Acylserotonins - a new class of plant lipids with antioxidant activity and potential pharmacological applications.

During analysis of components of baobab (Adansonia digitata) seed oil, several new fluorescent compounds were detected in HPLC chromatograms that were not found previously in any seed oils investigated so far. After preparative isolation of these compounds, structural analysis by NMR spectroscopy, UHPLC-HR-MS, GC-FID and spectroscopic methods were applied and allowed identification of these substances as series of N-acylserotonins containing saturated C22 to C26 fatty acids with minor contribution of C27 to C30 homologues. The main component was N-lignocerylserotonin and the content of odd carbon-atom-number fatty acids was unusually high among the homologues. The suggested structure of the investigated compounds was additionally confirmed by their chemical synthesis. Synthetic N-acylserotonins showed pronounced inhibition of membrane lipid peroxidation of liposomes prepared from chloroplast lipids, especially when the peroxidation was initiated by a water-soluble azo-initiator, AIPH. Comparative studies of the reaction rate constants of the N-acylserotonins and tocopherols with a stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in solvents of different polarity revealed that N-acylserotonins showed similar activity to δ-tocopherol in this respect. The described compounds have been not reported before either in plants or in animals. This indicates that we have identified a new class of plant lipids with antioxidant properties that could have promising pharmacological activities.

Integral methods for automatic quantification of fast-scan-cyclic-voltammetry detected neurotransmitters.

Modern techniques for estimating basal levels of electroactive neurotransmitters rely on the measurement of oxidative charges. This requires time integration of oxidation currents at certain intervals. Unfortunately, the selection of integration intervals relies on ad-hoc visual identification of peaks on the oxidation currents, which introduces sources of error and precludes the development of automated procedures necessary for analysis and quantification of neurotransmitter levels in large data sets. In an effort to improve charge quantification techniques, here we present novel methods for automatic selection of integration boundaries. Our results show that these methods allow quantification of oxidation reactions both in vitro and in vivo and of multiple analytes in vitro.

Simple and reliable serotonin assay in human serum by LC-MS/MS method coupled with one step protein precipitation for clinical testing in patients with carcinoid tumors.

Serotonin (5-hydroxytryptamine, 5-HT) is readily secreted in patients with carcinoid tumors, especially arising from the midgut. Although serotonin assay in human plasma or whole blood has been extensively studied, serotonin assay in human serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has received much less attention. In this study, a simple and reliable LC-MS/MS method based on one step protein precipitation for sample pretreatment was developed for clinical assay of serum serotonin. Namely, 150 µL of serum was mixed with 50 µL of internal standard (IS) and 200 µL of 4 wt.% 5-sulfosalicylic acid (SSA) solution for protein precipitation. The supernatant after centrifugation was analyzed directly without further treatment. This method was validated for consistent linearity from 0.94 to 240 ng/mL with CVs ≤ 11.7%, good recovery in the range of 87.5%-104%, excellent analyte stability and low carryover. No obvious matrix effect was observed. Intra- and inter-day imprecision were below 8.03% and 11.5% respectively. Dilution linearity was verified with satisfying linearly dependent coefficients (r2 = 0.9937). The reference interval of serotonin was established from 126 results derived from subjects without carcinoid tumors. Therefore, apart from development of a serum serotonin assay by the LC-MS/MS method, the reference interval (RI) of 5-HT has also been established for clinical testing in patients with carcinoid tumors. In addition, this method has been successfully used in our laboratory, indicating that this robust LC-MS/MS assay with simple sample preparation and short analysis time could offer inspiring potential for clinical testing of 5-HT in routine clinical laboratories.

Addressing the instability issue of dopamine during microdialysis: the determination of dopamine, serotonin, methamphetamine and its metabolites in rat brain.

Dopamine is a catecholamine neurotransmitter that degrades rapidly in aqueous solutions; hence, its analysis following brain microdialysis is challenging. The aim of the current study was to develop and validate a new microdialysis coupled LC-MS/MS system with improved accuracy, precision, simplicity and turnaround time for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine analysis in the brain. Dopamine degradation was studied with different stabilizing agents under different storage conditions. The modified microdialysis system was tested in vitro, and was optimized for best probe recovery, assessed by %gain. LC-MS/MS assay was developed and validated for the targeted compounds. Stabilizing agents (ascorbic acid, EDTA and acetic acid) as well as internal and cold standards were added on-line to the dialysate flow. Assay linearity range was 0.01-100 ng/mL, precision and accuracy passed criteria, and LOQ and LLOQ were 0.2 and 1.0 pg, respectively. The new microdialysis coupled LC-MS/MS system was used in Wistar rats striatum after 4 mg/kg subcutaneous methamphetamine. Methamphetamine rapidly distributed to rat striatum reaching an average ~200 ng/mL maximum, ~82.5 min post-dose. Amphetamine, followed by 4-hydroxymethamphetamine, was the most abundant metabolite. Dopamine was released following methamphetamine injection, while serotonin was not altered. In conclusion, we proposed and tested an innovative and simplified solution to improve stability, accuracy and turnover time to monitor unstable molecules, such as dopamine, by microdialysis.

A Strategy for Quality Control of Vespa magnifica (Smith) Venom Based on HPLC Fingerprint Analysis and Multi-Component Separation Combined with Quantitative Analysis.

As a folk medicine of the Jingpo minority in Yunnan province, the venom of Vespa magnifica has been commonly used for the treatment of rheumatoid arthritis. Quality standardization of the wasp venom is a necessary step for its pharmaceutical research and development. To control the quality of the wasp venom, a method based on high-performance liquid chromatography (HPLC) was developed for chemical fingerprint analysis. In the chromatographic fingerprinting, chemometrics procedures, including similarity analysis (SA), hierarchical clustering analysis (HCA), and principal component analysis (PCA), were applied to classify 134 batches (S1-S134) of wasp venom from different origins. The HPLC fingerprint method displayed good precision (Relative standard deviation, RSD < 0.27%), stability (in 16 h, RSD < 0.34%), and repeatability (RSD < 1.00%). Simultaneously, four compounds (VMS1, VMS2, VMS3, and VMS4) in the wasp venom were purified and identified. VMS1 was 5-hydroxytryptamine, and the other compounds were three peptides that were sequenced as follows: Gly-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg-Ile-Asp-NH2 (VMS2), Ile-Asn-Leu-Lys-Ala-Ile-Ala-Ala-Leu-Ala-Lys-Lys-Leu-Leu-NH2 (VMS3), and Phe-Leu-Pro-Ile-Ile-Gly-Lys-Leu-Leu-Ser-Gly-Leu-Leu-NH2 (VMS4). The quantifications for these components were 110.2 mg/g, 26.9 mg/g, 216.3 mg/g, and 58.0 mg/g, respectively. The results of this work indicated that the combination of the chemical fingerprint and quantitative analysis offers a reasonable way to evaluate the quality of wasp venom.

Investigation of the interaction between serotonin-related compounds and phenylboronate moieties in monolithic silica disk-packed spin columns.

Solid-phase extraction technologies are widely used for sample pretreatment in bioanalysis. Monolithic silica disk-packed spin columns modified with phenylboronate moieties have been developed for the selective extraction of cis-diol compounds such as catecholamines. However, in our preliminary studies, serotonin was found to also be extracted in this treatment, along with catecholamines. In this study, the interaction between serotonin-related compounds (serotonin, tryptophan, 5-hydroxy-tryptophan and 5-hydroxyindoleacetic acid) and phenylboronate moieties was investigated. We found that only serotonin was extracted with phenylboronate-modified monolithic silica, whereas tryptophan, 5-hydroxy-tryptophan and 5-hydroxyindoleacetic acid were not. Hydrophobic interactions rather than ionic interactions were the primary factor for the adsorption of serotonin to phenylboronate. Finally, the selective pretreatment procedure for catecholamines was improved: thus, the method could be applied for the pretreatment of bio-samples.

Mice Lacking Brain-Derived Serotonin Have Altered Swallowing Function.

The intricate sensorimotor neural circuits that control swallowing are heavily reliant on serotonin (5-hydroxytryptamine [5-HT]); however, the impact of 5-HT deficiency on swallow function remains largely unexplored. We investigated this using mice deficient in tryptophan-hydroxylase-2 (TPH2), the enzyme catalyzing the rate-limiting step in 5-HT synthesis. Videofluoroscopy was utilized to characterize the swallowing function of TPH2 knockout (TPH2-/-) mice as compared with littermate controls (TPH2+/+). Results showed that 5-HT deficiency altered all 3 stages of swallowing. As compared with controls, TPH2-/- mice had significantly slower lick and swallow rates and faster esophageal transit times. Future studies with this model are necessary to determine if 5-HT replacement may rescue abnormal swallowing function. If so, supplemental 5-HT therapy may have vast applications for a large population of patients with a variety of neurologic disorders resulting in life-diminishing dysphagia, particularly amyotrophic lateral sclerosis and Parkinson's disease, for which 5-HT deficiency is implicated in the disease pathogenesis.

Ratiometric Electrochemical Sensors Associated with Self-Cleaning Electrodes for Simultaneous Detection of Adrenaline, Serotonin, and Tryptophan.

Electrochemical sensors have long suffered from issues such as nonspecific adsorption, poor anti-interference ability, and internal and external disturbances. To address these challenges, we developed a facile electrochemical method, which integrated a ratiometric strategy with self-cleaning electrodes. In the novel sensing system, the self-cleaning electrode was realized via forming a hydrophobic layer on carbonized ZIF-67@ZIF-8 (cZIF) by polydimethylsiloxane (PDMS) precursor vaporization. As for ratiometry, it is worth to mention that the measurements were conducted by adding an interior reference (methylene blue) directly into electrolyte solution, which is more facile and flexible to operate compared with conventional ones. Sensing performance of the self-cleaning electrode as well as the newly established ratiometric strategy was explored fully, and it turned out that PDMS@cZIF nanocomposites provided decent electrocatalytic ability, superhydrophobic property, and stability. Furthermore, the ratiometric strategy significantly elevated the robustness and reproducibility of electrochemical sensing. Simultaneous detection of Adr, 5-HT, and Trp was performed under the optimum experimental conditions with wide linear ranges and low detection limits. Finally, the original ratiometric electrochemical sensor was successfully applied for monitoring the three target molecules in biological samples.

Inhibitory effect of moschamine isolated from Carthamus tinctorius on LPS-induced inflammatory mediators via AP-1 and STAT1/3 inactivation in RAW 264.7 macrophages.

Seeds of Carthamus tinctorius L. (Compositae) have been used in Korean traditional medicines for the treatment of cardiovascular and bone diseases. In this study, we investigated the anti-inflammatory effects of known serotonin derivatives (1-9) isolated from the ethyl acetate (EtOAc) soluble fraction from the seeds of C. tinctorius. Compound 2, identified as moschamine, most potently inhibited lipopolysaccharide (LPS)-induced production of prostaglandin E2 (PGE2) and nitric oxide (NO) in RAW 264.7 macrophages. Moschamine concentration-dependently inhibited LPS-induced PGE2 and NO production in RAW 264.7 macrophages. Consistent with these findings, moschamine suppressed the protein and mRNA levels of cyclooxygenase-2 (COX-2), microsomal prostaglandin E2 synthase (mPGES)-1, and inducible NO synthase (iNOS), interleukin (IL)-6, and IL-1ß. In addition, pretreatment of moschamine significantly inhibited LPS-stimulated the transcriptional activity of activator protein-1 (AP-1) and the phosphorylation of signal transducer and activator of transcription (STAT)1/3 in RAW 264.7 macrophages. Moreover, moschamine inhibited LPS-induced the phosphorylation of p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK), but it had no effect on c-Jun N-terminal kinase (JNK). These results suggest that the mechanism of anti-inflammatory activity of moschamine is associated with the downregulation of COX-2, mPGES-1, iNOS, IL-6, and IL-1ß expression through the suppression of AP-1 and STAT1/3 activation in LPS-induced RAW 264.7 macrophages.

Phytochemistry and Pharmacology of Carthamus tinctorius L.

Carthamus tinctorius L. is a multifunctional cash crop. Its flowers and seeds are extensively used in traditional herbal medicine in China, Korea, Japan, and other Asian countries, for treating various ailments such as gynecological, cardiovascular, and cerebrovascular diseases as well as blood stasis and osteoporosis. More than 100 compounds have been isolated and identified from C. tinctorius. Flavonoids and alkaloids, especially the quinochalcone c-glycoside hydroxysafflor yellow A, N-(p-Coumaroyl)serotonin, and N-feruloylserotonin, are responsible for most of the pharmacological activities of C. tinctorius. In this paper, comprehensive and up-to-date information on the phytochemistry and pharmacology of C. tinctorius is presented. This information will be helpful for further explorations of the therapeutic potential of C. tinctorius and may provide future research opportunities.

Quantitative analysis of serotonin secreted by human embryonic stem cells-derived serotonergic neurons via pH-mediated online stacking-CE-ESI-MRM.

A CE-ESI-MRM-based assay was developed for targeted analysis of serotonin released by human embryonic stem cells-derived serotonergic neurons in a chemically defined environment. A discontinuous electrolyte system was optimized for pH-mediated online stacking of serotonin. Combining with a liquid-liquid extraction procedure, LOD of serotonin in the Krebs'-Ringer's solution by CE-ESI-MS/MS on a 3D ion trap MS was0.15 ng/mL. The quantitative results confirmed the serotonergic identity of the in vitro developed neurons and the capacity of these neurons to release serotonin in response to stimulus.

The presence of serotonin in cigarette smoke - a possible mechanistic link to 5-HT-induced airway inflammation.

We previously reported the involvement of serotonin (5-HT) metabolism in cigarette smoke-induced oxidative stress in rat lung in vivo. Here, we report cigarette smoke as a source of serotonin (5-HT) to the airways and aim at investigating the effects of 5-HT on oxidative stress and inflammation in human bronchial epithelial cells (BEAS-2B). A 5-HT analog was identified to be present in aqueous phase cigarette smoke using the LC-MS/MS approach, which was later confirmed by a 5-HT enzyme-linked immune assay (EIA). Furthermore, exposure to 5-HT caused a time-dependent elevation of intracellular ROS level, which was blocked in the presence of apocynin (a NOX inhibitor). In support, the immunoblot analysis indicated that there was an increase in the expression of NOX2 time-dependently. 5-HT-induced elevation of IL-8 at both mRNA and protein levels was observed, which was inhibited by TEMPOL (a free radical scavenger), and inhibitors for p38 MAPK (SB203580) and ERK (U0126), in line with the time-dependent phosphorylation of p38 MAPK and ERK. In conclusion, our findings suggest that 5-HT presented in bronchial epithelium of smokers may be involved in cigarette smoke-induced oxidative stress and inflammation via activation of p38 MAPK and ERK pathway after the formation of free radicals.

[Serotonin and neuropeptide FMRFamide in the nervous system of Opisthioglyphe ranae (Trematoda: Plagiorchiidae). an immunocytochemical study].

The presence and localization of the serotoninergic and FMRFamidergic structures in the nervous system of the trematode Opisthioglyphe ranae, the marsh frog intestinal parasite, was studied using immunocytochemistry. The serotonin-immunoreactive nerve cells and fibers were revealed in the head ganglia, circular commissure, longitudinal nerve cords and their connective commissures, as well as around the oral and ventral suckers, oesophagus and genital pore. FMRF-specific immunoreactivity was observed in the head ganglia, longitudinal nerve cords and terminal parts of the reproductive system. The results obtained are discussed in light of the available data on the presence and functional significance of the above-mentioned neurotransmitters in trematodes.

Preparative Separation of N-Feruloyl Serotonin and N-(p-Coumaroyl) Serotonin from Safflower Seed Meal Using High-Speed Counter-Current Chromatography.

High-speed counter-current chromatography (HSCCC) was successfully applied for the preparative separation and purification of N-feruloyl serotonin (NF) and N-(p-coumaroyl) serotonin (NP) from safflower seed meal. After the measurement of partition coefficient of the two target compounds in the two-phase solvent systems, the HSCCC was performed well with a two-phase solvent system composed of CHCl3-methanol-0.1 M HCl at a volume ratio of 1 : 1 : 1, v/v. The upper phase was used as stationary phase and the lower phase was used as mobile phase. Under the optimized condition, 7.5 mg NF and 6.9 mg NP were separated from 40 mg crude sample with the purity of 98.8 and 97.3%, respectively. The structures of the isolated compounds were identified by (1)H NMR and (13)C NMR.

Electrochemical serotonin monitoring of poly(ethylenedioxythiophene):poly(sodium 4-styrenesulfonate)-modified fluorine-doped tin oxide by predeposition of self-assembled 4-pyridylporphyrin.

A 5,10,15,20-tetrakis(4-pyridyl)-21H,23H-porphyrin (TPyP)-modified self-assembled functional layer was prepared on a fluorine-doped tin oxide (FTO) substrate. We employed a bifunctional molecule, 3-iodopropionate (3IP), to covalently bind TPyP to the FTO substrate. The 3IP-monolayered FTO and the TPyP-3IP-bilayered FTO electrodes were characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and Fourier transform-infrared spectroscopy. Compared to conventional electropolymerized poly(ethylenedioxythiophene):poly(sodium 4-styrenesulfonate) (PEDOT:PSS) film on bare FTO, the PEDOT:PSS film on the TPyP-3IP-bilayered FTO showed better sensitivity and selectivity in monitoring serotonin in the presence of high concentrations of interfering agents such as ascorbic acid, urea, D-(+)-glucose, epinephrine, and L-3,4-dihydroxyphenylalanine. Both PEDOT:PSS films on the bare FTO and the TPyP-3IP-bilayered FTO showed electrocatalytic effects in serotonin detection, and only the TPyP-3IP-based PEDOT:PSS film acted as a pH resistant buffer layer in the selective detection of serotonin.

Comprehensive analysis of neurotransmitters from regenerating planarian extract using an ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring method.

RATIONALE: Absolute quantification of neurotransmitters (NTs) from biological systems is imperative to track how changes in concentration of active neurochemicals may affect biological behavior. A sensitive method for the absolute quantification of multiple NTs in a single method is highly needed. METHODS: A stable-isotope dilution ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC/MS/SRM) assay has been developed for a sensitive and quantitative assessment of NTs in planaria. We used this method for the simultaneous quantification of 16 NTs. All analytes showed a linear relationship between concentrations (0.78-50 ng/mL), regression coefficients higher than 0.97, accuracy (91-109%) and low coefficients of variation (CVs). The inter-day CVs for the lowest quality controls (1.56 ng/mL) were in the range between 2-11%. RESULTS: The levels of most of the NTs were similar in both sexual and asexual planarians except for glutamic acid, which was about two-fold higher in asexual compared to sexual planarians. We identified high levels of serotonin and failed to detect tryptamine suggesting that the pathway essential for the conversion of tryptophan into tryptamine is absent in planarians. Interestingly, we also found high levels of dopamine and L-DOPA in regenerating planarians suggesting their possible role in regeneration. CONCLUSIONS: For the first time, we developed novel methodology based on UHPLC/MS/SRM and quantified 16 NTs with high sensitivity and specificity from sexual and asexual strains of planarian Schmidtea mediterranea. This method will also have great application in quantifying various NTs with great precision in different model systems.

Production of indole alkaloids by metabolic engineering of the tryptophan pathway in rice.

Tryptophan decarboxylase (TDC) converts tryptophan (Trp) into tryptamine, consequently increasing the metabolic flow of tryptophan derivatives into the production of secondary metabolites such as indole alkaloids. We inserted an expression cassette containing OsTDC, a putative tryptophan decarboxylase gene from rice, into an expression plasmid vector containing OASA1D, the feedback-resistant anthranilate synthase alpha-subunit mutant (OASA1D). Overexpression of OASA1D has been reported to significantly increase Trp levels in rice. The co-expression of OsTDC and OASA1D in rice calli led to almost complete depletion of the Trp pool and a consequent increase in the tryptamine pool. This indicates that TDC inactivity is a contributory factor for the accumulation of Trp in rice transgenics overexpressing OASA1D. Metabolic profiling of the calli expressing OsTDC and OASA1D revealed the accumulation of serotonin and serotonin-derived indole compounds (potentially pharmacoactive ß-carbolines) that have not been reported from rice. Rice calli overexpressing OASA1D:OASA1D is a novel system for the production of significant amounts of pharmacologically useful indole alkaloids in rice.

Dual-templates molecularly imprinted monolithic columns for the evaluation of serotonin and histamine in CEC.

To extend the application of molecularly imprinted polymers, the dual-templates molecularly imprinted monolithic columns were developed in a capillary format. Two templates serotonin and histamine were simultaneously imprinted using two different functional monomers such as methacrylic acid (MAA) and methylenesuccinic acid (MSA) in a mixture of ethylene glycol dimethacrylate (EDMA) as a cross-linker and AIBN as polymerization initiator dissolved in DMF as porogen. The resulting molecular imprinted polymers (MIPs) were characterized based on their performance in the CEC separation of two imprinted templates. The optimization parameters such as pH, ACN composition, and concentration of the eluent were varied to achieve best resolution and efficiency for CEC separation of templates with each MIP column. It was found that the MIP monolith column fabricated using MSA offered better resolution and separation efficiency compared to column fabricated with MAA. This work utilized the dual-templates imprinting approach successfully and broadens the scope of multi-templates imprinting capabilities in capillary format in CEC application.

A carbon nanofiber based biosensor for simultaneous detection of dopamine and serotonin in the presence of ascorbic acid.

A biosensor based on an array of vertically aligned carbon nanofibers (CNFs) grown by plasma enhanced chemical vapor deposition is found to be effective for the simultaneous detection of dopamine (DA) and serotonin (5-HT) in the presence of excess ascorbic acid (AA). The CNF electrode outperforms the conventional glassy carbon electrode (GCE) for both selectivity and sensitivity. Using differential pulse voltammetry (DPV), three distinct peaks are seen for the CNF electrode at 0.13 V, 0.45 V, and 0.70 V for the ternary mixture of AA, DA, and 5-HT. In contrast, the analytes are indistinguishable in a mixture using a GCE. For the CNF electrode, the detection limits are 50 nM for DA and 250 nM for 5-HT.

Optimization for speed and sensitivity in capillary high performance liquid chromatography. The importance of column diameter in online monitoring of serotonin by microdialysis.

The speed of a separation defines the best time resolution possible in online measurements using chromatography. The desired time resolution multiplied by the flow rate of the stream of analyte being sampled defines the maximum volume of sample per injection. The best concentration sensitivity in chromatography is obtained by injecting the largest volume of sample that is consistent with achieving a satisfactory separation, and thus measurement accuracy. Taking these facts together, it is easy to understand that separation speed and concentration sensitivity are linked in this type of measurement. To address the problem of how to achieve the best sensitivity and shortest measurement time simultaneously, we have combined recent approaches to the optimization of the separation itself with an analysis of method sensitivity. This analysis leads to the column diameter becoming an important parameter in the optimization process. We use these ideas in one particular problem presented by online microdialysis sampling/liquid chromatography/electrochemical detection for measuring concentrations of serotonin in the dialysate. In this case the problem becomes the optimization of conditions to yield maximum signal for a given sample volume under the highest speed conditions with a certain required number of theoretical plates. It turns out that the observed concentration sensitivity at an electrochemical detector can be regulated by temperature, particle size, injection volume/column diameter, and void time. The theory was successfully used for optimization of neurotransmitter serotonin measurement by capillary HPLC when sampling from a microdialysis flow stream. The final conditions are: 150 µm i.d., 3.1cm long columns with 1.7 µm particle diameter working at a flow rate of 12 µL/min, an injection volume of 500 nL, and a temperature of 343 K. The retention time for serotonin is 22.7s, the analysis time is about 36 s (which allows for determination of 3-methoxytyramine), and the sampling time is about 0.8 min with a perfusion flow rate of 0.6 µL/min.