Blood Glutathione Concentrations Research Articles

Differential identification of GSH for acute coronary syndrome using a colorimetric sensor based on nanoflower-like artificial nanozymes

The ability to detect glutathione (GSH) concentrations in human blood offered a simple and non-invasive method to monitor changes associated with cardiovascular diseases, cancers and diabetes. We showed the potential of employing catalytically active protein-directed nanoflower-like artificial nanozymes (apo-TF-MnOx NFs) by bio-mineralization method to produce simple and visible colorimetric sensor for GSH. The experiments proved that apo-TF-MnOx NFs exhibited peroxidase, catalase- and superoxide dismutase-like activities, but the most notable feature was the excellent peroxidase-like activity, which could efficiently catalyze the oxidation reaction of 3,3′,5,5′- tetramethylbenzidine (TMB) in the existence of hydrogen peroxide (H2O2) to generate a blue product. Some outcomes also indicated that the apo-TF-MnOx NFs had stronger peroxidase-like activity, which was proved by the Michaelis-Menten constant (Km) and maximum initial velocity (Vmax). Hence, we used the peroxidase-like activity to develop a GSH colorimetric biosensor. Fortunately, the colorimetric platform exhibited a sensitive response to H2O2 and GSH in the range of 5 μМ to 300 μМ and 0.5 μМ to 35 μМ with a limit of detection of 3.29 μM and 0.15 μM (S/N = 3) under optimal conditions. The feasibility of the simple method was confirmed by qualitative detection of H2O2 and GSH in blood samples from acute coronary syndrome patients. A key outcome of our study was the ability to realized differential identification of GSH for acute coronary syndrome and healthy human without invasive treatment which was an advantage over other methods. This work not only proposed a new type of nanozymes, but also showed the multiple advantages of the apo-TF-MnOx NFs for the construction of biosensors. Thus, we believe that apo-TF-MnOx NFs with strong peroxidase-like activity can be employed as nanozymes and be widely applied in the fields of medicine and biological sensors.

Urinary Excretion of 5-L-Oxoproline (Pyroglutamic Acid) Is Increased during Recovery from Severe Childhood Malnutrition and Responds to Supplemental Glycine

We hypothesized that a limitation in the endogenous formation of glycine might constrain catch-up growth during recovery from severe childhood malnutrition. The urinary excretion of 5-L-oxoproline is increased when the glycine available for glutathione synthesis is limited. Urinary excretion of 5-L-oxoproline was measured throughout recovery in 12 children (aged 16 ± 6 mo) with severe malnutrition. Urinary 5-L-oxoproline was similar at admission and after recovery, but was increased significantly during rapid catch-up growth. There was a significant relationship between the rate of weight gain and 5-L-oxoproline excretion in urine. In nine children (aged 15 ± 5 mo), the effect of oral supplementation with glycine, [1.7 mmol/(kg·d) for 48 h] during rapid catch-up growth on 5-L-oxoprolinuria and blood glutathione concentration was determined. In seven of the nine children weight gain was less than 17 g/(kg·d) and following oral glycine supplements 5-L-oxoproline excretion was reduced up to 64% and blood glutathione concentration increased up to 100%. In the two children who were gaining weight at a rate >17 g/(kg·d), glycine supplementation was associated with a further increase in 5-L-oxoproline excretion and a decrease in blood glutathione. If 5-L-oxoproline is an index of the relative availability of glycine, then the data indicate that glycine may be limiting during rapid catch-up growth. This would have important implications for repletion of muscle and gain in height.

Antioxidant Capacity Is Decreased in Wilson's Disease and Correlates to Liver Function.

The metabolic disorder Wilson's disease (WD) is caused by copper accumulation in the tissues due to a biallelic pathogenic mutation of the gene ATP7B, encoding intracellular copper transporter ATPase-7B. As copper is a redox active metal; aberrations in its homeostasis may create favourable conditions for superoxide-yielding redox cycling and oxidative damage to the cells. We tried to characterise antioxidant defence in WD patients and to evaluate whether it is related to liver function.The blood glutathione concentration, the activity of manganese-SOD (MnSOD), catalase (Cat), glutathione peroxidase, and glutathione S-transferase glutathione (GST), and serum antioxidant potential (AOP-450) were measured in WD treatment-naive patients and healthy controls and correlated with clinical data.The blood glutathione concentration, the activity of MnSOD, Cat, glutathione peroxidase, and GST and AOP-450 are significantly decreased in WD patients. There was a positive correlation of AOP-450 with AST. Moreover, the Cat and GST activity as well as AOP-450 strongly correlated with parameters of synthetic liver function. MnSOD activity correlated positively with ALT and AST.The blood glutathione concentration, the activity of MnSOD, Cat, glutathione peroxidase, and GST and AOP-450 are significantly decreased in WD patients. There was a positive correlation of AOP-450 with AST. Moreover, the Cat and GST activity as well as AOP-450 strongly correlated with parameters of synthetic liver function. MnSOD activity correlated positively with ALT and AST.Liver injury in course of WD is linked with decreased antioxidant capacity.

Effects of N-Acetylcysteine Infusion in Patients with Sickle Cell Disease during Vaso-Occlusive Crises

Background: Our earlier studies showed that patients with sickle cell disease (SCD) have a high von Willebrand factor (VWF) activity index, which correlates with the extent of the hemolysis they experience [Chen et al Blood 2011; 117:3680‐3]. We are evaluating whether N-acetylcysteine (NAC), which dissociates VWF multimers, has a therapeutic role in SCD. Previously, we studied stable outpatients with SCD [Özpolat et al Blood Dec 2016, 128 (22) 1299]. In that study, we found that NAC infusion was safe and decreased red blood cell (RBC) fragments, dense cells, the size of VWF multimers, platelet activation, and the percentage of platelet-monocyte complexes. In addition, NAC infusion increased the total and free-thiol concentrations of cysteine and glutathione in blood, relieving oxidative stress in the patients at disease baseline. Given these findings, we have begun a pilot study of NAC in SCD patients admitted as inpatients with vaso-occlusive crisis (VOC) (NCT01800526) to examine the safety of NAC infusion and its effects on laboratory markers in that setting.Protocol and patients: Patients are approached for the study in the outpatient setting and consent is re-affirmed when they are admitted in VOC. We plan to enroll enough patients to treat 20 VOCs with NAC infusion, with each patient being treated up to two times in the study. Inclusion criteria: SCD patients with history of VOC requiring narcotics treatment. Exclusion criteria: SCD patients on chronic RBC exchange, expected to be treated by simple or exchange transfusion, inpatient with VOC in the previous 30 days, already had NAC infusion twice in our trial during VOC, and/or had oral NAC in the past 7 days. NAC (75 mg/kg/hr) is infused over 1 hr every 6 hr, for a total daily dose of 300 mg/kg. All patients also receive standard care. Blood is collected immediately before NAC infusion on the first day of admission, daily during admission, and at medical follow-ups during the first week after discharge. To date, we have completed NAC infusion in two patients with VOC under this protocol. Patient 1 is a 26-year-old male with Sβ0 thalassemia, a history of recurrent VOC and avascular necrosis (AVN) of both hips and the left shoulder, on hydroxyurea and oral narcotics. Patient 2 is a 58-year-old female with SS genotype, recurrent VOC and history of bilateral AVN of the hips and knees, pulmonary embolism, on hydroxyurea, oral narcotics, and rivaroxaban. We analyzed patient samples for dense cells, sickle cells (by imaging flow cytometry), VWF antigen and multimer distribution, ADAMTS13 activity and antigen. In addition, we will analyze the concentrations of NAC, cysteine and glutathione and their oxidized and mixed disulfide forms by mass spectrometry.Results: NAC infusions in the two SCD patients with VOC were safe and well tolerated. After NAC treatment, the number of dense cells were reduced (before vs after NAC treatment: patient 1; 1826/µl vs 46/µl, patient 2: 667/µl vs 36/µl), The VWF multimers migrated slightly faster during NAC infusion, indicating possible reduction of intra-chain disulfide bonds by NAC within VWF monomers [Chen J et al. JCI 2011, 121: 593]. The VWF concentrations were increased at the end of both NAC treatments. For patient 1, the ADAMTS13 activity was decreased during the NAC treatment, but the antigen remained unchanged. For patient 2, both the ADAMTS13 activity and antigen were increased with NAC treatment. For patient 1, phosphatidylserine positive microparticles (MP) and MP generated from RBCs and platelets were decreased with NAC treatment but there was no apparent trend for patient 2.Summary: In the first two SCD patients treated, NAC infusion during VOC was safe and well tolerated. NAC infusion decreased dense cells, and reduced intra-chain disulfide bonds in VWF. The full study should determine if NAC is useful therapy for SCD patients during VOC, acting through diverse mechanisms by improving RBC parameters and reducing adhesive activity of VWF. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of the glutathione concentration in whole blood of dairy Holstein cows

The objective of this study was to evaluate the concentrations of the total (T-GSH), oxidised (GSSG) and reduced (GSH) glutathione in the blood of dairy cows, assess the relationships of the phase and number of lactation, milk production, body condition score (BCS) and selected biochemical parameters on its concentrations. We analysed 79 samples of whole blood from dairy Holstein cows. The concentration of glutathione was assessed by the spectrophotometric enzymatic method. The whole dataset shows the mean concentration of T-GSH, GSSG and GSH of 803 ± 22 µmol/l, 23 ± 2.5 µmol/l and 757 ± 27 µmol/l, respectively. The GSH/GSSG ratio was 95 ± 17. The phase of the lactation had a significant impact on the levels of T-GSH and GSH, but the number of lactation and BCS had no effect. Dry cows had higher levels of T-GSH (938 ± 44 µmol/l) than the fresh (713 ± 46 µmol/l) and peak lactation (785 ± 45 µmol/l) cows. The fresh cows had significantly lower concentrations of GSH (618 ± 44 µmol/l) than the peak lactation (719 ± 46 µmol/l) and dry cows (827 ± 43 µmol/l). On the basis of a regression analysis, blood glutathione was affected mainly by the liver function and energy metabolism. Glutathione as the marker of oxidation stress seems to be a promising tool in monitoring the health and welfare of the herd, yet intensive research in this field remains necessary.

Are Silver Nanoparticles Useful for Treating Second-Degree Burns? An Experimental Study in Rats.

Purpose: In this work, the potential usefulness of silver nanoparticles (AgNPs) for treating burn wounds was examined. Methods: Second-degree burns were induced in male Wistar rats by touching the skin with a heated (70°C) metallic device for 10 s, after which the animals were randomly allocated to one of two groups: control (n=8, treated with sterile saline) and experimental (n=8, treated with AgNPs, 0.081 mg/mL; 50 µL applied to the burn surface). Seven, 14, 21 and 28 days after lesion induction two rats from each group were killed and blood samples were collected for a complete blood count and to assess oxidative stress. The livers were examined macroscopically and skin samples were collected for histological analysis. Results: Macroscopically, wound healing and skin remodeling in the experimental group were similar to the saline-treated rats. Likewise, there were no significant differences in the histological parameters between the two groups. However, treatment with AgNPs caused a persistent reduction in white blood cell (WBC) counts throughout the experiment, whereas platelet counts increased on days 7 and 28 but decreased on days 14 and 21; there was also an increase in the blood concentration of reduced glutathione on day 7 followed by a decrease on days 21 and 28. There were no significant changes in blood glutathione peroxidase (GSH-Px) and catalase (CAT) activities or in the serum concentration of thiobarbituric acid reactive substances. Conclusion: The findings of this study raise questions about the potential transitory effects of AgNPs based on the changes in WBC and platelet counts, blood glutathione concentrations and macroscopic hepatic alterations.

Effect of selenium sources in plant-based diets on antioxidant status and oxidative stress-related parameters in rainbow trout juveniles under chronic stress exposure

Selenium (Se) is an essential micronutrient required for normal development and antioxidant protection. Oxidative stress induced by stressful conditions has been shown to be reduced by dietary Se supplementation. However, replacing fishmeal, an important source of Se, with ingredients of plant origin usually results in decreased Se content in aquafeeds. The objective of the study was hence to assess the impact of dietary Se supplementation by an inorganic or organic source on the antioxidant defense system of rainbow trout (Oncorhynchus mykiss) juveniles fed practical plant-based feeds under normal or chronic stress conditions. Fish (initial mean weight: 42 ± 2 g) were maintained under normal (25 fish/tank, dissolved oxygen: 7.9 mg/l) or stressful conditions combining decreased water quality with hypoxia and increased stocking density (50 fish/tank, dissolved oxygen: 5.9 mg/l) for 12 weeks at 17 °C. Rainbow trout juveniles were fed three plant-derived protein-based diets containing 0.5 mg Se/kg diet supplemented or not with 0.3 mg Se/kg diet supplied as sodium selenite or Se-enriched yeast, Selsaf®. The apparent digestibility coefficients (ADC) of dry matter, protein, ash and Se were improved by Se-enriched yeast supplementation while the ADC of Se was reduced by sodium selenite supplementation and the ADC of dry matter, starch, energy, ash and phosphorus were reduced by chronic stress exposure. Dietary Se supplementation had no significant impact on growth performance contrary to chronic stress exposure which led to decreased final body weight and feed intake. Total Se content of fish was increased by both dietary Se supplementations but to a larger extent with Se-enriched yeast. Fish fed Se-enriched yeast displayed increased hepatic selenomethionine content and decreased muscle anisidine value. Blood glutathione content was reduced by chronic stress exposure. While hepatic catalase activity and transcript expression were increased by chronic stress exposure, both hepatic and renal glutathione peroxidase (GPX) activity and hepatic transcript expression of gpx1b2 and gpx4a2 were decreased in non-Se supplemented fish. These results suggest the necessity to supplement plant-based diets with Se for rainbow trout juveniles, independently of chronic stress exposure and highlight the superiority of organic form of Se to fulfil the dietary Se requirement and sustain the antioxidant status of fish.

Associations between folate and choline intake, homocysteine metabolism, and genetic polymorphism of MTHFR, BHMT and PEMT in healthy pregnant Polish women.

Physiological homocysteine (Hcy) concentrations depend on several factors, both dietary (including folate and choline intake) and biological (such as polymorphism of the genes involved in Hcy metabolism). This study aimed to thus test the associations between genes functionally linked with Hcy metabolism (MTHFR, BHMT and PEMT), folate and choline intakes, and total Hcy (tHcy) concentrations of healthy pregnant women. One hundred and three healthy Polish women aged 18-44 years, in the third trimester of pregnancy, were enrolled. Mean blood tHcy and glutathione (GSH) concentrations were 8.08 ± 3.25 μM and 4.84 ± 1.21 μM, respectively. Concentrations of tHcy were found to be lower in the women who were taking folic acid supplements than in those who did not take these supplements (7.42 ± 1.78 μM vs 9.28 ± 4.42 μM, P < 0.05). There were no associations found between the examined parameters and BHMT (rs7356530), MTHFR (rs1801133) and PEMT (rs12325817) alone. However, blood tHcy concentrations differed in the PEMT genotype subgroups when choline and folate intakes were considered: respectively, 25% and 20% lower levels were observed in the C allele carriers who met their needs of choline or folate than in those who did not take enough these nutrients (P < 0.05 for both associations). This study suggests that choline and folate intakes might interact with MTHFR, BHMT and PEMT polymorphisms to determine tHcy and GSH blood concentrations in healthy pregnant women.

The absorptive effects of orobuccal non-liposomal nano-sized glutathione on blood glutathione parameters in healthy individuals: A pilot study.

BackgroundGlutathione is an endogenous antioxidant found in oxidized (GSSG) and reduced (GSH) forms. Glutathione depletion is indicative of oxidative stress and occurs in various pathological conditions and following extreme exercise activity. Raising blood glutathione concentration has potential to attenuate and prevent chronic disease and also to improve recovery from exercise. There are a number of challenges to achieving this through traditional dietary supplements, and thus there is a need to develop optimized delivery methods to improve blood glutathione status. This study evaluated the effect of a novel glutathione formulation on blood glutathione parameters in healthy individuals.Methods15 (8 male) healthy individuals (25±5y old, 78.0±14.6kg) participated in a single-blinded randomized placebo-controlled crossover study, with a minimum one-week washout period between treatments. Participants were overnight fasted and administered 1mL of a non-liposomal nano-size glutathione solution (NLNG) containing 200mg of glutathione or 1mL of placebo lacking glutathione. The solution was held in the mouth for 90 seconds before the remainder was swallowed. Blood was collected at baseline, 5, 10, 30, 60 and 120 minutes post-treatment. Protein-bound plasma and erythrocyte lysate concentrations of GSH and GSSG were measured at all time points using previously validated procedures. Linear mixed effects models were used to compare differences between baseline and post-treatment glutathione concentrations between NLNG and placebo for each parameter.ResultsThere was a significant main effect for treatment type, such that increases in GSH concentration in erythrocyte lysate were greater following NLNG than placebo (p = 0.001). Similar significant main effects for treatment were also found for total (protein bound + erythrocyte lysate) GSH (p = 0.015) and GSSG (p = 0.037) concentration, as well as total blood glutathione pool (GSH+GSSG, p = 0.006).DiscussionNLNG increased multiple blood glutathione parameters compared to placebo. Future research should examine whether NLNG can attenuate oxidative stress.

The evaluation of glutathione concentration in whole blood of Holstein dairy calves

The aim of this study was to evaluate the concentrations of total (T-GSH), oxidized (GSSG) and reduced glutathione (GSH) in blood of clinically healthy calves, to assess the effect of sex and age together with the type of feeding on its concentrations, and to assess the relationships between glutathione and metabolic indices. A total of 117 Holstein calves at the age of 1 day to 6 months were divided according to age and together with the type of feeding into 4 groups: colostrum and transition milk (n = 20), native milk (n = 39), milk replacer (n = 29) and total mixed ration (n = 29). Blood serum was used for the assessment of the metabolic profile of calves. The concentrations of individual forms of glutathione (mean ± SEM) were as follows: T-GSH: 789.1 ± 19.4 µmol/l, GSSG: 54.2 ± 4.3 µmol/l, GSH: 681.6 ± 15.3 µmol/l, and GSH/GSSG: 41.1 ± 5.9, with the coefficients of variability of 26.6%, 85.3%, 24.3% and 157.4%, respectively. Age and the type of feeding affected GSSG (P≤ 0.01) and GSH/GSSG (P≤ 0.01). The relationships between glutathione and biochemical parameters were mostly weak. T-GSH correlated with creatinine (P≤ 0.05) and chlorides (P≤ 0.05); GSSG correlated with albumin (P≤ 0.01), creatinine (P≤ 0.01), cholesterol (P≤ 0.05), aspartate aminotransferase (P≤ 0.01), alanine aminotransferase (P≤ 0.01), gamma-glutamyltransferase (P≤ 0.05) and calcium (P≤ 0.05); GSH/GSSG ratio correlated with creatinine (P≤ 0.01), cholesterol (P≤ 0.05), aspartate aminotransferase (P≤ 0.05), gamma-glutamyltransferase (P≤ 0.01) and calcium (P≤ 0.05). The evaluation of individual forms of glutathione helps in estimation of redox status in healthy animals and their monitoring can detect the stress extent.

Effect of Intravenous and Oral N-Acetylcysteine Treatment in a Patient with Sickle Cell Disease at Disease Baseline

Background: We previously showed that patients with sickle cell disease (SCD) have elevated levels of von Willebrand factor (VWF) with high adhesive activity and the level of total active VWF correlated with hemolysis at disease baseline. These findings suggest that VWF plays a role in the pathophysiology of SCD [Chen et al, Blood. 2011; 117:3680‐3]. We also showed that N-acetylcysteine (NAC), a mucolytic and antioxidant drug, can reduce the level of large VWF multimers and platelet adhesion in human plasma in vitro and in mice in vivo [Chen et al, JCI, 2011, 121:522]. Based on these findings, we hypothesized that administration of NAC in SCD patients might reduce VWF multimers in vivo and reduce oxidative stress by scavenging reactive oxygen species (ROS). We started a pilot study to evaluate the effects of intravenous (IV) and oral NAC administration in SCD patients at disease baseline (NCT01800526). In this report, we administered NAC to one patient by IV and orally and compared the effect on biomarkers by the two routes of NAC administration.Patient and protocol: The subject is a 51-year-old male with SS genotype, a history of leg ulcers and acute chest syndrome, and who was not taking hydroxyurea at the time of the study. NAC was infused at a low dose (150 mg/kg over 8 hours) and infusion was repeated at high dose (300 mg/kg over 8 hrs) after 4 weeks. Blood samples were collected before (0), during (4 hr) and after the NAC infusions (8, 24 and 72 hr). Five months after the high dose IV infusion, during which parameters returned to baseline levels, oral NAC was administered for 4 weeks at 2.4g/day. Weekly blood samples were collected four weeks before the start of oral NAC, during oral NAC, and one week after oral NAC administration. We measured RBC parameters and RBC fragments, dense cells and percentage of platelet-monocyte complexes (PMC) by flow cytometry. We also analyzed VWF antigen, VWF multimers, ADAMTS13 antigen and activity levels in plasma. We also measured total, oxidized and reduced forms of NAC, cysteine and glutathione levels in plasma and whole blood by mass spectrometry.Results: 1) The subject did not experience adverse side effect during and after the treatment period. 2) RBC, hemoglobin and hematocrit levels did not significantly change with either IV or oral NAC treatment. Oral and IV NAC decreased dense cell and RBC fragments. NAC reduced the number of dense cells 13% during low dose IV, 79% during high dose IV and 73% with the oral administration. Compared to baseline, RBC fragments decreased 40% during low dose IV, 28% during high dose IV, and 27% with the oral NAC treatment. 3) NAC reduced formation of PMC from 71% to 24% (low dose IV), from 72% to 35% (high dose IV), and from 62% to 27% (oral). 4) VWF multimer size significantly decreased during low and high dose NAC infusions but did not change with oral NAC administration. VWF antigen and ADAMTS13 activity levels did not change with either routes of NAC administration. 5) The concentration of NAC in whole blood increased during treatments: elevated to 0.5 mM and 1 mM at the end of low- and high-dose IV infusions, respectively. At the conclusion of oral administration, the NAC concentration was 4 µM. Total glutathione concentrations in whole blood did not change significantly during and after NAC treatment at either dose or by either route. Free Cys concentrations in plasma increased compared to respective baseline levels 10-fold for low dose infusion (from 10 µM to 101 µM), 20-fold for high dose infusion (from 9 µM to 200 µM), but the level did not change significantly for oral administration (from 5 µM to 7 µM). Protein-bound Cys levels in plasma as markers of oxidative stress were significantly decreased with low- and high-dose IV infusion. However, the level did not change significantly with oral NAC administration.Summary: Both IV and oral administration of NAC were safe and well tolerated in the patient with SCD. Both IV and oral administration reduced the number of dense cells, RBC fragmentation, and formation of PMCs. IV, but not oral NAC, reduced VWF multimer size. These finding suggest that IV administration resulted in higher concentration and had more biologic effects; however both approaches may have a role in treatment of patients with SCD. DisclosuresKonkle:Sangamo: Research Funding; Gilead: Consultancy; Shire: Research Funding; Genentech: Consultancy; CSL Behring: Consultancy; Bioverativ: Research Funding; BioMarin: Consultancy; Spark: Consultancy, Research Funding; Pfizer: Research Funding.

Effects of a Saccharomyces cerevisiae fermentation product in receiving diets of newly weaned beef steers. I. Growth performance and antioxidant defense1.

To evaluate the effects of a Saccharomyces cerevisiae fermentation product (SCFP; Original XPC, Diamond V, Cedar Rapids, IA) on growth performance and antioxidant defense of newly weaned beef cattle, 180 single-source steers (278 ± 21 kg; SD) were used in a 56-d receiving study. Seven days after arrival, steers were blocked by body weight (BW) to pens of 6 and randomly assigned to treatments: SCFP at 0 (CON), 14 (SCFP14), or 28 (SCFP28) g·steer-1·d-1. Pen was the experimental unit (n = 10 per treatment). On day 0, steers were boostered against Bovine Viral Diarrhea Virus (BVDV) Type 1 and 2 (Vista Once, Merck, Madison, NJ). Weights were collected on days 1, 0, 14, 27, 42, 55, and 56. One steer per pen was bled on days 0, 14, 27, 42, and 56 for analysis of BVDV antibody titers; blood from days 0, 27, and 56 was analyzed for red blood cell lysate superoxide dismutase (SOD) activity and glutathione (total = tGSH, oxidized = GSSG, and reduced = GSH) concentrations, plasma malondialdehyde (MDA) concentrations, and serum lysozyme activity. Performance and blood data were analyzed as a randomized complete block design using Proc Mixed of SAS with fixed effects of treatment and block and random effect of pen. Linear and quadratic contrast statements were used. Antibody titers were log transformed and analyzed as repeated measures. There were no treatment by day interactions (P ≥ 0.16), and no linear or quadratic effects of SCFP on feedlot performance, antibody titers, or lysozyme activity (P > 0.10). Day 27 MDA concentrations tended to linearly increase (P = 0.09). A quadratic effect of SCFP on day 56 SOD activity (P = 0.004) was driven by lesser activity for SCFP14-fed steers. On day 27, a tendency for a quadratic effect of SCFP (P = 0.09) on GSH was driven by greater concentrations for SCFP14-fed steers resulting in a lesser GSSG:GSH ratio (P = 0.05). Greater GSH for SCFP14-fed steers caused a tendency for a quadratic effect on day 56 (P = 0.07); however, this did not result in an effect of SCFP on the GSSG:GSH ratio (P ≥ 0.25). A tendency for a linear effect of SCFP on tGSH was noted on day 56 (P = 0.09). Morbidity data were analyzed using Proc Glimmix of SAS. There was a quadratic effect of SCFP on percentage of respiratory treatments prior to day 14 (P = 0.04). These results could indicate lesser levels of oxidative stress for steers receiving SCFP at 14 vs. 0 or 28 g/d. Under the conditions of this study, no performance benefit of SCFP was noted.

Distribution of arsenic and oxidative stress in mice after rice ingestion

This study evaluated the effects of rice naturally contaminated with arsenic (As) and the same As-species added as solubilized-salt on the redox state and the As distribution in male mice. The total As amount in the Brazilian polished rice used in this study was 169.81±6.12ngg−1. Indeed, the concentrations of As species were 40.77ngg−1 (arsenite, As3+), 65.71ngg−1 (dimethylarsinic acid, DMA), 11.90ngg−1 (monomethylarsonic acid, MMA), and 25.96ngg−1 (arsenate, As5+). In this sense, animals were randomly divided into seven groups with six mice per group: I) control: habitual food; II) rice I: diet containing 10% of rice naturally containing As (4.08μg As3+/kg diet, 6.57μg DMA/kg diet, 1.19μg MMA/kg diet, and 2.60μg As5+/kg diet); III) rice II: diet containing 20% of rice naturally containing As (8.15μg As3+/kg diet, 13.14μg DMA/kg diet, 2.38μg MMA/kg diet, and 5.19μg As5+/kg diet); IV) rice III: diet containing 40% of rice naturally containing As (16.31μg As3+/kg diet, 26.28μg DMA/kg diet, 4.76μg MMA/kg diet, and 10.38μg As5+/kg diet); V) spiked feed I: diet containing As species as solubilized-salt (concentration equivalent to group II); VI) spiked feed II: diet containing As species as solubilized–salt (concentration equivalent to group III); VII) spiked feed III: diet containing As species as solubilized-salt (concentration equivalent to group IV). The time of treatment was 100days. After euthanasia, it was observed an increase in total As concentration in tissue samples of groups treated with diet containing rice naturally contaminated and diet containing As species. For instance, the highest As concentrations (higher than 330ngg−1) was observed in the bladder of animals belonging to Rice II and III and spiked feed III groups. Furthermore, it was verified the highest As concentrations in bladder > hair > lung > kidney > liver > blood. We also observed the presence of DMA and As5+ in liver and kidneys. Regarding oxidative stress biomarkers, we observed significant reduction of Glutathione (GSH) concentration in blood of animals belonging to groups IV, V, VI and VII. Besides, the antioxidant enzymes activities, Glutathione Peroxidase (GSH-Px), Catalase (CAT) and superoxide dismutase (SOD), increased significantly in blood of animals belonging to group VII. On the other hand, it was not observed differences in nitric oxide (NO) levels among all the groups used in this study and the control group. Thus, we conclude that some minor effects were found in mice exposed to the diet containing the highest amount of rice naturally contaminated with As species. These findings contribute to evaluate the safety of human dietary consumption.

Amyloid fibril systems reduce, stabilize and deliver bioavailable nanosized iron

Iron-deficiency anaemia (IDA) is a major global public health problem. A sustainable and cost-effective strategy to reduce IDA is iron fortification of foods, but the most bioavailable fortificants cause adverse organoleptic changes in foods. Iron nanoparticles are a promising solution in food matrices, although their tendency to oxidize and rapidly aggregate in solution severely limits their use in fortification. Amyloid fibrils are protein aggregates initially known for their association with neurodegenerative disorders, but recently described in the context of biological functions in living organisms and emerging as unique biomaterial building blocks. Here, we show an original application for these protein fibrils as efficient carriers for iron fortification. We use biodegradable amyloid fibrils from β-lactoglobulin, an inexpensive milk protein with natural reducing effects, as anti-oxidizing nanocarriers and colloidal stabilizers for iron nanoparticles. The resulting hybrid material forms a stable protein-iron colloidal dispersion that undergoes rapid dissolution and releases iron ions during acidic and enzymatic in vitro digestion. Importantly, this hybrid shows high in vivo iron bioavailability, equivalent to ferrous sulfate in haemoglobin-repletion and stable-isotope studies in rats, but with reduced organoleptic changes in foods. Feeding the rats with these hybrid materials did not result in abnormal iron accumulation in any organs, or changes in whole blood glutathione concentrations, inferring their primary safety. Therefore, these iron-amyloid fibril hybrids emerge as novel, highly effective delivery systems for iron in both solid and liquid matrices.

A Pilot Study of High-Dose N-Acetylcysteine Infusion in Patients with Sickle Cell Disease

▪We have completed a clinical trial evaluating the safety of N-acetylcysteine (NAC) infusion in adult patients with sickle cell disease (SCD) at disease baseline (NCT01800526). This trial was inspired by the hypothesis that von Willebrand factor (VWF) has an important role in the pathophysiology of vaso-occlusion and hemolysis in SCD and that NAC can reduce signs and symptoms of the disease by reducing the activity of VWF. A secondary hypothesis was that NAC could also act by reversing or preventing some of the oxidative changes associated with SCD.VWF is stored in the Weibel-Palade bodies of endothelial cells and α-granules of platelets and released from endothelial cells upon activation and mediates the attachment of platelets to the vessel wall, and secondarily, of erythrocytes and leukocytes. We previously showed that VWF levels and adhesive activity in SCD plasma were elevated and that total active VWF correlated with the extent of hemolysis (determined by plasma LDH levels), suggesting that VWF participates in the pathophysiology of SCD (Chen et al, Blood. 2011; 117:3680‐3). In another study, we showed that NAC was able to reduce VWF multimer size and platelet-binding activity ex vivo in human plasma and in vivo in mice (Chen et al, JCI, 2011, 121:522).In this clinical trial, we enrolled 5 subjects at disease baseline, 4 with SCD and 1 with sickle trait. All subjects received two high-dose NAC infusions: initially 150 mg/kg and 4 weeks later 300 mg/kg. NAC infusion was administered in subjects 1 and 2 as a bolus infusion of half of the dose in the 1sthour and the remaining half in 7 hr. Subjects 3-5 received NAC as constant infusion for 8 hr. Blood was collected immediately before infusion, during infusion (at 1 and 4 hr), immediately after infusion (8 hr), and 24 and 72 hr after infusion. We examined blood for red blood cell (RBC) fragments, dense cells, platelet activation status, platelet-monocyte complexes and the concentrations of reduced and oxidized glutathione. We also examined plasma for VWF concentration and multimer distribution, ADAMTS13 antigen and activity, and evaluated plasma redox status by measuring the concentrations of NAC and cysteine in their reduced, oxidized, and mixed disulfide forms, as well as the concentration of protein-bound cysteine. The small molecule thiols were measured by mass spectrometry.Results:1) All subjects tolerated NAC infusion well, except that subjects 1 and 2 experienced pruritus during the bolus infusion at the 300 mg/kg dose. We therefore changed the protocol to deliver the drug over 8 hr by continuous infusion for subjects 3 through 5. 2) During NAC infusion, the percentage of dense cells and concentration of RBC fragments decreased rapidly (average decrease: low dose 44%; high dose 31%) and the change persisted up to 72 hr. 3) Platelet activation also decreased, as determined by reduced P-selectin expression (low dose, 7.7 ± 0.7% to 5.0 ± 1.9%; high dose, 17.8 ± 0.07% to 7.3% ± 0.4), PAC-1 binding (evaluates activated αΙΙbβ3), and formation of platelet-monocyte complexes (monocytes with attached platelets: low dose, 52 ± 16% to 27 ± 12%; high dose, 77 ± 12% to 50 ± 17%). 4) The highest molecular weight VWF multimers transiently disappeared during the NAC infusion and recovered by 24 hr. There were no appreciable changes in VWF antigen or ADAMTS13 antigen or activity with NAC infusion. 5) With NAC infusion, the concentrations of Cys and NAC increased in plasma, both total and reduced forms, with a concomitant decrease in protein-bound Cys in the one patient studied (free thiol Cys increased 23 fold with the high dose infusion at 8 hr; protein-bound Cys decreased 87%). Similarly, the concentrations of reduced and oxidized glutathione increased in whole blood.Summary: NAC infusion in SCD patients at disease baseline appears safe and is well tolerated. NAC infusion decreases RBC fragments, dense cells, and the size of large VWF multimers, and inhibits platelet activation and formation of platelet-monocyte complexes. In addition, NAC infusion increases the total and free-thiol concentrations of cysteine and glutathione in blood, relieving oxidative stress in SCD. Our data suggest that NAC may be of benefit for SCD patients during vaso-occlusive crisis through a variety of mechanisms, which include improvements in red cell parameters, decreased platelet adhesion, reduced adhesive activity of VWF, and protection against oxidative damage. DisclosuresNo relevant conflicts of interest to declare.

The protective role of silymarin against methotrexate-induced gastrointestinal injury

The protective effect of silymarin was investigated against methotrexate-induced GI injury. Forty Swiss-Albino mice were divided into 4 groups as control (Normal saline), methotrexate (15 mg/kg), silymarin (100 mg/kg) and methotrexate+silymarin (100 mg/kg + 15 mg/kg respectively). Blood and tissue samples were collected from the animals for biochemical and pathological examinations. Plasma, intestinal and gastric tissue malondialdehyde and TSA levels in methotrexate group were significantly higher than in control whereas whole blood glutathione concentration was lower in methotraxe group than in control. Plasma, intestinal and gastric TSA levels in methotrexate group were significantly increased compared to control and methotrexate+silymarin groups. Gastric tissue of methotrexate treated mice showed degeneration, necrosis, desquamation and widespread edema. However, these alterations were less severe in methotrexate+silymarin group. In conclusion, silymarin could be of therapeutic value against methotrexate-induced GI injury.

Increased oxidative stress from early pregnancy in women who develop preeclampsia

Preeclampsia (PE) is a pregnancy-specific disorder, defined as new onset of maternal hypertension and proteinuria after 20 weeks of gestation. Our earlier study has shown increased maternal oxidative stress at delivery to be associated with poor birth outcome in PE. However, these results were observed when the pathology had progressed and may have been secondary to the effects of the disorder. To understand the role of antioxidant defense mechanisms in PE right from early pregnancy, in this prospective study, we measured malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione (GSH) concentrations in maternal blood at 3 time-points of gestation [16–20 weeks (T1), 26–30 weeks (T2), at delivery (T3)] and in cord blood. Gene expression of SOD and GPx and protein levels of endothelial nitric oxide synthase (eNOS) enzyme were also analyzed in the placenta. MDA levels were higher at T1 (p < 0.01) and T2 (p < 0.01) in women with PE as compared with control. GPx levels were higher at T3 (p < 0.05) while SOD levels were lower at T2 (p < 0.05), T3 (p < 0.01) and in cord (p < 0.01) in PE. GSH levels at T1 (p < 0.05) and expression of GPx in the placenta were lower in PE as compared with control. In conclusion, this study demonstrates that women who develop PE exhibit increased oxidative stress right from 16 to 20 weeks of gestation. This may alter placental development and lead to fetal programming of adult non-communicable disease in the offspring.

Repeated-dose oral N-acetylcysteine: Effect on brain and blood glutathione concentrations

CO RR EC TE D P RO OF practice in patients with MPS II. The records of twenty-one children with MPS II currently in our clinic were reviewed and the presence/ absence of the 7 key clinical markers of neurological function was noted (increased activity, behaviour difficulties, seizure-like behaviours, sleep problems, bladder training, bowel training and perseverative chewing). The score was repeated at sequential reviews from age at diagnosis until age 7 years or neurological involvement was obvious or until most recent assessment. Thirteen (65%) children developed CNS involvement. Median age at diagnosis due to symptoms was 30 months (range 10 to 72 months). Four children were diagnosed between birth and 8 months due to family history. Of the key clinical markers, behaviour and increased activity were the most frequently reported symptoms. The earliest features suggestive of neurological sub-type were sleep disturbance and aggressive behaviour. Increased activity and late bladder and bowel control were only observed in the neurological group. Abnormal chewing behaviour was never reported by parents. There were no seizure-like episodes in the whole cohort. This tool discriminated well between MPS II with and without neurological involvement in our patient cohort, however, the neurological phenotype was never diagnosed with clinical certainty before 3 years of age and more usually at 4 years. The presence of aggressive behaviour is likely a good indicator of impending neurological problems in MPS II. On the other hand several of the key factors of the scoring system such as seizure-like behaviour or bladder and bowel training were less helpful. Perseverative chewing was not present but this feature may not have been directly sought in the history. This study is limited by its historical review methodology and the scoring system should be tested prospectively. A scoring system does allow the paediatrician to make a focussed assessment of a newly diagnosed child. However, even with this focus it appears difficult to diagnose neurological MPS II clinically with any certainty before 3 years of age. Ultimately in order to benefit from new treatments aimed at the CNS, it is preferable that a biomarker is found. Holt J, Poe MD, Escolar ML: Early clinical markers of central nervous system involvement in mucopolysaccharidosis type II.: J Pediatr. 2011 Aug; 159(2): 320–6.

Improvement of pulmonary function with arm swing exercise in patients with type 2 diabetes.

[Purpose] Obesity and hyperglycemia play roles in the impairment of pulmonary function in type 2 diabetes mellitus (T2DM) patients. Low-intensity exercise is known to reduce body fat and improve hyperglycemia. The arm swing exercise (ASE), a low-intensity exercise, is easy and convenient to perform without any equipment and is suitable for daily practice. Therefore, we aimed to investigate the effects of ASE on lung function and obesity in overweight T2DM patients. [Subjects and Methods] Twenty-four subjects continued their daily life routines for 8 weeks (control period), and then performed ASE for 8 weeks (30 minutes per day, 3 days per week) (ASE period). Pulmonary function tests were performed, and fasting blood glucose, haemoglobin A1c (HbA1c), lipid profiles, high-sensitive C-reactive protein (HSCRP), insulin concentration, and anthropometric parameters were measured before and after each period. [Results] After the ASE period, the forced vital capacity, forced expiratory volume in the first second of expiration, and maximal voluntary ventilation were increased when compared with after the control period. HbA1c, a low-density lipoprotein, malondialdehyde, oxidized glutathione, and the percent body fat were significantly decreased when compared with after the control period. However, other parameters, such as lung volume, anthropometric parameters, and fasting blood glucose, insulin, high-density lipoprotein, triglycerides, total cholesterol and glutathione concentrations, showed no differences between the two periods. [Conclusion] These data suggest that there is improvement of pulmonary functions in T2DM patients after ASE training.

Effects of N-Acetylcysteine in Patients with Sickle Cell Disease

We previously showed that the plasma of patients with sickle cell disease (SCD) contains an elevated quantity of von Willebrand factor (VWF) with high specific adhesive activity (as measured by the binding of nanobody AU/VWFa-11). Total active VWF (VWF antigen multiplied by VWF specific activity) correlated with the extent of hemolysis in these patients (Chen J. et al., Blood, 2011, 117:3680). In another study, we showed that N-acetylcysteine (NAC) reduced VWF size and activity ex vivo and broke down platelet–VWF aggregates in vivo in mice (Chen J. et al, JCI, 2011, 121:522). Given the possibility that VWF is involved in the pathophysiology of sickle cell disease, we are examining whether NAC can benefit these patients. We are first conducting a pilot clinical trial to determine the safety profile of infused NAC and its effects on laboratory endpoints. Five SCD patients at disease baseline and not recently transfused will be enrolled. To date, two patients have completed intravenous infusion of NAC at a low dose of 150 mg/kg with a bolus infusion of 75 mg/kg for the 1st hr and 75 mg/kg for 7 hr. Approximately one month after the low-dose infusion, these patients received another infusion of 300 mg/kg given as a bolus infusion of 150 mg/kg for the 1st hr and 150 mg/kg for 7 hr. Blood was collected for analysis immediately before the infusion, at 1 hr (after bolus infusion), 8 hr, 24 hr, and 72 hr after infusion. Among the parameters examined were plasma VWF, red blood cell concentration, density, and size (to look for fragments), and the concentrations of NAC, cysteine and glutathione and their oxidized and mixed disulfide forms. In these two patients, the NAC infusion was well tolerated except that both patients experienced pruritus during the higher-dose bolus infusion. We measured the concentrations of reduced and oxidized NAC, cysteine, and glutathione in whole blood in the first study subject by mass spectrometry. NAC concentrations were 725 μM and 1.58 mM at the 1 hr time point at the low and high doses, respectively. Compared to the baseline (before NAC infusion), the concentration of total cysteine in blood was increased 2.4 fold for low dose and 2.9 fold for high dose at 1 hr and returned to baseline at 8 hr. The concentration of total glutathione in whole blood was increased 1.5 fold and 1.3 fold at 24, and 72 hr, respectively, for the high dose infusion but did not change much at the low dose infusion. The size of high molecular weight VWF multimers decreased with the high dose infusion, the effect being obvious at 1 hr. In addition, NAC infusion markedly reduced the concentration of red cell fragments and dense cells. Both of these effects were very rapid, being observable at the 1 hr time point. In summary, NAC infusion in sickle cell patients at disease baseline appears safe. NAC increases the concentrations of total cysteine and glutathione in blood, reduces high molecular weight VWF multimers, and decreases the number of dense red blood cells and the extent of red cell fragmentation. DisclosuresNo relevant conflicts of interest to declare.