Discovery of a Gut Bacterial Metabolic Pathway that Drives α-Synuclein Aggregation.

Parkinson's disease (PD) etiology is associated with aggregation and accumulation of α-synuclein (α-syn) proteins in midbrain dopaminergic neurons. Emerging evidence suggests that in certain subtypes of PD, α-syn aggregates originate in the gut and subsequently spread to the brain. However, mechanisms that instigate α-syn aggregation in the gut have remained elusive. In the brain, the aggregation of α-syn is induced by oxidized dopamine. Such a mechanism has not been explored in the context of the gastrointestinal tract, a niche harboring 46% of the body's dopamine reservoirs. Here, we report that Enterobacteriaceae, a bacterial family prevalent in human gut microbiotas, induce α-syn aggregation. More specifically, our in vitro data indicate that respiration of nitrate by Escherichia coli K-12, which results in production of nitrite that mediates oxidation of Fe2+ to Fe3+, creates an oxidizing redox potential. These oxidizing conditions enabled the formation of dopamine-derived quinones and α-syn aggregates. Exposing nitrite, but not nitrate, to enteroendocrine STC-1 cells induced aggregation of α-syn that is natively expressed in these cells, which line the intestinal tract. Taken together, our findings indicate that bacterial nitrate reduction may be critical for initiating intestinal α-syn aggregation.

Scoring system for diagnosis and pretreatment risk assessment of neuroblastoma using urinary biomarker combinations.

The urinary catecholamine metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA), are used for the adjunctive diagnosis of neuroblastomas. We aimed to develop a scoring system for the diagnosis and pretreatment risk assessment of neuroblastoma, incorporating age and other urinary catecholamine metabolite combinations. Urine samples from 227 controls (227 samples) and 68 patients with neuroblastoma (228 samples) were evaluated. First, the catecholamine metabolites vanillactic acid (VLA) and 3-methoxytyramine sulfate (MTS) were identified as urinary marker candidates through comprehensive analysis using liquid chromatography-mass spectrometry. The concentrations of these marker candidates and conventional markers were then compared among controls, patients, and numerous risk groups to develop a scoring system. Participants were classified into four groups: control, low risk, intermediate risk, and high risk, and the proportional odds model was fitted using the L2-penalized maximum likelihood method, incorporating age on a monthly scale for adjustment. This scoring model using the novel urine catecholamine metabolite combinations, VLA and MTS, had greater area under the curve values than the model using HVA and VMA for diagnosis (0.978 vs. 0.964), pretreatment risk assessment (low and intermediate risk vs. high risk: 0.866 vs. 0.724; low risk vs. intermediate and high risk: 0.871 vs. 0.680), and prognostic factors (MYCN status: 0.741 vs. 0.369, histology: 0.932 vs. 0.747). The new system also had greater accuracy in detecting missing high-risk neuroblastomas, and in predicting the pretreatment risk at the time of screening. The new scoring system employing VLA and MTS has the potential to replace the conventional adjunctive diagnostic method using HVA and VMA.

24-hour urine metanephrine excretion in patients diagnosed with adrenal incidentaloma: impact of commonly used drugs on a clinical decision.

INTRODUCTION: Incidentaloma is an adrenal tumor detected during diagnostic imaging performed for extra­adrenal causes. Evaluation of metanephrine concentrations in a 24­hour urine collection can be a significant challenge in patients with multiple medications and comorbidities. OBJECTIVES: The aim of this study was to evaluate the effect of commonly used groups of drugs on metanephrine levels in the 24­hour urine collection. PATIENTS AND METHODS: A total of 1051 patients with adrenal mass below 10 Hounsfield units on unenhanced computed tomography were included in the study. Patients diagnosed with Cushing or Conn syndrome, adrenal carcinoma, pheochromocytoma, active extra­adrenal malignant neoplasms, and exacerbation of severe illnesses were excluded. Metanephrine, normetanephrine, and 3­methoxytyramine in the 24­hour urine collection were measured by high­performance liquid chromatography with electrochemical detection. Information on concomitant medication (ß­blockers, calcium channel blockers [CCBs], loop diuretics, thiazide diuretics, potassium­sparing diuretics, α­blockers, angiotensin­converting enzyme inhibitors / angiotensin II receptor blockers, metformin, nonmetformin antidiabetic drugs [NMADs], lipid­lowering drugs, proton pump inhibitors, levothyroxine, thyreostatics, antidepressants, neuroleptics, benzodiazepines, glucocorticosteroids, inhaled B­receptor agonists, and ipratropium) was collected from each patient. RESULTS: The urinary excretion of normetanephrine was significantly higher in the patients on ß­blockers, CCBs, loop diuretics, α­blockers, NMADs, and neuroleptics. α­Blockers increased urine metanephrine concentration, and NMADs, antidepressants, and glucocorticosteroids lowered it. There was no association between the analyzed drugs and urinary 3­methoxytyramine level. CONCLUSIONS: Many drug groups interfere with the measurement of urinary fractionated metanephrines. These interactions should be taken into account during interpretation of a hormonal evaluation, as they can be crucial for further management, especially for making a decision on surgical treatment.

The pivotal role of neuronal nitric oxide synthase in the release of 6-nitrodopamine from mouse isolated vas deferens.

6-Nitrodopamine (6-ND) is released from rat and human vas deferens and is considered a major mediator of both tissues contractility. The contractions induced by 6-ND are selectively blocked by both tricyclic antidepressants and α1-adrenoceptor antagonists. Endothelial nitric oxide synthase (eNOS) is the major isoform responsible for 6-ND release in mouse isolated heart, however the origin of 6-ND in the vas deferens is unknown. Here it was investigated by LC-MS/MS the basal release of 6-ND from isolated vas deferens obtained from control, eNOS-/-, nNOS-/-, and iNOS-/- mice. In addition, it was evaluated in vitro vas deferens contractility following electric field stimulation (EFS). Basal release of 6-ND was significantly reduced in nNOS-/- mice compared to control mice, but not decreased when the vas deferens were obtained from either eNOS-/- or iNOS-/- mice. Pre-incubation of the vas deferens with tetrodotoxin (1 µM) significantly reduced the basal release of 6-ND from control, eNOS-/-, and iNOS-/- mice but had no effect on the basal release of 6-ND from nNOS-/- mice. EFS-induced frequency-dependent contractions of the vas deferens, which were significantly reduced when the tissues obtained from control, eNOS-/- and iNOS-/- mice, were pre-incubated with l-NAME, but unaltered when the vas deferens was obtained from nNOS-/- mice. In addition, the EFS-induced contractions were significantly smaller when the vas deferens were obtained from nNOS-/- mice. The results clearly demonstrate that nNOS is the main NO isoform responsible for 6-ND release in mouse vas deferens and reinforces the concept of 6-ND as a major modulator of vas deferens contractility.

Co-transplantation of autologous Treg cells in a cell therapy for Parkinson's disease.

The specific loss of midbrain dopamine neurons (mDANs) causes major motor dysfunction in Parkinson's disease, which makes cell replacement a promising therapeutic approach1-4. However, poor survival of grafted mDANs remains an obstacle to successful clinical outcomes5-8. Here we show that the surgical procedure itself (referred to here as 'needle trauma') triggers a profound host response that is characterized by acute neuroinflammation, robust infiltration of peripheral immune cells and brain cell death. When midbrain dopamine (mDA) cells derived from human induced pluripotent stem (iPS) cells were transplanted into the rodent striatum, less than 10% of implanted tyrosine hydroxylase (TH)+ mDANs survived at two weeks after transplantation. By contrast, TH- grafted cells mostly survived. Notably, transplantation of autologous regulatory T (Treg) cells greatly modified the response to needle trauma, suppressing acute neuroinflammation and immune cell infiltration. Furthermore, intra-striatal co-transplantation of Treg cells and human-iPS-cell-derived mDA cells significantly protected grafted mDANs from needle-trauma-associated death and improved therapeutic outcomes in rodent models of Parkinson's disease with 6-hydroxydopamine lesions. Co-transplantation with Treg cells also suppressed the undesirable proliferation of TH- grafted cells, resulting in more compact grafts with a higher proportion and higher absolute numbers of TH+ neurons. Together, these data emphasize the importance of the initial inflammatory response to surgical injury in the differential survival of cellular components of the graft, and suggest that co-transplanting autologous Treg cells effectively reduces the needle-trauma-induced death of mDANs, providing a potential strategy to achieve better clinical outcomes for cell therapy in Parkinson's disease.

Workplace Culture and Biomarkers of Health Risk.

Workplace culture has been studied for impact on health risk; however, connections with robust biologic markers of health remain to be established. We examined associations between the work environment and urinary levels of catecholamines and their metabolites as biomarkers of sympathetic nervous system activity, indicative of stress. We recruited participants (n = 219; 2018-2019) from a cardiovascular risk cohort to investigate workplace culture, well-being, and stress. Participants completed seven questionnaires. Urine samples were used to measure catecholamines and their metabolites by LC/MS/MS. Pearson correlation and linear regression models were used after adjusting for demographics and creatinine. Participants reporting higher well-being had lower urinary levels of dopamine, serotonin, and 3-methoxytyramine. Participants reporting a more engaged and more positive workplace had lower levels of dopamine and 3-methoxytyramine. Reported workplace isolation was correlated with higher levels of dopamine and 3-methoxytyramine. Given correlations between catecholamines, we used 3-methoxytyramine for linear regression. In fully adjusted models, in environments with a more positive culture, levels of 3-methoxytyramine remained lower (ß = -0.065 ± 0.025, p = 0.01) and indicated a positive association between workplace isolation and 3-methoxytyramine (ß = 0.064 ± 0.030, p = 0.04). These findings are consistent with an important relationship between workplace environment and sympathetic nervous system activity.

Release of 6-nitrodopamine modulates vascular reactivity of Pantherophis guttatus aortic rings.

6-Nitrodopamine (6-ND) is a novel catecholamine that is released from human umbilical cord vessels and Chelonoidis carbonaria aortic rings. The synthesis/release of 6-ND is inhibited by either pre-incubation of the vessels with the nitric oxide (NO) synthase inhibitor L-NAME or by mechanical removal of the endothelium. 6-ND causes powerful vasorelaxation, acting as a potent and selective dopamine D2-like receptor antagonist. Basal release of 6-ND from Panterophis guttatus endothelium intact and denuded aortic rings was quantified by LC-MS/MS. In order to evaluate the interaction of 6-ND with other catecholamines, aortic rings were suspended vertically between two metal hooks in 10-mL organ baths containing Krebs-Henseleit's solution and attached to isometric transducers. Endothelium intact aortic rings presented basal release of 6-ND, which was significantly reduced by previous incubation with L-NAME (100 µM). In endothelin-1 (3 nM) pre-contracted endothelium intact aortic rings, 6-ND (10pM-1 µM) and the dopamine D2-receptor antagonist L-761,626 (10 pM-1 µM) induced concentration-dependent relaxations, which were not affected by incubation with L-NAME but greatly reduced in endothelium-removed aortic rings. 6-ND (0.1-1 µM) produced significant rightward shifts of the concentration-response curves to dopamine in L-NAME pre-treated endothelium-intact (pA2 7.01) rings. Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND (1 µM). The EFS-induced contractions of L-NAME pre-treated endothelium-intact aortic rings were significantly inhibited by incubation with 6-ND (1 µM). The results indicate that 6-ND released from Pantherophis guttatus aortic rings is coupled to NO release and represents a new mechanism by which NO can modulate vascular reactivity independently of cGMP production.

Use of multibiomarker disease activity scores in biosimilarity studies for the treatment of patients with rheumatoid arthritis.

OBJECTIVES: This exploratory analysis investigated the potential use of the multibiomarker disease activity (MBDA) score to support biosimilarity assessments using data from two randomised controlled trials (RCTs) of biosimilar infliximab (IFX-qbtx) and biosimilar adalimumab (ADL-afzb) versus EU-sourced infliximab (Remicade; IFX-EU) and adalimumab (Humira; ADL-EU) reference products, respectively, both conducted in adult patients with active rheumatoid arthritis. METHODS: In one study, patients (N=650) were randomised 1:1 to IFX-qbtx or IFX-EU (3 mg/kg intravenous at weeks 0, 2 and 6, then every 8 weeks). In the other, patients (N=597) were randomised 1:1 to ADL-afzb or ADL-EU (40 mg subcutaneous every other week). All treatments were given with MTX. Mean values of MBDA scores were calculated at baseline (BL), based on the concentrations of 12 serum proteins using the Vectra disease activity algorithm, and at timepoints throughout treatment period 1 (TP1) of the IFX (weeks 6, 14, 30) and ADL (weeks 6, 12, 26) studies. Data were summarised using descriptive statistics for the intent-to-treat population, without imputation for missing data. RESULTS: At BL, mean (±SD) MBDA scores were 61.3 (±12.5) and 58.8 (±13.2) for IFX-qbtx (n=236) and IFX-EU (n=248), respectively, and 57.2 (±14.44) and 58.3 (±15.34) for ADL-afzb (n=292) and ADL-EU (n=293), respectively. Mean MBDA scores were highly comparable between IFX-qbtx and IFX-EU and between ADL-afzb and ADL-EU at all measured timepoints during TP1 in each study. CONCLUSIONS: These RCTs are the first to incorporate MBDA score as an exploratory assessment of biosimilarity. MBDA scores may provide objective, quantitative evidence of biosimilarity using an assessment of disease activity that is independent of the potential subjectivity inherent in joint counts, or in patient or physician global assessments. TRIAL REGISTRATION NUMBERS: NCT02222493 and NCT02480153.

Photo-oxidation and Photoreduction of Catechols by Chlorophyll Metabolites and Methylene Blue.

While plant-derived oxidants can protect cells from oxidative damage, limited research has examined the role of dietary chlorophyll. Photoreduction of ubiquinone by chlorophyll metabolites and red light has been reported in vitro and in animal models. Herein we examined photo-oxidation and photoreduction reactions of catechols, dopamine and hydrocaffeic acid. Photo-oxidation of dopamine by methylene blue and the chlorophyll metabolites pheophorbide A, chlorin e6 and sodium copper chlorophyllin was studied by monitoring aminochrome, the cyclized product of the dopamine o-quinone with its amine. Singlet oxygen scavengers including sodium azide, ascorbate and glutathione decreased aminochrome formation by methylene blue and pheophorbide A. Addition of EDTA, a tertiary amine electron donor, to the reaction of dopamine, photosensitizer and red light decreased aminochrome formation. Photoreduction of the dopamine o-quinone produced by mushroom tyrosinase was achieved by both methylene blue and pheophorbide A only when an electron donor was included. Due to limited solubility, photo-oxidation and photoreduction reactions by pheophorbide A required 5-7.5% dimethylformamide for optimal reactivity. Catalytic photoreduction of 2,3-dimethoxy-5-methyl-p-benzoquinone by methylene blue or pheophorbide A and tertiary amine electron donors was observed. Among the chlorophyll metabolites, pheophorbide A was more effective than chlorin e6 or sodium copper chlorophyllin in photo-oxidation of dopamine and photoreduction reactions. Singlet oxygen inhibited lactate dehydrogenase A activity, and higher molecular weight protein cross-links were observed on SDS-PAGE. Hydrocaffeic acid competed with lactate dehydrogenase A for reaction with singlet oxygen produced by methylene blue; however, no protection by hydrocaffeic acid (HCA) was observed when pheophorbide A was used. Cysteine modification of lactate dehydrogenase A by the o-quinone of hydrocaffeic acid was detected using a redox cycling stain. Inclusion of an electron donor decreased protein labeling.

6-nitrodopamine is a major endogenous modulator of human vas deferens contractility.

BACKGROUND: Rat isolated vas deferens releases 6-nitrodopamine (6-ND), and the spasmogenic activity of this novel catecholamine is significantly reduced by tricyclic compounds such as amitriptyline, desipramine, and carbamazepine and by antagonists of the α1 -adrenergic receptors such as doxazosin, tamsulosin, and prazosin. OBJECTIVES: To investigate the liberation of 6-ND by human epididymal vas deferens (HEVDs) and its pharmacological actions. METHODS: The in vitro liberation of 6-ND, dopamine, noradrenaline, and adrenaline from human vas deferens was evaluated by LC-MS/MS. The contractile effect of the catecholamines in HEVDs was investigated in vitro. The action of tricyclic antidepressants was evaluated on the spasmogenic activity ellicited by the catecholamines and by the electric-field stimulation (EFS). The tissue was also incubated with the inhibitor of nitric oxide (NO) synthase L-NAME and the release of catecholamines and the contractile response to EFS were assessed. RESULTS: 6-ND is the major catecholamine released from human vas deferens and its synthesis/release is inhibited by NO inhibition. The spasmogenic activity elicited by EFS in the human vas deferens was blocked by tricyclic antidepressants only at concentrations that selectively antagonize 6-ND induced contractions of the human vas deferens, without affecting the spasmogenic activity induced by dopamine, noradrenaline, and adrenaline in this tissue. Incubation of the vas deferens with L-NAME reduced both the 6-ND release and the contractions induced by EFS. DISCUSSION AND CONCLUSION: 6-ND should be considered a major endogenous modulator of human vas deferens contractility and possibly plays a pivotal role in the emission process of ejaculation. It offers a novel and shared mechanism of action for tricyclic antidepressants and α1 -adrenergic receptor antagonists.

6-NitroDopamine is an endogenous modulator of rat heart chronotropism.

6-Nitrodopamine (6-ND) is released by rat vas deferens and exerts a potent contractile response that is antagonized by tricyclic antidepressants and α1-, ß1- and ß1/ß2-adrenoceptor antagonists. The release of 6-ND, noradrenaline, adrenaline and dopamine from rat isolated right atria was assessed by tandem mass spectrometry. The effects of the catecholamines were evaluated in both rat isolated right atria and in anaesthetized rats. 6-ND was the major catecholamine released from the isolated atria and the release was significantly reduced in nitric oxide synthase inhibitor L-NAME pre-treated atria or in atria obtained from L-NAME chronically treated animals, but unaffected by tetrodotoxin. 6-ND (1 pM) significantly increased the atrial frequency, being 100 times more potent than noradrenaline and adrenaline. Selective ß1-blockers reduced the atrial frequency only at concentrations that prevented the increases in atrial frequency induced by 6-ND 1pM. Conversely, ß1-blockade did not affect dopamine (10 nM), noradrenaline (100 pM) or adrenaline (100 pM) effect. The reductions in atrial frequency induced by the ß1-adrenoceptor antagonists were absent in L-NAME pre-treated atria and in atria obtained from chronic L-NAME-treated animals. Tetrodotoxin did not prevent the reduction in atrial frequency induced by L-NAME or by ß1-blockers treated preparations. In anaesthetized rats, at 1 pmol/kg, only 6-ND caused a significant increase in heart rate. Inhibition of 6-ND synthesis by chronic L-NAME treatment reduced both atrial frequency and heart rate. The results indicate that 6-ND is a major modulator of rat heart chronotropism and the reduction in heart rate caused by ß1-blockers are due to selective blockade of 6-ND receptor.

ß1- and ß1/ß2-adrenergic receptor antagonists block 6-nitrodopamine-induced contractions of the rat isolated epididymal vas deferens.

6-Nitrodopamine (6-ND) is an endogenous modulator of the contractility in the rat isolated epididymal vas deferens (RIEVD) and considered to be the main peripheral mediator of the emission process. Use of selective and unselective ß-adrenergic receptor antagonists has been associated with ejaculatory failure. Here, the effects of selective ß1- and ß1/ß2-adrenergic receptor antagonists on RIEVD contractions induced by 6-ND, dopamine, noradrenaline, adrenaline, and electric-field stimulation (EFS) were investigated. The selective ß1-adrenergic receptor antagonists atenolol (0.1 and 1 µï»¿M), betaxolol (1 µï»¿M), and metoprolol (1 µï»¿M) and the unselective ß1/ß2-adrenergic receptor antagonists propranolol (1 and 10 µï»¿M) and pindolol (10 µï»¿M) caused significant rightward shifts of the concentration-response curve to 6-ND (pA2 6.41, 6.91, 6.75, 6.47, and 5.74; for atenolol, betaxolol, metoprolol, propranolol, and pindolol), but had no effect on dopamine-, noradrenaline-, and adrenaline-induced contractions. The effects of selective ß1- and ß1/ß2-adrenergic receptor antagonists at a higher concentration (atenolol 1 µï»¿M, betaxolol 1 µï»¿M, metoprolol 1 µï»¿M, propranolol 10 µï»¿M, and pindolol 10 µï»¿M) also reduced the EFS-induced RIEVD contractions in control, but not in RIEVD obtained from L-NAME-treated animals. The selective ß1-adrenoceptor agonist RO-363, the selective ß2-adrenoceptor agonist salbutamol, and the selective ß3-adrenoceptor agonist mirabegron, up to 300 µï»¿M, had no effect on the RIEVD tone. The results demonstrate that ß1- and ß1-/ß2-adrenoceptor receptor antagonists act as 6-ND receptor antagonists in RIEVD, further confirming the main role of 6-ND in the RIEVD contractility.

6-Nitrodopamine is an endogenous selective dopamine receptor antagonist in Chelonoidis carbonaria aorta.

Chelonoidis carbonaria aortic rings present endothelium-derived release of dopamine, noradrenaline, adrenaline and 6-nitrodopamine (6-ND). Here it was investigated whether 6-ND release is coupled to nitric oxide (NO) synthesis and its action on the vascular smooth muscle reactivity. Basal release of 6-ND from aortic rings in the absence and presence of the NO synthesis inhibitor L-NAME was quantified by LC-MS-MS. Aortic rings were suspended vertically between two metal hooks in 10-mL organ baths containing Krebs-Henseleit's solution and attached to isometric transducers. The tissues were allowed to equilibrate for 1 h before starting the experiments. The release of 6-ND was significantly reduced by previous incubation with L-NAME. 6-ND (up to 300 µM) had no contractile activity in the aortic rings. 6-ND (1, 3 and 10 µM) produced significant rightward shifts of the concentration-response curves to dopamine in endothelium-intact (pA2 6.09) and L-NAME pre-treated endothelium-intact (pA2 7.06) aortic rings. Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND. The EFS (16 Hz)-induced aortic contractions were significantly inhibited by incubation with 6-ND (10 µM). In the thromboxane A2 mimetic U-46619 (30 nM) pre-contracted endothelium intact aortic rings, 6-ND (1 nM-1 µM) and the dopamine D2-receptor antagonist haloperidol (1 nM-1 µM) induced concentration-dependent relaxations. The relaxations were not present in endothelium-removed aortic rings but they were not affected by incubation with L-NAME in endothelium-intact aortic rings. The results indicate that the synthesis of this novel catecholamine in Chelonoidis carbonaria aortic rings is coupled to NO release and that 6-ND acts as a highly selective dopamine D2-like receptor antagonist.

Dual-Template Magnetic Molecularly Imprinted Polymer for Simultaneous Determination of Spot Urine Metanephrines and 3-Methoxytyramine for the Diagnosis of Pheochromocytomas and Paragangliomas.

A novel dual-template magnetic molecularly imprinted polymer (MMIP) was synthesized to extract normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT) from spot urine samples. As the adsorbent of dispersive solid-phase extraction (d-SPE), the MMIP was prepared using dopamine and MN as dual templates, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the crosslinking reagent and magnetic nanoparticles as the magnetic core. NMN, MN, 3-MT and creatinine (Cr) in spot urine samples were selectively enriched by d-SPE and detected by HPLC-fluorescence detection/ultraviolet detection. The peak area (A) ratios of NMN, MN and 3-MT to Cr were used for the diagnosis of pheochromocytomas and paragangliomas (PPGLs). The results showed that the adsorption efficiencies of MMIP for target analytes were all higher than 89.0%, and the coefficient variation precisions of intra-assay and inter-assay for the analytes were within 4.9% and 6.3%, respectively. The recoveries of the analytes were from 93.2% to 112.8%. The MMIP was still functional within 14 days and could be reused at least seven times. The d-SPE and recommended solid-phase extraction (SPE) were both used to pretreat spot urine samples from 18 PPGLs patients and 22 healthy controls. The correlation coefficients of ANMN/ACr and AMN/ACr between d-SPE and SPE were both higher than 0.95. In addition, the areas under the receiver operator curves for spot urine ANMN/ACr, AMN/ACr and plasma free NMN and MN were 0.975, 0.773 and 0.990, 0.821, respectively, indicating the two methods had the similar performances. The d-SPE method took only 20 min, which was effective in clinical application.

Alternative pathway for dopamine production by acetogenic gut bacteria that O-Demethylate 3-Methoxytyramine, a metabolite of catechol O-Methyltransferase.

AIMS: The gut microbiota modulates dopamine levels in vivo, but the bacteria and biochemical processes responsible remain incompletely characterized. A potential precursor of bacterial dopamine production is 3-methoxytyramine (3MT); 3MT is produced when dopamine is O-methylated by host catechol O-methyltransferase (COMT), thereby attenuating dopamine levels. This study aimed to identify whether gut bacteria are capable of reverting 3MT to dopamine. METHODS AND RESULTS: Human faecal bacterial communities O-demethylated 3MT and yielded dopamine. Gut bacteria that mediate this transformation were identified as acetogens Eubacterium limosum and Blautia producta. Upon exposing these acetogens to propyl iodide, a known inhibitor of cobalamin-dependent O-demethylases, 3MT O-demethylation was inhibited. Culturing E. limosum and B. producta with 3MT afforded increased acetate levels as compared with vehicle controls. CONCLUSIONS: Gut bacterial acetogens E. limosum and B. producta synthesized dopamine from 3MT. This O-demethylation of 3MT was likely performed by cobalamin-dependent O-demethylases implicated in reductive acetogenesis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that gut bacteria can synthesize dopamine by O-demethylation of 3MT. Owing to 3MT being the product of host COMT attenuating dopamine levels, gut bacteria that reverse this transformation-converting 3MT to dopamine-may act as a counterbalance for dopamine regulation by COMT.

Multifunctional coating of cotton fabric via the assembly of amino-quinone networks with polyamine biomacromolecules and dopamine quinone.

Functional textiles with antibacterial properties and UV protection are essential for human health. However, the process of functional modification of textiles is usually done with the help of chemical cross-linking agents to improve the bonding fastness of functional finishing agents on textiles. The use of chemical cross-linking agents is not eco-friendly enough and is prone to chemical waste. In this study, some highly reactive polyamine biomolecules were combined with dopamine quinone, a super adhesive bionic material, to spontaneously construct amino-quinone networks (AQNs) coatings on the surface of cotton fabrics without the addition of chemical crosslinkers. The amino/quinone compounds (A/Q) self-crosslinking reaction is achieved by Michael addition and Schiff base reaction between the quinone group in dopamine quinone and the amino group in chitosan (CTS), chitooligosaccharide (COS) or ԑ-polylysine (ԑ-PL). The combination of polyamines and dopamine quinone during the cotton finishing process imparts antibacterial and UV protection to cotton fabric. The results showed that the AQNs coating modified fabrics had superb UV protection and antibacterial rates of over 96% against both E. coli and S. aureus. In addition, the AQNs coating modified fabrics had good resistance to washing and mechanical abrasion. This study proposes that self-assembled amino-quinone network multifunctional coatings of dopamine quinone and polyamine biomolecules are of guiding significance for the development of environmentally friendly bio-based materials.

Intelligence benefit of the 3-methoxytyramine to tyramine ratio in equine urine.

Equine urine analysis has evolved over time to detect thousands of urinary compounds for doping control in the horse racing industry. The longitudinal assessment of 3-methoxytyramine to tyramine ratio (3-MT/T) values in equine urine by GC-MS profiling was investigated to support the Racing NSW Equine Biological Passport (EBP) for detection of dopaminergic manipulation in racehorses. This involved comparison of routine urine samples to administration studies of Sinemet, a common Parkinson's disease medication containing levodopa. Using an endogenous reference compound (ERC) in a urinary ratio enabled greater confidence to provide intelligence of pharmaceutical manipulation as distinct from physiological variation. Population reference limits (PRLs) of 776 ng/ml for urinary 3-MT and 5.3 for 3-MT/T, together with the use of individual reference limits (IRLs), are proposed.

An automated magnetic bead extraction method for measuring plasma metanephrines and 3-methoxytyramine using liquid chromatography tandem mass spectrometry.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is used routinely in clinical diagnostics; however, automating the sample pretreatment is challenging. We established and evaluated an automated method based on the magnetic bead extraction principle (MBE) to measure normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (3-MT). The target analytes were extracted, purified, and concentrated using different solvents and chemical bond-modified magnetic beads transferred via a magnetic bar. The linearity, recovery, matrix effect, and precision of MBE were evaluated thoroughly, and compared with traditional solid-phase extraction (SPE) using 131 plasma samples. The chromatography peaks of metanephrines and 3-MT, extracted via MBE, are symmetrical, without interfering peaks. The linearity was excellent with correlation coefficient (r) > 0.99. The MBE exhibited good reproducibility with within-run coefficient variations (CVs) of 1.96-2.00%, 4.06-5.75%, and 3.89-4.90% for MN, NMN, and 3-MT, respectively. The total CVs for MN, NMN, and 3-MT were 1.96-2.80%, 5.12-5.75%, and 5.44-6.27%, respectively. The relative recoveries for MN, NMN, and 3-MT varied between 93.5 and 107.4%, whereas their biases were all within 10%. The results for MN, NMN, and 3-MT extracted via MBE compared with SPE exhibited excellent correlation, with r > 0.99; the mean bias% for MN, NMN, and 3-MT were small (-2.9%, -3.2%, and -3.2%, respectively). In conclusion, the automated MBE method for measuring plasma metanephrines and 3-MT can be applied in future routine clinical diagnostics, and the MBE principle may indicate a new era for LC-MS/MS in clinical application.