On the Accurate Determination of Serotonin in Human Plasma

doi:10.1006/bbrc.1993.2243

Biochemical and Biophysical Research Communications

Volume 196, Issue 1, 15 October 1993, Pages 260-266

https://doi.org/10.1006/bbrc.1993.2243 Get rights and content

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) in blood is stored in platelets and has vascular and platelet stimulating effects when released into plasma. Accurate measurements of 5-HT in plasma are complicated by inadvertent platelet activation causing sampling artifacts and by analytical problems when determining trace levels. We developed an assay for plasma 5-HT based on solid-phase extraction (Sep-Pak C₁₈), aqueous acetylation, pentafluoropropionylation, and negative ion chemical ionization gas chromatography-mass spectrometry. The method was able to recover 5-HT from plasma by >90% and to quantitate with a precision of 7.5% at a level of 0.5 nmol/l. It was used to define blood sampling and sample handling procedures giving low and consistent values for 5-HT. A good blood sampling technique, adequate platelet stabilization in the test tube, and rapid high speed centrifugation of the blood resulted in low plasma levels of both 5-HT and β-thromboglobulin (a platelet release product). Using these procedures plasma 5-HT levels in healthy volunteers were found to be 0.77 ± 0.38 (mean ± S.D.; range 0.27-1.49) nmol/l (n=18), which is 4-100-fold lower than previously reported values.

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The protocol with heat shocking of the platelets were inspired by the paper by Maurer-Spurej et al. [11], who demonstrated that moderate heat shock conditions (42 °C for 10 min) caused no signs of platelet activation, whereas room temperature causes marked activation of platelets and affected platelet serotonin content. Plasma serotonin concentration has been shown to increase by >300% after 40 min on ice compared to immediate centrifugation [12]. This motivated us to test EDTA-tubes with gel to achieve rapid separation of blood cells and plasma with only one centrifugation (1800×g for 15min, with temperatures 4 °C or 22 °C), as these gel tubes are commonly used in the clinic and would be convenient to implement in the daily routine.
Reported concentrations of serotonin in platelet-poor plasma (PPP) in healthy subjects vary widely due to different pre-analytical procedures.
To examine how different pre-analytical conditions affect the measured concentration of serotonin in PPP.
Six pre-analytical protocols were compared for preparation of PPP from EDTA whole blood for quantification of serotonin from nine healthy individuals. Three combinations of centrifugation with a mild centrifugation of gel-free EDTA tubes followed by a stronger centrifugation were compared to single-stage centrifugation of EDTA tubes with separator gel and heat shock treatment of blood prior to centrifugation. All samples were analysed using the same enzyme linked immunosorbent assay (ELISA) method.
Findings show that two consecutive centrifugations; first a mild centrifugation at 100 or 200×g followed by centrifugation at 4500 or 14500×g resulted in the lowest serotonin concentration in PPP.
Two successive centrifugations to produce PPP for serotonin analysis; first a mild centrifugation to avoid mechanical stress on the platelets, and next a stronger centrifugation to remove platelets, is superior to the use of gel tubes and heat shock treatment.
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Bromo-dragonfly is a benzodifuran derivative known as one of the most potent 5-HT_2A-receptor agonists within this chemical class, with long-lasting effects of up to 2–3 days. In addition to hallucinogenic effects, the drug is a potent vasoconstrictor, resulting in severe adverse effects, such as necrosis of the limbs. In some cases, intoxication has had fatal outcomes. Little is known about the metabolism of bromo-dragonfly. The aims of this study were to investigate the pharmacokinetics of bromo-dragonfly, determine the plasma protein binding, examine the human hepatic metabolism in vitro, and compare with those of its close analogue, 2C-B-fly. Additionally, we assayed the inhibition potency of both compounds on the monoamine oxidase (MAO) A- and B-mediated oxidative deamination of serotonin (5-HT) and dopamine, respectively. Liquid chromatography high-resolution mass spectrometry was used for metabolism studies in pooled human liver microsomes (HLM), pooled human liver cytosol (HLC) and recombinant enzymes. Inhibition studies of the deamination of 5-HT and dopamine were carried out using LC–MS/MS. Bromo-dragonfly was not metabolised in the tested in vitro systems. On the other hand, 2C-B-fly was metabolised in HLM by CYP2D6 and in HLC to some extent, with the main biotransformations being monohydroxylation and N-acetylation. Furthermore, MAO-A metabolised 2C-B-fly, producing the aldehyde metabolite, which was trapped in vitro with methoxyamine. Inhibition experiments revealed that bromo-dragonfly is a competitive inhibitor of MAO-A with a K_i of 0.352 μM. The IC₅₀ value for bromo-dragonfly indicated that the inhibition of MAO-A may be clinically relevant. However, more data are needed to estimate its impact on the increase of 5-HT in vivo.
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Considering both of these numbers are only approximate, they are in relatively good agreement. The concentrations of free 5-HT on the inside and outside of brain capillaries may also be comparable (Anderson et al., 1987b; Beck et al., 1993; Adell et al., 2002; Anderson, 2007), but these measurements pose major technical challenges and should be treated with caution. The exchange between serotonergic varicosities and blood platelets, direct or indirect, is currently hypothetical and has to be investigated experimentally.
Altered serotonin (5-hydroxytryptamine, 5-HT) signaling has been implicated in some developmental abnormalities of autism spectrum disorder (ASD). However, the presumed role of 5-HT in ASD raises new questions in fundamental neuroscience. Specifically, it is not clear if the current piecemeal approach to 5-HT signaling in the mammalian body is effective and whether new conceptual approaches may be required. This review briefly discusses 5-HT production and circulation in the central nervous system and outside of it, especially with regard to ASD, and proposes a more encompassing approach that questions the utility of the “neurotransmitter” concept. It then introduces the idea of a generalized 5-HT packet that may offer insights into possible links between serotonergic varicosities and blood platelets. These approaches have theoretical significance, but they are also well positioned to advance our understanding of some long-standing problems in autism research.
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Regular ArticleOn the Accurate Determination of Serotonin in Human Plasma

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Regular Article
On the Accurate Determination of Serotonin in Human Plasma