Simultaneous determination of three estrogens in human saliva without derivatization or liquid-liquid extraction for routine testing via miniaturized solid phase extraction with LC-MS/MS detection.

Accurate quantitation of estrogens (i.e, estrone (E1), estradiol (E2) and estriol (E3)) is valuable for clinical assessment of human health and disease. Alterations in estrogen levels have been implicated in numerous pathological conditions. However, inadequacies in sensitivity and specificity, cumbersome sample preparation and invasive specimen collection hamper the usability of available methods for clinical applications. Herein, a simple, rapid, highly sensitive and specific LC-MS/MS method was developed and validated for the simultaneous determination of three estrogens in human saliva providing a non-invasive alternative to conventional blood samples. For the first time, a 96-well hydrophilic-lipophilic-balanced (HLB) microplate was employed for clean-up and enrichment of estrogens in a single extraction without the requirements of derivatization, evaporation, liquid-liquid extraction and online extraction. A rapid LC chromatographic separation with a turnaround time of 5.0min was achieved on a BEH C18 XP column. The use of 0.1mM ammonium fluoride (NH4F) as LC additive, and integration of summated and scheduled multiple reaction monitoring (MRM) transitions substantially improved the sensitivity to 1pg/mL, allowing the accurate quantitation of trace levels of three estrogens in one run. The assay was fully validated with good performance for extraction efficiency (67.0-85.6%), matrix effect (89.6-100.2%), linearity (from 1.0pg/mL up to 1000pg/mL), accuracy (98.9-112.4%) and precision (≤7.4%). Additionally, the assay was unaffected by 34 structurally-similar, potentially interfering substances tested at high clinical concentrations. The applicability of the assay was demonstrated by assessing the reference intervals of authentic saliva samples from healthy adult males, pre- and post-menopausal females. The easy sample preparation, fast LC and multi-analyte MS/MS detection utilizing noninvasive saliva as a specimen delivers a simple, practical, sensitive and accurate tool suitable for the high throughput measurement of E1, E2 and E3 in clinical laboratories.

A simple dilute and shoot approach incorporated with pentafluorophenyl (PFP) column based LC-MS/MS assay for the simultaneous determination of trimethylamine N-oxide and trimethylamine in spot urine samples with high throughput.

Determination of trimethylamine N-oxide (TMAO) and trimethylamine (TMA) in biological and environmental samples has drawn great attention recently due to their increasing association with human health and disease. It remains a challenge to simultaneously quantify TMAO and TMA in a simple, fast and cost-effective manner due to pre-analytical and analytical constraints. For the first time, we describe a dilute and shoot approach combined with LC-MS/MS detection for the simultaneous measurement of the analytes in spot urine samples with high throughput. Compared to the existing methods, the merits of the proposed assay include the use of a simple dilute and shoot approach (100-fold), small sample volume (10µL), short LC run on a PFP column (4.0min) and multi-analyte MS detection without sample cleanup, derivatization, evaporation and a HILIC column. Dilution, LC and MS parameters were optimized in detail. Method validation yielded a wide linearity for TMAO (1.0-400µg/mL) and TMA (0.025-10µg/mL) with a respective limit of quantitation of 1.0 and 0.025µg/mL. The quantitation was not affected by 41 major urinary components, structurally-related drugs and metabolites. The intra- and inter-day assay precisions were ≤3.6% and recoveries were 93.3%-103.3% for spiked quality control samples. The clinical utility of the alternative spot urine sampling approach compared to conventional 24h urine collection was supported by a significant correlation between the two sampling strategies (n=20, p<0.0001, r=0.757-0.862; slope=0.687-1.170) and no statistical difference in day-to-day biological variability (n=20). The applicability and reliability of the assay was verified by the assessment of reference intervals in a cohort of 118 healthy people. The proposed assay would be beneficial for the rapid and accurate determination of the increasingly important TMAO and TMA demanded in clinical, environmental, pharmaceutical and nutritional fields.

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications.

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 µm) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

Simultaneous extraction and determination of monoamine neurotransmitters in human urine for clinical routine testing based on a dual functional solid phase extraction assisted by phenylboronic acid coupled with liquid chromatography-tandem mass spectrometry.

The major monoamine neurotransmitters, serotonin (5-HT) and catecholamines (i.e., norepinephrine (NE), epinephrine (E), and dopamine (DA)), are critical to the nervous system function, and imbalances of the neurotransmitters have been connected to a variety of diseases, making their measurement useful in a clinical setting. A simple, rapid, robust, sensitive, and specific LC-MS/MS method has been developed and validated for the simultaneous quantitation of urinary serotonin and catecholamines with low cost, which is ideal for routine clinical applications. A simple extraction from complex urine was accomplished using tailored solid phase extraction incorporating phenylboronic acid complexation on a 96-well HLB microplate for the sample extraction and resulted in significantly improved throughput, selectivity, and extraction recovery. Compared to 1-10 mL of urine typically used, this method required only 10 µL. A rapid chromatographic elution with a total cycle time of 6 min per sample compared to reported run times of 19-75 min was achieved on a PFP column. The sensitivity of l and 2 ng mL-1 for the detection of low abundant E and NE combined with the high coverage of 1024 ng mL-1 for DA enabled the multi-analyte detection of these biogenic amines in a single run. Good linearity (2.0-512, 1.0-512, 4.0-1024, and 4.0-1024 ng mL-1 for NE, E, DA, and 5-HT, respectively), accuracy (87.6-104.0%), precision (≤8.0%), extraction recovery (69.6-103.7%), and matrix effect (87.1-113.1% for catecholamines and 63.6-71.4% for 5-HT) were obtained. No autosampler carryover was observed. The analytes were stable for 5 days at 20 °C, 14 days at 4 °C, and 30 days at -20 °C and five freeze-thaw cycles. The easy sample preparation, rapid LC, and multi-analyte MS detection allow two 96-well plates of samples to be extracted within 2 h and analyzed on an LC-MS/MS system within 24 h. The applicability and reliability of the assay were demonstrated by assessment of the reference interval for authentic urine specimens from 90 healthy individuals. Graphical abstract A simple, rapid, robust, sensitive and specific LC-MS/MS method combined with a dual functional solid phase extraction has been developed and validated for the simultaneous extraction and quantitation of monoamine neurotransmitters in human urine with low cost.

Biomolecular aspects of depression: A retrospective analysis.

OBJECTIVE: The effects of psychological stress, oxidative stress, and chronic low grade inflammation on the neuro-immune connection have been implicated in the pathogenesis of depression. Thus, in the recent past, there has been a growing effort in determining the mechanism of this pathogenesis. While attempting to map out, this mechanism researchers and clinicians have searched for clinically relevant biomarkers for use in the diagnosis and for the assessment of those suffering from depression. In this study, we have performed a retrospective analysis of biomarkers with clinically relevant potentials, including peripheral catecholamines, chemokines, cytokines, and neurotransmitters. METHODS: The retrospective analysis was performed on data collected over a six-year period of time (July 2009 to July 2015), gathered from patients (N=1399; Mage=42, SD=13; 71% female, 29% male) who submitted samples with complaints of feeling hopeless, worthless, isolated, alone, general sadness, overwhelmed, and/or a lack of interest in things they once enjoyed. The data collected consisted of quantitative values of urinary catecholamines and neurotransmitters (peripheral dopamine, epinephrine, histamine, kynurenic acid, norepinephrine, ß-PEA, and serotonin), salivary hormones (peripheral cortisol and melatonin), and peripheral blood mononuclear cell secreted cytokines and chemokines (Interleukins 1ß, 6, 8, 10, MCP-1, GCSF, and TNFα). Statistical and clinical significance was assessed by comparison with a control group (N=2395; Mage=42, SD=13; 70% female, 30% male), calculating the percent mean difference, p value, and effect size (Cohen's ɗ) for each parameter between groups. RESULTS: The findings of this study suggested that, in a model of general depression, there is a dysregulation in the enzymatic production and degradation of catecholamines, neurotransmitters, hormones, and immunological proteins. A cycle of interaction was found between all of these biomolecules, where an increase or decrease in one marker could result in a stimulatory or inhibitory effect on others. The mechanism of this was proposed to occur through the interaction of psychological stress, inflammation, and oxidative stress pathways. All of these biomolecules were found to be significantly altered in the general depression group and are key components of the interaction between the neurological and immunological systems. CONCLUSIONS: This study serves to further elucidate the role of biomolecules in the regulation of affective disorders, such as depression. Resulting in providing a network of clinically relevant biomarkers to objectively assess and monitor general depression.

Pre-analytical and analytical validations and clinical applications of a miniaturized, simple and cost-effective solid phase extraction combined with LC-MS/MS for the simultaneous determination of catecholamines and metanephrines in spot urine samples.

It remains a challenge to simultaneously quantify catecholamines and metanephrines in a simple, sensitive and cost-effective manner due to pre-analytical and analytical constraints. Herein, we describe such a method consisting of a miniaturized sample preparation and selective LC-MS/MS detection by the use of second morning spot urine samples. Ten microliters of second morning urine sample were subjected to solid phase extraction on an Oasis HLB microplate upon complexation with phenylboronic acid. The analytes were well-resolved on a Luna PFP column followed by tandem mass spectrometric detection. Full validation and suitability of spot urine sampling and biological variation were investigated. The extraction recovery and matrix effect are 74.1-97.3% and 84.1-119.0%, respectively. The linearity range is 2.5-500, 0.5-500, 2.5-1250, 2.5-1250 and 0.5-1250ng/mL for norepinephrine, epinephrine, dopamine, normetanephrine and metanephrine, respectively. The intra- and inter-assay imprecisions are ≤9.4% for spiked quality control samples, and the respective recoveries are 97.2-112.5% and 95.9-104.0%. The Deming regression slope is 0.90-1.08, and the mean Bland-Altman percentage difference is from -3.29 to 11.85 between a published and proposed method (n=50). A correlation observed for the spot and 24h urine collections is significant (n=20, p<0.0001, r: 0.84-0.95, slope: 0.61-0.98). No statistical differences are found in day-to-day biological variability (n=20). Reference intervals are established for an apparently healthy population (n=88). The developed method, being practical, sensitive, reliable and cost-effective, is expected to set a new stage for routine testing, basic research and clinical applications.

An integrated liquid chromatography-tandem mass spectrometry approach for the ultra-sensitive determination of catecholamines in human peripheral blood mononuclear cells to assess neural-immune communication.

Catecholamines play a vital role in the interactions between the nervous and immune systems and their dysfunctions are implicated in various autoimmune and neurological diseases. However, accurate quantitation of catecholamines in the immune system presents a special analytical challenge. We proposed the first LC-MS/MS method for the determination of catecholamines in human peripheral blood mononuclear cells (PBMC) with significantly improved sensitivity, selectivity and throughput without requiring derivatization, evaporation and ion-pairing reagent. PBMC were separated by density gradient centrifugation and lysed with 0.2M acetic acid. The analytical novelty includes the first solid phase extraction on a 96-well hydrophilic-lipophilic-balanced (HLB) µElution plate upon complexation with phenylboronic acid (PBA), enabling specific clean-up and fivefold pre-concentration of catecholamines in a single extraction. LC chromatographic separation was obtained on a PFP column with 0.01% HCOOH as additive with enhanced signal response. Summation of five MRM transitions yielded three-four fold rise in sensitivity. The lower limit of quantification of 1pg/mL for epinephrine (E) and 5pg/mL for norepinephrine (NE) and dopamine (DA) represents a considerable sensitivity improvement over available methods. Less than 8.7% of intraday and interday precision, 91.8-111.3% of accuracy and successful assessment of reference intervals for 40 healthy donors suggested good reproducibility and reliability of the assay. The novel PBA-HLB-PFP-MRM summation approach allows rapid, sensitive and reliable determination of catecholamines in PBMC, which will facilitate better understanding of the new arena of neural-immune network. Additionally, the substantially improved method can be modified to quantify catecholamines and metabolites in other biological matrices.

Tailored 96-well µElution solid-phase extraction combined with UFLC-MS/MS: a significantly improved approach for determination of free 3-nitrotyrosine in human urine.

We developed and validated a simple and fast UFLC-MS/MS method for the accurate determination of 3-nitrotyrosine (3-NT) in human urine as a noninvasive biomarker for oxidative stress. The method, involving tailored 96-well µElution solid-phase extraction (SPE) combined with UFLC-MS/MS, allows 3-NT to be determined in biological samples without the need for hydrolysis, derivatization, evaporation, and two-dimensional LC for the first time. Using ammonium acetate (pH 9, 25 mM) as an elution buffer was found to improve SPE selectivity. Fast chromatographic elution of 3-NT with a total run time of 7 min was achieved on a PFPP column (150 mm × 2.1 mm, 3 µm). This fine-tuned integrated method delivered significantly improved throughput, specificity, and sensitivity while reducing the matrix effect, solvent usage, and waste disposal. Using this simple and rapid method, two plates of urine samples (n = 192) can be processed within 24 h. The lower limit of quantification for 3-NT is 10 pg/mL, which represents a notable sensitivity enhancement over reported methods. Less than 6.0 % variations for intraday and interday assay precisions and 97.7-106.3% for accuracies in terms of recovery were obtained. The applicability and reliability of the method were demonstrated by determining the reference range in human urine for 82 healthy people. Considering the noninvasive and inexpensive nature of urine sampling, this novel method could be used to re-evaluate the role of 3-NT as an oxidative stress biomarker in pre-clinical and clinical studies.

A novel mixed-mode solid phase extraction coupled with LC-MS/MS for the re-evaluation of free 3-nitrotyrosine in human plasma as an oxidative stress biomarker.

3-Nitrotyrosine (3-NT) has been widely adopted as a biomarker of oxidative stress. However, an enormous range of reported concentrations of 3-NT in biological matrices indicated an underlying methodological problem. Consequently, our understanding of tyrosine nitration in vivo and its significance as a biomarker may have been confounded. Here we report a fast, specific and sensitive LC-MS/MS method to accurately quantify free 3-NT in human plasma. For the first time, a single-step solid phase extraction of 3-NT using mixed-mode sorbent in a 96-well plate was developed after significant optimization. Complete chromatographic separation of 3-NT from other tyrosine analogs was achieved on a PFPP column (150 mm, 2.1 mm, 3 µm) with a cycle time of 10 min. The developed method was validated in terms of linearity, sensitivity, specificity, precision, accuracy, matrix effect, carryover, analyte stability and reference interval. The lower limit of quantification of the method was 5 pg/ml for plasma 3-NT, which represents a significant sensitivity improvement over reported methods. No artifactual 3-NT formation was observed, and the assay was not affected by 40 likely interferences. The average intra- and inter-assay variances were 3.4% and 3.7%, respectively. Of note, the reference interval for a healthy population was established to be 2.0-40.1 pg/ml (8.8-177 pM) with a mean of 11.1 pg/ml (49 pM). The mean value is at least 13-fold lower than previously reported mean values. Furthermore, the method was applied to the investigation of biological variation (n=15) over a three week period and no statistical differences were found. The solid phase extraction in a 96-well format and a fast LC-MS/MS delivered a practical, precise and accurate tool well-suited to quantify free plasma 3-NT in a large number of biological samples.

New insights into Lyme disease.

Lyme borreliosis is transmitted through the bite of a tick that is infected by the bacterial spirochete Borrelia burgdorferi. Clinical manifestation of the disease can lead to heart conditions, neurological disorders, and inflammatory disorders. Oxidative stress has been implicated in the pathogenesis of many human diseases. The aim of this study was to investigate the mechanisms of oxidative stress and intracellular communication in Lyme borreliosis patients. Mitochondrial superoxide and cytosolic ionized calcium was measured in peripheral blood mononuclear cells (PBMCs) of Lyme borreliosis patients and healthy controls. Mitochondrial superoxide levels were significantly higher (p<0.0001) in Lyme borreliosis patients (n=32) as compared to healthy controls (n=30). Significantly low (p<0.0001) levels of cytosolic ionized calcium were also observed in Lyme borreliosis patients (n=11) when compared to healthy controls (n=11). These results indicate that there is an imbalance of reactive oxygen species and cytosolic calcium in Lyme borreliosis patients. The results further suggest that oxidative stress and interrupted intracellular communication may ultimately contribute to a condition of mitochondrial dysfunction in the immune cells of Lyme borreliosis patients.

Development and validation of a specific and sensitive LC-MS/MS method for quantification of urinary catecholamines and application in biological variation studies.

Catecholamines are a class of biogenic amines that play an important role as neurotransmitters and hormones. We developed and validated a rapid, specific and sensitive LC-MS/MS method for quantitative determination of catecholamines in human urine. Linearity, specificity, sensitivity, precision, accuracy, matrix effect, carryover, analyte stability, method comparison and reference range were evaluated. The catecholamine measurements were not affected by 35 structurally-related drugs and metabolites. The outstanding specificity was achieved by use of a specific diphenylborate-based solid phase extraction and subsequent selective LC-MS/MS analysis. Excellent sensitivity, accuracy and precision (average intra-assay variations <2.9 % and inter-assay variations <4.6 %) were obtained. The method was successfully applied in the study of day-to-day biological within- and between-subject variations of 25 healthy people under free-living conditions over three consecutive days. We observed that catecholamine excretions for second morning sampling had least day-to-day within-subject variation and excellent reproducibility. This work is one of the rare studies on these topics and represents the first utilization of advanced LC-MS/MS technology. Additionally, we found significant correlations between spot and conventional 24 h collections of human urine (n = 22, r > 0.853, p < 0.0001). These findings suggest that determining the catecholamine concentrations in the second morning urine sample presents accurate, convenient and reliable measurement of catecholamine excretions. In addition, consistent and significant diurnal variations for norepinephrine and epinephrine excretions were observed during the three-day period, while dopamine did not exhibit a diurnal rhythm. The LC-MS/MS method presented here is rapid, sensitive and specific, which could be an advantage in clinical laboratories.

Validation of an ELISA for urinary dopamine: applications in monitoring treatment of dopamine-related disorders.

Dopamine is a catecholamine that serves as a neurotransmitter in the central and peripheral nervous system. Non-invasive, reliable, and high-throughput techniques for its quantification are needed to assess dysfunctions of the dopaminergic system and monitor therapies. We developed and validated a competitive ELISA for direct determination of dopamine in urine samples. The method provides high specificity, good accuracy, and precision (average inter-assay variation < 12%). The analysis is not affected by general urinary components and structurally related drugs and metabolites. The correlation between ELISA and LC-MS/MS analyses was very good (r = 0.986, n = 28). The reference range was 64-261 µg/g Cr (n = 64). Week-to-week biological variations of second morning urinary dopamine under free-living conditions were 23.9% for within- and 35.5% for between-subject variation (n = 10). The assay is applied in monitoring Parkinson's disease patients under different treatments. Urinary dopamine levels significantly increase in a dose-dependent manner for Parkinson's disease patients under l-DOPA treatment. The present ELISA provides a cost-effective alternative to chromatographic methods to monitor patients receiving dopamine restoring treatment to ensure appropriate dosing and clinical efficacy. The method can be used in pathological research for the assessment of possible peripheral biological markers for disorders related to the dopaminergic system.

Urinary neurotransmitters are selectively altered in children with obstructive sleep apnea and predict cognitive morbidity.

BACKGROUND: Pediatric obstructive sleep apnea (OSA) is associated with cognitive dysfunction, suggesting altered neurotransmitter function. We explored overnight changes in neurotransmitters in the urine of children with and without OSA. METHODS: Urine samples were collected from children with OSA and from control subjects before and after sleep studies. A neurocognitive battery assessing general cognitive ability (GCA) was administered to a subset of children with OSA. Samples were subjected to multiple enzyme-linked immunosorbent assays for 12 neurotransmitters, and adjusted for creatinine concentrations. RESULTS: The study comprised 50 children with OSA and 20 control subjects. Of the children with OSA, 20 had normal GCA score (mean ± SD) (101.2 ± 14.5) and 16 had a reduced GCA score (87.3 ± 13.9; P < .001). Overnight increases in epinephrine, norepinephrine, and γ-aminobutyric acid (GABA) levels emerged in children with OSA; taurine levels decreased. Using combinatorial approaches and cutoff values for overnight changes of these four neurotransmitters enabled prediction of OSA (area under the curve [AUC]: 0.923; P < .0001). Furthermore, GABA and taurine alterations, as well as overnight reductions in phenylethylamine, were more prominent in children with OSA and low GCA than in children with OSA and normal GCA (P < .001), and they reliably discriminated GCA status (AUC: 0.977; P < .0001). CONCLUSIONS: Pediatric OSA is associated with overnight increases in urinary concentrations of catecholamines indicative of heightened sympathetic outflow. Increases in GABA levels and decreases in taurine levels could underlie mechanisms of neuronal excitotoxicity and dysfunction. Combinatorial approaches using defined cutoffs in overnight changes in concentrations of selected neurotransmitters in urine may not only predict OSA but also the presence of cognitive deficits. Larger cohort studies appear warranted to confirm these findings.

An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi.

Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-g as a biomarker, we developed a new enzyme-linked immunospot method (iSpot Lyme™) to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.

Evaluation of a novel ELISA for serotonin: urinary serotonin as a potential biomarker for depression.

Depression is a common disorder with physical and psychological manifestations often associated with low serotonin. Since noninvasive diagnostic tools for depression are sparse, we evaluated the clinical utility of a novel ELISA for the measurement of serotonin in urine from depressed subjects and from subjects under antidepressant therapy. We developed a competitive ELISA for direct measurement of serotonin in derivatized urine samples. Assay performance was evaluated and applied to clinical samples. The analytical range of the assay was from 6.7 to 425 µg serotonin/g creatinine (Cr). The limit of quantification was 4.7 µg/g Cr. The average recovery for spiked urine samples was 104.4%. Average intra-assay variation was 4.4%, and inter-assay variation was <20%. The serotonin analysis was very specific. No significant interferences were observed for 44 structurally and nonstructurally related urinary substances. Very good correlation was observed between urinary serotonin levels measured by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS; ELISA = 1.16 × LC-MS/MS - 53.8; r = 0.965; mean % bias = 11%; n = 18). Serotonin was stable in acidified urine for 30 days at room temperature and at -20 °C. The established reference range for serotonin was 54-366 µg/g Cr (n = 64). Serotonin levels detected in depressed patients (87.53 ± 4.89 µg/g Cr; n = 60) were significantly lower (p < 0.001) than in nondepressed subjects (153.38 ± 7.99 µg/g Cr). Urinary excretion of serotonin in depressed individuals significantly increased after antidepressant treatment by 5-hydroxy-tryptophane and/or selective serotonin re-uptake inhibitor (p < 0.01). The present ELISA provides a convenient and robust method for monitoring urinary serotonin. It is suitable to monitor serotonin imbalances and may be particularly helpful in evaluating antidepressant therapies.

The Role of Th17 in Neuroimmune Disorders: A Target for CAM Therapy. Part III.

Abundant research has mapped the inflammatory pathways leading to autoimmunity and neuroinflammatory disorders. The latest T helper to be identified, Th17, through its proinflammatory cytokine IL-17, plays a pathogenic role in many inflammatory conditions. Today, healthcare providers have a wealth of anti-inflammatory agents from which to choose. On one hand, pharmaceutical companies market brand-name drugs direct to the public and physicians. Medical botanical knowledge, on the other hand, has been passed down from generation to generation. The demands for natural healing therapies have brought corresponding clinical and laboratory research studies to elucidate the medicinal properties of alternative practices. With a variety of options, it can be difficult to pinpoint the proper anti-inflammatory agent for each case presented. In this review, the authors highlight a vast array of anti-inflammatory medicaments ranging from drugs to vitamins and from botanicals to innate molecules. This compilation may serve as a guide for complimentary and alternative healthcare providers who need to target neuroinflammation driven by Th17 and its inflammatory cytokine IL-17. By understanding the mechanisms of anti-inflammatory agents, CAM practitioners can tailor therapeutic interventions to fit the needs of the patient, thereby providing faster relief from inflammatory complaints.

Novel ELISAs for screening of the biogenic amines GABA, glycine, beta-phenylethylamine, agmatine, and taurine using one derivatization procedure of whole urine samples.

The inhibitory neurotransmitters GABA, glycine and agmatine and neuromodulators beta-phenylethylamine (beta-PEA) and taurine are important biogenic amines of the sympathetic and parasympathetic nervous systems in the body. Abnormalities in the metabolism of these biomarkers have been implicated in a vast number of neurological diseases. Novel competitive immunoassays, using one unique whole urine derivatization procedure applicable for all five biomarkers, have been developed. The determination of these biomarkers was highly reproducible: the coefficient of variance of inter- and intra-assay variation is between 3.9% and 9.8% for all assays. The assays show a good linearity in urine samples within the range of 100-400 mg Cr/dL and specificity when urine samples are spiked with biogenic amines. The recoveries are between 76 and 154%. The correlation between HPLC and ELISA for glycine and taurine (n = 10) showed regression coefficients of 0.97 and 0.98, respectively. An in vivo study on the urinary clearance of beta-PEA, agmatine and taurine after oral intake by healthy individuals demonstrated the specificity and clinical significance of these new immunoassays. The immunoassays are useful for clinical and basic research where a fast and accurate assay for the screening of biogenic amines in urine is required, without preclearance of the sample.