Association between exposure to air pollution and blood lipids in the general population of Spain.

BACKGROUND AND AIMS: We aimed to assess the associations of exposure to air pollutants and standard and advanced lipoprotein measures, in a nationwide sample representative of the adult population of Spain. METHODS: We included 4647 adults (>18 years), participants in the national, cross-sectional, population-based di@bet.es study, conducted in 2008-2010. Standard lipid measurements were analysed on an Architect C8000 Analyzer (Abbott Laboratories SA). Lipoprotein analysis was made by an advanced 1 H-NMR lipoprotein test (Liposcale®). Participants were assigned air pollution concentrations for particulate matter <10 µm (PM10 ), <2.5 µm (PM2.5 ) and nitrogen dioxide (NO2 ), corresponding to the health examination year, obtained by modelling combined with measurements taken at air quality stations (CHIMERE chemistry-transport model). RESULTS: In multivariate linear regression models, each IQR increase in PM10 , PM2.5 and NO2 was associated with 3.3%, 3.3% and 3% lower levels of HDL-c and 1.3%, 1.4% and 1.1% lower HDL particle (HDL-p) concentrations (p < .001 for all associations). In multivariate logistic regression, there was a significant association between PM10 , PM2.5 and NO2 concentrations and the odds of presenting low HDL-c (<40 mg/dL), low HDL-p (

Plasma Expression of Carotid Plaque Presence-Related MicroRNAs Is Associated with Inflammation in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is associated with problems beyond the joints such as cardiovascular (CV) disease. MicroRNA-24, -146 and -Let7a are associated with carotid plaque presence in RA patients. We evaluated whether these microRNAs were involved in the inflammatory state of RA, and we studied their gene targets to understand their role in inflammation and atherosclerosis. A total of 199 patients with RA were included. Inflammatory variables such as disease activity score 28 (DAS28) and erythrocyte sedimentation rate (ESR) were quantified. MicroRNAs were extracted from plasma and quantified with qPCR. Multivariate models and classification methods were used for analysis. The multivariate models showed that diminished expression of microRNA-146 was associated with inferior levels of DAS28-ESR, and the decreased expression of microRNA-24, -146 and -Let7a were associated with lowered ESR in the overall cohort. When microRNAs were evaluated globally, a global increase was associated with increased DAS28-ESR and ESR in the overall cohort. Sex-stratified analyses showed different associations of these microRNAs with the inflammatory variables. Finally, random forest models showed that microRNAs have a pivotal role in classifying patients with high and low inflammation. Plasmatic expressions of microRNA-24, -146 and -Let7a were associated with inflammatory markers of RA. These microRNAs are associated with both inflammation and atherosclerosis and are potential therapeutic targets for RA.

A panel of plasma microRNAs improves the assessment of surrogate markers of cardiovascular disease in rheumatoid arthritis patients.

OBJECTIVE: Patients with RA present increased risk of cardiovascular (CV) disease compared with the general population. Moreover, CV risk factors that have a causal relationship with atherosclerosis do not seem to fully explain the accelerated process that they exhibit. We evaluated the association of a 10 microRNAs panel with surrogate markers of subclinical arteriosclerosis [carotid intima-media thickness (cIMT), carotid plaque presence (cPP), pulse wave velocity (PWV) and distensibility] in a cohort of RA patients. MATERIAL AND METHODS: A total of 199 patients with RA were included. Surrogate markers of arteriosclerosis were measured with My Lab 60 X-Vision sonographer. MicroRNAs were extracted from plasma and quantified with qPCR. Multivariate models and classification methods were performed. RESULTS: Multivariate models showed that microRNAs-24 (ß = 15.48), 125a (ß = 9.93), 132 (ß = 11.52), 146 (ß = 15.12), 191 (ß = 13.25) and 223 (ß = 13.30) were associated with cIMT globally. MicroRNA-24 [odds ratio (OR) = 0.41], 146 (OR = 0.36) and Let7a (OR = 0.23) were associated with cPP in men. Including the microRNAs in a partial least square discriminant analysis model properly classified men with and without cPP. MicroRNA-96 (ß = -0.28) was associated with PWV in male patients. Finally, several miRNAs were also associated with cIMT, cPP and arterial stiffness in the high DAS28 group and in the earlier tertile groups of disease duration. CONCLUSION: Plasmatic expression of microRNA-24, 96, 103, 125a, 132, 146, 191, 223 and Let7a were associated with surrogate markers of CV disease and could be predictors of CV risk in patients with RA.

Statistical mediation of the relationships between chronological age and lipoproteins by nonessential amino acids in healthy men.

Aging is a major risk factor for metabolic impairment that may lead to age-related diseases such as cardiovascular disease. Different mechanisms that may explain the interplay between aging and lipoproteins, and between aging and low-molecular-weight metabolites (LMWMs), in the metabolic dysregulation associated with age-related diseases have been described separately. Here, we statistically evaluated the possible mediation effects of LMWMs on the relationships between chronological age and lipoprotein concentrations in healthy men ranging from 19 to 75 years of age. Relative and absolute concentrations of LMWMs and lipoproteins, respectively, were assessed by nuclear magnetic resonance (NMR) spectroscopy. Multivariate linear regression and mediation analysis were conducted to explore the associations between age, lipoproteins and LMWMs. The statistical significance of the identified mediation effects was evaluated using the bootstrapping technique, and the identified mediation effects were validated on a publicly available dataset. Chronological age was statistically associated with five lipoprotein classes and subclasses. The mediation analysis showed that serine mediated 24.1% (95% CI: 22.9 - 24.7) of the effect of age on LDL-P, and glutamate mediated 17.9% (95% CI: 17.6 - 18.5) of the effect of age on large LDL-P. In the publicly available data, glutamate mediated the relationship between age and an NMR-derived surrogate of cholesterol. Our results suggest that the age-related increase in LDL particles may be mediated by a decrease in the nonessential amino acid glutamate. Future studies may contribute to a better understanding of the potential biological role of glutamate and LDL particles in aging mechanisms and age-related diseases.

Plasma expression of microRNA-425-5p and microRNA-451a as biomarkers of cardiovascular disease in rheumatoid arthritis patients.

To validate in a cohort of 214 rheumatoid arthritis patients a panel of 10 plasmatic microRNAs, which we previously identified and that can facilitate earlier diagnosis of cardiovascular disease in rheumatoid arthritis patients. We identified 10 plasma miRs that were downregulated in male rheumatoid arthritis patients and in patients with acute myocardial infarction compared to controls suggesting that these microRNAs could be epigenetic biomarkers for cardiovascular disease in rheumatoid arthritis patients. Six of those microRNAs were validated in independent plasma samples from 214 rheumatoid arthritis patients and levels of expression were associated with surrogate markers of cardiovascular disease (carotid intima-media thickness, plaque formation, pulse wave velocity and distensibility) and with prior cardiovascular disease. Multivariate analyses adjusted for traditional confounders and treatments showed that decreased expression of microRNA-425-5p in men and decreased expression of microRNA-451 in women were significantly associated with increased (ß = 0.072; p = 0.017) and decreased carotid intima-media thickness (ß = -0.05; p = 0.013), respectively. MicroRNA-425-5p and microRNA-451 also increased the accuracy to discriminate patients with pathological carotid intima-media thickness by 1.8% (p = 0.036) in men and 3.5% (p = 0.027) in women, respectively. In addition, microRNA-425-5p increased the accuracy to discriminate male patients with prior cardiovascular disease by 3% (p = 0.008). Additionally, decreased expression of microRNA-451 was significantly associated with decreased pulse wave velocity (ß = -0.72; p = 0.035) in overall rheumatoid arthritis population. Distensibility showed no significant association with expression levels of the microRNAs studied. We provide evidence of a possible role of microRNA-425-5p and microRNA-451 as useful epigenetic biomarkers to assess cardiovascular disease risk in patients with rheumatoid arthritis.

Title: Human Serum/Plasma Glycoprotein Analysis by 1H-NMR, an Emerging Method of Inflammatory Assessment.

Several studies suggest that variations in the concentration of plasma glycoproteins can influence cellular changes in a large number of diseases. In recent years, proton nuclear magnetic resonance (1H-NMR) has played a major role as an analytical tool for serum and plasma samples. In recent years, there is an increasing interest in the characterization of glycoproteins through 1H-NMR in order to search for reliable and robust biomarkers of disease. The objective of this review was to examine the existing studies in the literature related to the study of glycoproteins from an analytical and clinical point of view. There are currently several techniques to characterize circulating glycoproteins in serum or plasma, but in this review, we focus on 1H-NMR due to its great robustness and recent interest in its translation to the clinical setting. In fact, there is already a marker in H-NMR representing the acetyl groups of the glycoproteins, GlycA, which has been increasingly studied in clinical studies. A broad search of the literature was performed showing a general consensus that GlycA is a robust marker of systemic inflammation. The results also suggested that GlycA better captures systemic inflammation even more than C-reactive protein (CRP), a widely used classical inflammatory marker. The applications reviewed here demonstrated that GlycA was potentially a key biomarker in a wide range of diseases such as cancer, metabolic diseases, cardiovascular risk, and chronic inflammatory diseases among others. The profiling of glycoproteins through 1H-NMR launches an encouraging new paradigm for its future incorporation in clinical diagnosis.

The Circulating GRP78/BiP Is a Marker of Metabolic Diseases and Atherosclerosis: Bringing Endoplasmic Reticulum Stress into the Clinical Scenario.

BACKGROUND: Glucose-regulated protein 78/Binding immunoglobulin protein (GRP78/BiP) is a protein associated with endoplasmic reticulum stress and is upregulated by metabolic alterations at the tissue-level, such as hypoxia or glucose deprivation, and it is hyper-expressed in fat tissue of obese individuals. OBJECTIVE: To investigate the role of the GRP78/BiP level as a metabolic and vascular disease biomarker in patients with type 2 diabetes (DM), obesity and metabolic syndrome (MS). METHODS: Four hundred and five patients were recruited, of whom 52.5% were obese, 72.8% had DM, and 78.6% had MS. The intimae media thickness (cIMT) was assessed by ultrasonography. The plasma GRP78/BiP concentration was determined, and its association with metabolic and vascular parameters was assessed. Circulating GRP78/BiP was also prospectively measured in 30 DM patients before and after fenofibrate/niacin treatment and 30 healthy controls. RESULTS: In the cross-sectional study, the GRP78/BiP level was significantly higher in the patients with obesity, DM, and MS. Age-, gender- and BMI-adjusted GRP78/BiP was directly associated with LDL-cholesterol, non-HDL-cholesterol, triglycerides, apoB, and cIMT. GRP78/BiP was positively associated to carotid plaque presence in the adjusted model, irrespective of obesity, DM and MS. In the prospective study, nicotinic acid treatment produced a significant reduction in the GRP78/BiP levels that was not observed with fenofibrate. CONCLUSIONS: GRP78/BiP plasma concentrations are increased in patients with both metabolic derangements and subclinical atherosclerosis. GRP78/BiP could be a useful marker of metabolic and cardiovascular risk.

Effect of radiotherapy on the expression of cardiovascular disease-related miRNA-146a, -155, -221 and -222 in blood of women with breast cancer.

Breast cancer (BC) is one of the most important neoplasias among women. Many patients receive radiotherapy (RT), which involves radiation exposure of the thoracic zone, including the heart and blood vessels, leading to the development of cardiovascular disease (CVD) as a long-term side effect. The severity of CVD-related pathologies leads research on assessing novel CVD biomarkers as diagnostic, prognostic or therapeutic agents. Currently, the possible candidates include blood microRNAs (miRNAs). Previous studies have supported a role for miRNA-146a, -155, -221, and -222 in the progression of CVD. Our purpose was to evaluate the RT-induced modulation of the expression of these miRNAs in the blood of women with BC. Pre-RT control and post-RT blood samples were collected, and after miRNA isolation and reverse transcription, the levels of the selected miRNAs were measured by real-time PCR. Our results showed that miRNA-155 exhibited the lowest expression, while miRNA-222 exhibited the highest expression, followed by miRNA-221. The expression of each individual miRNA was positively correlated with that of the others both pre-RT control and post-RT and inversely correlated with age before RT. Furthermore, RT promoted the overexpression of the selected miRNAs. Their levels were also affected by CVD-linked clinical parameters, treatment and BC side. Modulation of the expression of the selected miRNAs together with other risk factors might be associated with the development of future cardiovascular pathologies. Further confirmatory studies are needed to assess their potential as possible biomarkers in the progression of or as therapeutic targets for RT-induced CVD in BC patients.

Palmitate decreases migration and proliferation and increases oxidative stress and inflammation in smooth muscle cells: role of the Nrf2 signaling pathway.

Fatty acids are essential to cell functionality and may exert diverging vascular effects including migration, proliferation, oxidative stress, and inflammation. This study examined the effect of palmitate on human coronary artery smooth muscle cell (HCASMC) function. An in vitro wound-healing assay indicated that palmitate decreased HCASMC migration in dose- and time-dependent manners. Furthermore, bromodeoxyuridine incorporation assays indicated that palmitate decreased HCASMC proliferation in a dose-response manner. Palmitate also increased reactive oxygen species formation, malondialdehyde content, and intracellular lipid droplets accompanied with increased fatty acid binding protein 4 expression. Moreover, palmitate induced gene expression (monocyte chemoattractant protein 1, matrix metalloproteinase-2, IL-1ß, IL-6, IL-8, and TNF-α) and intracellular protein content (plasminogen activator inhibitor-1 and urokinase plasminogen activator) of inflammatory mediators. Finally, we showed that palmitate activates the transcription factor Nrf2 and the upstream kinases ERK1/2 and Akt in HCASMCs. The inhibitor of Nrf2, trigonelline, significantly attenuated palmitate-induced HCASMC expression of the Nrf2 target gene NQO1. These findings indicate that palmitate might be critically related to HCASMC function by slowing cell migration and proliferation and inducing lipid-laden cells, oxidative stress, and inflammation in part by activation of the Nrf2 transcription factor. Palmitate's activation of proinflammatory Nrf2 signaling may represent a novel mechanism mediating the proatherogenic actions of saturated fatty acids.

Assessment of arterial stiffness variables in patients with rheumatoid arthritis: A mediation analysis.

We aimed to study arterial stiffness variables in patients with rheumatoid arthritis (RA), specifically considering their associations with path model mediation analysis. We examined arterial stiffness expressed by the pulse wave velocity (PVW), augmentation index (AIx), distensibility, and clinical and biochemical characteristics in a cohort of 214 RA patients. Variable associations were analysed using multivariate linear regression analysis. We also used path model mediation analysis for PWV variable. Our results indicate that age, systolic blood pressure (SBP), and body mass index (BMI) were significantly associated with PWV, and collectively accounted for 32% of PWV variability. The parallel mediation analysis showed that SBP and BMI accounted for 21% and 7% (a total of 28%) of the total effect of age on PWV, respectively, indicating a partial mediation effect. The associated variables with AIx were age and tender joint count, while those with distensibility were BMI and sex, overall accounting for 16.5% and 4.7% of the variation in AIx and distensibility, respectively. We observed no associations of arterial stiffness with inflammatory variables, disease activity and duration, or cholesterol levels. In conclusion, in our population of RA patients, age is the most important variable that determines the increase in PWV. We have also shown that a significant proportion of the negative effects of age on PWV occurs through increases in SBP and BMI. In our study, lipid and inflammation variables were not associated with an increase in arterial stiffness.

Effect of Radiation on the Expression of CVD-related miRNAs, Inflammation and Endothelial Dysfunction of HUVECs.

BACKGROUND/AIM: Radiotherapy (RT) can lead to cardiovascular disease (CVD). Evidence suggests that radiation modulates miRNA levels. Our purpose was to assess the acute response to radiation-induced modulation of the expression of miRNA-146a, miRNA-155, miRNA-221, and miRNA-222, inflammatory response and endothelial dysfunction on endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to 2 Gy RT, and intracellular levels of selected miRNAs were measured by real-time polymerase chain reaction at 2 and 24 h. Cytokine and adhesion molecule release were also assessed. RESULTS: Results showed that 2 Gy significantly increased the expression of miRNA-221 and miRNA-222, and reduced the level of miRNA-155 after 2 h; whereas miRNA-146a and miRNA-155 were significantly overexpressed and miRNA-222 was significantly down-regulated at 24 h. Interleukin-8 and soluble vascular cell adhesion molecule 1 levels were not affected by the studied RT. CONCLUSION: RT at 2 Gy modulated expression of selected miRNAs by endothelial cells after 2 and 24 h, which might be related to CVD development in patients who receive RT.

Characterization of 1H NMR Plasma Glycoproteins as a New Strategy To Identify Inflammatory Patterns in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease associated with a high index of morbidity and mortality from cardiovascular diseases. We used 1H NMR to characterize the plasma glycoprotein and lipoprotein profiles of a cohort of patients with RA ( n = 210) versus healthy individuals ( n = 203) to associate them with the RA disease and its severity. Using 1H NMR, we developed a line-shape method to characterize the two peaks associated with glycoproteins (GlycA and GlycB) and its derived variables: areas of GlycB (Area GlycB) and GlycA (Area GlycA), shape factors of these two peaks (H/W = height/width), and the distance between them (Distance GlycB-GlycA). We also used the advanced lipoprotein test Liposcale (CE) to characterize the lipoprotein subclasses. The standard lipid panel and traditional inflammatory markers such as C-reactive protein, the erythrocyte sedimentation rate, fibrinogen, the rheumatoid factor, anticitrullinated peptide antibodies, and the DAS28 index have also been determined. RA patients presented a significant 10.65% increase in the GlycA associated area compared with the control group ( p = 2.21 × 10-10). They also presented significantly higher H/W GlycA and GlycB ratios than the control population (H/W GlycB p = 7.88 × 10-8; H/W GlycA p = 5.61 × 10-8). The prediction model that uses the traditional inflammatory variables and the 1H NMR-derived parameters presented an AUC that was almost 10% higher than the model that only uses the traditional inflammatory variables (from 0.7 to 0.79 AUC). We have demonstrated that GlycA and GlycB variables derived from 1H NMR, along with classic inflammatory parameters, help to improve the classification of individuals with high RA disease activity.

Variables associated with subclinical atherosclerosis in a cohort of rheumatoid arthritis patients: Sex-specific associations and differential effects of disease activity and age.

OBJECTIVE: To advance the study of variables associated with subclinical atherosclerosis in rheumatoid arthritis (RA) with special consideration for the degree of disease activity, age and gender. METHODS: The carotid intima-media thickness (cIMT) and the presence of carotid atherosclerotic plaques along with clinical and biochemical characteristics were determined in 214 RA patients. RESULTS: Adjusted analysis reveals that men had a 0.059 mm significantly increased cIMT compared with women (p = 0.001; R2 = 3.8%) and that age was associated with cIMT (ß = 0.0048 mm; p = 0.0001; R2 = 16%). Interestingly, we observed a significant interaction between gender and age. Thus, the effect of age on cIMT was significantly increased (12%) in men compared with women (p-value for interaction term = 0.041). Moreover, adjusted multivariable linear regression analysis revealed that disease activity score (DAS28) was significantly associated with cIMT in women (ß = 0.021; p = 0.018: R2 = 0.03) but not men. In particular, women with high disease activity had a 0.079 mm increased cIMT compared with women in remission (p = 0.026). In addition, men in remission had a 0.134 mm increased cIMT compared with women in remission (p = 0.003; R2 = 8.7%). Active patients did not exhibit differences in cIMT values. Furthermore, 43% of patients presented carotid plaques. The variables independently associated with carotid plaques were age, smoking, health assessment questionnaire, erythrocyte sedimentation rate and rheumatoid factor (p<0.0001; R2 = 46%). CONCLUSION: In our cohort of patients with RA, DAS28 and age are differentially associated with cIMT in men and women. Our findings could explain the contradictory results that have previously been published in the literature.

Renal and hepatic effects following neonatal exposure to low doses of Bisphenol-A and 137Cs.

137-Cesium (137Cs) is one of the most important distributed radionuclides after a nuclear accident. Humans are usually co-exposed to various environmental toxicants, being Bisphenol-A (BPA) one of them. Exposure to IR and BPA in early life is of major concern, due to the higher vulnerability of developing organs. We evaluate the renal and hepatic effects of low doses of ionizing radiation (IR) and BPA. Sixty male mice (C57BL/6J) were randomly assigned to six experimental groups (n=10) and received a single subcutaneous dose of 0.9% saline solution, 137Cs and/or BPA on postnatal day 10: control, BPA (25 µg/kgbw), Cs4000 (4000 Bq 137Cs/kgbw), Cs8000 (8000 Bq 137Cs/kgbw), BPA/Cs4000 and BPA/Cs8000. At the age of two months, urines (24h) and blood samples were collected from animals of each group to determine biochemical parameters. Finally, kidneys and liver were removed to quantify DNA damage (8-OHdG), as well as to determine CYP1A2 mRNA expression. Data suggest that both BPA and 137Cs induced renal and liver damage evidenced by oxidative stress. However, when there is a co-exposure, it seems that there are compensatory mechanisms that may reverse the damage induced by each toxic itself. Notwithstanding, more studies are necessary to better understand the synergistic mechanisms behind.

Clinical and pathophysiological evidence supporting the safety of extremely low LDL levels-The zero-LDL hypothesis.

While the impact of very low concentrations of low-density lipoprotein cholesterol (LDL-C) on cardiovascular prevention is very reassuring, it is intriguing to know what effect these extremely low LDL-C concentrations have on lipid homoeostasis. The evidence supporting the safety of extremely low LDL levels comes from genetic studies and clinical drug trials. Individuals with lifelong low LDL levels due to mutations in genes associated with increased LDL-LDL receptor (LDLR) activity reveal no safety issues. Patients achieving extremely low LDL levels in the IMPROVE-IT and FOURIER, and the PROFICIO and ODYSSEY programs seem not to have an increased prevalence of adverse effects. The main concern regarding extremely low LDL-C plasma concentrations is the adequacy of the supply of cholesterol, and other molecules, to peripheral tissues. However, LDL proteomic and kinetic studies reaffirm that LDL is the final product of endogenous lipoprotein metabolism. Four of 5 LDL particles are cleared through the LDL-LDLR pathway in the liver. Given that mammalian cells have no enzymatic systems to degrade cholesterol, the LDL-LDLR pathway is the main mechanism for removal of cholesterol from the body. Our focus, therefore, is to review, from a physiological perspective, why such extremely low LDL-C concentrations do not appear to be detrimental. We suggest that extremely low LDL-C levels due to increased LDLR activity may be a surrogate of adequate LDL-LDLR pathway function.

Differential leucocyte RNA expression in the coronary arteries compared to systemic circulation discriminates between patients with and those without coronary artery disease / La expresión diferencial de ARN en leucocitos en las arterias coronarias comparada con la de leucocitos de circulación sistémica discrimina a pacientes con y sin enfermedad arterial coronaria

Aims: To test the hypothesis that the pattern of gene expression in circulating leukocytes may differ between vascular compartments, depending on the presence or absence of atherosclerosis, we evaluated the regional vascular differences in patterns of inflammatory cell activation. Methods: Patients (n = 8) with angiographically-established coronary artery disease (CAD+) and 8 without (CAD-) had blood samples taken from a peripheral vein as well as from left and right coronary arteries. Samples were pooled resulting in 4 CAD+ samples versus 4 CAD- samples and hybridised to a Whole Human Genome Microarray 4×44K. Results: CAD- patients had a similar gene expression profile across the different sites. CAD+ patients had statistically significant different gene expression patterns in venous vs. right and left coronary artery compartments. The expression pattern observed in the right coronary was where the most differences in gene expression were observed in CAD+ vs. CAD- patients. Overall, 1964 genes were differentially expressed between CAD+ and CAD−. Of these, 1052 were less expressed in CAD+ and 912 were more expressed in CAD+. Up to 12 of the 20 most differentially expressed genes appeared to reflect different phases of the atherosclerosis process: endothelial dysfunction, lipid accumulation, and smooth muscle cell proliferation. Conclusions: Gene expression of circulating leukocytes differentiates CAD+ from CAD- patients. Gene expression is significantly different between coronary arteries and the systemic circulation in CAD+ patients, but not in CAD- patients. Gene expression is significantly different between CAD+ and CAD- subjects, and appears to reflect the atherosclerosis process. These intra-individual differences may be an additional feature of established coronary artery disease

Objetivo: Para comprobar la hipótesis de que los patrones de expresión génica de leucocitos en circulación pueden ser diferentes entre los compartimentos vasculares dependiendo de la presencia o ausencia de arteriosclerosis, hemos evaluado en distintas regiones vasculares las diferencias entre los patrones de expresión y la activación de células inflamatorias. Métodos: Se extrajeron muestras de sangre de venas periféricas y de las arterias coronarias (derecha e izquierda) de pacientes con (n=8; CAD+) y sin (n=8; CAD−) enfermedad arterial coronaria establecida angiográficamente. Las muestras fueron hibridadas en dos pooles de 4 muestras (CAD+ vs CAD−) mediante el kit Whole Human Genome Microarray 4×44K. Resultados: Los pacientes CAD- tenían un perfil de expresión génica similar entre los distintos compartimentos vasculares. Los pacientes CAD+ tenían patrones de expresión génica significativamente diferentes entre los compartimentos venosos y las arterias coronarias derecha e izquierda. El patrón de expresión observado en la arteria coronaria derecha fue el que presentó más diferencias entre los pacientes CAD+ vs. CAD-. En conjunto, 1.964 genes estaban expresados diferencialmente entre CAD+ y CAD-. De estos, 1.052 estaban menos expresados en CAD+ i 912 estaban más expresados en CAD+. Hasta 12 de los 20 genes más diferencialmente expresados estaban relacionados con las diferentes fases del proceso arteriosclerótico: disfunción endotelial, acumulación lipídica y proliferación de células musculares lisas. Conclusiones: La expresión génica de leucocitos circulantes diferencia pacientes CAD+ de CAD-. La expresión genética es significativamente diferente entre arterias coronarias y circulación sistémica en pacientes CAD+, pero no en pacientes CAD-. Estas diferencias intraindividuales podrían ser una característica adicional en el diagnóstico de la enfermedad arterial coronaria

Unravelling and Quantifying the "NMR-Invisible" Metabolites Interacting with Human Serum Albumin by Binding Competition and T2 Relaxation-Based Decomposition Analysis.

Quantitative profiling of low-molecular-weight metabolites (LMWMs) by 1H NMR is routinely used in high-throughput serum metabolomics. First, the protein background is attenuated using a T2 filter; then, the LMWM signals are resolved by line-shape fitting. However, protein-binding modifies the motional properties of LMWM, and their signal partially attenuates with the T2 filter, along with the protein background. Consequently, the quantified LMWM signals do not reflect the total concentration in serum but the nonbinding part. Here we present a novel strategy based on binding competition to promote the release of the "NMR-invisible" metabolites from serum proteins and achieve quantifications closer to total concentrations. The study focuses on five clinically relevant amino acids with different binding properties (valine, isoleucine, leucine, tyrosine, and phenylalanine). We analyzed their binding affinity to human serum albumin (HSA) in serum mimic samples and promoted the release of their bound fraction by TSP titration. Furthermore, we used a novel combination of pseudo-2D CPMG and multivariate curve resolution analysis, allowing the separation of LMWM and protein signals and providing LMWM quantifications corrected for transverse relaxation effects. We found that TSP concentrations larger than 3 mM released most of the bound fraction and validated these findings in real serum/plasma samples.