been shown to be enzymatically transaminated to the selective glycine(B) receptor antagonist 7-chlorokynurenate. Since 4-chlorokynurenine, in contrast to 7-chlorokynurenate, readily penetrates the blood-brain barrier, the present study evaluated its neuroprotective properties after systemic administration in rats. Intrahippocampal injection of the NMDA receptor agonist quinolinate (15 nmol/l microl) was used as the neurotoxic paradigm. Serum and hippocampal tissue measurements confirmed that 4-chlorokynurenine serves as an effective pro-drug of 7-chlorokynurenate both in the periphery and in the brain. These studies and complementary hippocampal microdialysis experiments compared the effects of single and repeated injections of 4-chlorokynurenine (50 or 200 mg/kg, intraperitoneal (i.p.), 10 min prior to an intrahippocampal quinolinate injection; or 50 mg/kg, i.p., 10 min before and 30, 120 and 360 min after quinolinate). With the multiple-dosing regimen, extracellular 7-chlorokynurenate levels in the hippocampus reached a maximum of approximately 750 nM 7 h after quinolinate and gradually decreased with a half-life of about 3 h. In contrast, a single injection of 200 mg/kg 4-chlorokynurenine resulted in a considerably shorter rise in extracellular 7-chlorokynurenate without yielding higher peak levels. In separate animals, repeated treatment with 50 mg/kg 4-chlorokynurenine, but not a single injection of 200 mg/kg of the pro-drug, provided total protection against quinolinate-induced excitotoxicity. These data suggest that a prolonged and functionally relevant blockade of hippocampal glycine(B) receptors can be achieved after the systemic administration of 4-chlorokynurenine.

L-tryptophan-kynurenine pathway metabolite 3-hydroxyanthranilic acid induces apoptosis in macrophage-derived cells under pathophysiological conditions.

Accumulation of L-kynurenine and 3-hydroxyanthranilic acid (3HAA) occurs in the monocyte-derived cells following immune stimulation, and may derive from L-tryptophan following induction of indoleamine-2,3-dioxygenase. In the present study, we evaluate the possibility that 3HAA acts as an endogenous inducer of monocyte/macrophage apoptosis. Supplementation with 200 microM of 3HAA, but not other L-tryptophan metabolites tested, significantly increased the number of apoptotic cells in both THP-1 and U937 cells. Catalase, superoxide dismutase and manganese ions markedly enhanced apoptosis in the presence of 3HAA in these cells. The present results suggest that 3HAA induces the macrophage/monocyte apoptosis under certain conditions, which may be relevant to pathophysiology of inflammatory conditions.

Gamma-interferon-induced inhibition of the growth of Rickettsia prowazekii in fibroblasts cannot be explained by the degradation of tryptophan or other amino acids.

We examined the role of amino acid deprivation in gamma-interferon-induced (IFN-gamma) suppression of the growth of Rickettsia prowazekii in mouse L929 cells and human fibroblasts by measuring the amino acid pools in untreated and IFN-gamma-treated cells. In recombinant IFN-gamma-treated cultures of human fibroblasts, tryptophan was undetectable in both the intracellular pool and the extracellular medium. In contrast, tryptophan was not depleted from the intracellular pool or the extracellular medium of L929 cells treated with recombinant IFN-gamma or crude mouse lymphokines. None of the other amino acids measured was severely depleted in IFN-gamma-treated L929 cells and human fibroblasts. Extracts prepared from IFN-gamma-treated human fibroblasts exhibited indoleamine 2,3-dioxygenase activity, converting tryptophan into products that cochromatographed with N-formylkynurenine and kynurenine; however, extracts prepared from untreated human fibroblasts, untreated L929 cells, recombinant IFN-gamma-treated L929 cells, and mouse lymphokine-treated L929 cells did not degrade tryptophan. Human HeLa cells resembled the human fibroblasts in that they degraded tryptophan after IFN-gamma treatment. Similarly, mouse 3T3-A31 cells and mouse embryo fibroblasts resembled mouse L929 cells in that they did not degrade tryptophan. Supplementation of the extracellular medium with additional tryptophan reconstituted the tryptophan pool in mock-infected and R. prowazekii-infected, X-irradiated, IFN-gamma-treated human fibroblasts to values greater than those observed in untreated control cultures. However, reconstitution of the tryptophan pool did not relieve IFN-gamma-induced inhibition of rickettsial growth. Addition of kynurenine or N-formylkynurenine to rickettsia-infected human fibroblasts at concentrations four times the usual tryptophan concentration did not inhibit growth of R. prowazekii. We conclude that neither tryptophan depletion nor depletion of the other amino acids studied explains the inhibitory effect of IFN-gamma on rickettsial growth in mouse L929 cells. In IFN-gamma-treated human fibroblasts, either tryptophan depletion is not involved in the inhibition of rickettsial growth or tryptophan depletion and some other mechanism(s) together contribute to the inhibition of rickettsial growth.