In clinical and pharmaceutical proteomics, serum and plasma are frequently used for detection of early diagnostic biomarkers for therapeutic targets. Although obtaining these body fluid samples is non-invasive and easy, they contain some abundant proteins that mask other protein components present at low concentrations. The challenge in identifying serum biomarkers is to remove the abundant proteins, uncovering and enriching at the same time the low-abundance ones. The depletion strategies, however, could lead to the concomitant removal of some non-targeted proteins that may be of potential interest. In this study, we compared three different methods aimed to deplete high-abundance proteins from human serum, focusing on the identification of non-specifically bound proteins which might be eventually removed. A Cibacron blue-dye-based method for albumin removal, an albumin and IgG immunodepletion method and an immunoaffinity column (Multiple Affinity Removal System) that simultaneously removes a total of six high-abundance proteins, were investigated. The bound proteins were eluted, separated by two-dimensional gel electrophoresis and identified by Nano LC-CHIP-MS system. Flow-through fractions and bound fractions were also analysed with the ProteinChip technology SELDI-TOF-MS. Our results showed that the methods tested removed not only the targeted proteins with high efficiency, but also some non-targeted proteins. We found that the Multiple Affinity Removal Column improved the intensity of low-abundance proteins, displayed new protein spots and increased resolution. Notably, the column showed the lowest removal of untargeted proteins, proved to be the most promising depletion approach and a reliable method for serum preparation prior to proteomic studies.
OBJECTIVE -Reactive oxygen and nitric oxide (NO) have recently been considered to be involved in the cardiovascular complications of patients with type 2 diabetes, as NO is thought to lose its beneficial physiological effects in the presence of oxygen radicals. For this reason, we tested the effects of L-arginine (ARG) and N-acetylcysteine (NAC) administration in increasing NO bioavailability by reducing free radical formation. RESEARCH DESIGN AND METHODS-A double-blind study was performed on 24 male patients with type 2 diabetes and hypertension divided into two groups of 12 patients that randomly received either an oral supplementation of placebo or NAC ϩ ARG for 6 months.RESULTS -The NAC ϩ ARG treatment caused a reduction of both systolic (P Ͻ 0.05) and diastolic (P Ͻ 0.05) mean arterial blood pressure, total cholesterol (P Ͻ 0.01), LDL cholesterol (P Ͻ 0.005), oxidized LDL (P Ͻ 0.05), high-sensitive C-reactive protein (P Ͻ 0.05), intracellular adhesion molecule (P Ͻ 0.05), vascular cell adhesion molecule (P Ͻ 0.01), nitrotyrosine (P Ͻ 0.01), fibrinogen (P Ͻ 0.01), and plasminogen activator inhibitor-1 (P Ͻ 0.05), and an improvement of the intima-media thickness during endothelial postischemic vasodilation (P Ͻ 0.02). HDL cholesterol increased (P Ͻ 0.05). No changes in other parameters studied were observed.CONCLUSIONS -NAC ϩ ARG administration seems to be a potential well-tolerated antiatherogenic therapy because it improves endothelial function in hypertensive patients with type 2 diabetes by improving NO bioavailability via reduction of oxidative stress and increase of NO production. Our study's results give prominence to its potential use in primary and secondary cardiovascular prevention in these patients. Diabetes Care 31:940-944, 2008
Pseudoxanthoma elasticum (PXE) is a genetic disease characterized by calcification and fragmentation of elastic fibres of the skin, cardiovascular system and eye, caused by mutations of the ABCC6 gene, which encodes the membrane transporter MRP6. The pathogenesis of the lesions is unknown. Based on studies of similar clinical and histopathological damage present in haemolytic disorders, our working hypothesis is that PXE lesions may result from chronic oxidative stress occurring in PXE cells as a consequence of MRP6 deficiency. Our results show that PXE fibroblasts suffer from mild chronic oxidative stress due to the imbalance between production and degradation of oxidant species. The findings also show that this imbalance results, at least in part, from the loss of mitochondrial membrane potential (DeltaPsi(m)) with overproduction of H2O2. Whether mitochondrial dysfunction is the main factor responsible for the oxidative stress in PXE cells remains to be elucidated. However, mild chronic generalized oxidative stress could explain the great majority of structural and biochemical alterations already reported in PXE.
An increased oxidative stress is now considered one of the major risk factors in chronic renal failure (CRF) patients that may be exacerbated by dialysis. It has been postulated that this increased oxidative stress might cause an augmented red blood cell (RBC) membrane lipid peroxidation with the consequent alteration in membrane deformability. The aim of this study was to evaluate RBC susceptibility to an in vitro induced oxidative stress and RBC antioxidant potential in different groups of CRF patients undergoing different substitutive treatment modalities. Fifteen end-stage CRF patients were evaluated in conservative treatment, 23 hemodialysis (HD) patients, 15 continuous ambulatory peritoneal dialysis (CAPD) patients, 15 kidney transplanted patients, and 16 controls. Their RBCs were incubated with the oxidative stress-inducing agent tert-butylhydroperoxide both in the presence and in the absence of the catalase inhibitor sodium azide, and the level of malondialdehyde (MDA) (a product of lipid peroxidation), was measured at 0, 5, 10, 15, and 30 min of incubation. In addition, the RBC content of reduced glutathione (GSH) was measured by HPLC. As opposed to the controls, RBCs from end-stage CRF patients exhibited an increased sensitivity to oxidative stress induced in vitro, both in the absence and presence of a catalase inhibitor, as demonstrated by a significantly higher level of MDA production at all the incubation times (P < 0.05). Different substitutive treatments had different impacts on this phenomenon; CAPD and kidney transplantation were able to normalize this alteration while HD was not. GSH appeared to be related to the increase in RBC susceptibility to oxidative stress; its content being significantly elevated in end-stage CRF and HD patients as compared with CAPD and transplanted patients and controls (P < 0.05). No significant changes were observed in the RBC glutathione content during the HD session. The increase of GSH in RBCs of end-stage CRF and HD patients seems to indicate the existence of an adaptive mechanism under increased oxidative stress occurring in vivo. Unlike HD, the beneficial effect of CAPD on the anemia of dialysis patients might partly be due to a condition of lower oxidative stress that might in addition counterbalance the cardiovascular negative effects of dislipidemia of CAPD patients.
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