A study of tryptophan metabolism via serotonin in ventricular cerebrospinal fluid in HIV-1 infection using a neuroendoscopic technique.

In this paper we report the study of tryptophan metabolism via serotonin in ventricular CSF in HIV-1 infection in order to investigate the origin of tryptophan metabolites in the human brain. The patients (n=4) were affected with non-communicating hydrocephalus. One of these also was suffering from HIV-1 infection. The CSF was withdrawn from different sites of the cerebral cavity with a neuroendoscopic procedure which allows an accurate exploration of all the cerebral ventricles. The measurement of tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, and melatonin was carried out by HPLC with fluorometric detection. In HIV-1 infection the highest concentration of tryptophan is present in the CSF of the choroid plexus; however, the levels are markedly lower than those in hydrocephalic individuals (control group). 5-Hydroxytryptophan CSF content is higher in HIV-1 infection than in hydrocephalic controls in all districts examined. Regarding serotonin, a great difference appears in the choroid plexus and in the pituitary recess between the HIV-1 infected patient and the control group. The values of 5-hydroxyindoleacetic acid are much lower in the CSF of the HIV-1 infected patient than in hydrocephalic controls. Melatonin levels appear to fluctuate largely but, in the HIV-1 infection, a great variability is present among the sites of CSF withdrawal. The third ventricle contains the highest concentration of melatonin and the choroid plexus and the pituitary recess the lowest. All the melatonin concentrations in HIV-1 infection are largely different than in hydrocephalic controls. This is the first report on the measurement of tryptophan metabolites via serotonin in ventricular CSF in HIV-1 infection.

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.

Study of tryptophan metabolism via serotonin in cerebrospinal fluid of patients with noncommunicating hydrocephalus using a new endoscopic technique.

By a recent minimally invasive neuroendoscopic technique, the cerebral ventricles have been reached in a quick, reliable, and harmless way, making possible the study of cerebrospinal fluid (CSF) of the lateral ventricles and, above all, the CSF adjacent to the walls of the third ventricle. Tryptophan, 5-hydroxytryptophan, serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) were measured in CSF by HPLC equipment. Twenty-six patients affected with noncommunicating hydrocephalus were enrolled in the study and, as controls, 28 subjects not suffering from any neurological disease. The concentrations of tryptophan were higher in right ventricular CSF than in lumbar CSF (P < 0.01). 5-HT was detectable in the CSF of the right ventricle of hydrocephalic patients. 5-HIAA was higher in right ventricular CSF than in cisternal and lumbar CSF (P < 0.01), both in controls and in hydrocephalic patients. However, there was a higher concentration of 5-HIAA in right ventricular (P < 0.05) and cisternal (P < 0.01) CSF in hydrocephalic patients in comparison with controls. In the CSF samples withdrawn during neuroendoscopy, 5-HT presented the highest concentrations in the pineal recess. The highest amounts of 5-HIAA were found in the choroid plexus, third and right ventricles, pituitary recess, and aqueduct, and the lowest in pineal recess, subarachnoid space, infundibulum, and interpeduncolar cistern. These results provide new insight into the fate of tryptophan and its metabolites via serotonin in the CSF and suggest the feasibility of the new neuroendoscopic technique for brain metabolic studies.

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.

Search for melanoma markers in plasma and serum samples.

Fourteen blood samples from patients with melanoma and eleven blood samples from healthy subjects were analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The study was focussed on species of low molecular weight, in the range 800-5000 Da, present in plasma and sera. While for healthy subjects plasma samples lead to the production of a higher number of ionic species, for melanoma patients a high number of diagnostic ions, present with high frequency and with quite high relative abundance, are present in particular in serum samples and to a lesser extent also in plasma. Since plasma samples are obtained more easily in comparison to sera, it is possible to suggest that also plasma can be used for these studies.

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.

An investigation on the role of 3-hydroxykynurenine in pigment formation by matrix-assisted laser desorption/ionization mass spectrometry.

In order to investigate the role of tryptophan and its metabolites in biogenesis of melanins, a study on the enzymatic reaction of 3-hydroxykynurenine with tyrosinase and peroxidase was performed. The reaction at different pH values was monitored by sampling at different times, with ultrafiltration used before analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The data obtained in this way showed that oligomerization processes take place with both enzymes, but with different behaviour, also depending on pH. 3-Hydroxykynurenine in the presence of tyrosinase at pH 6.0 leads to formation of xanthommatin, and at pH 8.0 hydroxanthommatin is formed in the first step of the reaction followed by formation of black-brown pigments. In contrast, the formation of oligomerization products by peroxidase action is observed in high yields under both acidic and basic conditions; however, at pH 6.0, a more extensive oligomerization process is observed. Thus peroxidase is able to activate oligomerization analogous to that observed in the case of tyrosinase without depending on the variation of pH. Due to the early formation of decarboxylated hydroxykynurenine, hydroxanthommatin and decarboxylated hydroxanthommatin, the enzymatic reaction leads to mixed oligomers, which can be considered as precursors of new pathways in pigment production.

Enzyme activities of tryptophan metabolism along the kynurenine pathway in various species of animals.

The purpose of this study was to investigate variations in the enzyme activities of the kynurenine pathway in various mammals (rabbit, mouse, rat, guinea-pig). 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 were analysed. Small intestine superoxide dismutase activity and free and total serum tryptophan were also measured. Liver tryptophan 2,3-dioxygenase was present as both holoenzyme and apoenzyme only in rat, while in the other species only holoenzyme activity was observed. Also, small intestine indole 2,3-dioxygenase activity was more abundant in rat than in the other animals studied. The highest activity of small intestine superoxide dismutase was found in rat, and the lowest in rabbit. Liver and kidney kynurenine 3-monooxygenase activity was very elevated and higher in mouse, followed by rat; rabbit showed the lowest activity. Kynureninase activity appeared to be much lower among the enzymes of the kynurenine pathway. However, guinea-pig showed higher activity in both liver and kidney in comparison with other species. With regard to kynurenine-oxoglutarate transaminase, all species examined here presented more abundant enzyme activity in kidney, the value being similar between rat and mouse. Guinea-pig was the animal with the lowest activity. 3-Hydroxyanthranilate 3,4-dioxygenase showed the highest activity of all the enzymes evaluated in the study, but with different levels in liver and kidney, varying among species. The most elevated activity of aminocarboxymuconate-semialdehyde decarboxylase was present in kidney of guinea-pig, and the lowest in rabbit. Serum concentrations of tryptophan were higher in rat, followed by mouse, rabbit and guinea-pig. In conclusion, the present study demonstrates that the enzyme activities of the kynurenine pathway are very active in tissues of the four species of mammals investigated. The proposed method of in vitro enzyme determination represents a valid alternative to study of the tryptophan metabolic route.

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.

Tryptophan metabolism along the kynurenine pathway in rats.

Enzyme activities along the kynurenine pathway, 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, involved in the catabolism of tryptophan, were studied in male adult Wistar albino rats. Intestine superoxide dismutase and serum tryptophan were also determined. Hepatic tryptophan 2,3-dioxygenase is present both as holoenzyme and apoenzyme, but the total activity is inferior to that of intestine indole 2,3-dioxygenase which, therefore, actively oxidizes tryptophan in rats. However, this activity is inhibited by scavengers for the superoxide anion, such as superoxide dismutase, which also shows to be active in small intestine of rat. However, the more active enzymes appeared to be kynurenine 3-monooxygenase and 3-hydroxyanthranilate 3,4-dioxygenase. The former is equally active in both liver and kidney, the latter is more active in liver. Kynurenine-oxoglutarate transaminase is much more active in kidney than in liver, and much more active than kynureninase, which shows similar activities in both tissues. In contrast to the high activity of 3-hydroxyanthranilate 3,4-dioxygenase, aminocarboxymuconate-semialdehyde decarboxylase is 30-35 times less active, showing the efficiency of conversion of tryptophan to NAD. These data demonstrate that rat is a useful animal model for studying tryptophan metabolism along the kynurenine pathway. Serum tryptophan appeared to be 90% bound to proteins. Results demonstrate that, in rat, tryptophan is mainly metabolised along the kynurenine pathway. Therefore, rat is a suitable animal model for studying tryptophan metabolism in the pathological field.

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.

Antiproliferative effect of indigoid pigments isolated from human urine.

Two indigoid pigments, red indirubin and blue indigotin, were isolated from the urine of healthy male subjects. The two pigments were assayed on HL60 (human promyelocytic cell line) cells. Preliminary results revealed the antiproliferative effect of the pigments. Indigotin seemed more effective than indirubin.

Involvement of 5-hydroxytryptophan in melanogenesis.

Tyrosine is generally considered to be the physiological precursor of melanins and tyrosinase the enzyme responsible. However, recent studies have shown that also peroxidases are involved in the biosynthesis of melanins. These enzymes use hydrogen peroxide to oxidise various phenol substrates. In this paper, we used a substrate other than tyrosine, i.e. 5-hydroxytryptophan, to verify if its peroxidase/H2O2-mediated oxidation gave rise to the formation of melanin. We also subjected 5-hydroxytryptophan to the action of tyrosinase, for comparison purposes. We observed that both enzymes converted this substrate to melanin and that peroxidase, in the presence of hydrogen peroxide, was much more effective than tyrosinase in catalysing the oxidative polymerization of 5-hydroxytryptophan, with the formation of insoluble black melanin-like pigments. Samples deriving from the reaction-substrate enzyme were ultrafiltered at different times through an Amicon ultrafiltration cell equipped with an Amicon Diaflo XM-50 membrane, in order to remove the enzyme, and immediately lyophilised. The resulting samples were analysed by matrix assisted laser desorption/ionisation (MALDI) mass spectrometry, which clearly identified several oligomer species in the reaction mixture. This work was undertaken to investigate the possible precursors of neuromelanin and the enzyme responsible for melanogenesis in brain, since although the central nervous system does not contain tyrosinase, it is rich in peroxidase and hydrogen peroxide.