5-Hydroxytryptophol and 5-hydroxyindoleacetic acid levels in rat brain: effects of various drugs affecting serotonergic transmitter mechanisms.

The effect of various pharmacological treatments on the levels of free and total 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) in rat brain were investigated using a gas chromatographic-mass spectrometric technique. The disappearance of 5-HTOL following monoamine oxidase (MAO) inhibition induced by pargyline was more rapid (t1/2 10-15 min) than that of 5-HIAA (t1/2 30-40 min) in all regions investigated, indicating a rapid turnover of 5-HTOL. The selective MAO-A inhibitor chlorgyline produced a more pronounced reduction of 5-HTOL than of 5-HIAA, while the MAO-B inhibitor deprenyl was without effect on both serotonin metabolites. The MAO-A inhibitor amiflamine which is selective for serotonin neurons, was also more effective in reducing free 5-HTOL levels than of 5-HIAA levels, suggesting that the formation of 5-HTOL is closely associated with serotonin neurons. Neonatal treatment with the serotonin neurotoxin 5, 7-dihydroxytryptamine (5,7-HT) led to a more pronounced reduction of 5-HTOL levels in cerebral cortex than that of 5-HIAA levels, while the increase of 5-HTOL levels in pons-medulla was more marked than of 5-HIAA levels. Probenecid treatment increased several fold both conjugated 5-HTOL and 5-HIAA levels in brain tissue. An increase was also noted for free 5-HTOL, although of less magnitude. Treatment with the serotonin receptor active agents methiothepin and d-lysergic acid diethylamide produced similar alterations of free 5-HTOL and 5-HIAA. The present results have demonstrated that free 5-HTOL has a rapid turnover in rat brain and that free 5-HTOL levels may serve as a useful index for serotonin turnover.

5-Hydroxyindoleacetic acid and 5-hydroxytryptophol levels in rat brain: effects of ethanol, pyrazole, cyanamide and disulfiram treatment.

The two serotonin metabolites 5-hydroxyindoleacetic acid (5HIAA) and 5-hydroxytryptophol (5HTOL) were measured in two regions of rat brain (pons medulla and diencephalon) using a gas chromatographic-mass spectrometric (GC-MS) method. Acute ethanol intoxication effected an elevation of 5-hydroxytryptophol levels, while 1 week of treatment with ethanol appeared to have no effect on either metabolite when measured 24 h after the last dose. Disulfiram and cyanamide treatment produced an approximately 2-fold increase in 5-hydroxytryptophol and a slight reduction in 5-hydroxyindole-acetic acid. Pyrazole treatment produced an increase in both metabolites. This effect was, however, counteracted by the simultaneous administration of ethanol.

Measurement of 5-hydroxytryptophol and 5-hydroxyindoleacetic acid in human and rat brain and plasma.

The levels of 5-hydroxyindoleacetic acid (5-HIAA) and free and total 5-hydroxytryptophol (5-HTOL) in human and rat brain regions and plasma were determined by a specific capillary column gas chromatographic--mass spectrometric method. The human brains were obtained 2-3 hours post mortem, and the levels of 5-HIAA were in the range of 0.48-31.3 nmoles/g in the regions investigated. The levels of free and total 5-HTOL were 10.9-387 pmoles/g and 14.5-821 pmoles/g, respectively. The ratio of total 5-HTOL to 5-HIAA was in the range of 0.6-5.5%. In human plasma the levels of free and total 5-HTOL were 0.9 +/- 0.3 and 2.9 +/- 0.8 pmoles/ml +/- S.E.M., respectively. In regions of rat brain, the 5-HIAA levels ranged from 0.37-2.84 nmoles/g. Free and total 5-HTOL were in the range of 11.4-56.1 and 16.2-77.1 pmoles/g, respectively. The ratio of total 5-HTOL and 5-HIAA ranged from 2.3-5.1%. Higher levels of 5-HIAA and 5-HTOL occurred in the rat pineal gland. In rat plasma the levels of free and total 5-HTOL were 1.34 +/- 0.06 and 21.6 +/- 1.6 pmoles/ml +/- S.E.M., respectively.

Occurrence of 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline in tissues and body fluids of rat.

A capillary column gas chromatographic-mass spectrometric method was used to identify and quantitate 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline (6OMTHBC) in rat. The excretion rate in urine was 0.73 +/- 0.20 nmoles per 24 hr and in faeces 0.18 +/- 0.03 nmoles per 24 hr. In urine, about 90% of the 6OMTHBC was in a conjugated form, whereas in faeces most (approximately 75%) of the 6OMTHBC was in a free form. The compound was detectable in liver (11.1 +/- 3.6 pmoles/g), kidney (2.1 +/- 0.9 pmoles/g) and plasma (0.52 +/- 0.15 pmoles/ml), but not in brain (less than 0.3 pmoles/g). When 6OMTHBC was injected to rats, 75% of the injected amount was excreted in urine during the first 10 hr. The plasma level of 6OMTHBC declined with a half-life of 1.5 hr.

Presence of formaldehyde in biological media and organic solvents: artifactual formation of tetrahydro-beta-carbolines.

A gas chromatography-mass spectrometry method is described for the identification and quantitation of 6-hydroxy-1,2,3,4-tetrahydro-beta-carboline (6OHTHBC). During the analysis of 6OHTHBC in human platelets, the artifactual formation of 6OHTHBC was observed and shown to be due to the presence of formaldehyde in both organic solvents and in platelet homogenates. Formaldehyde was facilely removed from the organic solvent via a Pictet-Spengler reaction; however, removal from tissue homogenates was not possible by using conventional aldehyde-trapping agents. The solution of these problems and their consequences for persons attempting to establish the in vivo presence of tetrahydro-beta-carbolines or biochemically study tissue preparations are discussed.

5-hydroxytryptophol in the cerebrospinal fluid and urine of alcoholics and healthy subjects.

The serotonin metabolite 5-hydroxytryptophol was determined in cerebrospinal fluid and urine of alcoholics and healthy subjects, by a glass capillary gas chromatographic-mass spectrometric method. The urinary excretion rate (14.6 +/- 2.9 pmoles/mumoles creatinine) and urine (109 +/- 20 pmoles/ml) and cerebrospinal fluid (4.12 +/- 0.21 pmoles/ml) concentrations in healthy subjects were established. Only 1% of the 5-hydroxytryptophol in urine occurred in free form. Ethanol ingestion (80, 120 g) by healthy subjects lead to a 20--100-fold increase in the urinary excretion rate of 5-hydroxytryptophol. In cerebrospinal fluid the increase was about 60%. Alcoholics had increased urinary excretion rates and cerebrospinal fluid levels during intoxication, which were in the same range as in intoxicated healthy subjects. During recovery from intoxication, the 5-hydroxytryptophol level in alcoholics decreased, but the CSF levels were still higher than in healthy subjects.

Identification and measurement of 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline by gas chromatography-mass spectrometry.

A glass capillary gas chromatographic-mass spectrometric method for the analysis of 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline in urine, plasma and blood platelets was developed. The method involves the use of a deuterated analogue as internal standard, addition of semicarbazide, extraction with methylene chloride and formation of pentafluoropropionyl derivatives. The 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline was found to occur in the urine of controls and alcoholics as both the free and conjugated compound. In the controls the mean level of free and conjugated 60MTHBC was 75 +/- 20 nmole/l. When intoxicated, the alcoholics had a statistically higher excretion rate of the conjugate(s) than the controls. The compound was not detectable in blood platelets or plasma.

Concentration of 5-methoxyindoles in the human pineal gland.

The pineal gland concentration of the four 5-methoxyindoles: melatonin, 5-methoxytryptamine, 5-methoxytryptophol and 5-methoxyindole-3-acetic acid, were measured by gas chromatography-mass spectrometry. The pineal glands were obtained from 11 subjects 2-3 hours post mortem. The concentration of 5-methoxytryptophol ranged from 4-93 pmoles/g, 5-methoxyindole-3-acetic acid from 79-560 pmoles/g and melatonin from 24-6860 pmoles/g. 5-methoxytryptamine was present only in trace amounts (0-12 pmoles/g). A variation over time of these compounds was not evident from the data obtained.

5-Methoxyindoles in pineal gland of cow, pig, sheep and rat.

The occurrence and concentration of the four 5-methoxyindoles: 5-methoxtryptamine, 5-methoxytryptophol, 5-methyoxyindole-3-acetic acid and melatonin in the pineal gland of pig, cow, sheep and rat was investigated. The analytical method involved the use of deuterated analogues as internal standards and capillary column gas chromatography - mass spectrometry. The analyses of pineal glands obtained during the morning hours revealed the presence of 5-methoxyindole-3-acetic acid and melatonin in nmoles/g and 5-methyoxytryptophol and 5-methoxytryptamine in pmoles/g amounts in pig, cow and sheep. In Wistar and Sprague-Dawley strain of rat, melatonin was present at a concentration of about 0.5 pmoles/pineal, a level which was elevated more than five times by treatment with a monoamine oxidase inhibitor. 5-Methoxytryptamine was found at a concentration of about 0.03 pmoles/pineal, and was elevated by monoamine oxidase inhibition.