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The Human Plasma Proteome has always been the most investigated compartment in proteomics-based biomarker discovery, and is considered the largest and deepest version of the human proteome, reflecting the state of the body in health and disease. Even if efforts have been always dedicated to the refinement of proteomic approaches to investigate more deeply the plasma proteome, it should not be forgotten that also highly abundant plasma proteins, like human serum albumin (HSA), often neglected in these studies, might provide fundamental physiological functions in plasma, and should be better considered. This review summarizes the important roles of HSA in the context of cardiovascular diseases (CVD), and in particular in heart failure. Notwithstanding much attention has been historically directed toward the association of HSA levels and CVD risk, the advances in the field of mass spectrometry research allow also a better characterization of the effects of oxidative modifications that could alter not only the structure but also the function of HSA.

Human serum albumin (HSA) represents the most abundant plasma protein, with relevant antioxidant activity due to the presence of the sulfhydryl group on cysteine at position 34 (Cys34), the latter being one of the major target sites for redox-dependent modifications leading to the formation of mixed disulfide linkages with low molecular weight thiols. Thiolated forms of HSA (Thio-HSA) may be useful as markers of an unbalanced redox state and as a potential therapeutic target. Indeed, we have previously reported that albumin Cys34 can be regenerated in vitro by N-Acetylcysteine (NAC) through a thiol-disulfide breaking mechanism, with a full recovery of the HSA antioxidant and antiplatelet activities. With this case study, we aimed to assess the ability of NAC to regenerate native mercaptoalbumin (HSA-SH) and the plasma antioxidant capacity in subjects with redox unbalance, after oral and intravenous administration. A placebo-controlled crossover study, single-blinded, was performed on six hypertensive subjects, randomized into two groups, on a one-to-one basis with NAC (600 mg/die) or a placebo, orally and intravenously administered. Albumin isoforms, HSA-SH, Thio-HSA, and glutathione levels were evaluated by means of mass spectrometry. The plasma antioxidant activity was assessed by a fluorimetric assay. NAC, orally administered, significantly decreased the Thio-HSA levels in comparison with the pre-treatment conditions (T0), reaching the maximal effect after 60 min (−24.7 ± 8%). The Thio-HSA reduction was accompanied by a concomitant increase in the native HSA-SH levels (+6.4 ± 2%). After intravenous administration of NAC, a significant decrease of the Thio-HSA with respect to the pre-treatment conditions (T0) was observed, with a maximal effect after 30 min (−68.9 ± 10.6%) and remaining significant even after 6 h. Conversely, no effect on the albumin isoforms was detected with either the orally or the intravenously administered placebo treatments. Furthermore, the

Advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs), particularly carboxymethyl-lysine (CML), have been largely proposed as factors involved in the establishment and progression of heart failure (HF). Despite this evidence, the current literature lacks the comprehensive identification and characterization of the plasma AGEs/ALEs involved in HF (untargeted approach). This work provides the first ex vivo high-resolution mass spectrometry (HR-MS) profiling of AGEs/ALEs occurring in human serum albumin (HSA), the most abundant protein in plasma, characterized by several nucleophilic sites and thus representing the main protein substrate for AGE/ALE formation. A set of AGE/ALE adducts in pooled HF-HSA samples was defined, and a semi-quantitative analysis was carried out in order to finally select those presenting in increased amounts in the HF samples with respect to the control condition. These adducts were statistically confirmed by monitoring their content in individual HF samples by applying a targeted approach. Selected AGEs/ALEs proved to be mostly CML derivatives on Lys residues (i.e., CML-Lys12, CML-Lys378, CML-Lys402), and one deoxy-fructosyl derivative on the Lys 389 (DFK-Lys 389). The nature of CML adducts was finally confirmed using immunological methods and in vitro production of such adducts further confirmed by mass spectrometry.

Significance: Atherosclerotic cardiovascular diseases (ACVDs) continue to be a primary cause of mortality worldwide in adults aged 35-70 years, occurring more often in countries with lower economic development, and they constitute an ever-growing global burden that has a considerable socioeconomic impact on society. The ACVDs encompass diverse pathologies such as coronary artery disease and heart failure (HF), among others. Recent Advances: It is known that oxidative stress plays a relevant role in ACVDs and some of its effects are mediated by lipid oxidation. In particular, lipid peroxidation (LPO) is a process under which oxidants such as reactive oxygen species attack unsaturated lipids, generating a wide array of oxidation products. These molecules can interact with circulating lipoproteins, to diffuse inside the cell and even to cross biological membranes, modifying target nucleophilic sites within biomolecules such as DNA, lipids, and proteins, and resulting in a plethora of biological effects. Critical Issues: This review summarizes the evidence of the effect of LPO in the development and progression of atherosclerosis-based diseases, HF, and other cardiovascular diseases, highlighting the role of protein adduct formation. Moreover, potential therapeutic strategies targeted at lipoxidation in ACVDs are also discussed. Future Directions: The identification of valid biomarkers for the detection of lipoxidation products and adducts may provide insights into the improvement of the cardiovascular risk stratification of patients and the development of therapeutic strategies against the oxidative effects that can then be applied within a clinical setting.

Human serum albumin (HSA) is associated with several physiological functions, such as maintaining oncotic pressure and microvascular integrity, among others. It also represents the major and predominant antioxidant in plasma due to the presence of the Cys34 sulfhydryl group. In this study, we assessed qualitative and quantitative changes in HSA in patients with heart failure (HF) and their relationship with the severity of the disease. We detected by means of mass spectrometry a global decrease of the HSA content in the plasma of HF patients in respect to control subjects, a significant increase of thio-HSA with a concomitant decrease in the reduced form of albumin. Cysteine and, at a lesser extent, homocysteine represent the most abundant thiol bound to HSA. A strong inverse correlation was also observed between cysteine-HSA and peak VO2/kg, an index of oxygen consumption associated with HF severity. Moreover, in HL-1 cardiomyocytes incubated with H2O2, we showed a significant decrease of cell viability in cells treated with thioHSA in respect to restored native-HSA. In conclusion, we found for the first time that S-thiolation of albumin is increased in the plasma of HF patients and induced changes in the structure and antioxidant function of HSA, likely contributing to HF progression.

Physiological cholesterol metabolism implies the generation of a series of oxidized derivatives, whose oxysterols are by far the most investigated ones for their potential multifaceted involvement in human pathophysiology. In this regard, noteworthy is the broad antiviral activity displayed by defined side chain oxysterols, in particular 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol (27HC). Although their antiviral mechanism(s) may vary depending on virus/host interaction, these oxysterols share the common feature to hamper viral replication by interacting with cellular proteins. Here reported is the first analysis of the modulation of a cell proteome by these two oxysterols, that, besides yielding additional clues about their potential involvement in the regulation of sterol metabolism, provides novelinsights about the mechanism underlying the inhibition of virus entry and trafficking within infected cells. We show here that both 25HC and 27HC can down-regulate the junction adhesion molecule-A (JAM-A) and the cation independent isoform of mannose-6-phosphate receptor (MPRci), two crucial molecules for the replication of all those viruses that exploit adhesion molecules and the endosomal pathway to enter and diffuse within target cells

In the last years, the interest in non-alcoholic grape products has considerably increased. Table grapes are largely produced in the Mediterranean area and their consumption has raised worldwide. Beside the positive pattern of nutrients, table grapes could provide benefits on human health. Among the health-promoting substances contained in table grapes, flavonoids (mainly anthocyanins, flavan-3-ols and flavonols) seem to be the most interesting. The aim of this study was the characterization of the phenolic pattern of sixteen grape varieties, and the evaluation of the associated antioxidant activity. The methods were: 1) Folin-Cocalteau's assay for the quantification of total polyphenol content; 2) DPPH (1,1,-diphenil-2—picrylhydrazyl) assay and 3) ORAC (Oxygen Radical Absorbance Capacity) spectrophotometric assays for the assessment of radical scavenging activity; 4) High Performance Liquid Chromatography (HPLC) method, coupled with electrospray ionization mass spectrometer (ESI-MS) and DAD detector was developed in order to obtain the phenolic pattern of grape samples. Data obtained in this study underline that some table grape varieties can show interesting phenolic pattern independently from the presence or not of seeds. This observation suggests that selected varieties of seedless grapes could represent an interesting source of healthy compounds, satisfying consumers' preferences and reducing concerns versus alcoholic beverages.

Carnosine is a natural dipeptide able to react with reactive carbonyl species, which have been recently associated with the onset and progression of several human diseases. Herein, we report an intervention study in overweight individuals. Carnosine (2g/day) was orally administered for twelve weeks in order to evaluate its bioavailability and metabolic fate. Two carnosine adducts were detected in the urine samples of all subjects. Such adducts are generated from a reaction with acrolein, which is one of the most toxic and reactive compounds among reactive carbonyl species. However, neither carnosine nor adducts have been detected in plasma. Urinary excretion of adducts and carnosine showed a positive correlation although a high variability of individual response to carnosine supplementation was observed. Interestingly, treated subjects showed a significant decrease in the percentage of excreted adducts in reduced form, accompanied by a significant increase of the urinary excretion of both carnosine and carnosine-acrolein adducts. Altogether, data suggest that acrolein is entrapped in vivo by carnosine although the response to its supplementation is possibly influenced by individual diversities in terms of carnosine dietary intake, metabolism and basal production of reactive carbonyl species.

Objective Carnosine is a naturally present dipeptide in humans and an over-the counter food additive. Evidence from animal studies supports the role for carnosine in the prevention and treatment of diabetes and cardiovascular disease, yet there is limited human data. This study investigated whether carnosine supplementation in individuals with overweight or obesity improves diabetes and cardiovascular risk factors. Methods In a double-blind randomized pilot trial in nondiabetic individuals with overweight and obesity (age 43 +/- 8 years; body mass index 31 +/- 4 kg/m2), 15 individuals were randomly assigned to 2 g carnosine daily and 15 individuals to placebo for 12 weeks. Insulin sensitivity and secretion, glucose tolerance (oral glucose tolerance test), blood pressure, plasma lipid profile, skeletal muscle (1H-MRS), and urinary carnosine levels were measured. Results Carnosine concentrations increased in urine after supplementation (P < 0.05). An increase in fasting insulin and insulin resistance was hampered in individuals receiving carnosine compared to placebo, and this remained significant after adjustment for age, sex, and change in body weight (P = 0.02, P = 0.04, respectively). Two-hour glucose and insulin were both lower after carnosine supplementation compared to placebo in individuals with impaired glucose tolerance (P < 0.05). Conclusions These pilot intervention data suggest that carnosine supplementation may be an effective strategy for prevention of type 2 diabetes.Copyright © 2016 The Obesity Society.

Obesity, type 2 diabetes (T2DM) and cardiovascular disease (CVD) are the most common preventable causes of morbidity and mortality worldwide. They represent major public health threat to our society. Increasing prevalence of obesity and T2DM contributes to escalating morbidity and mortality from CVD and stroke. Carnosine (beta-alanyl-l-histidine) is a dipeptide with anti-inflammatory, antioxidant, anti-glycation, anti-ischaemic and chelating roles and is available as an over-the-counter food supplement. Animal evidence suggests that carnosine may offer many promising therapeutic benefits for multiple chronic diseases due to these properties. Carnosine, traditionally used in exercise physiology to increase exercise performance, has potential preventative and therapeutic benefits in obesity, insulin resistance, T2DM and diabetic microvascular and macrovascular complications (CVD and stroke) as well as number of neurological and mental health conditions. However, relatively little evidence is available in humans. Thus, future studies should focus on well-designed clinical trials to confirm or refute a potential role of carnosine in the prevention and treatment of chronic diseases in humans, in addition to advancing knowledge from the basic science and animal studies.Copyright © 2016 Springer-Verlag Wien.