5-Hydroxytryptamine assay

Biogenic amines are of great interest to researchers because of their potential roles in several psychiatric and neurological disorders. They include dopamine (DA), noradrenaline (NA), 5-hydroxytryptamine (5-HT, serotonin), histamine, and trace amines such as 2-phenylethylamine (PEA), tyramine, octopamine, phenylethanolamine, and tryptamine (Coutts and Baker, 1982). Although GC assays for DA, NA, and 5-HT are available, HPLC analysis with electrochemical detection has for many years now been the method of choice for analysis of these neurotransmitter amines. 

The use of HPLC to analyze biogenic amines and their acid metabolites is well documented. HPLC assays for classical biogenic amines such as norepinephrine (NE), epinephrine (E), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin) and their acid metabolites are based on several physicochemical properties that include a catechol moiety (aryl 1,2-dihydroxy), basicity, easily oxidized nature, and/or native fluorescence characteristics (Anderson, 1985). Based on these characteristics, various types of detector systems can be employed to assay low concentrations of these analytes in various matrices such as plasma, urine, cerebrospinal fluid (CSE), tissue, and dialysate. 

Lakshmana MK, Trichur TR. 1997. An isocratic assay for norepinephrine, dopamine and 5-hydroxytryptamine using their native fluorescence by high-performance liquid chromatography with fluorescence detection in discrete brain areas of rat. Anal Biochem 246 166-170. 

Hows ME, Lacroix L, Heidbreder C, Organ AJ, Shah AJ. 2004. High-performance liquid chromatography/tandem mass spectrometric assay for the simultaneous measurement of dopamine, norepinephrine, 5-hydroxytryptamine and cocaine in biological samples. J Neurosci Methods 138 123. Hows ME, Organ AJ, Murray S, Dawson LA, Foxton R, et al. 

Cussac D, Newman-Tancredi A, Duqueyroix D, Pasteau V, Millan MJ. Differential activation of Gq/11 and Gi(3) proteins at 5-hydroxytryptamine(2C) receptors revealed by antibody capture assays influence of receptor reserve and relationship to agonist-directed trafficking. Mol Pharmacol 2002 62 578-589. 

The HPLC method described here allows the assay of both isoforms in the same run by using 5-hydroxytryptamine and 3-methoxy-4-hydroxybenzyl-amine as highly selective substrates for the A and B isoforms, respectively. The product of 5-hydroxytryptamine that is quantitated is 5-hydroxyindole-2-carboxylic acid, which is obtained by including aldehyde dehydrogenase in the assay mixture to oxidize the intermediate aldehyde. The product of monoamine oxidase B is 3-methoxy-4-hydroxybenzaldehyde. The internal standards used are 5-hydroxyindole-2-carboxylic acid and 3,4-dihydroxyben-zoic acid. 

Figure 3.11. Activation 5-hydroxytryptamine (=serotonin) receptors by various agonists. a Principle of the assay. Cell membranes carrying the receptors and associated G-proteins were in-...

Indolealkylamines. GC-MS methods applied in studies of the biochemical pharmacology of indoleamines parallel work on the catecholamines. SIM assays for serotonin (5-hydroxytryptamine), 5-methoxytryptamine, JV-acetylserotonin and melatonin (5-methoxy-N-acetyltryptamine) in rat pineal and brain tissue have been described [453,469]. Pentafluoro-propionyl derivatives and structural homologue standardisation were employed with detection limits in the subpicomole range. Estimation of central indoleamine turnover in man currently depends upon metabolite determination in CSF. Ion monitoring determination of indole-3-acetic acid [454] a metabolite of tryptamine, and isotope dilution assays for 5-hydroxyindoleacetic acid (5-HIAA) [455,458] have been reported. Serotonin is converted by central monoamine oxidase to 5-HIAA and the measurement of this metabolite, formerly by fluorimetry, is of interest in patients with CNS disorders [470]. GC-MS has also contributed to the identification of N,N-dimethyltryptamine in vitro [471] and isotope dilution technique has been applied to the measurement of this metabolite in control subjects and in psychiatric patients [472]. 

Assays utilizing coelenterazine-charged aequorin as a probe for intracellular calcium level have been used to investigate a wide variety of receptors endothehn ETa, angiogensin ATn, tyrosine-releasing hormone (TRH), and neurokinin NKi [170] 5-hydroxytryptamine 2A [171], and human EPl prostanoid receptor [172]. 

Blood pressure can be regulated by receptor, enzyme, endogerous substance, and cell, such as bradykinin B2-receptor, ACE, 5-Hydroxytryptamine, and endothelia cell. Based on these facts, the human plasma assay [131], pulmonary hypertension assay [132], coronary arteries (CA) constriction assay [133], MCAO and HSPG assay [134], and temperature assay in awake subjects [135] are established. 

Marsden, C. A., 1981, Effect of L-tryptophan on mouse brain 5-hydroxytryptamine comparison of values obtained using a fluorometric assay and a liquid chromatographic assay with electrochemical detection,/. Neurochem. 36 1621-1626. 

Finally, the possible role of endogenous histamine in the CNS has been investigated. This histamine comes from the histidine which has been transformed in situ by cerebral histidine decarboxylase, since histamine cannot cross the blood-brain barrier. It is difficult to determine exactly how much histamine the CNS contains since assays are rendered imprecise by chemical interference, but it appears that histamine behaves at this level like the catecholamines and S-hydroxytryptamine under the influence of reserpine. The hypothalamus is rich in histidine decarboxylase and in histamine, but the way in which the histamine is distributed is altogether different from that of the other amines. This histamine is governed by the action of methyltransferase, which in turn is inhibited by chlorpromazine. The question of whether histamine is somehow related to schizophrenia is still being debated. 

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