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Plasma levels of endocannabinoids (eCBs) are very dynamic and variable in different circumstances and pathologies. The aim of the study was to determine the levels of the main eCBs and N-acylethanolamines (NAEs) in COVID-19 patients during the acute and post-acute phase of SARS-CoV-2 infection. Samples collected before December 31, 2020 were used for the determination of circulating eCB levels by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The association between plasma eCB measurements and biochemical and hematological parameters, as well as serum IL-6 levels, was evaluated. Samples of 64 individuals were analysed, n = 18 healthy donors, n = 30 acute, and n = 16 post-acute patients. Plasma levels of 2-arachidonoylglycerol (2-AG), were significantly elevated in COVID-19 patients when compared to healthy individuals. Plasma N-palmitoylethanolamide (PEA) and N-arachidonoylethanolamide (AEA) levels were found to be decreased in post-acute patient samples. These results suggest that 2-AG plays an important role in the inflammatory cascade in COVID-19 disease; in addition, eCBs might be involved in the post-acute pathogenesis of COVID-19. This study provides evidence of altered levels of circulating eCBs as a consequence of SARS-CoV-2 infection.
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Ácidos Araquidónicos , COVID-19 , Endocannabinoides , Glicéridos , Inflamación , SARS-CoV-2 , Humanos , Endocannabinoides/sangre , COVID-19/sangre , COVID-19/virología , Masculino , Femenino , Persona de Mediana Edad , Adulto , Ácidos Araquidónicos/sangre , Inflamación/sangre , Glicéridos/sangre , Alcamidas Poliinsaturadas/sangre , Etanolaminas/sangre , Anciano , Interleucina-6/sangre , Ácidos Palmíticos/sangre , Espectrometría de Masas en Tándem , Amidas , Cromatografía LiquidaRESUMEN
The aim of this study was to investigate the effect of 3 and 6 mg of caffeine per kg of body mass (mg/kg) on whole-body substrate oxidation during an incremental cycling exercise test in healthy active women. Using a double-blind placebo-controlled counterbalanced experimental design, 14 subjects performed three identical exercise trials after the ingestion of 3 or 6 mg/kg of caffeine or placebo. The exercise trials consisted of an incremental test on a cycle ergometer with 3-min stages at workloads from 30 to 70% of maximal oxygen uptake (VO2max). Substrate oxidation rates were measured by indirect calorimetry. During exercise, there was a significant effect of substance (F = 5.221; p = 0.016) on fat oxidation rate. In comparison to the placebo, 3 mg/kg of caffeine increased fat oxidation rates at 30 to 60% of VO2max (all p < 0.050) and 6 mg/kg at 30 to 50% of VO2max (all p < 0.050). There was also a significant effect of substance (F = 5.221; p = 0.016) on carbohydrate oxidation rate (F = 9.632; p < 0.001). In comparison to placebo, both caffeine doses decreased carbohydrate oxidation rates at 40 to 60% VO2max (all p < 0.050). The maximal rate of fat oxidation with placebo was 0.24 ± 0.03 g/min, which increased with 3 mg/kg to 0.29 ± 0.04 g/min (p = 0.032) and to 0.29 ± 0.03 with 6 mg/kg of caffeine (p = 0.042). Acute intake of caffeine improves the utilization of fat as a fuel during submaximal aerobic exercise in healthy active women with an effect of similar magnitude after the intake of 3 and 6 mg of caffeine per kg of body mass. Thus, the use of 3 mg/kg of caffeine would be more recommended than 6 mg/kg for women seeking increased fat utilization during submaximal exercise.
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The endocannabinoid system (ECS), a signalling network with immunomodulatory properties, is a potential therapeutic target in multiple sclerosis (MS). Dimethyl fumarate (DMF) is an approved drug for MS whose mechanism of action has not been fully elucidated; the possibility exists that its therapeutic effects could imply the ECS. With the aim of studying if DMF can modulate the ECS, the endocannabinoids 2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) were determined by liquid chromatography-mass spectrometry in peripheral blood mononuclear cells from 21 healthy donors (HD) and 32 MS patients at baseline and after 12 and 24 months of DMF treatment. MS patients presented lower levels of 2-AG and PEA compared to HD. 2-AG increased at 24 months, reaching HD levels. AEA and PEA remained stable at 12 and 24 months. OEA increased at 12 months and returned to initial levels at 24 months. Patients who achieved no evidence of disease activity (NEDA3) presented the same modulation over time as EDA3 patients. PEA was modulated differentially between females and males. Our results show that the ECS is dysregulated in MS patients. The increase in 2-AG and OEA during DMF treatment suggests a possible role of DMF in ECS modulation.
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Endocannabinoides , Esclerosis Múltiple , Masculino , Femenino , Humanos , Dimetilfumarato/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Leucocitos MononuclearesRESUMEN
p-Synephrine is the principal alkaloid of bitter orange (Citrus aurantium). Several recent investigations have found that the intake of 2-3 mg/kg of p-synephrine raises fat oxidation rate during exercise of low-to-moderate intensity. However, these investigations have been carried out only with samples of male participants or mixed men/women samples. Therefore, the aim of this investigation was to study the effect of p-synephrine intake on fat oxidation during exercise of increasing intensity in healthy women. Using a double-blind, randomized experiment, 18 healthy recreationally active women performed two identical exercise trials after the ingestion of (a) 3 mg/kg of p-synephrine and (b) 3 mg/kg of a placebo (cellulose). The exercise trials consisted of a ramp test (from 30 to 80% of maximal oxygen uptake; VO2max) on a cycle ergometer while substrate oxidation rates were measured at each workload by indirect calorimetry. In comparison to the placebo, the intake of p-synephrine increased resting tympanic temperature (36.1 ± 0.5 vs. 36.4 ± 0.4 °C p = 0.033, d = 0.87) with no effect on resting heart rate (p = 0.111) and systolic (p = 0.994) and diastolic blood pressure (p = 0.751). During exercise, there was no significant effect of p-synephrine on fat oxidation rate (F = 0.517; p = 0.484), carbohydrate oxidation rate (F = 0.730; p = 0.795), energy expenditure rate (F = 0.480; p = 0.833), heart rate (F = 4.269; p = 0.068) and participant's perceived exertion (F = 0.337; p = 0.580). The maximal rate of fat oxidation with placebo was 0.26 ± 0.10 g/min and it was similar with p-synephrine (0.28 ± 0.08 g/min, p = 0.449, d = 0.21). An acute intake of 3 mg/kg of p-synephrine before exercise did not modify energy expenditure and substrate oxidation during submaximal aerobic exercise in healthy active women. It is likely that the increase in resting tympanic temperature induced by p-synephrine hindered the effect of this substance on fat utilization during exercise in healthy active women.
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Citrus , Sinefrina , Femenino , Humanos , Carbohidratos , Celulosa , Citrus/química , Suplementos Dietéticos , Metabolismo Energético , Ejercicio Físico/fisiología , Oxidación-Reducción , Oxígeno , Consumo de Oxígeno , Extractos Vegetales/farmacología , Sinefrina/farmacología , Método Doble CiegoRESUMEN
BACKGROUND: The complex pathophysiology of Alzheimer's disease (AD) hampers the development of effective treatments. Attempts to prevent neurodegeneration in AD have failed so far, highlighting the need for further clarification of the underlying cellular and molecular mechanisms. Neuroinflammation seems to play a crucial role in disease progression, although its specific contribution to AD pathogenesis remains elusive. We have previously shown that the modulation of the endocannabinoid system (ECS) renders beneficial effects in a context of amyloidosis, which triggers neuroinflammation. In the 5xFAD model, the genetic inactivation of the enzyme that degrades anandamide (AEA), the fatty acid amide hydrolase (FAAH), was associated with a significant amelioration of the memory deficit. METHODS: In this work, we use electrophysiology, flow cytometry and molecular analysis to evaluate the cellular and molecular mechanisms underlying the improvement associated to the increased endocannabinoid tone in the 5xFAD mouse- model. RESULTS: We demonstrate that the chronic enhancement of the endocannabinoid tone rescues hippocampal synaptic plasticity in the 5xFAD mouse model. At the CA3-CA1 synapse, both basal synaptic transmission and long-term potentiation (LTP) of synaptic transmission are normalized upon FAAH genetic inactivation, in a CB1 receptor (CB1R)- and TRPV1 receptor-independent manner. Dendritic spine density in CA1 pyramidal neurons, which is notably decreased in 6-month-old 5xFAD animals, is also restored. Importantly, we reveal that the expression of microglial factors linked to phagocytic activity, such as TREM2 and CTSD, and other factors related to amyloid beta clearance and involved in neuron-glia crosstalk, such as complement component C3 and complement receptor C3AR, are specifically upregulated in 5xFAD/FAAH-/- animals. CONCLUSION: In summary, our findings support the therapeutic potential of modulating, rather than suppressing, neuroinflammation in Alzheimer's disease. In our model, the long-term enhancement of the endocannabinoid tone triggered augmented microglial activation and amyloid beta phagocytosis, and a consequent reversal in the neuronal phenotype associated to the disease.
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Enfermedad de Alzheimer/metabolismo , Amidohidrolasas/deficiencia , Péptidos beta-Amiloides/metabolismo , Plasticidad Neuronal/fisiología , Transmisión Sináptica/fisiología , Enfermedad de Alzheimer/patología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/fisiologíaRESUMEN
Ensuring healthy lives and well-being constitutes one of the Sustainable Development Goals of the UN 2030 agenda. Consequently, research into how natural products may promote health is essential for the new generation of nutraceuticals and functional foods that are in high demand today. Grape juice is a natural foodstuff composed of water, sugars, minerals, vitamins and a wide array of polyphenols. Polyphenols are bioactive compounds of great interest due to their antioxidant properties and benefits to health, supporting antimicrobial, anti-aging, and anticarcinogenic activity. The majority of grape juice produced in the world is used for the production of wine, although a small part is used in the food industry, mainly in baby food and sports drinks. The aim of this work is to determine the polyphenol content in the natural and concentrated juice of Airen grapes, the main white grape variety produced in Spain. For this, fresh juices from five grape varietals (Airen, Sauvignon Blanc, Gewürztraminer, Verdejo and Tempranillo) and concentrated Airen juice were analyzed and compared. Results showed similar contents of phenolic acids and stilbenes in all grape varietals studied, although the Airen variety demonstrated a higher concentration of two flavonoids: quercetin and catechin. It can be concluded that the grape juice concentration process negatively affects the stability of these compounds, causing a reduction in the polyphenol content that ranges between 54-71%, with the exception of quercetin and catechin.
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We pursued here the identification of specific signatures of proteins and metabolites in urine which respond to atherosclerosis development, acute event and/or recovery. An animal model (rabbit) of atherosclerosis was developed and molecules responding to atherosclerosis silent development were identified. Those molecules were investigated in human urine from patients suffering an acute coronary syndrome (ACS), at onset and discharge. Kallikrein1 (KLK1) and zymogen granule protein16B (ZG16B) proteins, and l-alanine, l-arabitol, scyllo-inositol, 2-hydroxyphenilacetic acid, 3-hydroxybutyric acid and N-acetylneuraminic acid metabolites were found altered in response to atherosclerosis progression and the acute event, composing a molecular panel related to cardiovascular risk. KLK1 and ZG16B together with 3-hydroxybutyric acid, putrescine and 1-methylhydantoin responded at onset but also showed normalized levels at discharge, constituting a molecular panel to monitor recovery. The observed decreased of KLK1 is in alignment with the protective mechanism of the kallikrein-kinin system. The connection between KLK1 and ZG16B shown by pathway analysis explains reduced levels of toll-like receptor 2 described in atherosclerosis. Metabolomic analysis revealed arginine and proline metabolism, glutathione metabolism and degradation of ketone bodies as the three main pathways altered. In conclusion, two novel urinary panels of proteins and metabolites are here for the first time shown related to atherosclerosis, ACS and patient's recovery.
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Diabetic nephropathy (DN) is a major complication of diabetes mellitus and the most frequent cause of end-stage renal disease. DN progresses silently and without clinical symptoms at early stages. Current noninvasive available markers as albuminuria account with severe limitations (late response, unpredictable prognosis, and limited sensitivity). Thus, it urges the discovery of novel markers to help in diagnosis and outcome prediction. Tissue proteomics allows zooming-in where pathophysiological changes are taking place. We performed a differential analysis of renal tissue proteome in a rat model of early DN by 2-dimensional differential gel electrophoresis and mass spectrometry. Confirmation was performed by Western blot, immunohistochemistry (IHC), and selected reaction monitoring (SRM). Rat urine samples were collected and exosomes were isolated from urine to evaluate if these microvesicles reflect changes directly occurring at tissue level. The protein showing maximum altered expression in rat tissue in response to DN was further analyzed in human kidney tissue and urinary exosomes. Regucalcin protein or senescence marker protein-30 (SMP30) (Swiss-Prot Q03336) was found to be strongly downregulated in DN kidney tissue compared with healthy controls. The same trend was observed in exosomes isolated from urine of control and DN rats. These data were further confirmed in a pilot study with human samples. IHC revealed a significant decrease of regucalcin in human kidney disease tissue vs control kidney tissue, and regucalcin was detected in exosomes isolated from healthy donors' urine but not from kidney disease patients. In conclusion, regucalcin protein expression is reduced in DN kidney tissue and this significant change is reflected in exosomes isolated from urine. Urinary exosomal regucalcin represents a novel tool, which should be explored for early diagnosis and progression monitoring of diabetic kidney disease.
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Proteínas de Unión al Calcio/metabolismo , Nefropatías Diabéticas/metabolismo , Regulación hacia Abajo , Exosomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Riñón/metabolismo , Proteómica , Anciano , Anciano de 80 o más Años , Animales , Hidrolasas de Éster Carboxílico , Estudios de Casos y Controles , Nefropatías Diabéticas/orina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ratas , Ratas Endogámicas WKYRESUMEN
Mustard is a condiment added to a variety of foodstuffs and a frequent cause of food allergy. A new strategy for the detection of mustard allergen in food products is presented. The methodology is based on liquid chromatography analysis coupled to mass spectrometry. Mustard allergen Sin a 1 was purified from yellow mustard seeds. Sin a 1 was detected with a total of five peptides showing a linear response (lowest LOD was 5ng). Sin a 1 was detected in mustard sauces and salty biscuit (19±3mg/kg) where mustard content is not specified. Sin a 1, used as an internal standard, allowed quantification of this mustard allergen in foods. A novel LC/MS/MS SRM-based method has been developed to detect and quantify the presence of mustard. This method could help to detect mustard allergen Sin a 1 in processed foods and protect mustard-allergic consumers.
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Antígenos de Plantas/análisis , Cromatografía Liquida/métodos , Análisis de los Alimentos/métodos , Espectrometría de Masas/métodos , Planta de la Mostaza/química , Proteínas de Plantas/análisis , Antígenos de Plantas/inmunología , Hipersensibilidad a los Alimentos/inmunología , Planta de la Mostaza/inmunología , Proteínas de Plantas/inmunologíaRESUMEN
Urine is a source of potential markers of disease. In the context of renal disease, urine is particularly important as it may directly reflect kidney injury. Current markers of renal dysfunction lack both optimal specificity and sensitivity, and improved technologies and approaches are needed. There is no clear consensus about the best sample pretreatment procedure for 2DE analysis of the urine proteome. Sample pretreatment conditions spots resolution and detection sensitivity, critically. As a first goal, we exhaustively compared eight different sample cleaning and protein purification methodologies for 2DE analysis of urine from healthy individuals. Oasis® HLB cartridges allowed the detection of the highest number of low molecular weight proteins; while PD10 desalting columns resulted in the highest number of detected spots in the high molecular weight area. Sample pretreatment strategies were also explored in the context of proteinuria, a clinical condition often associated to renal damage. Testing of urine samples from 13 patients with hypertension or kidney disease and different levels of proteinuria identified Oasis® HLB cartridge purification in combination with albumin depletion by ProteoPrep kit as the best option for urine proteome profiling from patients with proteinuric (> 30 mg/L albumin in urine) renal disease.
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Biomarcadores/orina , Electroforesis en Gel Bidimensional/métodos , Enfermedades Renales/metabolismo , Proteoma/análisis , Proteómica/métodos , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas/química , Proteínas/aislamiento & purificación , Proteinuria/metabolismo , Proteoma/químicaRESUMEN
The prevalence of chronic kidney disease (CKD) is increasing and frequently progresses to end-stage renal disease. There is an urgent demand to discover novel markers of disease that allow monitoring disease progression and, eventually, response to treatment. To identify such markers, and as a proof of principle, we determined if a metabolite signature corresponding to CKD can be found in urine. In the discovery stage, we analyzed the urine metabolome by NMR of 15 patients with CKD and compared that with the metabolome of 15 healthy individuals and found a classification pattern clearly indicative of CKD. A validation cohort of urine samples from an additional 16 patients with CKD and 15 controls was then analyzed by (Selected Reaction Monitoring) liquid chromatography-triple quadrupole mass spectrometry and indicated that a group of seven urinary metabolites differed between CKD and non-CKD urine samples. This profile consisted of 5-oxoproline, glutamate, guanidoacetate, α-phenylacetylglutamine, taurine, citrate, and trimethylamine N-oxide. Thus, we identified a panel of urine metabolites differentially present in urine that may help identify and monitor patients with CKD.
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Biomarcadores/orina , Fallo Renal Crónico/orina , Metaboloma , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana EdadRESUMEN
Diabetic nephropathy (DN) is a major complication of diabetes mellitus (DM), the most frequent cause of end-stage renal disease (ESRD). Exosomes isolated from urine are considered a rich non-invasive source of markers for renal events. Proteinuria associated with DN patients at advanced stages may result in "contamination" of exosomal fraction by co-precipitation of high abundance urine proteins, making it enormously difficult to obtain a reliable comparison of healthy individuals and DN patients and to detect minor proteins. We evaluated different protocols for urinary exosome isolation (ultracentrifugation-based and Exoquick® reagent-based) in combination with an easy and quick depletion procedure of contaminating high abundance proteins (albumin). The optimal methodology was then applied to investigate the proteome of human urinary exosomes in DN and controls using spectral counting LC-MS/MS analysis followed by selected reaction monitoring (SRM) confirmation. A panel of 3 proteins (AMBP, MLL3, and VDAC1) is differentially present in urinary exosomes from DN patients, opening a new field of research focused on improving diagnosis and follow-up of this pathology. BIOLOGICAL SIGNIFICANCE: Diabetic nephropathy (DN) is a progressive proteinuric kidney disease, a major complication of diabetes mellitus, and the most frequent cause of end-stage renal disease. Current markers of disease (i.e. creatinine and urinary albumin excretion) have proven limitations (i.e. some patients regress to normoalbuminuria, kidney damage may be already present in recently diagnoses microalbuminuric patients and renal function may decrease in the absence of significant albuminuria). We show here the first study on human DN proteome of urinary exosomes. Proteinuria associated to DN patients resulting in contamination of exosomal fraction and the associated difficulty to reliably compare healthy and disease conditions, are here overcome. A combined methodology pointed to increase exosomal proteome recovery and depletion of high-abundance proteome was here set-up. A total of 352 proteins were here identified for the first time associated to human urinary exosomes. Label-free quantitative comparison of DN urinary exosomes vs control group and SRM further validation, resulted in the discovery of a panel of three proteins (AMBP, MLL3 and VDAC1) which changes in DN, opening a new field of research focused to improve diagnosis and follow-up of this pathology.
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alfa-Globulinas/orina , Proteínas de Unión al ADN/orina , Nefropatías Diabéticas/orina , Proteoma/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/orina , Adulto , Anciano , Albuminuria/orina , Exosomas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Discovery of new biomarkers is critical for early diagnosis of acute coronary syndrome (ACS). Recent advances in metabolomic technologies have drastically enhanced the possibility of improving the knowledge of its physiopathology through the identification of the altered metabolic pathways. In this study, analyses of peripheral plasma from non-ST segment elevation ACS patients and healthy controls by gas chromatography-mass spectrometry (GC-MC) permitted the identification of 15 metabolites with statistical differences (p < 0.05) between experimental groups. Additionally, validation by GC-MC and liquid chromatography-MC permitted us to identify a potential panel of biomarkers formed by 5-OH-tryptophan, 2-OH-butyric acid and 3-OH-butyric acid. This panel of biomarkers reflects the oxidative stress and the hypoxic state that suffers the myocardial cells and consequently constitutes a metabolomic signature of the atherogenesis process that could be used for early diagnosis of ACS.
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Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.
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Exosomas/química , Túbulos Renales Proximales/citología , Osteoprotegerina/análisis , Osteoprotegerina/orina , Insuficiencia Renal Crónica/orina , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Línea Celular , Citocina TWEAK , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Ligando Inductor de Apoptosis Relacionado con TNF/análisis , Factores de Necrosis Tumoral/análisisRESUMEN
With the aim of studying a wide cohort of erythrocyte samples in a clinical setting, this chapter details a novel approach that allows the analysis of both human cytosolic and membrane sub-proteomes. Despite their simple structure, the high content of hemoglobin present in the red blood cells (RBCs) makes their proteome analysis enormously difficult. Careful investigation of different strategies for isolation of the membrane and cytosolic fractions from erythrocytes and their influence on proteome profiling by 2-DE was carried out, paying particular attention to hemoglobin removal. As result, a simple, quick, and satisfactory approach for hemoglobin depletion of erythrocyte cells based on HemogloBind™ reagent is shown here to satisfactorily analyze the cytosolic sub-proteome by 2-DE without major interference. For membrane proteome, a novel combined strategy based on hypotonic lysis isolation and further purification on minicolumns is described, allowing detection of high-molecular-weight proteins (i.e., spectrin, ankyrin) and well-resolved 2-DE patterns. The analysis of the membrane fraction by nano-LC coupled to an LTQ-Orbitrap mass spectrometer results in the identification of a total of 188 unique proteins.
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Citosol/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/citología , Proteínas de la Membrana/metabolismo , Proteómica/métodos , Recolección de Muestras de Sangre , Separación Celular , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Hemoglobinas/aislamiento & purificación , Humanos , Proteínas de la Membrana/aislamiento & purificación , Nanotecnología , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
The molecular basis of the reduced half-life of chronic kidney disease (CKD) erythrocytes is unclear. The erythrocyte membrane plays a key role in the erythrocyte mechanical properties and survival. The aim of the present work is to uncover erythrocyte membrane proteins whose expression could be altered in CKD. The erythrocyte membrane subproteome was analyzed by a non-biased approach where the whole set of proteins was simultaneously investigated by 2D fluorescence difference gel electrophoresis without preselection of potential targets. Proteins significantly altered in CKD were identified by mass spectrometry (MS) and results validation was performed by Western blot and confocal microscopy. Nine differentially expressed spots among healthy individuals, non-dialyzed CKD and erythropoietin/dialysis-treated CKD patients were identified by MS/MS corresponding to 5 proteins (beta-adducin, HSP71/72, tropomodulin-1, ezrin, and radixin). Ezrin and radixin were higher in dialyzed CKD patients than in the other 2 groups. Beta-adducin was increased in CKD patients (dialyzed or not). Three spots were normalized in patients on the dialysis/erythropoietin combination compared with non-dialyzed CKD. Among these, a spot corresponding to tropomodulin 1, was found to be of higher abundance in non-dialyzed CKD patients compared with controls or dialyzed CKD. In conclusion, this study identifies changes in erythrocyte membrane proteins in CKD, which may be relevant for the pathogenesis of red cell abnormalities in uremia.
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Membrana Eritrocítica/química , Eritrocitos/química , Proteínas de la Membrana/análisis , Proteoma , Insuficiencia Renal Crónica/patología , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Estudios de Casos y Controles , Electroforesis en Gel Bidimensional , Femenino , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Diálisis Renal , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/terapia , Adulto JovenRESUMEN
AIMS: Early detection of cardiovascular diseases and knowledge of underlying mechanisms is essential. Tissue secretome studies resemble more closely to the in vivo situation, showing a much narrower protein concentrations dynamic range than plasma. This study was aimed to the analysis of human arterial tissue secretome and to the quantitative comparison of healthy and atherosclerotic secretome to discover proteins with key roles in atherosclerosis development. METHODS AND RESULTS: Secretomes from three biological replicates of human atherosclerotic coronary arteries (APC), preatherosclerotic coronaries (PC) and mammaries (M) were analyzed by LC-MS/MS. The identified proteins were submitted to Ingenuity Pathway Analysis (IPA) tool. Label-free MS/MS based quantification was performed and validated by immunohistochemistry. 64 proteins were identified in the 3 replicates of at least one of the 3 groups and 15 secreted proteins have not been previously reported in plasma. Four proteins were significantly released in higher amounts by mammary tissue: gelsolin, vinculin, lamin A/C and phosphoglucomutase 5. CONCLUSION: The study of tissue secretome reveals key proteins involved in atherosclerosis which have not been previously reported in plasma. Novel proteins are here highlighted which could be potential therapeutic targets in clinical practice. This article is part of a Special Issue entitled: Proteomics: The clinical link.