Search

Two patients with exogenous ochronosis, an unfamiliar side effect of hydroquinone-containing bleaching creams, are presented. This disorder is characterized by progressive darkening of the area to which the cream is applied. Histologically there is deposition of yellow-brown pigment in the dermis, in addition to degeneration of collagen and elastic fibres. The chemical composition of the ochronotic material and the pathogenesis of this paradoxical effect of hydroquinone are unknown. Hydroquinone containing creams should not be used for longer than two years. If there is no improvement within six months, application should be discontinued.

A new sensitive method for the determination of catechol O-methyltransferase activity has been developed. The method is based on the O-methylation of the indolic intermediates of melanin metabolism. The substrate, 5,6-dihydroxyindole-2-carboxylic acid, is converted by the enzyme to two O-methylated products, which can be separated by high-performance liquid chromatography and measured with fluorimetric detection. The physiological presence of both substrate and products could be detected in crude melanoma cell extracts. The limit of sensitivity for detection of the O-methylated products is less than 0.5 pmol per injection. The method was compared with an earlier described HPLC method which makes use of uv detection of O-methylated products of 3,4-dihydroxybenzoic acid. The described method will be used to study the importance of catechol O-methyltransferase as a protective enzyme in (malignant) melanocytes.

Cultured pineal ependymal cells from rat fetuses aged 17 to 19 days post-coitum release into their media a substance that has hydroosmotic, antidiuretic and rat uterine activities. Paper chromatography of concentrates from culture media demonstrates that the substance possessing all the above activities was eluted from a slow moving region (Rf 0.15-0.35) with a peak at 0.20-0.30. This region is not significantly different from the Rf of synthetic arginine vasotocin (AVT) used as standard. Neither hydroosmotic nor rat uterine activities could be detected in the chromatographic eluates from the fast moving region corresponding to the Rf of synthetic oxytocin (0.55-0.65) used as standard. The chromatographic mobility of biological activities from culture media, the ratio of the activities as well as their susceptibility to tryptic digestion, demonstrates the presence of a basic peptide indistinguishable from synthetic AVT. The total amount of AVT released into the medium during 43 days of incubation is about 40 times greater than the amount contained in non-incubated pineal glands of the same age, strongly suggesting de novo synthesis of AVT.

Both the pineal nonapeptide hormone arginine vasotocin (AVT) (2.5 micrograms) administered intra-nasally and the pineal indole melatonin (50 mg) administered intravenously to three male narcoleptics (two with auxiliary symptoms and one with sleep attacks only), dramatically increased the amount of REM sleep and decreased REM sleep latency. The duration of the sleep onset REM periods in the two narcoleptics with auxiliary symptoms increased by more than 100 percent after AVT and melatonin administration. In the narcoleptic with sleep attacks only both AVT and melatonin induced REM periods at sleep onset. The hypothesis is advanced that narcolepsy represents an impairment of the melatonin-AVT control in the induction and circadian organization of REM sleep associated with an immaturity of REM triggering centers.

In the supernatant of melanoma cell culture SK mel 25 three indolic compounds - 5-hydroxy-6-methoxyindole, 5-hydroxy-6-methoxyindolyl-2-carboxylic, and 6-hydroxy-5-methoxyindolyl-2-carboxylic acids - have been identified. The supernatants were extracted with ethyl acetate, derivatized, and analyzed by gas chromatography-mass spectrometry. The significance of this finding is discussed.

Arginine vasotocin (AVT), in doses of 1.5 microgram, administered intranasally to two narcoleptics (a male aged 42 years and a female aged 35 years), as well as to 5 symptomatic hypersomniacs (3 females and 2 males), aged 25-57 years, induced very rapidly in most instances in sleep with REM periods at onset and markedly increased in amount of REM sleep during a fixed period of 180 min of sleep recording. After AVT administration there was a significant shortening of the time of occurrence of the first REM sleep period both in narcoleptics (t test = 0.05 greater than P greater than 0.025) and in symptomatic hypersomniacs (t test = 0.02 greater than P greater than 0.01).

A new possible pathogenesis of gallstones is described. The theory is based on the presence of indolic melanin precursors in biological fluids. It is proposed that the increased concentration of these compounds in bile could lead to the formation of melanin polymer as a network nucleus for a future concrement. An activation of the pigmentary system (e.g. insolation, gravidity) and the presence of hydrolytic enzymes (beta-glucuronidase and/or arylsulphatase) in biliary tract might belong to the risk factors of gallstone formation. The detection of 5-hydroxy-6-methoxyindolyl-2-carboxylic acid in bile and 5,6-dihydroxyindole in the extract of an alkali fused gallstone provided the first support of this theory.

The pigmented form of malignant melanoma is a tumor with an exceptionally high production of melanin. It is therefore comprehensible that the precursors of melanin and/or their metabolites are excreted in elevated amounts during the course of this disease. Using gas chromatography--mass spectrometry we were able to identify five indolic compounds in the urine of melanoma patients: 5,6-dihydroxyindole (5,6DHI), 5-hydroxy-6-methoxyindole (5H6MI), 6-hydroxy-5-methoxyindole (6H5MI), and 5-hydroxy-6-methoxyindolyl-2-carboxylic (5H6MI2C) and 6-hydroxy-5-methoxyindolyl-2-carboxylic acids (6H5MI2C). For their quantitative mass fragmentographic analyses, deuterium-labeled analogs have been synthesized. The first experience with these measurements is presented.

Extremely small amounts (10(-4) pg) of the pineal nonapeptide arginine vasotocin (AVT), injected into the pineal recess of freely moving cats, decreased the levels of 5-hydroxyindole acetic acid (5-HIAA) in the raphe dorsalis nucleus and induced slow wave sleep. In cats with lesions in the lateral habenula, 10(-4) pg AVT injected into the pineal recess, failed to decrease raphe dorsalis 5-HIAA levels and to induce slow wave sleep. The GABA antagonist picrotoxin (1 ng) injected into the pineal recess 15 min before the administration of AVT (10(-4) pg), completely prevented AVT from decreasing raphe dorsalis 5-HIAA levels and from inducing slow wave sleep. A highly significant correlation could be established between the decrease of raphe dorsalis 5-HIAA levels and the induction of slow wave sleep. No changes in raphe dorsalis 5-HIAA levels could be detected in cats injected with 10(-4) pg AVT into the lateral or into the fourth ventricle. Neither arginine vasopressin nor oxytocin (10(-4) pg) injected into the pineal recess, could alter raphe dorsalis 5-HIAA levels. It is concluded that AVT induces slow-wave sleep in cats by activating an inhibitory GABAergic lateral habenula-raphe dorsalis pathway.

The authors point out that melanogenuria, i.e. the excretion of specific phenolic and, particulary, indole metabolites in melanoma, is undoubtedly due to elevated tyrosinase activity in this disease. Other enzymes, in particular glucuronyltransferase, catechol-O-methyltransferase and possibly hydroxyindole-O-methyltransferase, also evidently participate in the further biochemical conversion of the resultant metabolites, however.

A qualitative gas chromatographic analysis of trimethylsilylated ethyl acetate extracts of melanotic urine revealed 5 indolic compounds, which have been identified as substituted 5,6-dihydroxyindoles. Ethyl acetate extracts of melanotic urines at pH 2.0 contained isomeric 5-hydroxy-6-methoxy and 6-hydroxy-5-methoxy-indolyl-2-carboxylic acids which were not separable under the conditions used. A careful hydrolysis of melanotic urine with a Helix pomatia preparation followed by extraction at pH 6.5 in a nitrogen atmosphere released 3 additional indolic compounds from their conjugated form. Using gas chromatographic-mass spectrometric analysis they were identified as 5-hydroxy-6-methoxy, 6-hydroxy-5-methoxyindole and 5,6-dihydroxyindole.

Two simple spectrophotometric assays have been employed for the measurement of dopa oxidase activity and ceruloplasmin polyphenol oxidase activity in the sera from normal hamsters and hamsters bearing melanotic melanoma. Both activities were found to be augmented in tumor animals, the dopa oxidase activity much more prominently. The levels of the enzymes tested increased proportionally to the tumor mass.

Cultured pineal ependymal cells from rat fetuses release into their incubation media immunoreactive neurophysin. The presence of neurophysin was assessed by radioimmunoassay. The culture medium was found to contain 440 pg neurophysin per mg protein.

The pineal indole melatonin, injected into the third ventricle of unanesthetized cats, induced NREM sleep within 11-15 min and suppressed REM sleep for about 3 hr. A dose-related effect could be established between 1, 10 and 100 ng melatonin. The reappearance of REM sleep at about 3 hr following melatonin administration occurred with a marked rebound. The present results further support the hypothesis that melatonin represents the releasing hormone for pineal arginine vasotocin.

The human metabolism of 4-hydroxyanisole was investigated by the analysis of urine samples from melanoma patients treated with this substance. The samples were hydrolyzed with glucoronidase and/or arylsulphatase, extracted with ethyl acetate, and, after derivatization with pentafluoropropionylanhydride, analyzed by gas chromatography-mass spectrometry. We were able to identify peaks by their retention times and mass spectra corresponding to 4-hydroxyanisole, 3,4-dihydroxyanisole, and two of its o-methyl derivatives, namely, 3-hydroxy-4-methoxy- and 4-hydroxy-3-methoxyanisole. We also detected a peak of hydroquinone, which may have originated (at least partly) from 4-hydroxyanisole. All the above-mentioned compounds were excreted predominantly as sulphates and glucuronides. Only a small proportion of the substances was present in urine in an unconjugated form. Our results demonstrate that 3,4-dihydroxyanisole is the most important metabolite of 4-hydroxyanisole.

Lumbar cerebrospinal fluid (CSF) was obtained from healthy newborns, infants, and children of both sexes between 23-777 days of age. Bioassay of the CSF revealed relatively high antidiuretic and hydroosmotic activities. Rat uterine activities could be detected only in the CSF of newborns, and these activities were increased in the presence of magnesium. The antidiuretic and hydroosmotic activities of CSF from newborns were significantly higher than the same CSF activities of infants (P less than 0.01) and children (P less than 0.001). No sex differences were observed in the above biological activities of CSF. The ratio of these activities, their susceptibility to tryptic digestion, and the increase of the rat uterine activity by magnesium demonstrate the presence in CSF of a basic peptide indistinguishable from synthetic arginine vasotocin. In contrast to the CSF of healthy newborns and infants, neither antidiuretic nor hydroosmotic activities could be detected in the CSF of a boy, aged 413 days, with a pineal tumor and precocious puberty.

The isomeric metabolites, 5-hydroxy-6-methoxy-2-indolylcarboxylic and 6-hydroxy-5-methoxy-2-indolylcarboxylic acids have been identified in the urine of hamsters bearing transplantable melanotic melanoma. Their identification was carried out by spectral and chromatographic comparison of the two isomers isolated from urine with the same compounds achieved synthetically.

The Thormählen positive melanogen previously determined as "A" has been isolated from melanotic urine by means of column chromatography on Amberlite IR 45, DEAE cellulose and Dowex 50. According to its behavior during a study with 1H--NMR spectra it has been found that the isolatd compound is 6-methoxy-5-indolyglucosiduronate, the metabolite of 5,6-dihydroxyindole. The possible formation and biotransformation of this compound is discussed.

The pineal nonapeptide hormone arginine vasotocin (AVT) (100 ng/kg) administered intra-nasally (IN) to healthy prepubertal boys, dramatically increased the amount of REM sleep, decreased REM sleep latency, and induced REM periods at sleep onset. Neither arginine vasopressin (AVP) nor oxytocin administered IN at the dose of 100 ng/kg was able to reproduce the effects of AVT, demonstrating its high specificity. Methergoline, a selective central 5-hydroxytryptamine (5-HT) receptor blocker, administered IN at the dose of 100 ng/kg, completely prevented AVT induction of REM sleep. Fluoxetine, a specific 5-HT uptake inhibitor, administered IN at the dose of 25 microgram/kg, 10 min after AVT, greatly potentiated the effects of AVT in inducing REM periods at sleep onset and in increasing the amount of REM sleep and the percentage of dream reports. It is suggested that AVT induces REM sleep in prepubertal boys by interfering with 5-HT neurotransmission and that the high sensitivity of prepubertal boys to AVT reflects an immaturity of REM triggering centers.

A single injection of 10(-6) pg synthetic arginine vasotocin (AVT), corresponding to about 600 molecules AVT, into the third ventricle of unanesthetized cats, induced slow-wave sleep 5 min after the injection. An equivalent amount, of a partially purified pineal AVT injected into the third ventricle, produced the same effects. After incubation with trypsin, pineal AVT completely lost its ability to induce slow-wave sleep. The slow-wave sleep induced by 10(-6) pg synthetic AVT injected intraventricularly could be matched by 1 microgram synthetic AVT injected intraperitoneally. Neither synthetic arginine vasopressin, nor synthetic oxytocin, injected intraventricularly in the amount of 10(-6) pg, was able to induce slow-wave sleep. Whereas in the control animals injected with pineal AVT after incubation with trypsin, or in the control animals injected with vasopressin or oxytocin, the paradoxical sleep averaged 21.9--22.8% of the sleep time, during a total recording time of 5 hr, in the cats injected with synthetic or pineal AVT, the paradoxical sleep was completely suppressed.

The pineal nonapeptide hormone arginine vasotocin (AVT) is synthesized by the ependymal cells of the pineal recess and subcommissural organ and stored in so far undefined cells of the pineal gland proper. AVT is first released into the cerebrospinal fluid (CSF) and reaches the blood only secondarily after its absorption from CSF. It displays a diurnal rhythm in the pineal and CSF, suggesting its release into the CSF during the night in the dark. Melatonin represents its releasing hormone. AVT exerts both its endocrine and non-endocrine effects by a unique mechanism involving the activation of serotonin neurotransmission in the brain with resultant inhibition of release of hypothalamic releasing and inhibiting hormones and induction of sleep. It produces both its endocrine effects and sleep at concentrations equivalent to only several hundreds of molecules, being thus by far the most active hormone so far known. Midbrain raphe nuclei or some structures intimately correlated with these cell bodies, most contain the extremely sensitive and specific AVT receptors in the mammalian brain. In contrast with its natural analogues arginine vasopressin and oxytocin which are mainly blood hormones, AVT is a CSF hormone whose major if not the sole site of action is the brain itself.

A single dose (10 ng/kg) of the nonapeptide arginine vasotocin (AVT) administered intranasally to healthy young men, significantly decreased 5-HIAA levels in the lumbar cerebrospinal fluid (CSF) 8 hr after its administration. So far, this represents the smallest amount of an active substance able to alter CSF 5-HIAA levels in man. It is suggested that the decrease of CSF 5-HIAA levels after AVT administration reflects an AVT-induced reduction of the brain 5-HT turnover.

The identification of the entire structure of the Thormählen-positive compound B from melanotic urine is described. The compound was separated from other Thormählen-positive compounds using DEAE-cellulose column chromatography. On the basis of differential enzymic hydrolysis followed by gas chromatography-mass spectrometry analysis and comparison with synthetically prepared compounds it is possible to conclude that the Thormählen-positive compound B is a mixture of O-sulphate 5-hydroxy-6-methoxyindole and 6-hydroxy-5-methoxyindole with predominance of the latter.

It has previously been shown that enzymatically hydrolyzed urine of patients with malignant melanoma contains 5,6-dihydroxyindole (5, 6DHI ). In this study we describe the elucidation of the entire structure of urinary 5, 6DHI -conjugate. Differential hydrolysis of melanotic urine revealed that, in contrast to beta-glucuronidase, sulfatase can liberate 5, 6DHI from its conjugated form. 5, 6DHI -sulfate was synthetized by reacting 5, 6DHI with sulfur trioxide trimethylamine complex. Thin-layer chromatography (TLC) documented its close similarity to the Thorm ahlen -positive compound usually entitled "C." Gas chromatographic-mass spectrometric (GC-MS) analysis of methylated and subsequently hydrolyzed synthetic 5, 6DHI -sulfate showed that the synthetic product consisted of a mixture of 5-hydroxy-6-indolyl-O-sulfate and 6-hydroxy-5-indolyl-O-sulfate (with a certain amount of 5, 6DHI -disulfate). 5, 6DHI -sulfate was purified with use of DEAE-cellulose column chromatography from melanotic urine. Methylation of this conjugate with deuterated dimethylsulfate and subsequent GC-MS analysis of the hydrolyzed product provided evidence that 5, 6DHI from melanotic urine was almost exclusively sulfated in position 6. It was concluded (1) that 5, 6DHI is excreted as a 6-O-sulfate, and (2) that this compound is consistent with Thorm ahlen -positive compound "C".

Lumbar cerebrospinal fluid (CSF) of seven healthy male volunteers contains detectable levels of arginine vasotocin (AVT) when CSF was removed after awakening from rapid eye movement (REM) sleep. No detectable levels of AVT were found in the CSF of the same subjects when CSF WAS removed after awakening from non rapid eye movement (NREM) sleep. The amount of AVT detected in CSF after REM sleep was significantly higher if the subjects experienced vivid and emotive dreams. The present results provide the first evidence for a REM sleep dependent release of AVT into CSF of man.

A specific arginine vasotocin (AVT) antiserum (10 microliter) injected undiluted into the lateral ventricle of unanesthetized cats, induced sleep onset REM periods, markedly increased the number of REM periods, and dramatically decreased REM sleep latency. The amount of total REM sleep significantly increased and the amount of total NREM sleep significantly decreased. Neither a specific arginine vasopressin or oxytocin antiserum nor a normal rabbit serum injected undiluted into the lateral ventricle in the same volume (10 microliter) was able to reproduce the effects of the AVT antiserum. It is concluded that pineal AVT is physiologically involved in the induction and/or circadian organization of REM sleep.

The pineal content of arginine vasotocin in the male rat is subject to diurnal changes, having a high value around noon and a low value around midnight. Since in rats exposed to 24 h constant light the pineal content in arginine vasotocin decreases, and in the rats exposed to 24 h constant darkness the pineal content in arginine vasotocin increases, it appears that during the night both the synthesis and release of arginine vasotocin is enhanced.

A single injection of 1 pg synthetic arginine vasotocin (AVT) into the third ventricle of the mouse completely prevented the pituitary MSH increase which occurs seven days after pinealectomy or 24 h after exposure to constant light. The same concentration of AVT injected into the third ventricle of normal mice markedly decreased the pituitary MSH content at 20 min following the injection. The present results suggest that AVT injected into the third ventricle of the mouse inhibits synthesis and/or release of MSH-release inhibiting factor (MIF).