A multi-marker integrative analysis reveals benefits and risks of bariatric surgery.

Bariatric surgery (BS) is an effective intervention for severe obesity and associated comorbidities. Although several studies have addressed the clinical and metabolic effects of BS, an integrative analysis of the complex body response to surgery is still lacking. We conducted a longitudinal data study with 36 patients with severe obesity who were tested before, 6 and 12 months after restrictive BS for more than one hundred blood biomarkers, including clinical, oxidative stress and metabolic markers, peptide mediators and red blood cell membrane lipids. By using a synthetic data-driven modeling based on principal component and correlation analyses, we provided evidence that, besides the early, well-known glucose metabolism- and weight loss-associated beneficial effects of BS, a tardive, weight-independent increase of the hepatic cholesterol metabolism occurs that is associated with potentially detrimental inflammatory and metabolic effects. Canonical correlation analysis indicated that oxidative stress is the most predictive feature of the BS-induced changes of both glucose and lipids metabolism. Our results show the power of multi-level correlation analysis to uncover the network of biological pathways affected by BS. This approach highlighted potential health risks of restrictive BS that are disregarded with the current practice to use weight loss as surrogate of BS success.

Young transgenic hMTH1 mice are protected against dietary fat-induced metabolic stress-implications for enhanced longevity.

hMTH1 protects against mutation during oxidative stress. It degrades 8-oxodGTP to exclude potentially mutagenic oxidized guanine from DNA. hMTH1 expression is linked to ageing. Its downregulation in cultured cells accelerates RAS-induced senescence, and its overexpression in hMTH1-Tg mice extends lifespan. In this study, we analysed the effects of a brief (5 weeks) high-fat diet challenge (HFD) in young (2 months old) and adult (7 months old) wild-type (WT) and hMTH1-Tg mice. We report that at 2 months, hMTH1 overexpression ameliorated HFD-induced weight gain, changes in liver metabolism related to mitochondrial dysfunction and oxidative stress. It prevented DNA damage as quantified by a comet assay. At 7 months old, these HFD-induced effects were less severe and hMTH1-Tg and WT mice responded similarly. hMTH1 overexpression conferred lifelong protection against micronucleus induction, however. Since the canonical activity of hMTH1 is mutation prevention, we conclude that hMTH1 protects young mice against HFD by reducing genome instability during the early period of rapid growth and maximal gene expression. hMTH1 protection is redundant in the largely non-growing, differentiated tissues of adult mice. In hMTH1-Tg mice, expression of a less heavily mutated genome throughout life provides a plausible explanation for their extended longevity.

Advanced Raman Spectroscopy Detection of Oxidative Damage in Nucleic Acid Bases: Probing Chemical Changes and Intermolecular Interactions in Guanosine at Ultralow Concentration.

DNA/RNA synthesis precursors are especially vulnerable to damage induced by reactive oxygen species occurring following oxidative stress. Guanosine triphosphates are the prevalent oxidized nucleotides, which can be misincorporated during replication, leading to mutations and cell death. Here, we present a novel method based on micro-Raman spectroscopy, combined with ab initio calculations, for the identification, detection, and quantification of oxidized nucleotides at low concentration. We also show that the Raman signature in the terahertz spectral range (<100 cm-1) contains information on the intermolecular assembly of guanine in tetrads, which allows us to further boost the oxidative damage detection limit. Eventually, we provide evidence that similar analyses can be carried out on samples in very small volumes at very low concentrations by exploiting the high sensitivity of surface-enhanced Raman scattering combined with properly designed superhydrophobic substrates. These results pave the way for employing such advanced spectroscopic methods for quantitatively sensing the oxidative damage of nucleotides in the cell.

Toxicity of the readily leachable fraction of urban PM2.5 to human lung epithelial cells: Role of soluble metals.

Fine airborne particulate matter (PM2.5) has been repeatedly associated with adverse health effects in humans. The PM2.5 soluble fraction, and soluble metals in particular, are thought to cause lung damage. Literature data, however, are not consistent and the role of leachable metals is still under debate. In this study, Winter and Summer urban PM2.5 aqueous extracts, obtained by using a bio-compatible solution and different contact times at 37 °C, were used to investigate cytotoxic effects of PM2.5 in cultured lung epithelial cells (A549) and the role played by the leachable metals Cu, Fe, Zn, Ni, Pb and Cd. Cell viability and migration, as well as intracellular glutathione, extracellular cysteine, cysteinylglycine and homocysteine concentrations, were evaluated in cells challenged with both PM2.5 extracts before and after ultrafiltration and artificial metal ion solutions mimicking the metal composition of the genuine extracts. The thiol oxidative potential was also evaluated by an abiotic test. Results demonstrate that PM2.5 bioactive components were released within minutes of PM2.5 interaction with the leaching solution. Among these are i) low MW (<3 kDa) solutes inducing oxidative stress and ii) high MW and/or water-insoluble compounds largely contributing to thiol oxidation and to increased homocysteine levels in the cell medium. Cu and/or Ni ions likely contributed to the effects of Summer PM2.5 extracts. Nonetheless, the strong bio-reactivity of Winter PM2.5 extracts could not be explained by the presence of the studied metals. A possible role for PM2.5 water-extractable organic components is discussed.

Vitamins (A, C and E) and oxidative status of hemodialysis patients treated with HFR and HFR-Supra.

BACKGROUND: Hemodiafiltration with on-line endogenous reinfusion (HFR) is an extracorporeal dialytic method that combines diffusion, convection and adsorption. HFR-Supra (HFR-S) is a second-generation system with increased convective permeability and adsorption capability. Previous studies suggested that HFR reduces oxidative stress compared to standard haemodialysis. The principal aim of the present study was to compare antioxidant vitamins behavior and oxidative status of hemodialysis patients treated with HFR and HFR-S. METHODS: The study was designed as a multicenter, randomized, crossover trial. Forty-one patients were recruited from 19 dialysis centers and after a 4-month washout stabilization period in on-line hemodiafiltration (ol-HDF), each patient was randomized to a sequence of treatments (HFR-S followed by HFR or viceversa) with each treatment applied over 6 months. Plasma levels of Advanced Oxidation Protein Products, Total Antioxidant Status, vitamins C, A and E and their ligands (Retinol Binding Protein and total lipids) were measured at baseline and at the end of each treatment period. RESULTS: Results show that the higher convective permeability of HFR-S with respect to HFR did not produce additional beneficial effects on the patients' oxidative status, a slight decrease of both Vitamin A and Retinol Binding Protein being the only difference registered in the long-term. However, as compared to ol-HDF, both the re-infusive techniques allowed to reduce the intradialytic loss of Vitamin C and, in the long-term, improve the patients' oxidative status and increase Retinol Binding Protein plasma values. No significant differences were found between the Vitamin C concentration of pre- and post cartridge UF neither in HFR-S nor in HFR showing that the sorbent resin does not adsorb Vitamin C. CONCLUSION: HFR-S and HFR are almost equivalent in term of impact on antioxidant vitamins and oxidative status of hemodialysis patients. Nonetheless, as compared to ol-HDF, both treatments produced a sensible sparing of Vitamin C and may represent a new approach for reducing oxidative stress and related complications in dialysis patients. Long-term effects of re-infusive treatments on patients' cardiovascular morbidity and mortality need to be evaluated. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01492491 , retrospectively registered in 10 December 2011.

Ultraweak Photon Emission from the Seed Coat in Response to Temperature and Humidity-A Potential Mechanism for Environmental Signal Transduction in the Soil Seed Bank.

Seeds beneath the soil sense the changing environment to time germination and seedling emergence with the optimum time of year for survival. Environmental signals first impact with the seed at the seed coat. To investigate whether seed coats have a role in environmental sensing we investigated their ultraweak photon emission (UPE) under the variable temperature, relative humidity and oxygen conditions they could experience in the soil seed bank. Using a custom-built luminometer we measured UPE intensity and spectra (300-700 nm) from Phaseolus vulgaris seeds, seed coats and cotyledons. UPE was greatest from the internal surface of the seed coat. Seed coat UPE increased concomitantly with both increasing temperature and decreasing relative humidity. Emission was oxygen dependent and it was abolished by treatment with dinitrophenylhydrazine, demonstrating the key role of seed coat carbonyls in the phenomenon. We hypothesize that beneath the soil surface the attenuation of light (virtual darkness: low background noise) enables seeds to exploit UPE for transducing key environmental variables in the soil (temperature, humidity and oxygen) to inform them of seasonal and local temperature patterns. Overall, seed coats were found to have potential as effective transducers of key fluctuating environmental variables in the soil.

Setup and validation of a convenient sampling procedure to promptly and effectively stabilize vitamin C in blood and plasma specimens stored at routine temperatures.

Vitamin C (ascorbic acid, AA) is very labile in nature and decays quickly after blood withdrawal. To ensure AA stability, the current procedure prescribes immediate plasma acidification followed by sample storage at ultra-low temperature. The aim of this study was to set up a pre-analytical procedure to promptly stabilize AA at routine temperatures while minimizing both specimen manipulation and instrumental requirement. Blood from healthy subjects was collected in lithium-heparin gel separator tubes containing or not different reducing agents (dithioerythritol, tris(2-carboxyethyl)phosphine, n-acetylcysteine and sodium thiosulfate). Plasma AA stability during blood and plasma storage at routine temperatures was evaluated. Plasma AA concentration was assayed by RP-HPLC-UV under ion suppression conditions. Each of the reductants tested was able to slow down the ex vivo degradation of plasma AA; dithioerythritol was the most effective. Five to 10 mmol/L dithioerythritol did not interfere with blood separation and allowed plasma AA to be stabilized up to 6 h, 24 h and 60 days at room temperature, +4 °C and -25 °C, respectively. The method worked well even in case of delayed blood separation and/or incomplete vacutainer filling. The procedure is feasible and reliable. Of special usefulness in clinical and epidemiological studies, prompt plasma manipulation after blood withdrawal or special storage equipments are not required. Graphical Abstract Collecting blood in tubes containing a reducing agent is a feasible method to promptly and effectively stabilize plasma vitamin C at routine temperature.

Estimated glomerular filtration rate, all-cause mortality and cardiovascular diseases incidence in a low risk population: the MATISS study.

BACKGROUND: Chronic kidney disease (CKD) independently increases the risk of death and cardiovascular disease (CVD) in the general population. However, the relationship between estimated glomerular filtration rate (eGFR) and CVD/death risk in a general population at low risk of CVD has not been explored so far. DESIGN: Baseline and longitudinal data of 1465 men and 1459 women aged 35-74 years participating to the MATISS study, an Italian general population cohort, were used to evaluate the role of eGFR in the prediction of all-cause mortality and incident CVD. METHODS: Bio-bank stored sera were used to evaluate eGFR at baseline. Serum creatinine was measured on thawed samples by means of an IDMS-calibrated enzymatic method. eGFR was calculated by the CKD-EPI formula. RESULTS: At baseline, less than 2% of enrolled persons had eGFR < 60 mL/min/1.73 m(2) and more than 70% had a 10-year cardiovascular risk score < 10%. In people 60 or more years old, the first and the last eGFR quintiles (<90 and ≥109 mL/min/1.73 m(2), respectively) were associated to an increased risk for both all-cause mortality (hazard ratio 1.6, 95% confidence interval 1.2-2.1 and 4.3, 1.6-11.7, respectively) and incident CVD (1.6, 1.0-2.4 and 7.0, 2.2-22.9, respectively), even if adjusted for classical risk factors. CONCLUSIONS: These findings strongly suggest that in an elderly, general population at low risk of CVD and low prevalence of reduced renal filtration, even a modest eGFR reduction is related to all-cause mortality and CVD incidence, underlying the potential benefit to this population of considering eGFR for their risk prediction.

Coenzyme Q metabolism is disturbed in high fat diet-induced non-alcoholic fatty liver disease in rats.

Oxidative stress is believed to be a major contributory factor in the development of non alcoholic fatty liver disease (NAFLD), the most common liver disorder worldwide. In this study, the effects of high fat diet-induced NAFLD on Coenzyme Q (CoQ) metabolism and plasma oxidative stress markers in rats were investigated. Rats were fed a standard low fat diet (control) or a high fat diet (57% metabolizable energy as fat) for 18 weeks. The concentrations of total (reduced + oxidized) CoQ9 were increased by >2 fold in the plasma of animals fed the high fat diet, while those of total CoQ10 were unchanged. Reduced CoQ levels were raised, but oxidized CoQ levels were not, thus the proportion in the reduced form was increased by about 75%. A higher percentage of plasma CoQ9 as compared to CoQ10 was in the reduced form in both control and high fat fed rats. Plasma protein thiol (SH) levels were decreased in the high fat-fed rats as compared to the control group, but concentrations of lipid hydroperoxides and low density lipoprotein (LDL) conjugated dienes were unchanged. These results indicate that high fat diet-induced NAFLD in rats is associated with altered CoQ metabolism and increased protein, but not lipid, oxidative stress.

High fat diet-induced non alcoholic fatty liver disease in rats is associated with hyperhomocysteinemia caused by down regulation of the transsulphuration pathway.

BACKGROUND: Hyperhomocysteinemia (HHcy) causes increased oxidative stress and is an independent risk factor for cardiovascular disease. Oxidative stress is now believed to be a major contributory factor in the development of non alcoholic fatty liver disease, the most common liver disorder worldwide. In this study, the changes which occur in homocysteine (Hcy) metabolism in high fat-diet induced non alcoholic fatty liver disease (NAFLD) in rats were investigated. METHODS AND RESULTS: After feeding rats a standard low fat diet (control) or a high fat diet (57% metabolisable energy as fat) for 18 weeks, the concentration of homocysteine in the plasma was significantly raised while that of cysteine was lowered in the high fat as compared to the control diet fed animals. The hepatic activities of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGS), the enzymes responsible for the breakdown of homocysteine to cysteine via the transsulphuration pathway in the liver, were also significantly reduced in the high fat-fed group. CONCLUSIONS: These results indicate that high fat diet-induced NAFLD in rats is associated with increased plasma Hcy levels caused by down-regulation of hepatic CBS and CGL activity. Thus, HHcy occurs at an early stage in high fat diet-induced NAFLD and is likely to contribute to the increased risk of cardiovascular disease associated with the condition.

Microenvironmental pH is a key factor for exosome traffic in tumor cells.

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.

Di(2-ethylhexyl)phthalate stimulates Ca(2+) entry, chemotaxis and ROS production in human granulocytes.

Di(2-ethylhexyl)phthalate (DEHP), a plasticizer widely used in polyvinyl chloride-based medical devices, is known to interfere with immune functions but the underlying molecular mechanisms remain elusive. In this in vitro study, we investigated DEHP effect on intracellular calcium concentration ([Ca(2+)](i)), chemotaxis and reactive oxygen species (ROS) production in human granulocytes. DEHP increased [Ca(2+)](i) by inducing a Ca(2+) influx from the extracellular medium. The effect was slow and inhibitable both by the membrane cation channel blockers SKF96365, econazole and 2-aminoethoxy diphenylborate and by the protein kinase C activator phorbol myristate acetate. The plasticizer stimulated both cell chemotaxis and ROS production; however, only the latter effect was dependent on DEHP-induced Ca(2+) influx. Collectively, our results indicate that DEHP interaction with human granulocytes leads to multiple and independent effects, each potentially contributing to inappropriate cell activation.

Homocysteinemia correlates with plasma thiol redox status in patients with end-stage renal disease.

BACKGROUND/AIMS: In end-stage renal disease (ESRD), hyperhomocysteinemia is a common finding associated with increased cardiovascular risk. However, the pathogenic role of homocysteine is still unclear. In vitro studies show that thiol redox status affects endothelial cell functions. We therefore investigated the possible association between homocysteinemia and plasma thiol redox status in ESRD patients. METHODS: Total plasma homocysteine (Hcy), cysteine (Cys) and free thiols (SH) were measured both before and after a dialytic session in 54 ESRD patients receiving (n = 15) or not receiving (n = 39) folate supplementation, and 17 control subjects. RESULTS: High predialysis levels of both Hcy and Cys were found to be negatively correlated with low SH levels both in supplemented (r = -0.680, p < 0.01 and r = -0.624, p < 0.02, respectively) and unsupplemented (r = -0.698, p < 0.001 and r = -0.445, p < 0.01, respectively) patients. Following dialysis, SH values returned to normal and the above correlations were no longer appreciable. CONCLUSION: A strong, folate therapy-insensitive association between homocysteinemia and plasma free thiol levels was found in ESRD patients. These results support a role for oxidative stress in ESRD-related hyperhomocysteinemia and suggest the plasma thiol redox status alteration as a possible pathogenic mechanism underlying the cardiovascular toxicity of hyperhomocysteinemia in these patients.

Reliability of oxidative stress biomarkers in hemodialysis patients: a comparative study.

BACKGROUND: Oxidative stress (OS) is considered to play a major role in the development of end-stage renal disease (ESRD) complications. However, conflicting and inconsistent data have been reported on OS in ESRD patients. Our aim was to investigate the reliability of the most popular non-enzymatic plasma OS biomarkers in ESRD. METHODS: Vitamins A (VitA), E and C (VitC), uric acid, plasma antioxidant and ferric-reducing potential (PAP and PRP), thiols (SH), malondialdehyde (MDA) and lipid hydroperoxides (HPO) were determined before and after dialysis in plasma from 33 ESRD patients on hemodialysis, hemodiafiltration or peritoneal dialysis and 20 control subjects. RESULTS: In ESRD patients, high PRP and normal PAP values were positively correlated with VitC levels. After dialysis, PRP levels decreased, while unchanged PAP levels correlated positively with high VitA and transiently recovered SH values. All patients showed high levels of both MDA and cholesterol-normalized HPO. However, while the former significantly decreased after dialysis, the latter were unaffected by treatment. Paradoxical correlations of MDA with both VitA and HPO were found. CONCLUSIONS: Plasma PRP and MDA levels may be dramatically affected by both uremia and dialysis; their use in ESRD patients may therefore lead to OS misevaluation and should be avoided. More reliable results can be obtained using physiologically relevant OS functional tests, such as PAP, and early biomarkers of OS damage, such as SH and HPO.

The partial head decondensation test is a new, quick method to assess acrosome status in human spermatozoa.

OBJECTIVE: To develop a fast method for assessing acrosome status in human spermatozoa. DESIGN: Development of a new in vitro test to assess acrosome reaction in human spermatozoa. SETTING: Academic medical institution. PATIENT(S): Normozoospermic subjects. INTERVENTION(S): Spermatozoa were isolated from fresh semen samples, capacitated, and stimulated or not with P or ionomycin. Acrosome reactions were evaluated by phase-contrast microscopy after a brief sperm incubation in a decondensing solution. The results were compared with those obtained by scanning electron microscopy and fluoresceinated lectin staining. MAIN OUTCOME MEASURE(S): Percentage of intact acrosomes. RESULT(S): The new procedure allowed intact acrosomes to be easily identified and quantified by phase-contrast microscopy. In unstimulated and ionomycin-treated spermatozoa, a very good agreement was found among the new test, scanning electron microscopy, and fluoresceinated lectin staining. In P-treated spermatozoa, the proposed method allowed a significantly higher percentage of reacted acrosomes to be resolved, likely due to its ability to detect the very initial stages of the acrosome reaction. CONCLUSION(S): The new test allows acrosome-intact and acrosome-reacted spermatozoa to be unambiguously singled out and quantified. The method is rapid, reliable, sensitive, and easy to perform, which makes it of profitable use in both basic research and diagnostic practice.