Hydroxytyrosol, a Promising Supplement in the Management of Human Stroke: An Exploratory Study.

Hydroxytyrosol (HT) is a bioactive olive oil phenol with beneficial effects in a number of pathological situations. We have previously demonstrated that an HT-enriched diet could serve as a beneficial therapeutic approach to attenuate ischemic-stroke-associated damage in mice. Our exploratory pilot study examined this effect in humans. Particularly, a nutritional supplement containing 15 mg of HT/day was administered to patients 24 h after the onset of stroke, for 45 days. Biochemical and oxidative-stress-related parameters, blood pressure levels, serum proteome, and neurological and functional outcomes were evaluated at 45 and 90 days and compared to a control group. The main findings were that the daily administration of HT after stroke could: (i) favor the decrease in the percentage of glycated hemoglobin and diastolic blood pressure, (ii) control the increase in nitric oxide and exert a plausible protective effect in oxidative stress, (iii) modulate the evolution of the serum proteome and, particularly, the expression of apolipoproteins, and (iv) be beneficial for certain neurological and functional outcomes. Although a larger trial is necessary, this study suggests that HT could be a beneficial nutritional complement in the management of human stroke.

Tyrosol: Repercussion of the Lack of a Hydroxyl-Group in the Response of MCF-7 Cells to Hypoxia.

In solid tumors, such as breast cancer, hypoxic microenvironment worsens patient prognoses. We have previously reported in MCF-7 breast cancer cells that, under hypoxic conditions, hydroxytyrosol (HT) downregulates the level of reactive oxygen species, reduces the expression of hypoxia inducible factor-1α (HIF-1α), and, at high concentrations, can bind to the aryl hydrocarbon receptor (AhR). With this background, the present study investigated whether the most abundant extra virgin olive oil (EVOO) phenolic compound tyrosol (TYR), with a chemical structure similar to HT but with only one hydroxyl group, exerts comparable effects. Our results revealed that, although TYR did not show any antioxidant activity in hypoxic MCF-7 cells, it inhibited the PI3K/Akt/mTOR/S6 kinase (S6K) pathway and reduced the expression of HIF-1α and some of its target genes. Besides, TYR showed a lower binding affinity with the cytosolic transcription factor AhR, and even reduced its transcriptional activity. Some of these results are positive to control tumor progression in a hypoxic environment; however, they are observed at doses unachievable with diet intake or nutraceutical presentations. Considering that EVOO phenols can have synergistic effects, a mixture of low doses of TYR and other phenols could be useful to achieve these beneficial effects.

New Strategies for Stroke Therapy: Nanoencapsulated Neuroglobin.

Stroke is a global health and socio-economic problem. However, no efficient preventive and/or palliative treatments have yet been found. Neuroglobin (Ngb) is an endogen neuroprotective protein, but it only exerts its beneficial action against stroke after increasing its basal levels. Therefore, its systemic administration appears to be an efficient therapy applicable to stroke and other neurodegenerative pathologies. Unfortunately, Ngb cannot cross the blood-brain barrier (BBB), making its direct pharmacological use unfeasible. Thus, the association of Ngb with a drug delivery system (DDS), such as nanoparticles (NPs), appears to be a good strategy for overcoming this handicap. NPs are a type of DDS which efficiently transport Ngb and increase its bioavailability in the infarcted area. Hence, we previously built hyaluronate NPS linked to Ngb (Ngb-NPs) as a therapeutic tool against stroke. This nanoformulation induced an improvement of the cerebral infarct prognosis. However, this innovative therapy is still in development, and a more in-depth study focusing on its long-lasting neuroprotectant and neuroregenerative capabilities is needed. In short, this review aims to update the state-of-the-art of stroke therapies based on Ngb, paying special attention to the use of nanotechnological drug-delivering tools.

[Usefulness of COVID-19 patients first analysis made at the Emergency Department as a prediction tool for mortality (PMCovid Score).] / Utilidad de la primera analítica de Urgencias realizada en pacientes con COVID-19 como predictora de mortalidad (PMCOVID SCORE).

OBJECTIVE: The COVID-19 pandemic has generated a high demand for hospital resources taking the national health system to its limit. In order to reduce this over burden and to avoid a potential system collapse, it would be beneficial to generate scientific evidence for the prognosis of the disease and to count with models that are able to forecast the mortality and progression of the disease. Identify mortality risk factors in COVID-19 patients from analytic data obtained from the Emergency Service at our hospital and to elaborate a prognostic score for predicting 30-day mortality (PMCovid Score) that will be included in the report submitted by the Emergency Clinic Laboratory. METHODS: Transversal descriptive study in a population that came to the Emergency Service at the University Hospital of Jaén between March 8th and April 7th 2020. We obtained the variables for the prognosis by a univariate data analysis. On this basis, we applied a multivariate analysis of the logistical regression of the mortality after 30 days in order to generate a prognostic score which was validated subsequently by the TRIPOD method. RESULTS: 298 patients were included. PMCovid Score assigns 1 point to patients age ≥77 years old; 1 point to patients with a urea level ≥49 mg/dL, 1 point to erythrocyte values <4.6x106/µL, 2 points to platelet values <165x103/µL; 1 point to patients with a percentage of lymphocytes below 18.1%; 1 point to those with a % of monocytes <6.8% and 2 points if the % of eosinophils is <0.4%.Our score had a predictive accuracy of 88.6% (AUC 0.886 IC at 95%; 0.842-0.931), with a sensibility of 91.7% (IC at 95% 82.810-100) and a specificity of 69.7% (IC at 95% 63.840-75.680). CONCLUSIONS: PMCovid Score provides the doctor with information on the prognosis of the positive COVID-19 patient along with the usual first analysis data, the necessary parameters for the calculations are available at all Emergency Laboratory Clinics. This information can be very useful for the management of this kind of patients and their classification based on the risk supplied by the PMCovid Score.

OBJETIVO: La pandemia por COVID-19 ha generado una alta demanda de recursos hospitalarios llevando al límite al sistema nacional de salud. Es por ello que para reducir dicha sobrecarga y el posible colapso del sistema, sería beneficioso generar evidencia científica sobre el pronóstico de la enfermedad y disponer de modelos que permitan predecir la mortalidad y la progresión de la enfermedad. El objetivo de este trabajo fue identificar factores de riesgo de mortalidad en pacientes COVID-19 a partir de los datos analíticos solicitados por el Servicio de Urgencias del hospital y elaborar un score pronóstico de mortalidad a 30 días. METODOS: Estudio transversal descriptivo en pacientes con COVID-19 que acudieron al Servicio de Urgencias del Hospital Universitario de Jaén entre el 8 de marzo y el 7 de abril de 2020. Las variables con utilidad para el pronóstico se obtuvieron mediante un análisis univariante de los datos, a partir de ellas se aplicó un análisis multivariante de regresión logística de mortalidad a los 30 días para generar el score pronóstico que posteriormente fue validado mediante el método TRIPOD. RESULTADOS: Se incluyeron 298 pacientes. PMCovid Score asigna 1 punto a pacientes con edad ≥77 años; 1 punto a los pacientes con niveles de urea ≥49 mg/dL, 1 punto a valores de hematíes <4,6x106/µL, 2 puntos a valores de plaquetas <165x103/µL; 1 punto a pacientes con un porcentaje de linfocitos inferior al 18,1%; 1 punto a los que tenga un porcentaje de monocitos menores a 6,8% y 2 puntos si el porcentaje de eosinófilos es menor del 0,4%. La capacidad pronóstica de nuestro score fue del 88,6% (AUC 0,886 IC al 95%; 0,842-0,931), con una sensibilidad del 91,7% (IC al 95% 82,810-100) y una especificidad del 69,7% (IC al 95% 63,840-75,680). CONCLUSIONES: PMCovid Score proporciona al clínico información acerca del pronóstico del paciente con COVID-19 positivo con los datos habituales de la primera analítica, los parámetros necesarios para el cálculo están disponibles en la totalidad de los Laboratorios Clínicos de Urgencias.

Adrenomedullin Is a Diagnostic and Prognostic Biomarker for Acute Intracerebral Hemorrhage.

Hemorrhagic stroke remains an important health challenge. Adrenomedullin (AM) is a vasoactive peptide with an important role in cardiovascular diseases, including stroke. Serum AM and nitrate-nitrite and S-nitroso compounds (NOx) levels were measured and compared between healthy volunteers (n = 50) and acute hemorrhagic stroke patients (n = 64). Blood samples were taken at admission (d0), 24 h later (d1), and after 7 days or at the time of hospital discharge (d7). Neurological severity (NIHSS) and functional prognosis (mRankin) were measured as clinical outcomes. AM levels were higher in stroke patients at all times when compared with healthy controls (p < 0.0001). A receiving operating characteristic curve analysis identified that AM levels at admission > 69.0 pg/mL had a great value as a diagnostic biomarker (area under the curve = 0.89, sensitivity = 80.0%, specificity = 100%). Furthermore, patients with a favorable outcome (NIHSS ≤ 3; mRankin ≤ 2) experienced an increase in AM levels from d0 to d1, and a decrease from d1 to d7, whereas patients with unfavorable outcome had no significant changes over time. NOx levels were lower in patients at d0 (p = 0.04) and d1 (p < 0.001) than in healthy controls. In conclusion, AM levels may constitute a new diagnostic and prognostic biomarker for this disease, and identify AM as a positive mediator for hemorrhagic stroke resolution.

Biological Implications of a Stroke Therapy Based in Neuroglobin Hyaluronate Nanoparticles. Neuroprotective Role and Molecular Bases.

Exogenous neuroprotective protein neuroglobin (Ngb) cannot cross the blood-brain barrier. To overcome this difficulty, we synthesized hyaluronate nanoparticles (NPs), able to deliver Ngb into the brain in an animal model of stroke (MCAO). These NPs effectively reached neurons, and were microscopically identified after 24 h of reperfusion. Compared to MCAO non-treated animals, those treated with Ngb-NPs showed survival rates up to 50% higher, and better neurological scores. Tissue damage improved with the treatment, but no changes in the infarct volume or in the oxidative/nitrosative values were detected. A proteomics approach (p-value < 0.02; fold change = 0.05) in the infarcted areas showed a total of 219 proteins that significantly changed their expression after stroke and treatment with Ngb-NPs. Of special interest, are proteins such as FBXO7 and NTRK2, which were downexpressed in stroke, but overexpressed after treatment with Ngb-NPs; and ATX2L, which was overexpressed only under the effect of Ngb. Interestingly, the proteins affected by the treatment with Ngb were involved in mitochondrial function and cell death, endocytosis, protein metabolism, cytoskeletal remodeling, or synaptic function, and in regenerative processes, such as dendritogenesis, neuritogenesis, or sinaptogenesis. Consequently, our pharmaceutical preparation may open new therapeutic scopes for stroke and possibly for other neurodegenerative pathologies.

Hydroxytyrosol as a Promising Ally in the Treatment of Fibromyalgia.

Fibromyalgia (FM) is a chronic and highly disabling syndrome, which is still underdiagnosed, with controversial treatment. Although its aetiology is unknown, a number of studies have pointed to the involvement of altered mitochondrial metabolism, increased oxidative stress and inflammation. The intake of extra virgin olive oil, and particularly of one of its phenolic compounds, hydroxytyrosol (HT), has proven to be protective in terms of redox homeostatic balance and the reduction of inflammation. In this context, using a proteomic approach with nanoscale liquid chromatography coupled to tandem mass spectrometry, the present study analysed: (i) Changes in the proteome of dermal fibroblasts from a patient with FM versus a healthy control, and (ii) the effect of the treatment with a nutritional relevant dose of HT. Our results unveiled that fibroblast from FM show a differential expression in proteins involved in the turnover of extracellular matrix and oxidative metabolism that could explain the inflammatory status of these patients. Moreover, a number of these proteins results normalized by the treatment with HT. In conclusion, our results support that an HT-enriched diet could be highly beneficial in the management of FM.

Crosstalk between hydroxytyrosol, a major olive oil phenol, and HIF-1 in MCF-7 breast cancer cells.

Olive oil intake has been linked with a lower incidence of breast cancer. Hypoxic microenvironment in solid tumors, such as breast cancer, is known to play a crucial role in cancer progression and in the failure of anticancer treatments. HIF-1 is the foremost effector in hypoxic response, and given that hydroxytyrosol (HT) is one of the main bioactive compounds in olive oil, in this study we deepen into its modulatory role on HIF-1. Our results in MCF-7 breast cancer cells demonstrate that HT decreases HIF-1α protein, probably by downregulating oxidative stress and by inhibiting the PI3K/Akt/mTOR pathway. Strikingly, the expression of HIF-1 target genes does not show a parallel decrease. Particularly, adrenomedullin and vascular endothelial growth factor are up-regulated by high concentrations of HT even in HIF-1α silenced cells, pointing to HIF-1-independent mechanisms of regulation. In fact, we show, by in silico modelling and transcriptional analysis, that high doses of HT may act as an agonist of the aryl hydrocarbon receptor favoring the induction of these angiogenic genes. In conclusion, we suggest that the effect of HT in a hypoxic environment is largely affected by its concentration and involves both HIF-1 dependent and independent mechanisms.

Hyaluronate Nanoparticles as a Delivery System to Carry Neuroglobin to the Brain after Stroke.

Therapies against stroke can restore the blood supply but cannot prevent the ischemic damage nor stimulate the recovery of the infarcted zone. The neuroglobin protein plays an important role in the neuro-regeneration process after stroke; however, the method for its effective systemic application has not been identified yet, as neuroglobin is unable to pass through the blood-brain barrier. Previously, we developed different types of sodium hyaluronate nanoparticles, which successfully cross the blood-brain barrier after stroke. In this work, these nanoparticles have been used to carry neuroglobin through the bloodstream to the nerve cells in rats submitted to stroke. We have biosynthesized rat-recombinant neuroglobin and determined the formulation of sodium hyaluronate nanoparticles loaded with neuroglobin, as well as its size and ζ-potential, encapsulation efficiently, in vitro release, and its kinetic of liberation. The results show that the formulation achieved is highly compatible with pharmaceutical use and may act as a delivery system to transport neuroglobin within the blood. We have found that this formulation injected intravenously immediately after stroke reached the damaged cerebral parenchyma at early stages (2 h). Neuroglobin colocalizes with its nanocarriers inside the nerve cells and remains after 24 h of reperfusion. In conclusion, the systemic administration of neuroglobin linked to nanoparticles is a potential neuroprotective drug-delivery strategy after stroke episodes.

Tyrosol, a simple phenol from EVOO, targets multiple pathogenic mechanisms of neurodegeneration in a C. elegans model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder involving α-synuclein (α-syn) aggregation, oxidative stress, dysregulation of redox metal homeostasis, and neurotoxicity. Different phenolic compounds with known antioxidant or antichelating properties have been shown to also interfere with aggregation of amyloid proteins and modulate intracellular signaling pathways. The present study aims to investigate for the first time the effect of tyrosol (TYR), a simple phenol present in extra-virgin olive oil, on α-syn aggregation in a Caenorhabditis elegans model of PD and evaluate its potential to prevent α-syn toxicity, neurodegeneration, and oxidative stress in this model organism. Our results show that TYR is effective in reducing α-syn inclusions, resulting in a lower toxicity and extended life span of treated nematodes. Moreover, TYR delayed α-syn-dependent degeneration of dopaminergic neurons in vivo. TYR treatment also reduced reactive oxygen species level and promoted the expression of specific chaperones and antioxidant enzymes. Overall, our study puts into perspective TYR potential to be considered as nutraceutical that targets pivotal causal factors in PD.

Hypoxia modulates the antioxidant effect of hydroxytyrosol in MCF-7 breast cancer cells.

Although cancer is multifactorial, a strong correlation between this pathology and increased oxidative stress has long been stablished. Hypoxia, inherent to solid tumors, increases reactive oxygen species and should be taken into account when analyzing the response of tumor cells to antioxidants. The Mediterranean diet has been related to a lower incidence of cancer, and particularly of breast cancer. Given that hydroxytyrosol (HT) is largely responsible for the antioxidant properties of olive oil, we have performed a comprehensive and comparative study of its effect on the oxidative stress response of the human breast cancer cell line MCF-7 in hypoxia and normoxia. Our results demonstrate that the antioxidant action of HT is particularly effective in a hypoxic environment. Moreover, we have observed that this polyphenol modulates the transcription and translation of members of the PGC-1α/ERRα and PGC-1α/Nrf2 pathways. However, while the transcriptional effects of HT are similar in normoxic and hypoxic conditions, its translational action is less prominent and partially attenuated in hypoxia, and therefore cannot completely explain the antioxidant effect of HT. Consequently, our results underscore that the hypoxic environment of tumor cells should be considered when analyzing the effect of bioactive compounds. Besides, this study also points to the importance of assessing the regulatory role of HT at both mRNA and protein level to get a complete picture of its effects.

Insight into the biological pathways underlying fibromyalgia by a proteomic approach.

Fibromyalgia (FM) is a form of non-articular rheumatism difficult to diagnose and treat because its etiology remains still elusive. Proteomics makes possible the systematic analysis of hundreds of proteins in clinical samples. Consequently, it has become a key tool for finding altered molecular pathways in different diseases. In this context, the present study analyzes changes in the plasma proteome of patients with FM by nanoscale liquid chromatography coupled to tandem mass spectrometry. Deregulated proteins were studied using Ingenuity Pathways Analysis (IPA) and Kyoto Encyclopedia of Genes and Genomes. Conventional analytical methods were used to validate selected proteins. We found a total of 33 proteins differentially expressed in patients with FM. Haptoglobin and fibrinogen showed the highest FM/control ratio. IPA analysis revealed that the top enriched canonical pathways were acute-phase response signaling, Liver-X Receptor/Retinoid-X Receptor activation, Farnesoid-X Receptor/Retinoid-X Receptor activation, and coagulation and complement systems. The importance of inflammation in FM was corroborated by the increase in erythrocyte sedimentation rate. In conclusion, our results support the existence of a plasma protein signature of FM that involves different biological pathways all of them related to inflammation, and point to haptoglobin and fibrinogen as plausible biomarker-candidates for future studies. SIGNIFICANCE: The etiology of fibromyalgia (FM) remains elusive making its diagnosis and treatment difficult. The characterization of the proteome signature of this syndrome will improve its understanding. However, to date proteomic analyses in FM are scarce. The goal of the present work is to analyse, for the first time, changes in plasma protein profiles of patients with FM in comparison to control subjects, using label free relative protein quantification by nanoscale liquid chromatography coupled to tandem mass spectrometry. Our data demonstrate the existence of a common protein signature in the plasma of patients with FM that could explain some of the symptoms associated to this syndrome. The analysis of the 33 proteins differentially expressed corroborates the crucial role of inflammation in the pathogenesis of this syndrome. The interplay of the complement and coagulation cascades contributes to the inflammatory process, while the activation of Liver-X Receptor/Retinoid-X Receptor and Farnesoid-X Receptor/Retinoid-X Receptor could attempt to alleviate it. Finally, we have identified two proteins, haptoglobin and fibrinogen, as potential biomarker-candidates of FM for future studies.

Temporal profiles of blood pressure, circulating nitric oxide, and adrenomedullin as predictors of clinical outcome in acute ischemic stroke patients.

Stroke remains an important health and social challenge. The present study investigated whether blood pressure (BP) parameters and circulating levels of nitric oxide metabolites (NOx) and adrenomedullin (AM) may predict clinical outcomes of stroke. Patients (n=76) diagnosed with acute ischemic stroke were admitted to the stroke unit and clinical history data and monitored parameters were recorded. Blood plasma was collected at days 1, 2, and 7 to measure NOx and AM levels. Infarct volume, neurological severity [on the National Institutes of Health Stroke Scale (NIHSS)], and functional prognosis (on the Rankin scale) were measured as clinical outcomes. Patients with higher BP had more severe symptoms (NIHSS >3; P<0.01) and BP variability predicted neurological severity and growth of infarct volume. NOx values were significantly lower in stroke patients than in healthy controls (P<0.01). An increase in NOx levels from day 1 to day 2 was beneficial for the patients as measured by NIHSS at 7 days and 3 months, and by Rankin at 3 months [odds ratio (OR), 0.91] whereas a steep increase from day 2 to day 7 was detrimental and associated with an increase in infarct volume (OR, 35.3). AM levels were significantly higher in patients at day 1 and 2 than in healthy individuals (P<0.01) and these levels returned to normal at day 7. Patients with high AM levels at day 2 had significantly higher NIHSS scores measured at day 1 (P<0.05) and 7 (P<0.01). A receiving operating characteristic curve analysis identified that AM levels at day 2 of >522.13 pg/ml predicted increased neurological severity at day 7 (area under the curve=0.721). Multivariate logistic regression indicated that AM levels at day 2 predicted increased neurological severity at 7 days and at 3 months. BP parameters and changing levels for NOx and AM predicted long­term clinical outcomes as measured by infarct volume, neurological severity scale, and functional prognosis.

Hydroxytyrosol decreases the oxidative and nitrosative stress levels and promotes angiogenesis through HIF-1 independent mechanisms in renal hypoxic cells.

In the kidney, tissue oxygen tension is comparatively low and this renders this organ more prone to hypoxic injury. In fact, hypoxia has a central role in the development and progression of renal disease. The recovery from this situation is dependent on the degree to which sublethally damaged cells restore normal function. The master regulator of the hypoxic response is hypoxia-inducible factor-1 (HIF-1). HIF-1 activity depends on the HIF-1α subunit level which is regulated by oxygen, nitric oxide (NO), reactive oxygen species and mTOR. Given the antioxidant and antinitrosative properties ascribed to hydroxytyrosol (HT), this study evaluates the impact of this olive oil polyphenol on the response to hypoxia in kidney cells. For this purpose, the human embryonic kidney HEK293T cell line was treated with HT and cultured under sublethal hypoxic conditions. Our results demonstrate that HT treatment decreases both, post-hypoxic reactive oxygen species and NO levels and, consequently, HIF-1α accumulation. However, HT does not affect mTOR activation or the factor inhibiting HIF level but promotes the expression of angiogenic proteins, suggesting that HT activates an adaptive response to hypoxia in a HIF-1α-independent pathway. In fact, this effect could be ascribed to the up-regulation of estrogen-related receptor α. In conclusion, our results suggest that in renal hypoxia, HT treatment might act as an effective preventive therapeutic approach to decrease stress and to improve the adaptive response to this pathological situation.

Effects of a human milk oligosaccharide, 2'-fucosyllactose, on hippocampal long-term potentiation and learning capabilities in rodents.

Human milk oligosaccharides (HMOs) are unique with regard to their diversity, quantity and complexity, particularly in comparison to bovine milk oligosaccharides. HMOs are associated with functional development during early life, mainly related to immunity and intestinal health. Whether HMOs elicit a positive effect on cognitive capabilities of lactating infants remains an open question. This study evaluated the role of the most abundant HMO, 2'-fucosyllactose (2'-FL), in synaptic plasticity and learning capabilities in rodents. Mice and rats were prepared for the chronic recording of field excitatory postsynaptic potentials evoked at the hippocampal CA3-CA1 synapse. Following chronic oral administration of 2'-FL, both species showed improvements in input/output curves and in long-term potentiation (LTP) evoked experimentally in alert behaving animals. This effect on LTP was related to better performance of animals in various types of learning behavioral tests. Mice were tested for spatial learning, working memory and operant conditioning using the IntelliCage system, while rats were submitted to a fixed-ratio schedule in the Skinner box. In both cases, 2'-FL-treated animals performed significantly better than controls. In addition, chronic administration of 2'-FL increased the expression of different molecules involved in the storage of newly acquired memories, such as the postsynaptic density protein 95, phosphorylated calcium/calmodulin-dependent kinase II and brain-derived neurotrophic factor in cortical and subcortical structures. Taken together, the data show that dietary 2'-FL affects cognitive domains and improves learning and memory in rodents.

Study of tamoxifen urinary metabolites in rat by ultra-high-performance liquid chromatography time-of-flight mass spectrometry.

Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug used for the treatment of breast cancer. It is also included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this work, the excretion of urinary metabolites of TMX after a single therapeutic dose administration in rats has been studied using ultra-high-performance liquid chromatography electrospray time-of-flight mass spectrometry (UHPLC-TOFMS). A systematic strategy based on the search of typical biotransformations that a xenobiotic can undergo in living organisms, based on their corresponding molecular formula modification and accurate mass shifts, was applied for the identification of TMX metabolites. Prior to UHPLC-TOFMS analyses, a solid-phase extraction step with polymeric cartridges was applied to urine samples. Up to 38 TMX metabolites were detected. Additional collision induced dissociation (CID) MS/MS fragmentation was performed using UHPLC-QTOFMS. Compared with recent previous studies in human urine and plasma, new metabolites have been reported for the first time in urine. Metabolites identified in rat urine include the oxygen addition, owing to different possibilities for the hydroxylation of the rings in different positions (m/z 388.2271), the incorporation of two oxygen atoms (m/z 404.2220) (including dihydroxylated derivatives or alternatives such as epoxidation plus hydroxylation or N-oxidation and hydroxylation), epoxide formation or hydroxylation and dehydrogenation [m/z 386.2114 (+O -H2 )], hydroxylation of the ring accompanied by N-desmethylation (m/z 374.2115), combined hydroxylation and methoxylation (m/z 418.2377), desaturated TMX derivate (m/z 370.2165) and its N-desmethylated derivate (m/z 356.2009), the two latter modifications not previously being reported in urine. These findings confirm the usefulness of the proposed approach based on UHPLC-TOFMS.

Detection of main urinary metabolites of ß2-agonists clenbuterol, salbutamol and terbutaline by liquid chromatography high resolution mass spectrometry.

Clenbuterol, terbutaline and salbutamol are B2-agonists drugs included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this article, the excretion of urinary metabolites of clenbuterol, terbutaline and salbutamol have been studied using liquid chromatography electrospray time-of-flight mass spectrometry (LC-TOFMS), after a single therapeutic dose administration in rats. Urine collected was processed with solid-phase extraction prior to LC-TOFMS analyses using electrospray in the positive ion mode and pseudo MS/MS experiments from in-source collision induced dissociation (CID) fragmentation (without precursor ion isolation). The strategy applied for the identification of metabolites was based on the search of typical biotransformations with their corresponding accurate mass shift and the use of common diagnostic fragment ions from the parent drugs. The approach was satisfactory applied, achieving the identification of 11 metabolites (5 from clenbuterol, 4 from salbutamol and 3 from terbutaline), 4 of them not previously reported in urine. Novel metabolites identified in rat urine included N-oxide-salbutamol, hydroxy-salbutamol, methoxy-salbutamol glucuronide and terbutaline N-oxide, which are all reported here for the first time.

Combined data mining strategy for the systematic identification of sport drug metabolites in urine by liquid chromatography time-of-flight mass spectrometry.

The development of comprehensive methods able to tackle with the systematic identification of drug metabolites in an automated fashion is of great interest. In this article, a strategy based on the combined use of two complementary data mining tools is proposed for the screening and systematic detection and identification of urinary drug metabolites by liquid chromatography full-scan high resolution mass spectrometry. The proposed methodology is based on the use of accurate mass extraction of diagnostic ions (compound-dependent information) from in-source CID fragmentation without precursor ion isolation along with the use of automated mass extraction of accurate-mass shifts corresponding to typical biotransformations (non compound-dependent information) that xenobiotics usually undergo when metabolized. The combined strategy was evaluated using LC-TOFMS with a suite of nine sport drugs representative from different classes (propranolol, bumetanide, clenbuterol, ephedrine, finasteride, methoxyphenamine, methylephedrine, salbutamol and terbutaline), after single doses administered to rats. The metabolite identification coverage rate obtained with the systematic method (compared to existing literature) was satisfactory, and provided the identification of several non-previously reported metabolites. In addition, the combined information obtained helps to minimize the number of false positives. As an example, the systematic identification of urinary metabolites of propranolol enabled the identification of up to 24 metabolites, 15 of them non previously described in literature, which is a valuable indicator of the usefulness of the proposed systematic procedure.

Tyrosol, a main phenol present in extra virgin olive oil, increases lifespan and stress resistance in Caenorhabditis elegans.

Extra virgin olive oil (EVOO) consumption has been traditionally related to a higher longevity in the human population. EVOO effects on health are often attributed to its unique mixture of phenolic compounds with tyrosol and hydroxityrosol being the most biologically active. Although these compounds have been extensively studied in terms of their antioxidant potential and its role in different pathologies, their actual connection with longevity remains unexplored. This study utilized the nematode Caenorhabditis elegans to investigate the possible effects of tyrosol in metazoan longevity. Significant lifespan extension was observed at one specific tyrosol concentration, which also induced a higher resistance to thermal and oxidative stress and delayed the appearance of a biomarker of ageing. We also report that, although tyrosol was efficiently taken up by these nematodes, it did not induce changes in development, body length or reproduction. In addition, lifespan experiments with several mutant strains revealed that components of the heat shock response (HSF-1) and the insulin pathway (DAF-2 and DAF-16) might be implicated in mediating tyrosol effects in lifespan, while caloric restriction and sirtuins do not seem to mediate its effects. Together, our results point to hormesis as a possible mechanism to explain the effects of tyrosol on longevity in C. elegans.

Nitric oxide modulates hypoxia-inducible factor-1 and poly(ADP-ribose) polymerase-1 cross talk in response to hypobaric hypoxia.

The physiological response to hypobaric hypoxia represents a complex network of biochemical pathways in which the nitrergic system plays an important role. Previous studies have provided evidence for an interplay between the hypoxia-inducible factor-1 (HIF-1) and poly(ADP-ribose) polymerase-1 (PARP-1) under hypoxia. Here, we evaluate the potential involvement of nitric oxide (NO) in the cross talk between these two proteins. With this aim, we studied comparatively the effect of pharmacological inhibitors of NO production or PARP activity in the response of the mouse cerebral cortex to 4 h of exposure to a simulated altitude of 31,000 ft. Particularly, we analyzed the NO and reactive oxygen species production, the expression of NO synthase (NOS) isoforms, PARP-1 activity, HIF-1α expression and HIF-1 transcriptional activity, the protein level of the factor inhibiting HIF, and, finally, beclin-1 and fractin expression, as markers of cellular damage. Our results demonstrate that the reduction of NO level did not affect reactive oxygen species production but significantly 1) dampened the posthypoxic increase in neuronal NOS and inducible NOS expression without altering endothelial NOS protein level; 2) prevented PARP activation; 3) decreased HIF-1α response to hypoxia; 4) achieved a higher long-term HIF-1 transcriptional activity by reducing factor inhibiting HIF expression; and 5) reduced hypoxic damage. The pharmacological inhibition of PARP reproduced the NOS expression pattern and the HIF-1α response observed in NOS-inhibited mice, supporting its involvement in the NO-dependent regulation of hypoxia. As a whole, these results provide new data about the molecular mechanism underlying the beneficial effects of controlling NO production under hypobaric hypoxic conditions.