Both red and blond orange juice intake decreases the procoagulant activity of whole blood in healthy volunteers.

AIM: Numerous epidemiological studies suggest that exposure to flavonoid-rich fruits has beneficial influence on risk factors for cardiovascular disease. We investigated whether intake of orange juice (OJ) could affect whole blood (WB) procoagulant activity. METHODS: 17 healthy subjects (aged 31 ± 1.5 SEM 10 males) were randomized to receive, according to a cross-over design, either red or blond OJ, enriched or free of anthocyanins, respectively. After one week run-in period on a controlled diet, the subjects were randomly allocated to receive either type of OJ for 4 weeks, with a 4-week wash-out period. Venous blood was collected on citrate before and at the end of each treatment period. WB was incubated with or without an inflammatory stimulus (tumor necrosis factor-α or bacterial endotoxin LPS). Procoagulant activity was evaluated by a one-stage clotting assay. Tissue factor (TF) and TF pathway inhibitor (TFPI) were measured in plasma by ELISA. RESULTS: Intake of either type of OJ caused a prolongation of unstimulated and stimulated WB clotting times, without any difference between the two treatments. Intake of OJ did not modify TF levels. On the contrary, an increase in circulating TFPI antigen was detected following either treatment. CONCLUSIONS: Orange juice intake by healthy volunteers decreases procoagulant activity, possibly through mechanisms independent of its anthocyanin content.

Proteomics: bases for protein complexity understanding.

In the post genomic era we became aware that the genomic sequence and protein functions cannot be correlated. One gene can encode multiple protein functions mainly because of mRNA splice variants, post translational modifications (PTM) and moonlighting functions. To study the whole population of proteins present in a cell to a specific time point and under defined conditions it is necessary to investigate the proteome. Comprehensive analysis of the proteome requires the use of emerging high technologies because of the complexity and wide dynamic range of protein concentrations. Proteomics provides the tools to study protein identification and quantitation, protein-protein interactions, protein modifications and localization. The most widespread strategy for studying global protein expression employs two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) allowing thousands of proteins to be resolved and their expression quantified. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has emerged as a high throughput technique for protein identification and characterization because of its high sensitivity, precision and accuracy. LC-MS/MS is well suited for accurate quantitation of protein expression levels, post-translational modifications and comparative and absolute quantitative analysis of peptides. Bioinformatic tools are required to elaborate the growing number of proteomic data. Here, we give an overview of the current status of the wide range of technologies that define and characterize the modern proteomics.

Postprandial cell inflammatory response to a standardised fatty meal in subjects at different degree of cardiovascular risk.

A fatty meal may represent a challenge of in vivo acute inflammatory reaction. We evaluated the acute effects of a standardised fatty meal administration on leukocytes and platelets and on their interactions on 61 subjects at different degree of cardiovascular risk, without any clinical event. Before and 2 hours after a fatty meal, blood cells were counted and markers of leukocyte (intracellular myeloperoxidase [MPO] and Mac-1) and platelet (P-selectin and microparticles) activation and mixed platelet-leukocyte conjugates measured by flow-cytometry. After the fatty meal, both white blood cell and platelet count significantly increased, more markedly in subjects with lower cardiovascular risk score. Mac-1 expression too increased (from 32.2 ± 27.2% to 45.6 ± 29.0%, p=0.0016), while MPO decreased (from 83.1 ± 16.3% to 64.5 ± 23.1%, p<0.0001). A trend for increased platelet activation and interaction with leukocytes was also observed. Women were more markedly susceptible to fatty meal challenge, as compared to men, while age did not seem to affect any cell response to fatty meal. Waist-to-hip ratio and body mass index influenced polymorphonuclear cells (PMN) degranulation and platelet count increase, respectively. Cellular responses to the fatty meal, in particular PMN degranulation, were attenuated in subjects at higher degree of cardiovascular risk, who showed a basal mild inflammatory activation status. In conclusion, a fatty meal consumption may represent a model of acute inflammatory response and appears to be modulated by different demographic and cardiovascular risk degree. This model could be applied to study the effect of food-derived antioxidants or nutritional supplements, but its relevance remains to be demonstrated.

Four-week ingestion of blood orange juice results in measurable anthocyanin urinary levels but does not affect cellular markers related to cardiovascular risk: a randomized cross-over study in healthy volunteers.

PURPOSE: Blood orange juice (OJ) is an important source of anthocyanins (ACN). The latter molecules are endowed with antioxidant activity and might thus modulate different cell function. Our aim was to investigate ACN absorption following a 1-month daily supplementation of blood OJ and their potential effects on cell markers of platelet and leukocyte activation and interaction. METHODS: Eighteen healthy subjects (10 men and 8 women) were supplemented for 4 weeks with 1 L/day of either blood OJ or blond OJ (that contains no ACN), following a cross-over design. Blood samples were obtained from fasting participants both at baseline and after each week of treatment to measure plasma ACN concentration. At the same time-intervals, 24-h urinary excretion of these molecules was also measured. At the beginning and the end of each 4-week intervention period, platelet and leukocyte markers and mixed cell conjugates were assessed both in basal condition and upon in vitro collagen/ADP activation. RESULTS: After 1 week supplementation with blood OJ, 24-h urinary excretion of ACN reached average levels of 11.47 ± 5.63 nmol that significantly differed from baseline and remained substantially unchanged until the end of treatment. No plasma accumulation of ACN following blood OJ supplementation was observed. Cellular markers were not significantly affected by either OJ after 4-week supplementation. CONCLUSIONS: Following supplementation of healthy volunteers with 1 L/day of blood OJ for 4 weeks, the ACN plasma levels reached were insufficient to significantly modify cell markers of platelet and leukocyte activation and interaction.

Platelet proteome in healthy volunteers who smoke.

Smoking accelerates atherosclerosis and is a well-known risk factor for acute cardiovascular complications; however, the mechanisms of these effects have not been completely clarified. Recently developed proteomic approaches may offer new clues when combined with well-established functional tests. Platelet proteome of healthy smokers and non-smokers was resolved by two-dimensional difference gel electrophoresis, compared by Decyder software and identified by mass spectrometry analysis (nano-LC-MS/MS). In smokers, three proteins (Factor XIII-A subunit, platelet glycoprotein IIb and beta-actin) were significantly up-regulated, whereas WDR1 protein and chaperonine HSP60 were down-regulated. Furthermore, the highest scored network derived by Ingenuity Pathway Analysis using the modulated proteins as input showed the involvement of several proteins to be related to inflammation and apoptosis. Platelet function tests and the levels of markers of platelet and leukocyte activation were not different in smokers vs. non-smoker subjects. The platelet proteomic approach confirms that cigarette smoking triggers several inflammatory reactions and may help clarify some of the molecular mechanisms of smoke effect on cellular systems relevant for vascular integrity and human health.

Influence of harvest method and period on olive oil composition: an NMR and statistical study.

The influence of harvest period and harvest method on olive oil composition was investigated by nuclear magnetic resonance (NMR) spectroscopy and by some quality parameters such as free acidity, peroxide value, and UV spectrophotometric indices. This work focuses on two secondary factors (harvest period and harvest method) and investigated their interactions with primary (genetic and pedoclimatic) and secondary (agronomic practices and technological procedures) factors. To avoid misinterpretation, the general linear model analysis (GLM) was used to adjust the result obtained from the analysis of variance (ANOVA). In this way, the effect of the factor of interest was corrected for the effects of the other factors that might influence the variable under investigation. The weight of each factor was evaluated by the variance component analysis (VCA). Finally, multivariate statistical analyses, namely, principal component analysis (PCA) and linear discriminant analysis (LDA), were applied. Samples were grouped according to the harvest period and harvest method. Volatile compounds, that is, hexanal and trans-2-hexenal, as well as the sn-1,3-diglycerides and squalene, significantly decreased during the ripening. The relative value of the ΔK parameter and the hexanal amount were higher in the olive oils obtained from olives harvested by one type of hand-held machine (shaker), whereas the unsaturated fatty chains in the olive oils were higher when another type (comb) was used.

Standardized sample preparation phases for a quantitative measurement of plasma peptidome profiling by MALDI-TOF.

A growing body of literature defines MALDI-TOF MS as a technique for studying plasma and serum, thus enabling the detection of proteins, and the generation of reproducible protein profile mass spectra, potentially able to discriminate correctly different biological systems. In this work, the different steps of the pre-analytical phase that may affect the reproducibility of plasma proteome analysis have been carefully considered. The results showed that the method is highly accurate (9.1%) and precise (8.9%) and the calibration curve for the ACTH (18-39), in human plasma, gave a good correlation coefficient (r>0.99 and r(2)>0.98). The limit of detection (LOD) and the limit of quantification (LOQ), relative intensity, were of 0.5 x 10(-)(9)M and 1.0 x 10(-)(9)M respectively. Thus, an assay has been developed for the detection of low-abundant and low molecular weight proteins, from human plasma, aiming at the identification of new potential biomarkers. The method was tested on plasma from patients with a first diagnosis of pelvic mass. Statistical analysis of plasma profile generated a sub-profile of 17 peptides with their relative abundance able to discriminate patients bearing malignant or benign tumors. The sensitivity and specificity were 85.7% and 80.0% respectively.

1H NMR-based protocol for the detection of adulterations of refined olive oil with refined hazelnut oil.

A (1)H NMR analytical protocol for the detection of refined hazelnut oils in admixtures with refined olive oils is reported according to ISO format. The main purpose of this research activity is to suggest a novel analytical methodology easily usable by operators with a basic knowledge of NMR spectroscopy. The protocol, developed on 92 oil samples of different origins within the European MEDEO project, is based on (1)H NMR measurements combined with a suitable statistical analysis. It was developed using a 600 MHz instrument and was tested by two independent laboratories on 600 MHz spectrometers, allowing detection down to 10% adulteration of olive oils with refined hazelnut oils. Finally, the potential and limitations of the protocol applied on spectrometers operating at different magnetic fields, that is, at the proton frequencies of 500 and 400 MHz, were investigated.