Antenatal creatine supplementation reduces persistent fetal lung inflammation and oxidative stress in an ovine model of chorioamnionitis.

Chorioamnionitis is a common antecedent of preterm birth and induces inflammation and oxidative stress in the fetal lungs. Reducing inflammation and oxidative stress in the fetal lungs may improve respiratory outcomes in preterm infants. Creatine is an organic acid with known anti-inflammatory and antioxidant properties. The objective of the study was to evaluate the efficacy of direct fetal creatine supplementation to reduce inflammation and oxidative stress in fetal lungs arising from an in utero proinflammatory stimulus. Fetal lambs (n = 51) were instrumented at 90 days gestation to receive a continuous infusion of creatine monohydrate (6 mg·kg-1·h-1) or saline for 17 days. Maternal chorioamnionitis was induced with intra-amniotic lipopolysaccharide (LPS; 1 mg, O55:H6) or saline 7 days before delivery at 110 days gestation. Tissue creatine content was assessed with capillary electrophoresis, and inflammatory markers were analyzed with Luminex Magpix and immunohistochemistry. Oxidative stress was measured as the level of protein thiol oxidation. The effects of LPS and creatine were analyzed using a two-way ANOVA. Fetal creatine supplementation increased lung creatine content by 149% (PCr < 0.0001) and had no adverse effects on lung morphology. LPS-exposed groups showed increased levels of interleukin-8 in the bronchoalveolar lavage (PLPS < 0.0001) and increased levels of CD45+ leukocytes (PLPS < 0.0001) and MPO+ (PLPS < 0.0001) cells in the lung parenchyma. Creatine supplementation significantly reduced the levels of CD45+ (PCr = 0.045) and MPO+ cells (PCr = 0.012) in the lungs and reduced thiol oxidation in plasma (PCr < 0.01) and lung tissue (PCr = 0.02). In conclusion, fetal creatine supplementation reduced markers of inflammation and oxidative stress in the fetal lungs arising from chorioamnionitis.NEW & NOTEWORTHY We evaluated the effect of antenatal creatine supplementation to reduce pulmonary inflammation and oxidative stress in the fetal lamb lungs arising from lipopolysaccharide (LPS)-induced chorioamnionitis. Fetal creatine supplementation increased lung creatine content and had no adverse effects on systemic fetal physiology and overall lung architecture. Importantly, fetuses that received creatine had significantly lower levels of inflammation and oxidative stress in the lungs, suggesting an anti-inflammatory and antioxidant benefit of creatine.

Temporal tracking of cysteine 34 oxidation of plasma albumin as a biomarker of muscle damage following a bout of eccentric exercise.

PURPOSE: Exercise-induced muscle damage (EIMD) results in the generation of reactive oxygen species (ROS), but little is known about the temporal profile of change in ROS post-EIMD and how ROS levels relate to the onset of and recovery from EIMD. Our primary aim was to examine the effect of EIMD on the pattern of change in the blood level of thiol-oxidised albumin, a marker of oxidative stress. METHODS: Seven male participants were subjected on separate days to eccentric muscle contraction to cause EIMD or a no-exercise condition. After each session, the participants collected daily dried blood spots to measure thiol-oxidised albumin and returned to the laboratory every 2 days for the assessment of indirect markers of EIMD, namely maximal voluntary contraction (MVC), delayed onset muscle soreness (DOMS), creatine kinase (CK), and myoglobin. RESULTS: Eccentric exercise resulted in a significant decrease in MVC and increase in DOMS, CK, myoglobin, and thiol-oxidised albumin with the latter reaching above baseline level within 24-48 h post-exercise. All the markers of EIMD returned to baseline level within 6 days post-exercise, but not the level of thiol-oxidised albumin which remained elevated for 10 days after exercise. There was a moderate correlation between changes in thiol-oxidised albumin and DOMS, but no significant relationship between any other markers of muscle damage. CONCLUSION: The levels of thiol-oxidised albumin increase in response to EIMD and remain elevated for several days post-exercise. The temporal pattern of change in the level of thiol-oxidised albumin suggests that this may be a useful biomarker of muscle repair post-EIMD.

Hypochlorous acid exposure impairs skeletal muscle function and Ca2+ signalling: implications for Duchenne muscular dystrophy pathology.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease characterised by severe muscle wasting. The mechanisms underlying the DMD pathology likely involve the interaction between inflammation, oxidative stress and impaired Ca2+ signalling. Hypochlorous acid (HOCl) is a highly reactive oxidant produced endogenously via myeloperoxidase; an enzyme secreted by neutrophils that is significantly elevated in dystrophic muscle. Oxidation of Ca2+ -handling proteins by HOCl may impair Ca2+ signalling. This study aimed to determine the effects of HOCl on skeletal muscle function and its potential contribution to the dystrophic pathology. Extensor digitorum longus (EDL), soleus and interosseous muscles were surgically isolated from anaesthetised C57 (wild-type) and mdx (dystrophic) mice for measurement of ex vivo force production and intracellular Ca2+ concentration. In whole EDL muscle, HOCl (200 µM) significantly decreased maximal force and increased resting muscle tension which was only partially reversible by dithiothreitol. The effects of HOCl (200 µM) on maximal force in slow-twitch soleus were lower than found in the fast-twitch EDL muscle. In single interosseous myofibres, HOCl (10 µM) significantly increased resting intracellular Ca2+ concentration and decreased Ca2+ transient amplitude. These effects of HOCl were reduced by the application of tetracaine, Gd3+ or streptomycin, implicating involvement of ryanodine receptors and transient receptor potential channels. These results demonstrate the potent effects of HOCl on skeletal muscle function potentially mediated by HOCl-induced oxidation to Ca2+ signalling proteins. Hence, HOCl may provide a link between chronic inflammation, oxidative stress and impaired Ca2+ handling that is characteristic of DMD and presents a potential therapeutic target for DMD. KEY POINTS: Duchenne muscular dystrophy is a fatal genetic disease with pathological mechanisms which involve the complex interaction of chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations. Hypochlorous acid can be endogenously produced by neutrophils via the enzyme myeloperoxidase. Both neutrophil and myeloperoxidase activity are increased in dystrophic mice. This study found that hypochlorous acid decreased muscle force production and increased cytosolic Ca2+ concentrations in isolated muscles from wild-type and dystrophic mice at relatively low concentrations of hypochlorous acid. These results indicate that hypochlorous acid may be key in the Duchenne muscular dystrophy disease pathology and may provide a unifying link between the chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations observed in Duchenne muscular dystrophy. Hypochlorous acid production may be a potential target for therapeutic treatments of Duchenne muscular dystrophy.

Isotope-Coded Maleimide Affinity Tags for Proteomics Applications.

Isotope-coded affinity tags (ICATs) are valuable tools for mass spectrometry-based quantitative proteomics, in particular, for comparison of protein (cysteine-residue) thiol oxidation state in normal, stressed, and diseased tissue. However, the iodoacetamido electrophile used in most commercial ICATs suffers from poor thiol-selectivity and modest rates of adduct formation, which can lead to spurious results. Hence, we designed and synthesized three ICATs containing thiol-selective N-alkylmaleimide electrophiles (isotope-coded maleimide affinity tags = ICMATs) and assessed these as mass spectrometry probes for ratiometric analysis of lysozyme and muscle proteomes. Two ICMAT pairs containing butylene/D8-butylene linkers were effective MS probes, but not ideal for typical proteomics workflows, because peptides bearing these tags frequently did not coelute with HPLC. A switch to a phenylene/13C6-phenylene linker solved this issue without compromising the efficiency of adduct formation.

Investigation of the effect of taurine supplementation on muscle taurine content in the mdx mouse model of Duchenne muscular dystrophy using chemically specific synchrotron imaging.

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle wasting disorder, which currently has no cure. Supplementation with the drug taurine has been shown to offer therapeutic benefit in the mdx model for DMD, however the mechanism by which taurine protects dystrophic muscle is not fully understood. Mdx muscle is deficient in taurine, however it is not known if this deficiency occurs in the extracellular space, in other cells present in the tissue (such as immune cells) or in the myofibre itself. Likewise, the tissue location of taurine enrichment in taurine treated mdx muscle is not known. In this study we applied X-ray absorption near edge spectroscopy (XANES) at the sulfur K-edge in an imaging format to determine taurine distribution in muscle tissue. XANES is the only technique currently capable of imaging taurine directly in muscle tissue, at a spatial resolution approaching myocyte cell size (20-50 µm). Using a multi-modal approach of XANES imaging and histology on the same tissue sections, we show that in mdx muscle, it is the myofibres that are deficient in taurine, and taurine supplementation ameliorates this deficiency. Increasing the taurine content of mdx myofibres was associated with a decrease in myofibre damage (as shown by the percentage of intact myofibres) and inflammation. These data will help drive future studies to better elucidate the molecular mechanisms through which taurine protects dystrophic muscle; they also support the continued investigation of taurine as a therapeutic intervention for DMD.

Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation.

Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (∼14%) of gels and proteolytic digestion buffers (∼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.

Pre-clinical evaluation of N-acetylcysteine reveals side effects in the mdx mouse model of Duchenne muscular dystrophy.

KEY POINTS: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation and oxidative stress. The antioxidant N-acetylcysteine (NAC) has been proposed as a therapeutic intervention for DMD boys, but potential adverse effects of NAC have not been widely investigated. We used young (6 weeks old) growing mdx mice to investigate the capacity of NAC supplementation (2% in drinking water for 6 weeks) to improve dystrophic muscle function and to explore broader systemic effects of NAC treatment. NAC treatment improved normalised measures of muscle function, and decreased inflammation and oxidative stress, but significantly reduced body weight gain, muscle weight and liver weight. Unexpected significant adverse effects of NAC on body and muscle weights indicate that interpretation of muscle function based on normalised force measures should be made with caution and careful consideration is needed when proposing the use of NAC as a therapeutic treatment for young DMD boys. ABSTRACT: Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disease characterised by severe muscle weakness, necrosis, inflammation and oxidative stress. The antioxidant N-acetylcysteine (NAC) has been proposed as a potential therapeutic intervention for DMD boys. We investigated the capacity of NAC to improve dystrophic muscle function in the mdx mouse model of DMD. Young (6 weeks old) mdx and non-dystrophic C57 mice receiving 2% NAC in drinking water for 6 weeks were compared with untreated mice. Grip strength and body weight were measured weekly, before the 12 week old mice were anaesthetised and extensor digitorum longus (EDL) muscles were excised for functional analysis and tissues were sampled for biochemical analyses. Compared to untreated mice, the mean (SD) normalised grip strength was significantly greater in NAC-treated mdx [3.13 (0.58) vs 4.87 (0.78) g body weight (bw)-1 ; P < 0.001] and C57 mice [3.90 (0.32) vs 5.32 (0.60) g bw-1 ; P < 0.001]. Maximum specific force was significantly greater in NAC-treated mdx muscles [9.80 (2.27) vs 13.07 (3.37) N cm-2 ; P = 0.038]. Increased force in mdx mice was associated with reduced thiol oxidation and inflammation in fast muscles, and increased citrate synthase activity in slow muscle. Importantly, NAC significantly impaired body weight gain in both strains of young growing mice, and reduced liver weight in C57 mice and muscle weight in mdx mice. These potentially adverse effects of NAC emphasise the need for caution when interpreting improvements in muscle function based on normalised force measures, and that careful consideration be given to these effects when proposing NAC as a potential treatment for young DMD boys.

Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy.

KEY POINTS: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation, oxidative stress and myofibre necrosis. Cysteine precursor antioxidants such as N-acetyl cysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) reduce dystropathology in the mdx mouse model for DMD, and we propose this is via increased synthesis of the amino acid taurine. We compared the capacity of OTC and taurine treatment to increase taurine content of mdx muscle, as well as effects on in vivo and ex vivo muscle function, inflammation and oxidative stress. Both treatments increased taurine in muscles, and improved many aspects of muscle function and reduced inflammation. Taurine treatment also reduced protein thiol oxidation and was overall more effective, as OTC treatment reduced body and muscle weight, suggesting some adverse effects of this drug. These data suggest that increasing dietary taurine is a better candidate for a therapeutic intervention for DMD. ABSTRACT: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease for which there is no widely available cure. Whilst the mechanism of loss of muscle function in DMD and the mdx mouse model are not fully understood, disruptions in intracellular calcium homeostasis, inflammation and oxidative stress are implicated. We have shown that protein thiol oxidation is increased in mdx muscle, and that the indirect thiol antioxidant l-2-oxothiazolidine-4-carboxylate (OTC), which increases cysteine availability, decreases pathology and increases in vivo strength. We propose that the protective effects of OTC are a consequence of conversion of cysteine to taurine, which has itself been shown to be beneficial to mdx pathology. This study compares the efficacy of taurine with OTC in decreasing dystropathology in mdx mice by measuring in vivo and ex vivo contractile function and measurements of inflammation and protein thiol oxidation. Increasing the taurine content of mdx muscle improved both in vivo and ex vivo muscle strength and function, potentially via anti-inflammatory and antioxidant effects of taurine. OTC treatment increased taurine synthesis in the liver and taurine content of mdx muscle, improved muscle function and decreased inflammation. However, OTC was less effective than taurine treatment, with OTC also decreasing body and EDL muscle weights, suggesting that OTC had some detrimental effects. These data support continued research into the use of taurine as a therapeutic intervention for DMD, and suggest that increasing dietary taurine is the better strategy for increasing taurine content and decreasing severity of dystropathology.

Protein thiol oxidation does not change in skeletal muscles of aging female mice.

Oxidative stress caused by reactive oxygen species is proposed to cause age related muscle wasting (sarcopenia). Reversible oxidation of protein thiols by reactive oxygen species can affect protein function, so we evaluated whether muscle wasting in normal aging was associated with a pervasive increase in reversible oxidation of protein thiols or with an increase in irreversible oxidative damage to macromolecules. In gastrocnemius muscles of C57BL/6J female mice aged 3, 15, 24, 27, and 29 months there was no age related increase in protein thiol oxidation. In contrast, there was a significant correlation (R (2) = 0.698) between increasing protein carbonylation, a measure of irreversible oxidative damage to proteins, and loss of mass of gastrocnemius muscles in aging female mice. In addition, there was an age-related increase in lipofuscin content, an aggregate of oxidised proteins and lipids, in quadriceps limb muscles in aging female mice. However, there was no evidence of an age-related increase in malondialdehyde or F2-isoprostanes levels, which are measures of oxidative damage to lipids, in gastrocnemius muscles. In summary, this study does not support the hypothesis that a pervasive increase in protein thiol oxidation is a contributing factor to sarcopenia. Instead, the data are consistent with an aging theory which proposes that molecular damage to macromolecules leads to the structural and functional disorders associated with aging.

Oxidised Albumin Levels in Plasma and Skeletal Muscle as Biomarkers of Disease Progression and Treatment Efficacy in Dystrophic mdx Mice.

Redox modifications to the plasma protein albumin have the potential to be used as biomarkers of disease progression and treatment efficacy in pathologies associated with inflammation and oxidative stress. One such pathology is Duchenne muscular dystrophy (DMD), a fatal childhood disease characterised by severe muscle wasting. We have previously shown in the mdx mouse model of DMD that plasma albumin thiol oxidation is increased; therefore, the first aim of this paper was to establish that albumin thiol oxidation in plasma reflects levels within mdx muscle tissue. We therefore developed a method to measure tissue albumin thiol oxidation. We show that albumin thiol oxidation was increased in both mdx muscle and plasma, with levels correlated with measures of dystropathology. In dystrophic muscle, albumin content was associated with areas of myonecrosis. The second aim was to test the ability of plasma thiol oxidation to track acute changes in dystropathology: we therefore subjected mdx mice to a single treadmill exercise session (known to increase myonecrosis) and took serial blood samples. This acute exercise caused a transient increase in total plasma albumin oxidation and measures of dystropathology. Together, these data support the use of plasma albumin thiol oxidation as a biomarker to track active myonecrosis in DMD.

Longitudinal analysis of protein glycosylation and ß-casein phosphorylation in term and preterm human milk during the first 2 months of lactation.

Human milk proteins provide term and preterm infants with both nutrition and protection. The objective of the present study was to examine longitudinal changes in the protein composition of term and preterm milk during the first 2 months of lactation, focusing on protein phosphorylation and glycosylation. Using gel electrophoresis, the relative concentration and glycosylation status of lactoferrin, secretory Ig A, ß-casein, α-lactalbumin, serum albumin, bile salt-stimulated lipase, xanthine oxidoreductase, tenascin and macrophage mannose receptor 1 were measured in milk collected on days 7, 10, 14, 18, 21, 28 and 60 postpartum from preterm mothers (28-32 weeks gestation, n 17). The phosphorylation status of ß-casein was also investigated. To determine if these variables differ in term and preterm milk, samples from term mothers (38-41 weeks gestation, n 8) collected on days 7, 14 and 30 of lactation were also analysed. The concentration of the abundant milk proteins decreased during lactation in term and preterm milk (P <0·05). No difference in protein glycosylation was observed, except for the glycoproteins serum albumin and tenascin. The phosphorylation of ß-casein varied significantly between term and preterm milk. Further investigation is required to determine whether these modifications affect protein function and are clinically important to preterm infants.

Muscle Pathology in Dystrophic Rats and Zebrafish Is Unresponsive to Taurine Treatment, Compared to the mdx Mouse Model for Duchenne Muscular Dystrophy.

Inflammation and oxidative stress are strongly implicated in the pathology of Duchenne muscular dystrophy (DMD), and the sulphur-containing amino acid taurine ameliorates both and decreases dystropathology in the mdx mouse model for DMD. We therefore further tested taurine as a therapy using dystrophic DMDmdx rats and dmd zebrafish models for DMD that have a more severe dystropathology. However, taurine treatment had little effect on the indices of dystropathology in both these models. While we and others have previously observed a deficiency in taurine in mdx mice, in the current study we show that the rat and zebrafish models had increased taurine content compared with wild-type, and taurine treatment did not increase muscle taurine levels. We therefore hypothesised that endogenous levels of taurine are a key determinate in potential taurine treatment efficacy. Because of this, we felt it important to measure taurine levels in DMD patient plasma samples and showed that in non-ambulant patients (but not in younger patients) there was a deficiency of taurine. These data suggest that taurine homeostasis varies greatly between species and may be influenced by age and disease progression. The potential for taurine to be an effective therapy may depend on such variables.

Dimethyl fumarate modulates the dystrophic disease program following short-term treatment.

New medicines are urgently required to treat the fatal neuromuscular disease Duchenne muscular dystrophy (DMD). Dimethyl fumarate (DMF) is a potent immunomodulatory small molecule nuclear erythroid 2-related factor 2 activator with current clinical utility in the treatment of multiple sclerosis and psoriasis that could be effective for DMD and rapidly translatable. Here, we tested 2 weeks of daily 100 mg/kg DMF versus 5 mg/kg standard-care prednisone (PRED) treatment in juvenile mdx mice with early symptomatic DMD. Both drugs modulated seed genes driving the DMD disease program and improved force production in fast-twitch muscle. However, only DMF showed pro-mitochondrial effects, protected contracting muscles from fatigue, improved histopathology, and augmented clinically compatible muscle function tests. DMF may be a more selective modulator of the DMD disease program than PRED, warranting follow-up longitudinal studies to evaluate disease-modifying impact.

Proteome mapping of human skim milk proteins in term and preterm milk.

The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. However, relatively little is known about the expression of the low abundance proteins that are present in human milk because of the technical difficulties associated with their detection. We used a combination of electrophoretic techniques, ProteoMiner treatment, and two-dimensional liquid chromatography to examine the proteome of human skim milk expressed between 7 and 28 days postpartum by healthy term mothers and identified 415 in a pooled milk sample. Of these, 261 were found in human skim milk for the first time, greatly expanding our understanding of the human skim milk proteome. The majority of the proteins identified were involved in either the immune response (24%) or in cellular (28%) or protein (16%) metabolism. We also used iTRAQ analysis to examine the effects of premature delivery on milk protein composition. Differences in protein expression between pooled milk from mothers delivering at term (38-41 weeks gestation) and preterm (28-32 weeks gestation) were investigated, with 55 proteins found to be differentially expressed with at least 90% confidence. Twenty-eight proteins were present at higher levels in preterm milk, and 27 were present at higher levels in term milk.

The location of protein oxidation in dystrophic skeletal muscle from the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.

A Blood Biomarker for Duchenne Muscular Dystrophy Shows That Oxidation State of Albumin Correlates with Protein Oxidation and Damage in Mdx Muscle.

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle wasting disease with no cure. While the precise mechanisms of progressive dystropathology remain unclear, oxidative stress caused by excessive generation of oxidants is strongly implicated. Blood biomarkers that could track oxidant levels in tissues would be valuable to measure the effectiveness of clinical treatments for DMD; our research has focused on developing such biomarkers. One target of oxidants that has the potential to be harnessed as a clinical biomarker is the thiol side chain of cysteine 34 (Cys34) of the blood protein albumin. This study using the mdx mouse model of DMD shows that in plasma, albumin Cys34 undergoes thiol oxidation and these changes correlate with levels of protein thiol oxidation and damage of the dystrophic muscles. A comparison with the commonly used biomarker protein carbonylation, confirmed that albumin thiol oxidation is the more sensitive plasma biomarker of oxidative stress occurring in muscle tissue. We show that plasma albumin oxidation reflects muscle dystropathology, as increased after exercise and decreased after taurine treatment of mdx mice. These data support the use of albumin thiol oxidation as a blood biomarker of dystropathology to assist with advancing clinical development of therapies for DMD.

N-Acetyl Cysteine Restores Sirtuin-6 and Decreases HMGB1 Release Following Lipopolysaccharide-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Mice.

Inflammation and neonatal hypoxia-ischemia (HI) are important etiological factors of perinatal brain injury. However, underlying mechanisms remain unclear. Sirtuins are a family of nicotinamide adenine dinucleotide (NAD)+-dependent histone deacetylases. Sirtuin-6 is thought to regulate inflammatory and oxidative pathways, such as the extracellular release of the alarmin high mobility group box-1 (HMGB1). The expression and role of sirtuin-6 in neonatal brain injury are unknown. In a well-established model of neonatal brain injury, which encompasses inflammation (lipopolysaccharide, LPS) and hypoxia-ischemia (LPS+HI), we investigated the protein expression of sirtuin-6 and HMGB1, as well as thiol oxidation. Furthermore, we assessed the effect of the antioxidant N-acetyl cysteine (NAC) on sirtuin-6 expression, nuclear to cytoplasmic translocation, and release of HMGB1 in the brain and blood thiol oxidation after LPS+HI. We demonstrate reduced expression of sirtuin-6 and increased release of HMGB1 in injured hippocampus after LPS+HI. NAC treatment restored sirtuin-6 protein levels, which was associated with reduced extracellular HMGB1 release and reduced thiol oxidation in the blood. The study suggests that early reduction in sirtuin-6 is associated with HMGB1 release, which may contribute to neonatal brain injury, and that antioxidant treatment is beneficial for the alleviation of these injurious mechanisms.

Detecting changes in the thiol redox state of proteins following a decrease in oxygen concentration using a dual labeling technique.

Cells are routinely exposed to hyperoxic conditions when cultured in the presence of 95% air and 5% carbon dioxide. Hyperoxic conditions can increase the generation of reactive oxygen species and cause oxidative stress. Oxidative stress has been proposed to cause cells in culture to behave differently from cells in vivo. One route by which oxidative stress could affect cellular function is through alterations in protein function caused by the oxidation of thiol groups (-SH) of redox-sensitive cysteine residues. To test whether changes in oxygen concentration were sufficient to cause changes in the thiol redox state of proteins, we developed a sensitive method involving the labeling of reduced and oxidized cysteine residues with fluorescent tags. Using this dual labeling method, we found 62 of 411 protein spots that were significantly more reduced following a 30 min decrease in oxygen concentration. We conclude that the elevated oxygen concentration characteristic of typical cell culture conditions has the potential to affect cellular behavior through changes in the thiol redox state of proteins.

Ethanol decreases cellular protein content and mitochondrial membrane potential of cultured neonatal rat cardiomyocytes: microassays with fluorometric and spectrometric plate readers.

BACKGROUND: Ethanol decreases protein synthesis and mitochondrial respiration of the heart. We developed a new time-saving, non-radioactive, small-scale method to determine ethanol-induced alteration in cellular protein content of the cells cultivated in a 96-well plate. To verify the utility of our small-scale method, we used endothelin-1 (ET-1) as a positive control. METHODS: Neonatal rat cardiomyocytes were cultivated in a 96-well plate and exposed to ethanol (10, 50 and 100 mM) or ET-1 (100 nM). The protein content per cell was determined by measuring both the DNA (Hoechst 33258) and protein amounts of the same well with fluorometric and spectrometric plate readers. We also examined ultrastructure, mitochondrial membrane potential (deltapsim) using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide), anaerobic glycolysis measured by lactate amount released to the medium, and superoxide production using dihydroethidium. RESULTS: As expected, ethanol dose- and time-dependently decreased protein content per cell of cardiomyocytes at 24 and 48 h, while ET-1 time-dependently increased it. Ultrastructurally, ethanol decreased rough endoplasmic reticulum at 24 h, whereas ET-1 increased it as well as Golgi apparatus. Among ethanol groups, only 100 mM ethanol affected deltapsim: hyperpolarization just after exposure but slight depolarization at 24 h. ET-1 significantly depolarized deltapsim at 24 h, but the depolarization was within the physiological fluctuation. Ethanol did not change the lactate release at 3 and 24 h. Ethanol did not increase superoxide production at 24 h, but rather 10 mM ethanol significantly decreased it. CONCLUSION: Our small-scale method was able to demonstrate that ethanol and ET-1 induced expected alterations in protein content per cell with dose- and time-dependencies and statistically significant differences compared with the control, thereby validating the practical use of this method. Ultrastructural alterations supported the biochemical results. The ethanol-decreased cellular protein content might partly result from energy shortage attributable to reduced mitochondrial respiration, which was not compensated by anaerobic glycolysis.