Free Thiol Levels Research Articles

Development of a Novel Label-Free Subunit HILIC-MS Method for Domain-Specific Free Thiol Identification and Quantitation in Therapeutic Monoclonal Antibodies.

Cysteine residues are crucial for the formation of conserved disulfide bonds in therapeutic monoclonal antibodies (mAbs), which are essential for their folding and structural stability. The presence of free thiols in mAbs can indicate incomplete disulfide bond formation, potentially impacting the molecule's conformational stability. Free thiol quantitation has been achieved using labeling-based strategies such as maleimide and haloalkyl derivatives at both intact and peptide levels. However, intact-level measurement only provides total free thiol levels, while peptide-level measurement is time-consuming and more prone to assay-induced artifacts. In this study, we present a novel label-free HILIC-MS method that separates free thiol species at the subunit level, followed by free thiol localization by the MS2 fragmentation pattern. This allows for facile identification and quantitation of intrachain free thiols at domain-specific resolution. Compared to bottom-up approaches, this subunit HILIC-MS method excels in simpler sample preparation and higher throughput and enables chain-specific free thiol analysis for bispecific mAbs. This method can be readily applied for screening mAb candidates with elevated levels of free thiols in early-stage developability assessment and facilitating an effective comparability evaluation of mAb samples during process development.

P454 Systemic free thiols associate with clinical and biochemical disease activity in Inflammatory Bowel Disease: a prospective diagnostic validation study

Abstract Background In the pathophysiology of inflammatory bowel diseases (IBD), oxidative stress plays an important role. Lower levels of plasma free thiols (FT) reflect a higher oxidative stress burden and correlate with IBD disease activity. However, their clinical significance as biomarker for disease activity remains unclear. This study aimed to prospectively validate the utility of plasma FT levels as biomarker for clinical, biochemical, and endoscopic disease activity in patients with IBD. Methods In an ongoing prospective diagnostic trial aiming to include 100 IBD patients, we conducted an interim analysis of plasma FT levels in 52 patients with IBD (24 Crohn's disease [CD], 21 ulcerative colitis [UC], and 7 patients with undetermined IBD). Clinical disease activity was quantified by the Harvey-Bradshaw Index (HBI) for CD and Simple Clinical Colitis Activity Index (SCCAI) for UC. Biochemical disease activity was determined by measuring blood C-reactive protein [CRP] and faecal calprotectin [fCal]). Endoscopic disease activity was quantified with the Mayo endoscopic subscore for UC and the Simple Endoscopic Score (SES-CD) for CD. Plasma FT levels were assessed for correlations with disease activity parameters using linear and logistic regressions models. Discriminative capacity of biomarkers was evaluated through calculating receiver operating characteristics (ROC) statistics. Results Plasma FT levels were significantly negatively correlated in clinical parameters, like HBI (r = -0.53, p < 0.05) (Fig. 1A). Other inflammatory biomarkers, like CRP, were also negatively correlated with plasma FT levels and were reduced in patients with active IBD (r = -0.43, p < 0.05) (Fig. 1B). Plasma FT levels accurately discriminated between CD patients with clinically quiescent (HBI < 5) and active disease (HBI ≥ 5) (AUC = 0.80, p = 0.02, Fig. 1C), better than fCal (AUC = 0.55, p = 0.83). Plasma FT were not significantly different for biochemically quiescent (CRP < 5 mg/l) and active (CRP ≥ 5mg/l) disease (AUC = 0.69, p = 0.20, Fig. 1D). Both Mayo and SES-CD scores, after adjusting for confounders, also exhibited a trend towards negative association with plasma FT levels (Fig. 1E). Figure 1 Conclusion Further completion of the trial will reveal whether systemic FT levels will reflect endoscopically observed disease activity, and this interim analysis shows strong correlations FT levels and parameters of clinically and biochemically active disease. Measuring systemic FTs in IBD may be a promising, minimally invasive strategy to monitor IBD disease activity. Funding None

P241 Systemic redox status associates with disease activity and clinical phenotypes in Inflammatory Bowel Disease

Abstract Background Oxidative stress is a key pathophysiological mechanism in inflammatory bowel diseases (IBD). Systemic levels of oxidative stress are reflected by reduced levels of free thiols (FT) in circulating proteins, especially those in albumin, which associates with disease activity in IBD. Yet, clinal value of circulating FT level as biomarkers of disease and therapy response remains largely unexplored. Here we investigated the association between plasma FT levels and clinical parameters, the plasma inflammatory proteome and medication use. Methods Plasma samples from 1,028 patients with IBD (567 Crohn's disease [CD] and 461 ulcerative colitis [UC]), and 500 healthy controls (HCs) participating in the 1000IBD and LifeLines projects were profiled for free thiols (FT), uric acid, bilirubin and 92 inflammation-related proteins (Olink Inflammation panelÒ). All biomarkers were associated with clinical phenotypes using general linear models adjusting for age, sex, body mass index, smoking and medication use. Results Plasma FT levels were significantly lower in IBD compared to HCs (p<0.05) (Figure 1A), with patients with UC showing even lower levels than patients with CD (p<0.05). Patients with UC on induction therapy had lower FT levels compared to patients on maintenance therapy (p<0.05). Furthermore, FT levels of patients with IBD were strongly associated with systemic inflammation (C-reactive protein [CRP], p<0.05) (Figure 1B). In both patients withCD and UC, reduced FT levels were associated with elevated levels of inflammation-, apoptosis-, and growth factor-related proteins, including C-X-C motif chemokine 9 (CXCL9) (Figure 1C-D), CUB domain-containing protein 1 (CDCP1) and caspase-8 (CASP8), proteins previous associated with preclinical IBD1-3. Furthermore, specifically for patients with UC, reduced FT levels were associated with elevated eotaxin-1 (CCL11) (Figure 1C), monocyte chemotactic protein-1 (MCP-1), and several cytokine biomarkers like Interleukine-6 (IL6) and Interleukin-17A (IL17A). Conclusion Systemic FT levels associate with inflammatory disease activity and clinical therapy and may offer potential utility in clinical assessments. This study highlights the intricate involvement of oxidative stress in various inflammatory pathways and components, particularly in innate/adaptive immune balance, cell damage, and apoptosis. These findings contribute to a deeper understanding of the interplay between oxidative stress, inflammation, and IBD. Funding: This study was supported by Janssen Research & Development LLC.

Systemic Oxidative Stress in Severe Early-Onset Fetal Growth Restriction Associates with Concomitant Pre-Eclampsia, Not with Severity of Fetal Growth Restriction.

Placental insufficiency is an important mechanism underlying early-onset fetal growth restriction (eoFGR). Reduced placental function causes impaired metabolic and gaseous exchange. This unfavorable placental environment is among other processes characterized by increased oxidative stress. Systemic free thiols (FT) are known for their reactive oxygen species scavenging capacity, and higher plasma levels of FT are associated with a better outcome in a multitude of ischemic and inflammatory diseases. We aimed to investigate the relationships between systemic FT levels and maternal and perinatal clinical characteristics and outcomes. In a post hoc analysis of the Dutch Strider study, a cohort of women with eoFGR, we investigated the association between the maternal redox status (FT) levels at study inclusion, placental biomarkers, and maternal and neonatal outcomes in 108 patients. FT were significantly lower in pregnancies complicated with eoFGR with concurrent maternal hypertensive disorders (pregnancy-induced hypertension; ρ = -0.281 p = 0.004, pre-eclampsia; ρ = -0.505 p = 0.000). In addition, lower FT levels were significantly associated with higher systolic (ρ = -0.348 p = 0.001) and diastolic blood pressure (ρ = -0.266 p = 0.014), but not with the severity of eoFGR. FT levels were inversely associated with sFlt (ρ = -0.366, p < 0.001). A strong relation between systemic FT levels and PlGF levels was observed in women with pre-eclampsia at delivery (ρ = 0.452, p = 0.002), which was not found in women without hypertensive disorders (ρ = 0.008, p = 0.958). In women with pregnancies complicated with eoFGR, FT levels reflect the severity of maternal disease related to the underlying placental insufficiency rather than the severity of the placental dysfunction as reflected in eoFGR or perinatal outcomes.

High resolution imaging and analysis of extracellular vesicles using mass spectral imaging and machine learning

AbstractExtracellular vesicles (EVs) are potentially useful biomarkers for disease detection and monitoring. Development of a label‐free technique for imaging and distinguishing small volumes of EVs from different cell types and cell states would be of great value. Here, we have designed a method to explore the chemical changes in EVs associated with neuroinflammation using Time‐of‐Flight Secondary Ion Mass spectrometry (ToF‐SIMS) and machine learning (ML). Mass spectral imaging was able to identify and differentiate EVs released by microglia following lipopolysaccharide (LPS) stimulation compared to a control group. This process requires a much smaller sample size (1 µL) than other molecular analysis methods (up to 50 µL). Conspicuously, we saw a reduction in free cysteine thiols (a marker of cellular oxidative stress associated with neuroinflammation) in EVs from microglial cells treated with LPS, consistent with the reduced cellular free thiol levels measured experimentally. This validates the synergistic combination of ToF‐SIMS and ML as a sensitive and valuable technique for collecting and analysing molecular data from EVs at high resolution.

Biomarkers of Oxidative Stress in Systemic Lupus Erythematosus Patients with Active Nephritis.

Oxidative stress plays an important role in systemic lupus erythematosus (SLE) and especially in lupus nephritis (LN). The aim of this study was to compare redox-related biomarkers between patients with active LN, quiescent SLE (Q-SLE) and healthy controls (HC) and to explore their association with clinical characteristics such as disease activity in patients. We investigated levels of plasma free thiols (R-SH, sulfhydryl groups), levels of soluble receptor for advanced glycation end products (sRAGE) and levels of malondialdehyde (MDA) in SLE patients with active LN (n = 23), patients with quiescent SLE (n = 47) and HC (n = 23). Data of LN patients who previously participated in Dutch lupus nephritis studies and longitudinal samples up to 36 months were analyzed. Thiol levels were lower in active LN at baseline and Q-SLE patients compared to HC. In generalized estimating equation (GEE) modelling, free thiol levels were negatively correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) over time (p < 0.001). sRAGE and MDA were positively correlated with the SLEDAI over time (p = 0.035 and p = 0.016, respectively). These results indicate that oxidative stress levels in LN patients are increased compared to HC and associated with SLE disease activity. Therefore, interventional therapy to restore redox homeostasis may be useful as an adjunctive therapy in the treatment of oxidative damage in SLE.

Evaluation of the salivary level of glutathione reductase, catalase and free thiol in patients with oral lichen planus

ObjectiveOral lichen planus (OLP) is a usual chronic inflammatory disease of the oral mucosa with malignant capacity, whose pathogenesis is not yet well known. Free radicals and reactive oxygen species may have a vital role in the pathogenesis of oral lichen planus. This study aimed to assess Glutathione reductase, catalase, and free thiol levels in the saliva of OLP patients and compare it with healthy people.MethodsIn this cross-sectional study, 35 patients with OLP and 20 healthy people were involved. Five mL of whole, unstimulated saliva samples were collected in the morning, and the salivary levels of antioxidants were measured by ELISA technique. In this experiment, sex, age and OLP types were also evaluated.ResultsThere was a significant decrease in the patients’ salivary level of Glutathione reductase (0.2043 mU/ml in patients and 0.3901 mU/ml in the control group) and catalase (0.1525 mU/ml in patients and 0.2700 mU/ml in the control group) (p = 0.001). But there were no differences between the two groups regarding free-thiol levels (0.0586 mU/ml in patients and 0.0569 mU/ml in the control group) (p = 0.7). However, there was no correlation between age and gender with the antioxidants’ contents. There was a significant decrease in glutathione reductase and catalase in the erosive type than in the non-erosive type.ConclusionsIn this study, we found that the salivary levels of Glutathione reductase and Catalase were lower in OLP patients than in the healthy group, which means these antioxidants were affected by OLP and also associated with the type of it. So salivary Glutathione reductase and Catalase levels may be used as biomarkers for OLP monitoring and treatment.

Dinitrosyl iron complexes with thiol-containing ligands as sources of universal cytotoxins - nitrosonium cations

It was shown that the release of a half of nitrosyl ligands from dinitrosyl iron units in the form of nitrosonium cation (NO+) from binuclear dinitrosyl iron complexes with thiol-containing ligands (B-DNIC) during DNIC decomposition in acid solutions is increased with the decrease of the stability of these complexes and completely blocked with the increase of the concentrations of free thiols (non-included into B-DNIC) up to the level that was two times and more than that of dinitrosyl iron units. It was demonstrated that the less stable B-DNIC with mercaptosuccinate degrade in an acidic environment at ambient temperature, while the decomposition of more stable B-DNIC with glutathione was only marked when the solution was heated at 80°C. The inhibition of NO+ release from B-DNIC in the presence of elevated free thiol level in the solution was due to the ability of free thiols to induce the reduction of NO+ to NO.

Thiol-Mediated Uptake of a Cysteine-Containing Nanobody for Anticancer Drug Delivery.

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that specifically recognizes B cell lymphoma and breast cancer in a thiol-dependent manner. CB2 binding strictly requires the presence of a nonconserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 via thiol-mediated endocytosis which can be exploited to deliver cytotoxic agents. CB2 internalization combined with functionalization forms the basis for a wide range of diagnostic and therapeutic applications, rendering thiol-reactive nanobodies promising tools for targeting cancer.

A Sex-Specific Comparative Analysis of Oxidative Stress Biomarkers Predicting the Risk of Cardiovascular Events and All-Cause Mortality in the General Population: A Prospective Cohort Study.

Oxidative stress plays a pivotal role in cardiovascular (CV) disease, but current biomarkers used to predict CV events are still insufficient. In this study, we comparatively assessed the utility of redox-related biomarkers in predicting the risk of CV events and all-cause mortality in male and female subjects from the general population. Subjects (n = 5955) of the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) population-based cohort study were included. Blood homocysteine, gamma-GT, HDL cholesterol, bilirubin and protein-adjusted free thiol (R-SH, sulfhydryl groups) levels were quantified at baseline and were prospectively analyzed in association with the risk of CV events and all-cause mortality. After adjustment for potentially confounding factors, protein-adjusted R-SH and homocysteine levels were significantly associated with the risk of CV events in men (HR 0.63 [0.40-0.99], p = 0.045 and HR 1.58 [1.20-2.08], p = 0.001, respectively). Protein-adjusted R-SH and HDL cholesterol levels were significantly associated with the risk of all-cause mortality in men (HR 0.52 [0.32-0.85], p = 0.009 and HR 0.90 [0.85-0.94], p < 0.001, respectively), while the same was observed for bilirubin and homocysteine levels in women (HR 0.68 [0.48-0.98], p = 0.040 and HR 2.30 [1.14-3.76], p < 0.001, respectively). Lower levels of protein-adjusted R-SH were robustly associated with an increased risk of CV events and all-cause mortality in men. Our results highlight the value of R-SH levels in cardiovascular risk assessment and their potential significance as being amenable to therapeutic intervention, while reaffirming the importance of other oxidative stress-related biomarkers, such as homocysteine, HDL cholesterol and bilirubin.

METABOLOMICS ANALYSIS IDENTIFIED AN ORALLY ACTIVE METABOLITE FOR TREATING CROHN’S DISEASE-ASSOCIATED INTESTINAL FIBROSIS

Abstract BACKGROUND Intestinal fibrosis is a debilitating complication of Crohn’s disease (CD) patients. Surgical resection is used to treat intestinal strictures, but this approach may adversely affect the patients' quality of life. Based on the findings of our groups and others, we hypothesize that intestinal metabolites are associated with intestinal fibrosis. We aimed to discover novel anti-fibrogenic metabolites. METHODS We compared the intestinal metabolite profiles between non-IBD, stricturing CD, and non-stricturing CD patients in an inflammatory bowel disease (IBD) multi-omics database to identify CD stricture-related metabolites. We compared the fecal metabolite profiles between trinitrobenzene sulfonic acid (TNBS)-treated mice with and without anti-fibrogenic CSA13 treatment to identify mouse intestinal fibrosis-related metabolites. RESULTS Compared to non-stricturing CD patients and non-IBD patients, stricturing CD patients had reduced fecal levels of inosine. Compared to normal mice, TNBS-treated mice had reduced fecal levels of inosine. CSA13-exposed TNBS-treated mice had increased fecal inosine levels. Oral administration of inosine (10 mg/kg/day for 14 days) effectively reduced ileal fibrosis in fibrotic 40-week-old SAMP1/YitFc mice with 96-100% reduction of intestinal collagen mRNA expression and 87% reduction of overall ileal disease activity. Although inosine did not directly inhibit collagen expression in primary human ileal fibroblasts from stricturing CD patients (CD-HIF), it reduced collagen mRNA expression in fresh ileal tissues from stricturing CD patients. RNA-seq and proteomics identified that peroxiredoxin 2 (PRDX2) is negatively associated with intestinal strictures in CD patients. PRDX2 is a thiol-specific antioxidant gene that reduces peroxides to alcohols. Inosine increased the secretion of peroxiredoxin 2 (PRDX2) in primary human intestinal epithelial cells (HPEC). The inosine-mediated PRDX2 secretion was inhibited by the pretreatment of adenosine 2A receptor (A2AR) inhibitor ZM241385. PRDX2 reduced cell stress, induced cell stress-related protein (cyclooxygenase 2/COX-2), and inhibited collagen expression in the CD-HIF. Inhibition of COX2 by indomethacin reversed the anti-fibrogenic effect of PRDX2 in the CD-HIF. Inhibition of PRDX2 caused increased collagen expression in the CD-HIF. Lentiviral overexpression of Prdx2 ameliorated ileal fibrosis in the fibrotic SAMP1/YitFc mice. Fibrotic SAMP1/YitFc mice had reduced circulating free thiol, reflecting increased systemic oxidative stress. Inosine restored circulating free thiol levels in SAMP1/YitFc mice. CONCLUSIONS Inosine is an orally active anti-fibrogenic metabolite that promotes PRDX2 expression from intestinal epithelial cells. PRDX2 is a newly identified anti-fibrogenic protein, which may inhibit collagen expression by reducing cell stress.

Promotion of Protein Solubility and Reduction in Stiffness in Human Lenses by Aggrelyte-1: Implications for Reversing Presbyopia.

With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for presbyopia. In this study, we tested aggrelyte-1 (S,N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58-75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44-66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia.

The effect of riboflavin supplementation on the systemic redox status in healthy volunteers: A post-hoc analysis of the RIBOGUT trial.

Riboflavin is a redox-active vitamin that plays a pivotal role in human energy metabolism. Riboflavin may have beneficial health effects by increasing extracellular antioxidant capacity, thereby alleviating oxidative stress. Reduced levels of free thiols in blood reflect systemic oxidative stress, since they are readily oxidized by reactive species. In this study, we aimed to study the potential of riboflavin supplementation to improve the systemic redox status in healthy volunteers. This study was a post-hoc analysis of the RIBOGUT study, a randomized, double-blind, placebo-controlled human intervention trial that investigated the effect of riboflavin supplements on the gut microbiota composition of healthy individuals. Serum free thiols were quantified before and after intervention and adjusted to serum albumin levels. Changes in albumin-adjusted free thiols were analyzed, as well as potential associations with routine laboratory parameters and faecal bacterial quantification by fluorescence in-situ hybridization (FISH). Participants were randomized to either placebo (n=34), riboflavin 50mg daily (n=32), or riboflavin 100mg daily (n=33). At baseline, no significant differences in albumin-adjusted serum free thiols were observed. After intervention with either placebo or riboflavin, albumin-adjusted serum free thiols did not significantly change (P>0.05), however, observed changes were inversely associated with changes in C-reactive protein (CRP) levels (r=-0.22, P<0.05). At baseline, albumin-adjusted serum free thiols were positively associated with faecal relative abundances of Faecalibacterium prausnitzii (P<0.01). Riboflavin did not change the systemic redox status in healthy individuals as reflected by serum free thiols, but observed changes in albumin-adjusted free thiol levels were negatively associated with changes in CRP levels. Strikingly, albumin-adjusted free thiols were independently associated with relative abundances of faecal F. prausnitzii, which may suggest a potential host redox-microbiota interaction.

Systemic oxidative stress associates with new-onset hypertension in the general population

BackgroundOxidative stress is known to be involved in the development of hypertension, but accurate redox biomarkers predicting the risk of developing hypertension are scarce. Serum free sulfhydryl groups (R–SH, free thiols) have been shown to accurately reflect systemic oxidative stress in various conditions. In this study, we aimed to investigate associations between serum free thiols and the risk of developing new-onset hypertension in a population-based cohort study. MethodsSubjects (n = 3,575) who participated in the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) study, a prospective, population-based cohort study in the Netherlands, were included. Baseline protein-adjusted serum free thiols were studied for their associations with the development of hypertension, defined as a systolic blood pressure (SBP) of at least 140 mmHg, a diastolic blood pressure (DBP) of at least 90 mmHg, or the first usage of antihypertensive medication. Subjects with hypertension at baseline were excluded from the study. ResultsMean protein-adjusted serum free thiols at baseline was 5.16 μmol/g of protein (range: 1.62–8.41 μmol/g). Protein-adjusted serum free thiols were significantly associated with the risk of incident hypertension (hazard ratio [HR] per doubling 0.60 [95% confidence interval [CI]: 0.49–0.72, P < 0.001), also after adjustment for age and sex (HR 0.81 [95% CI: 0.66–0.91], P < 0.05), but not after additional adjustment for relevant confounding factors (HR 0.90 [95% CI: 0.70–1.15], P = 0.382). ConclusionHigher levels of serum free thiols, i.e. less oxidative stress, are associated with a decreased risk of developing incident hypertension in subjects from the general population.

Local and Systemic Oxidative Stress Biomarkers for Male Infertility: The ORION Study.

Infertility problems occur in around 10% of all couples worldwide, with male-factor infertility as the sole contributor in 20–30% of these cases. Oxidative stress (OS) is suggested to be associated with the pathophysiology of male infertility. In spermatozoa, OS can lead to damage to the cell membrane, resulting in disruption of DNA integrity and a decrease in motility. Established biomarkers for OS include free thiols and malondialdehyde (MDA), both representing different components of the reactive species interactome (RSI). This exploratory study aimed to investigate seminal plasma-free thiol and MDA levels in relation to semen parameters as defined by the World Health Organization (WHO) to determine if these markers are adequate to define local OS status. Furthermore, this study investigated if there is a relation between systemic and local OS status by comparing seminal concentrations of free thiol (R-SH, sulfhydryl groups, representing the extracellular redox status) and MDA (lipid peroxidation product) levels to those measured in serum. Free thiol and MDA measurements in both serum and semen plasma were performed in 50 males (18–55 y) of couples seeking fertility treatment. A significant positive correlation was found between seminal plasma-free thiol levels and sperm concentration and progressive motility (r = 0.383, p = 0.008 and r = 0.333, p = 0.022, respectively). In addition, a significant positive correlation was found between MDA levels in seminal plasma and sperm concentration (r = 0.314, p = 0.031). This study supports that seminal plasma-free thiols may be promising as local OS biomarkers. No associations were observed between local and systemic OS biomarker concentrations.

Plasma Free Thiol Levels during Early Sepsis Predict Future Renal Function Decline.

Sepsis is a life-threatening syndrome characterized by acute organ dysfunction due to infection. In particular, acute kidney injury (AKI) is common among patients with sepsis and is associated with increased mortality and morbidity. Oxidative stress is an important contributor to the pathogenesis of sepsis-related AKI. Plasma free thiols (R-SH) reflect systemic oxidative stress since they are readily oxidized by reactive species and thereby serve as antioxidants. Here, we aimed to assess the concentrations of serum free thiols in sepsis and associate these with major adverse kidney events (MAKE). Adult non-trauma patients who presented at the emergency department (ED) with a suspected infection were included. Free thiol levels and ischemia-modified albumin (IMA), a marker of oxidative stress, were measured in plasma at baseline, at the ward, and at three months, and one year after hospitalization. Plasma free thiol levels were lower at the ED visit and at the ward as compared to three months and one year after hospital admission (p < 0.01). On the contrary, plasma levels of IMA were higher at the ED and at the ward compared to three months and one year after hospital admission (p < 0.01). Furthermore, univariate logistic regression analyses showed that plasma free thiol levels at the ED were inversely associated with long-term renal function decline and survival at 90 days (MAKE90) and 365 days (MAKE365) (OR 0.43 per standard deviation [SD] [0.22–0.82, 95% CI], p = 0.011 and OR 0.58 per SD [0.34–0.96, 95% CI], p = 0.035, respectively). A multivariate regression analysis revealed an independent association of plasma free thiols at the ED (OR 0.52 per SD [0.29–0.93, 95% CI], p = 0.028) with MAKE365, even after adjustments for age, eGFR at the ED, SOFA score, and cardiovascular disease. These data indicate the clear role of oxidative stress in the pathogenesis of sepsis-AKI, as reflected in the lower plasma free thiol levels and increased levels of IMA.

Oxidative Stress Predicts Post-Surgery Complications in Gastrointestinal Cancer Patients

IntroductionAn excessive perioperative inflammatory reaction can lead to more postoperative complications in patients treated for gastrointestinal cancers. It has been suggested that this inflammatory reaction leads to oxidative stress. The most important nonenzymatic antioxidants are serum free thiols. The purpose of this study was to evaluate whether high preoperative serum free thiol levels are associated with short-term clinical outcomes.MethodsBlood samples were drawn before, at the end of, and 1 and 2 days after surgery of a consecutive series of patients with gastrointestinal cancer. Serum free thiols were detected using a colorimetric detection method using Ellman’s reagent. Short-term clinical outcomes were defined as 30-day complications (Clavien-Dindo ≥2) and length of hospital stay. Logistic regression was applied to examine the association between serum free thiol levels and short-term patient outcomes.ResultsEighty-one patients surgically treated for gastrointestinal cancer were included in the study. Median age was 68 (range 26–87) years, and 28% were female. Patients in the lowest tertile of preoperative serum free thiols had a threefold higher risk to develop postoperative complications (odds ratio [OR]: 3.4; 95% confidence interval [CI]:1.1–10.7) and a fourfold higher risk to have an increased length of stay in the hospital (OR 4.0; 95% CI 1.3–12.9) compared with patients in the highest tertile.ConclusionsPatients with lower preoperative serum free thiol levels, indicating a decrease in extracellular antioxidant capacity and therefore an increase in systemic oxidative stress, are more likely to develop postoperative complications and show a longer in hospital stay than patients with higher serum free thiol levels.

Mode of action of the sesquiterpene lactones eupatoriopicrin and estafietin on Trypanosoma cruzi

Background: The sesquiterpene lactones (STLs) eupatoriopicrin (EP) and estafietin (ES), isolated from Stevia alpina Griseb. (Asteraceae) and Stevia maimarensis (Hieron.) Cabrera (Asteraceae) respectively, have previously showed promising trypanocidal activity, both in vitro and in vivo. Purpose: In this work, using biochemical studies and electron microscopy, we aimed at characterizing the mode of action of both STLs on Trypanosoma cruzi. Methods: The interaction of STLs with hemin was examined by measuring modifications in the Soret absorption band of hemin; the thiol groups interaction was determined spectrophotometrically through its reaction with 5,5’-dithiobis-2-nitrobenzoate; the effect on cruzipain activity was also assayed by spectrophotometry. The synthesis of sterols were qualitatively and quantitatively tested by TLC. Mitochondrial functionality was assessed by measuring mitochondrial membrane potential and the activity of NADH-cytochrome c reductase and succinate-cytochrome c reductase enzymes. The status of the antioxidant system was assessed by quantifying the level of free thiols by spectrophotometry, together with the intracellular oxidative state by flow cytometry. Ultrastructural changes were analyzed by transmission electron microscopy. Results: EP and ES were found to impair the functionality and the redox status of the parasite. ES produced a greater decrease in the activity of succinate dehydrogenase than eupatoriopicrin, affecting the functioning of the respiratory chain and the Krebs cycle. EP increased the formation of triglycerides leading to the presence of cytoplasmic lipid droplets. By electron microscopy, alterations in the kinetoplast and the appearance of large translucent vacuoles in the cytoplasm were observed for both compounds. Conclusions: Both sesquiterpenelactones proved to act additively on T. cruzi, supporting the hypothesis that each compound would be acting on different primary targets.. The treatment combining eupatoriopicrin and estafietin could be considered a promising alternative for the treatment of Chagas’ disease.

Serum free sulfhydryl status associates with new-onset chronic kidney disease in the general population

BackgroundSerum sulfhydryl groups (R-SH, free thiols) reliably reflect the systemic redox status in health and disease. As oxidation of R-SH occurs rapidly by reactive oxygen species (ROS), oxidative stress is accompanied by reduced levels of free thiols. Oxidative stress has been implicated in the pathophysiology of chronic kidney disease (CKD), in which redox imbalance may precede the onset of CKD. Therefore, we aimed to investigate associations between serum free thiols and the risk of incident CKD as defined by renal function decline and albuminuria in a population-based cohort study. MethodsSubjects without CKD (n = 4,745) who participated in the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) study, a prospective, population-based cohort study in the Netherlands, were included. Baseline protein-adjusted serum free thiols were studied for their associations with the development of CKD, defined as a composite outcome of an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73m2, urinary 24-h albumin excretion (UAE) > 30 mg/24-h, or both. ResultsMedian level of protein-adjusted serum free thiols at baseline was 5.14 μmol/g of protein (interquartile range [IQR]: 4.50–5.75 μmol/g) and median eGFR was 96 mL/min/1.73 m2 [IQR: 85–106]. Protein-adjusted serum free thiols were significantly associated with incident CKD (hazard ratio [HR] per doubling 0.42 [95% confidence interval [CI]: 0.36–0.52, P < 0.001), even after adjustment for traditional risk factors (HR 0.67 [95% CI: 0.47–0.94], P=0.022). In secondary analyses, the highest tertile of protein-adjusted serum free thiols was inversely associated with incident UAE >30 mg/24-h after full adjustment for confounding factors (HR per doubling 0.70 [95% CI: 0.51–0.96], P=0.028). ConclusionHigher levels of serum R-SH, reflecting less oxidative stress, are associated with a decreased risk of developing CKD in subjects from the general population. This association is primarily driven by incident CKD as defined by UAE.

Metabolic Changes in Seed Embryos of Hypoxia-Tolerant Rice and Hypoxia-Sensitive Barley at the Onset of Germination.

Rice (Oryza sativa L.) and barley (Hordeum vulgare L.) are the cereal species differing in tolerance to oxygen deficiency. To understand metabolic differences determining the sensitivity to low oxygen, we germinated rice and barley seeds and studied changes in the levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), activities of the enzymes involved in their scavenging, and measured cell damage parameters. The results show that alcohol dehydrogenase activity was higher in rice than in barley embryos providing efficient anaerobic fermentation. Nitric oxide (NO) levels were also higher in rice embryos indicating higher NO turnover. Both fermentation and NO turnover can explain higher ATP/ADP ratio values in rice embryos as compared to barley. Rice embryos were characterized by higher activity of S-nitrosoglutathione reductase than in barley and a higher level of free thiols in proteins. The activities of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase) in imbibed embryos were higher in rice than in barley, which corresponded to the reduced levels of ROS, malonic dialdehyde and electrolyte leakage. The observed differences in metabolic changes in embryos of the two cereal species differing in tolerance to hypoxia can partly explain the adaptation of rice to low oxygen environments.