John A. Monti

9-Diazomethylanthracene as a new fluorescence and ultraviolet label for the spectrometric detection of picomole quantities of fatty acids by high-pressure liquid chromatography.

Abstract 9-Diazomethylanthracene reacts with carboxyl groups to give an ester derivative which can be used as either a fluorescence or ultraviolet label for fatty acid analysis by high-pressure liquid chromatography. The limit of detection by ultraviolet spectroscopy was demonstrated to be approximately 150 pg/μl of the individual fatty acid esters. Fluorescence detection showed a limit of approximately 15 pg/μl. The fluorescence detector response was linear from 0.49 to 14.2 pmol/μl. Thus, derivatization of fatty acids with 9-diazomethylanthracene provides a new and very sensitive method for the quantification of picomole quantities of fatty acids by high-pressure liquid chromatographic techniques using either ultraviolet or fluorescence detection.

Identification and quantification of 1,2,3,4-tetrahydro-β-carboline, 2-methyl-1,2,3,4-tetrahydro-β-carboline, and 6-methoxy-1,2,3,4-tetrahydro-β-carboline as in vivo constituents of rat brain and adrenal gland☆☆☆

Abstract The identification and quantification of three 1,2,3,4-tetrahydro-β-carbolines as normal constitutents of rat brain and adrenal gland, using combined gas chromatographic/mass spectrometric techniques, are reported. Qualitative analyses of these tissues led to the identification of 1,2,3,4-tetrahydro-β-carboline (THBC), 2-methyl-THBC (2-MTHBC) and 6-methoxy-THBC (6-MeOTHBC), as determined by observed peak retention times, mass fragments and ion mass ratios. Quantitative analyses, using deuterated internal standards, gave the following results: THBC (ng/g wet wt) in brain = 17.5 ± 4.86, adrenal = 500.3 ± 163. 6-MeOTHBC (ng/g wet wt) in brain = 35.6 ± 16.6, adrenal = 1113.7 ± 300. Mechanisms for the formation of these β-carbolines as well as their possible function in vivo are discussed.

N, N-dimethyltryptamine: an endogenous hallucinogen.

Publisher Summary This chapter reviews the biosynthesis, metabolism, pharmacology, and properties of N,N-dimethyltryptamine (DMT), leading to a conclusion that DMT may be a neurotransmitter in the mammalian brain. The identification of DMT and other hallucinogens in man explains the experience of hallucinatory phenomena in general. Data is presented in the chapter to illustrate that DMT is a normal constituent of mammalian brain and other tissues. Enzymes capable of synthesizing DMT from tryptamine (TA) and -N-methyltransfera (NMT) are also described. These enzymes are apparently controlled by small peptide-like compounds as well as by feedback inhibition from substrate and product. A cyclic metabolic pathway for DMT is offered. There is also evidence that DMT is taken up into synaptosomes and stored in vesicles by mechanisms identical to those described for known neurotransmitter substances. Specific binding sites for DMT are suggested and DMT is shown to lead to the production of cyclic adenosine monophosphate. (cAMP), a secondary receptor messenger. As evidence of its electrophysiological activity, it has been shown that, DMT stimulates fluid secretion from the salivary glands of blowflies, changes the transepithelial and intracellular potential of the gland, and increases the production of cAMP. Thus, DMT fulfills the criteria for consideration as a neurotransmitter or a neuromodulator per se.

Metabolism of the hallucinogen N,N-dimethyltryptamine in rat brain homogenates☆

Abstract The metabolism of the hallucinogen N , N -dimethyltryptamine (DMT) in whole rat brain homogenate is reported. Studies were conducted using tritiated DMT and DMT- N -oxide (DMT-NO), and metabolites were identified and quantified using thin-layer chromatography and liquid scintillation counting techniques. Metabolite confirmation was obtained by incubation of α,α,β,β-tetradeutero-DMT (DDMT) with whole brain homogenate followed by combined gas chromatographic/mass spectrometric analyses. The metabolites of DMT were identified as indoleacetic acid (IAA), DMT-NO, N -methyltryptamine (NMT), 2-methyl-1,2,3,4-tetrahydro-β-carboline (2-MTHBC), tryptamine (TA) and 1,2,3,4- tetrahydro-β-carboline (THBC). DMT-NO was metabolized to give DMT, NMT, IAA and 2-MTHBC. Formation of these metabolites from DMT-NO was stimulated by anaerobic incubation. Mechanisms for the formation of β-carbolines from DMT and DMT-NO are discussed. The effects of the monamine oxidase inhibitor iproniazid phosphate on DMT metabolism were also studied. Iproniazid inhibited the formation of IAA from DMT by 83 per cent. However, the formation of NMT and DMT-NO was inhibited by 90 per cent under these conditions. Thus, the reported extension of half-life and potentiation of DMT behavioral effects by iproniazid may be due to inhibition of NMT and DMT-NO formation rather than inhibition of monoamine oxidase. A cyclic pathway for the synthesis and metabolism of DMT in brain tissue is proposed.

Effects of dolichol on membrane permeability

Small vesicles containing the tetra-anionic fluorescent probe calcein were prepared by sonication of mixtures of plant phosphatidylethanolamine, plant phosphatidylcholine, and dolichol. Following chromatography, the isolated vesicles were found to retain entrapped calcein over the temperature range of 15 to 40 degrees C. Utilizing an assay measuring the fluorescence quenching of entrapped calcein by cobalt ions, the presence of dolichol in the membranes was found to promote the permeability of the phospholipid bilayers to the divalent cation. The permeability was shown to be dependent on temperature with an increase in rate of 17-fold between 15 and 35 degrees C although the plant phospholipids used in these experiments have no known phase transition within this temperature range. The incorporated dolichol was distributed uniformly throughout the vesicle population. Similar vesicles prepared from phosphatidylethanolamine and phosphatidylcholine without added dolichol, from phosphatidylcholine alone, or with phosphatidylcholine and dolichol were far less permeable to the divalent cation under the same assay conditions. These results demonstrate that dolichols have significant effects on the permeability properties of phospholipid bilayers that contain phosphatidylethanolamine.

Abnormalities of one-carbon metabolism in psychiatric disorders: Study of methionine adenosyltransferase kinetics and lipid composition of erythrocyte membranes

Two independent lines of inquiry have implicated some disturbance of one-carbon cycle metabolism in affective disorders. Folic acid deficiency commonly leads to depression, and S-adenosylmethionine has been reported to have antidepressant properties. Methionine adenosyltransferase has been reported to be underactive in depression and schizophrenia and overactive in mania. This study reports the effects on erythrocyte methionine adenosyltransferase (MAT) kinetics (Vmax) of a 2-week treatment in a population of patients housed on a psychiatric research ward. The drug-free schizophrenic patients and depressives had, upon admission, low Vmax values, and the drug-free manic patients had high Vmax values on admission. After 2 weeks of appropriate treatment, the values for all three patient samples showed significant normalization (i.e., the levels rose in schizophrenics and depressives and fell in manics). We have further shown that pretreatment low levels of erythrocyte membrane phosphatidylcholine in depressives and high levels in manics show statistically significant normalization following 2 weeks of pharmacotherapy. The significance of these results is discussed.

The in vitro identification of dimethyltryptamine (DMT) in mammalian brain and its characterization as a possible endogenous neuroregulatory agent.

Abstract The psychotomimetic agent dimethyltryptamine (DMT) has been identified as an endogenous compound in the central nervous system of rodents using a sensitive electron capture gas chromatographic technique. DMT along with its proposed precursor, tryptamine, were identified and quantitated as the heptafluorobutyryl derivatives. A specific high affinity binding site on synaptosomal membranes has been proposed for DMT. This proposal is based on equilibrium dialysis experiments which indicate that DMT at a concentration of 1 × 10−5 m will displace d-LSD on isolated membranes but will not displace bound serotonin at the same concentration. When DMT interacts with the synaptosomal membranes at a concentration of 5 × 10−10 m , the membrane-bound enzyme adenylate cyclase is stimulated such that adenosine3′, 5′-monophosphate (cAMP) is produced at a rate of 100 pmole/min/mg of protein (2.3 times the endogenous rate). It has also been shown that its presumed precursor, tryptamine, inhibits this process. LSD appears to exhibit a high affinity for the proposed DMT binding site but seems to have a low intrinsic activity. From data obtained in this study it has been postulated that DMT may have in vivo activity similar to those proposed for neurotransmitters or other neuroregulatory agents. These data further suggest that at least one mode of action of d-LSD may be the displacement of DMT from its binding site on the neuron.

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Abstract Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10 −10 , 5 × 10 −9 , 1 × 10 −8 , and 5 × 10 −8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10 −5 M fluphenazine, while 1 × 10 −5 M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10 −8 M 5-HT by 30 percent, 80 percent, and 57 percent respectively.

Gas chromatographic/mass spectrometric evidence for the identification of 6,7-dihydroxy-1, 2,3,4-tetrahydroisoquinoline as a normal constituent of rat brain: Its quantification and comparison to rat whole brain levels of dopamine

Gas chromatographic/mass spectrometric data are presented which demonstrate the presence of 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (DHTIQ) as a normal constituent of rat brain. The level of DHTIQ was calculated to be 10.0 +/- 3.0 ng/g wet weight (+/- S.D., N = 9) of brain tissue while the level of dopamine (DA) was measured as 1.22 +/- 0.22 microg/g (N = 14). The ratio of DHTIQ:DA was thus observed to be approximately 1:100. The possible formation of DHTIQ in alcoholism and schizophrenia is discussed.

One-carbon metabolism disturbances in affective disorders: A preliminary report

Methionine adenosyltransferase (MAT) activity (Vmax) and the relative amount of phosphatidylcholine (% PC) were measured in erythrocytes of up to 30 DSM-III diagnosed manic, 17 unipolar depressed patients, and 28 normal controls. Manic subjects had significantly higher and depressed subjects significantly lower MAT Vmax than normals. The relative amount of PC was in the low range for the depressives, and in the high range for the manics. Depressive patients present, in these tests, similar abnormalities to those seen previously in schizophrenic patients. Clinical and diagnostic implications of these findings are discussed.