Enantiomeric Resolution and Enantiomer Isolation of H2 TPPS4†J-Aggregate from Aqueous Solution Is Enabled by Vortexes.

Protonated achiral H2 TPPS4 spontaneously self-arranges at acids pH and high ionic strength to build mesoscopic J-aggregates that are intrinsically chiral. According to the symmetry rule aggregation leads to a racemate that, however, can be unbalanced by chemical (chiral pollutants) or physical stimuli (as vortexing the solution). Vortexing the title racemate, in principle, might either induce chiral separation or chiral enrichment. Indeed, herein it is shown that vortices enable the resolution of this racemic solution exploiting the tendency to deposit, onto the quartz cuvette walls, of the enantiomer favored by the stirring sense. Simultaneously, over time, it was found that the opposite chiral conformation becomes prevalent in solution realizing a significant enantiomeric resolution. Therefore, after removing all stirring-favored chiral J-aggregate from the solution, the recovering and isolating of the desired enantiomers from the cuvette walls was successfully obtained without complex procedures. In this sense, it has been demonstrated that the stirring forces are executively able to fulfil the chiral separation in H2 TPPS4 J-aggregates, employed as model of a self-assembled system in aqueous solution.

Effects of PEG-interferon alpha plus ribavirin on tryptophan metabolism in patients with chronic hepatitis C.

The currently recommended therapy for chronic hepatitis C (HCV) is a combination of pegylated interferon-alpha (PEG-IFN alpha) and ribavirin. Psychiatric disorders, including depression, are frequent in HCV patients under therapy. We investigated the effect of the antiviral treatment on tryptophan (Trp) metabolism along both serotonin pathway (via 5-hydroxytryptophan, 5-HTP) and kynurenine (Kyn) pathway and on the onset of depressive symptoms in patients with HCV. The key enzyme of the Kyn pathway is indoleamine 2,3-dioxygenase (IDO), an intracellular haem protein enzyme expressed in several tissues. It was also investigated the influence of the therapy with PEG-IFN-alpha-2a or PEG-IFN-alpha-2b plus oral ribavirin and possible differences between genders. Free and total Trp, 5-hydroxytryptophan (5-HTP) and Kyn serum concentrations and the presence of depressive symptoms [Beck Depression Inventory (BDI) scores] were evaluated in 45 patients with HCV infection treated with PEG-IFN alpha-2a or -2b at four different times: baseline (before treatment), 1 and 6 months during therapy, and 3 months after the end of therapy. The concentration of serum total TRP (free+protein bound) as well as that of 5-HTP significantly decreased after 1 and 6 months of therapy, and then returned to baseline values 3 months after the end of therapy, while the levels of free TRP did not vary significantly during and after the therapy. On the contrary, the time course of Kyn markedly arose during treatment, paralleled by a significant increase of [Kyn/Trp]×10(3) ratio, an index used to measure IDO activity. No significant difference was detected between males and females neither between PEG-IFN-alpha-2a or -2b treatment. The BDI scores significantly increased during therapy, and returned to baseline values 3 months after the end of therapy. Our results support the hypothesis that the increased IDO-mediated tryptophan metabolism along the Kyn pathway, leading to plasma Trp depletion and a decline of serotonin pathway, concurs to the development of depressive symptoms observed in HCV patients undergoing IFN-alpha therapy.

The protein profile of Theobroma cacao L. seeds as obtained by matrix-assisted laser desorption/ionization mass spectrometry.

The water-soluble protein profile of the seeds of green, red, and yellow Theobroma cacao L. fruits has been determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). The seeds were powdered under liquid nitrogen and defatted. The residues were dialyzed and lyophilized. The obtained samples were suspended in the matrix solution of sinapinic acid. The obtained MALDI mass spectra showed the presence of a wide number of proteins with molecular weight ranging from 8000 to 13,000 Da and a cluster of peaks centered at 21,000 Da that were attributed to albumin. The abundance of this peak was found to depend on the different portion of the seed (husk, apical and cortical parts); however, the MALDI mass spectra obtained from the different varieties of cocoa were practically superimposable. Changes in the protein profiles were also observed after the cocoa seeds were treated by fermentation and roasting, which are processes usually employed for the commercial production of cocoa.

First report of the lace bug Neoplerochila paliatseasi (Rodrigues, 1981) (Hemiptera: Tingidae) infesting cultivated olive trees in South Africa, and its complete mitochondrial sequence.

Olive lace bugs are small phytophagous Hemipteran insects known to cause agricultural losses in olive production in South Africa. Plerochila australis (Distant, 1904) has been reported as the species responsible for damage to olive trees; however, the diversity of olive lace bug species in the region has lacked attention. Adult olive lace bugs were collected incidentally from wild and cultivated olive trees in the Western Cape Province, and identified as P. australis and Neoplerochila paliatseasi (Rodrigues, 1981). The complete mitochondrial genome of a representative specimen of N. paliatseasi was sequenced, and used for comparative mitogenomics and phylogenetic reconstruction within the family. Furthermore, the value of DNA barcodes for species identification in Tingidae was assessed using genetic clustering and estimates of genetic divergence. The patterns of genetic clustering and genetic divergence of COI sequences supported the morphological identification of N. paliatseasi, and the utility of DNA barcoding methods in Tingidae. The complete mitogenome sequence had the typical Metazoan gene content and order, including 13 PCGs, 22 tRNAs, two rRNAs, and an AT-rich non-coding region. A+T content was high, as commonly found in Tingidae. The phylogenetic reconstruction recovered Agramma hupehanum (Drake Maa 1954) as basal to Tingini, and as a sister species to N. paliatseasi. Stephanitis Stål 1873 and Corythucha Stål 1873 were monophyletic, but Metasalis populi (Takeya 1932) was not recovered as sister to Tingis cardui (Linnaeus 1746), as expected. The mitochondrial phylogeny of the family Tingidae has been recovered inconsistently across different studies, possibly due to sequence heterogeneity and high mutation rates. Species diversity of olive lace bugs in South Africa was previously underestimated. The presence of P. australis was confirmed in both wild and cultivated olives, and N. paliatseasi is reported in cultivated olives for the first time. These results warrant further investigation on the diversity and distribution of olive lace bugs in the Western Cape to inform pest control strategies.

The complete mitochondrial genome of Bactrocera biguttula (Bezzi) (Diptera: Tephritidae) and phylogenetic relationships with other Dacini.

Bactrocera biguttula is an African olive fruit fly that does not attack cultivated olives but rather develops in the fruits of wild species of Olea and Noronhia. The complete mitochondrial genome of an individual specimen was characterized in comparison to other Bactrocera. The phylogenetic relationships of B. biguttula with other Dacini were investigated, with special focus on B. oleae, an agricultural pest known to attack cultivated and wild olives. The sequence had a total length of 15,829 bp, and included the typical features of insect mitogenomes, similarly to the other Bactrocera analysed. Start codons included ATG, ATC, ATT, and TCG (in COI). The majority of stop codons (TAA) were fully encoded, whereas in some cases only TA or T were present. The complete sequence was biased towards A + T, with a positive AT-skew and a negative GC-skew. The predicted cloverleaf structure of tRNASer1 showed absence of the DHU arm, a common feature in insects and other Metazoans. Phylogenetic reconstruction showed that B. biguttula and B. oleae are sister species, having diverged from a common ancestor < 10 Myr ago. This result warrants future genomic comparisons between these two closely related species for investigating the specific adaptations to the different hosts.

Influence of Antisynthetase Antibodies Specificities on Antisynthetase Syndrome Clinical Spectrum Time Course.

Antisynthetase syndrome (ASSD) is a rare clinical condition that is characterized by the occurrence of a classic clinical triad, encompassing myositis, arthritis, and interstitial lung disease (ILD), along with specific autoantibodies that are addressed to different aminoacyl tRNA synthetases (ARS). Until now, it has been unknown whether the presence of a different ARS might affect the clinical presentation, evolution, and outcome of ASSD. In this study, we retrospectively recorded the time of onset, characteristics, clustering of triad findings, and survival of 828 ASSD patients (593 anti-Jo1, 95 anti-PL7, 84 anti-PL12, 38 anti-EJ, and 18 anti-OJ), referring to AENEAS (American and European NEtwork of Antisynthetase Syndrome) collaborative group's cohort. Comparisons were performed first between all ARS cases and then, in the case of significance, while using anti-Jo1 positive patients as the reference group. The characteristics of triad findings were similar and the onset mainly began with a single triad finding in all groups despite some differences in overall prevalence. The "ex-novo" occurrence of triad findings was only reduced in the anti-PL12-positive cohort, however, it occurred in a clinically relevant percentage of patients (30%). Moreover, survival was not influenced by the underlying anti-aminoacyl tRNA synthetase antibodies' positivity, which confirmed that antisynthetase syndrome is a heterogeneous condition and that antibody specificity only partially influences the clinical presentation and evolution of this condition.

A Response Surface Methodology Approach to Investigate the Effect of Sulfur Dioxide, pH, and Ethanol on DbCD and DbVPR Gene Expression and on the Volatile Phenol Production in Dekkera/Brettanomyces bruxellensis CBS2499.

Dekkera/Brettanomyces bruxellensis, the main spoilage yeast in barrel-aged wine, metabolize hydroxycinnamic acids into off-flavors, namely ethylphenols. Recently, both the enzymes involved in this transformation, the cinnamate decarboxylase (DbCD) and the vinylphenol reductase (DbVPR), have been identified. To counteract microbial proliferation in wine, sulfur dioxide (SO2) is used commonly to stabilize the final product, but limiting its use is advised to preserve human health and boost sustainability in winemaking. In the present study, the influence of SO2 was investigated in relation with pH and ethanol factors on the expression of DbCD and DbVPR genes and volatile phenol production in D. bruxellensis CBS2499 strain under different model wines throughout a response surface methodology (RSM). In order to ensure an exact quantification of DbCD and DbVPR expression, an appropriate housekeeping gene was sought among DbPDC, DbALD, DbEF, DbACT, and DbTUB genes by GeNorm and Normfinder algorithms. The latter gene showed the highest expression stability and it was chosen as the reference housekeeping gene in qPCR assays. Even though SO2 could not be commented as main factor because of its statistical irrelevance on the response of DbCD gene, linear interactions with pH and ethanol concurred to define a significant effect (p < 0.05) on its expression. The DbCD gene was generally downregulated respect to a permissive growth condition (0 mg/L mol. SO2, pH 4.5 and 5% v/v ethanol); the combination of the factor levels that maximizes its expression (0.83-fold change) was calculated at 0.25 mg/L mol. SO2, pH 4.5 and 12.5% (v/v) ethanol. On the contrary, DbVPR expression was not influenced by main factors or by their interactions; however, its expression is maximized (1.80-fold change) at the same conditions calculated for DbCD gene. While no linear interaction between factors influenced the off-flavor synthesis, ethanol and pH produced a significant effect as individual factors. The obtained results can be useful to improve the SO2 management at the grape harvesting and during winemaking in order to minimize the D./B. bruxellensis spoilage.

A mass spectrometric investigation on the possible role of tryptophan and 7-hydroxytryptophan in melanogenesis.

The activity of tyrosinase and peroxidase + H2O2 in promoting melanogenesis from tryptophan (Trp) and 7-hydroxytryptophan (7-HTP) has been investigated. The reaction samples have been drawn at different reaction times and analysed by MALDI mass spectrometry. The data obtained showed that tryptophan undergoes, under tyrosinase and peroxidase action, an oligomerization process mainly due to the reaction of anthranilic acid (AA) and Trp. However, analysing the UV and fluorescence data, it is seen that the oligomers cannot belong to the melanin pattern, but their possible role in melanogenesis is not to be excluded. Once it reacts with the two enzymes, 7-hydroxytryptophan leads to dark brown products, indicating its possible role in melanin production. In contrast to what was observed in the case of 5-hydroxytryptophan, for which oligomers were constituted by 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT) units, the MALDI data indicate a sharply different behaviour for 7-HTP. In fact, in the case of 5-hydroxytryptophan, oligomerization takes place through the formation of 5-hydroxytryptamine and the oligomerization products are due to mixed 5-HTP-5-HT oligomers. In the case of 7-hydroxytryptophan, the formation of 7-hydroxytryptamine (7-HT) is also observed, but it does not seem to play any role; the only oligomerization products formed are due to the reaction of 7-hydroxytryptophan and AA. The data so obtained indicate that 7-hydroxytryptophan acts like an effective melanin precursor in the presence of both tyrosinase and peroxidase + H2O2.

An investigation on the possible role of melatonin in melanogenesis.

The possible role of melatonin in melanogenesis was investigated by performing reactions of melatonin with peroxidase + H2O2 or H2O2 only, in the presence or absence of UV irradiation. Samples of the reaction mixtures were drawn at different times (from 15 to 480 min), the enzyme (when present) was removed by ultrafiltration and the samples so obtained were analyzed by MALDI/MS. The results show that melatonin undergoes oligomerization reaction with peroxidase + H2O2, leading to heptameric species. For high reaction times the MALDI/MS data do not show the formation of larger oligomers, but UV-vis spectroscopy indicates that the oligomerization processes proceed. The failure of MALDI-TOF in the identification of larger oligomers was related to the chemical-physical and morphological behavior of melanins. In the case of UV irradiation, the formation of species originating from the O- and O2 addition to melatonin, which activate new oligomerization channels, has been observed.

Cloricromene effect on the enzyme activities of the tryptophan-nicotinic acid pathway in diabetic/hyperlipidemic rabbits.

Since alterations of tryptophan metabolism have been reported in diabetes and atherosclerosis, it was thought of interest to investigate any role of cloricromene through the influence on the oxidative metabolism of the amino acid by using diabetic/hyperlipidemic rabbits. Male 4-month-old New Zealand white rabbits, fed a diet enriched with 1% cholesterol and 10% corn oil, were made diabetic with alloxan. During the hyperlipidemic diet, a group of rabbits was treated with cloricromene (10 mg/kg/day subcutaneously plus 1.5 mg/kg/day intravenously, for 5 weeks). The other group received saline. Normometabolic New Zealand rabbits fed standard diet, treated or not with cloricromene, were used as control. The specific activities of liver tryptophan 2,3-dioxygenase and small intestine indole 2,3-dioxygenase were not significantly changed by the drug treatment. Also the specific activities of other enzymes of the kynurenine pathway in the liver and kidneys, specifically kynurenine 3-monooxygenase, kynureninase and kynurenine-oxoglutarate transaminase, did not show any significant difference in both tissues between the two groups of rabbits. On the contrary, 3-hydroxyanthranilate 3,4-dioxygenase activity in the liver of diabetic/hyperlipidemic rabbits and control rabbits treated with cloricromene showed a slight increase in comparison with untreated animals. Conversely, the specific activity of the enzyme in kidneys was not affected by the drug treatment in diabetic/hyperlipidemic animals but was reduced in controls. Aminocarboxymuconate-semialdehyde decarboxylase specific activity remained unchanged in the liver following cloricromene treatment, instead the specific activity of the enzyme in the kidneys of the diabetic/hyperlipidemic rabbits was significantly increased by the drug, with a value more than double in comparison to untreated animals. The activity of the scavenger enzyme Cu/Zn superoxide dismutase (Cu/Zn SOD) in the small intestine was also determined and found significantly increased of about twice as much in the group of diabetic/hyperlipidemic rabbits treated with cloricromene. In conclusion, in diabetic/hyperlipidemic rabbits, cloricromene appeared to influence the enzymes involved in the last steps of tryptophan oxidative metabolism through the kynurenine pathway. This, together with the antioxidant action through the activation of Cu/Zn SOD, might deserve further investigation for evaluating any link between the observed experimental findings at the level of the kynurenine pathway and the clinical effect of the drug.

Enzyme activities along the tryptophan-nicotinic acid pathway in alloxan diabetic rabbits.

Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits. The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits. These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid-->nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.

The effect of age on the enzyme activities of tryptophan metabolism along the kynurenine pathway in rats.

BACKGROUND: Quinolinic acid and other kynurenine metabolites of the oxidative metabolism of tryptophan play an important role in several pathophysiological conditions. We aimed to study the effect of age on the enzyme activities of tryptophan metabolism along the kynurenine pathway. METHODS: Enzyme activity was investigated in liver, kidneys and small intestine obtained from Sprague-Dawley rats of various ages (1 week, 2-3, 12 and 18 months). RESULTS: We found age-related differences in the liver tryptophan 2,3-dioxygenase, small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase activities, which decreased significantly with age. Also liver kynureninase activity declined with age, while the activity in kidneys did not show an evident age-related pattern from 2-3 months to 18 months of age. Liver kynurenine oxoglutarate transaminase was quite similar through all considered age groups, while the activity in kidneys was significantly lower in newborn rats and progressively increased up to 12 months, then significantly decreased at 18 months of age. Liver and kidney 3-hydroxyanthranilate 3,4-dioxygenase progressively and significantly increased from newborns to 12 months of age; in the group of rats aged 18 months, the enzyme activity tended to diminish, although not significantly. The liver aminocarboxymuconate-semialdehyde decarboxylase activity increased up to 12 months of age, then tended to decrease at 18 months, while in the kidneys, in which the activity was higher than in the liver at all the considered ages, the activity remained constantly elevated from 2-3 months to 18 months of age. CONCLUSIONS: A progressive decline in the enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rat tissues was found with age, except for aminocarboxymuconate-semialdehyde decarboxylase, which, on the contrary, was increased after 2-3 months to the other older groups of age. The altered metabolism of tryptophan with ageing can lead to a decreased biosynthesis of nicotinic acid, tryptophan being the major source of body stores of NAD coenzymes, which are involved in almost all biogenetic and biosynthetic pathways of the organism.

Influence of age on Cu/Zn-superoxide dismutase and indole 2,3-dioxygenase activities in rat tissues.

The aim of this study was to investigate in vitro the variations with age of the activities of the two antioxidant enzymes Cu/Zn-superoxide dismutase (SOD) and indole 2,3-dioxygenase (IDO) in metabolically active tissues of rats of various ages. In rats aged one week and 2-3 months the highest Cu/Zn-SOD activity was found in the liver and the lowest in the small intestine. At 12 and 18 months of age, the activity was higher in the brain and kidneys, when compared to the small intestine, lungs and liver. Cu/Zn-SOD activity decreased significantly after 2-3 months of age with advancing age in all tissues examined. In newborn rats IDO activity was present only in the small intestine. In the group of rats aged 2-3 months, the highest specific activity was observed in the small intestine and the lowest in the lungs and kidneys, whereas at 12 months of age, the highest IDO activity was found in the brain, with kidneys presenting the lowest activity. At 18 months, IDO returned to be more elevated in the small intestine. At 12 months of age the values of IDO in the tissues varied slightly, while at 18 months similar activities were found between the lungs and brain and between the small intestine and kidneys. In relation to age, IDO specific activity declined in the small intestine, after 2-3 months of age. In the lungs, the activity remained unchanged; in the brain and in the kidneys activity decreased significantly from 2-3 to 18 months of age. In conclusion, this study demonstrates an age-related decline in Cu/Zn-SOD and IDO activities, the two enzymes responsible for scavenging O2*-.

Tryptophan metabolism along the kynurenine pathway in diet-induced and genetic hypercholesterolemic rabbits.

BACKGROUND: The activity of nicotinic acid in hypercholesterolemia has been poorly understood. In man, nicotinic acid derives for the most part from tryptophan along the tryptophan-nicotinic acid pathway, also called the kynurenine pathway, kynurenine being the key metabolite in this process. In the present paper, we investigated if, in animals with hypercolesterolemia, degradation of tryptophan to nicotinic acid along the kynurenine pathway was perturbated. METHODS: Liver, kidney and intestine enzyme activities of the tryptophan-nicotinic acid pathway in normolipidemic, diet-induced hyperlipidemic New Zealand and heritable hypercholesterolemic Watanabe (WHHL) rabbits were determined. RESULTS: Liver tryptophan 2,3-dioxygenase (TDO) activity was present only as a holoenzyme and was higher in the controls than in the hyperlipidemic and Watanabe rabbits, but no difference was present between the group fed an atherogenic hyperlipidic diet and the WHHL rabbits. Small intestine indole 2,3-dioxygenase (IDO) did not vary significantly among the three groups but was higher in comparison with liver TDO activity. In liver, kynurenine 3-monooxygenase and kynurenine-oxoglutarate transaminase activities did not show any significant difference among the three groups of rabbits. Kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase activities per g of fresh tissue decreased significantly in the group of hyperlipidemic and in WHHL rabbits. In the kidneys, kynurenine 3-monooxygenase and kynureninase activity did not change significantly in the three groups of rabbits; kynurenine-oxoglutarate transaminase activity per g of fresh tissue decreased in both hyperlipidemic groups, but no significant difference was observed between hyperlipidemic and Watanabe rabbits. 3-Hydroxyanthranilate 3,4-dioxygenase activity in kidney was decreased markedly in hyperlipidemic and Watanabe rabbits, but there was no difference between the two hypercholesterolemic groups. Aminocarboxymuconate-semialdehyde decarboxylase activity did not change. Thus 3-hydroxyanthranilate 3,4-dioxygenase may be an important regulatory mechanism in the control of the flow of tryptophan along the kynurenine pathway to NAD in hypercholesterolemic rabbits. CONCLUSIONS: This study first demonstrates that in rabbits, hypercholesterolemia, both diet- or genetically induced, can influence the enzyme activities of the tryptophan-nicotinic acid pathway leading to a decreased formation of nicotinic acid, and thus NAD.

Enzyme activities along the kynurenine pathway in mice.

Tryptophan metabolism was studied in adult male Swiss mice by determining enzyme activities along the kynurenine pathway. The following enzymes were assayed: liver tryptophan 2,3-dioxygenase, small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Liver tryptophan 2,3-dioxygenase was present only as a holoenzyme: similar results were obtained in the absence or in the presence of the cofactor haematin. The specific activity of small intestine indole 2,3-dioxygenase was higher than that of tryptophan 2,3-dioxygenase. As superoxide dismutase was very active in mouse intestine, this enzyme may be one of the rate controlling factors of the indole 2,3 dioxygenase activity. Kynurenine 3-monooxygenase appeared to be very active. Kidneys showed higher activity than liver. Instead, kynureninase was more active in liver, but activity was lower than that demonstrated by the other enzymes of the kynurenine pathway. Conversely, kynurenine-oxoglutarate transaminase was much more active in kidney than in liver. However, the most active enzyme along the kynurenine pathway was 3-hydroxyanthranilate 3,4-dioxygenase, with liver showing the highest activity; aminocarboxymuconate-semialdehyde decarboxylase, which showed similar values in both liver and kidney, showed activity markedly lower than 3-hydroxyanthranilate 3,4-dioxygenase. Serum tryptophan appeared to be 87% bound to proteins. Results demonstrate that, in mouse, tryptophan is mainly metabolised along the kynurenine pathway. Therefore, mouse is a suitable animal model for studying tryptophan metabolism in the pathological field.

The kynurenine pathway enzymes in healthy and hyperlipidemic rabbits.

Tryptophan metabolism along the kynurenine pathway in normolipidemic and diet-induced hyperlipidemic New Zealand rabbits was studied. The activities of liver tryptophan 2,3-dioxygenase small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate semialdehyde decarboxylase (picolinic carboxylase) were determined. Liver tryptophan 2,3-dioxygenase (TDO) was present only as a holoenzyme. In fact, similar results were found in the absence or presence of the cofactor haematin. In addition, TDO activity was higher in normolipidemic than in hyperlipidemic rabbits. Small intestine indole 2,3-dioxygenase did not change significantly among the two groups of animals, but was higher than liver TDO. Liver and kidney kynurenine 3-monooxygenase activities did not change significantly whereas kynurenine-oxoglutarate transaminase activity decreased only per g of fresh kidney in hyperlipidemic rabbits. Kynureninase activity decreased significantly per g of fresh tissue only in liver. 3-Hydroxyanthranilate 3,4-dioxygenase, both as specific activity and per g of fresh tissue and aminocarboxymuconate-semialdehyde decarboxylase activities showed significantly lower values per g of fresh kidney in hyperlipidemic rabbits compared with controls. Therefore, hyperlipidemia can influence enzyme activities along the kynurenine pathway, leading to a reduction in the biosynthesis of NAD coenzymes.

Metabolism of tryptophan along the kynurenine pathway in alloxan diabetic rabbits.

Enzyme activities along the kynurenine pathway were assayed in the tissues of New Zealand white rabbits made diabetic with alloxan treatment and hypercholesterolemic with a high-cholosterol diet. Activities are expressed as nmoles of product forming per min per mg of protein and per g of fresh tissue. Liver tryptophan 2,3-dioxygenase (TDO) was present only in holoenzyme form. This activity decreased in diabetic-hyperlipidemic and hyperlipidemic rabbits in comparison with healthy animals. Small intestine indole 2,3-dioxygenase was markedly higher than liver TDO in all rabbit groups, but did not show any significant difference in the values among the three groups. Mitochondrial kynurenine 3-monooxygenase activity was higher in liver than in kidney, but were unchanged with respect to controls. Kynureninase showed similar specific activities in the liver and kidney among groups, whereas the activity per g of fresh tissue was significantly lower in the liver of hyperlipidemic and kidney of diabetic-hyperlipidemic rabbits than in healthy animals. Kynurenine-oxoglutarate transaminase and kynureninase showed lower values in kidney, but not in liver, of diabetic-hyperlipidemic rabbits. However, 3-hydroxyanthranilate 3,4-dioxygenase activity was reduced in both liver and kidney of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared with controls. Instead, aminocarboxymuconate-semialdehyde decarboxylase (picolinic carboxylase) activity was significantly higher in diabetic-hyperlipidermic rabbits in comparison with hyperlypidemic and control rabbits. Therefore, in diabetic rabbits, there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid --> nicotinic acid step, which has the effect of reducing the biosynthesis of NAD in diabetes.

Kynurenine pathway enzymes in different species of animals.

Kynurenine pathway enzyme activities, liver tryptophan 2,3-dioxygenase (TDO), small intestine indole 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase, were assayed in rabbits, rats, mice and guinea pigs. Their activities varied among species. Especially, TDO was present as both holoenzyme and apoenzyme only in rat, while the other species, rabbit, mouse and guinea pig, only showed holoenzyme activity. Mitochondrial liver and kidney kynurenine 3-monooxygenase activities were much higher in mouse and rat, with rabbit showing the lowest activity. Kynureninase activity showed similar values in both liver and kidney in each species. However, lower activity was present in rabbit. As regards kynurenine-oxoglutarate transaminase, the highest activity appeared in kidney, in all species studied. 3-Hydroxyanthranilate 3,4-dioxygenase activity showed different behaviour in the four species. In rabbit, its activity was higher in kidney than in liver; in rat and mouse, it was viceversa; and in guinea pig, both liver and kidney had similar activity. Instead, the activity of aminocarboxymuconate-semialdehyde decarboxylase was higher in kidney than in liver only in guinea pig. Serum tryptophan concentrations were also determined. Rabbit and guinea pig showed similar values, whereas in rat and mouse, serum tryptophan levels were higher, rat having the highest concentrations. In all species assayed, the free fraction was present as 11-12% of total tryptophan.

Kynurenine pathway enzymes in guinea pigs.

In some animals, the administration of repeated doses of tryptophan can cause death. It has been reported that guinea pig does not survive repeated doses of tryptophan, due to the absence of the hormonal induction mechanism of liver tryptophan 2,3-dioxygenase (TDO). Therefore, it was of interest to investigate if guinea pig is an animal model suitable for studying tryptophan metabolism. The activities of the enzymes of the kynurenine pathway were determined. Liver TDO was present only as a holoenzyme; kynurenine 3-monooxygenase showed similar, but very high, activity in both liver and kidney. Liver and kidney kynureninase values were also similar, whereas kynurenine-oxoglutarate transaminase activity was higher in kidney than in liver. 3-Hydroxyanthranilate 3,4-dioxygenase gave similar, but very high, values in both liver and kidney, whereas aminocarboxymuconate-semialdehyde decarboxylase activity was double in kidney with respect to liver, but much lower than that of 3-hydroxyanthranilate 3,4-dioxygenase. Total and free tryptophan concentrations in serum were also determined. The free fraction was about 10% of total tryptophan.

Tryptophan metabolism in rabbits.

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway were studied in male New Zealand white rabbits. Activities are expressed both as specific activity and per g of fresh tissue. Liver tryptophan 2,3-dioxygenase activity (TDO), when assayed in either the absence (holoenzyme) or presence of added haematin (apoenzyme), did not change. Therefore, in rabbit, TDO was present only in holoenzyme form. Small intestine indole 2,3-dioxygenase was significantly higher than liver TDO. Mitochondrial kynurenine 3-monooxygenase was higher in liver than in kidney. Kynureninase activity was similar in both tissues, whereas kynurenine-oxoglutarate transaminase was markedly higher in kidney than in liver. 3-Hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase activities were higher in kidney than in liver. However, the former enzyme showed much higher activity than the latter. These findings suggest that, in rabbit, tryptophan is mainly metabolised along the kynurenine pathway although the apo-TDO enzyme is lacking, as high indole 2,3-dioxygenase activity can obviate this lack.