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Glioblastoma (GBM) represents an aggressive brain tumor, characterized by intra- and inter-tumoral heterogeneity and therapy resistance, leading to unfavourable prognosis. An increasing number of studies pays attention on the regulation of ferroptosis, an iron-dependent cell death, as a strategy to reverse drug resistance in cancer. However, the debate on whether this strategy may have important implications for the treatment of GBM is still ongoing. In the present study, we used ferric ammonium citrate and erastin to evaluate ferroptosis induction effects on two human GBM cell lines, U-251 MG, with proneural characteristics, and T98-G, with a mesenchymal profile. The response to ferroptosis induction was markedly different between cell lines, indeed T98-G cells showed an enhanced antioxidant defence, with increased glutathione levels, as compared to U-251 MG cells. Moreover, using bioinformatic approaches and analysing publicly available datasets from patients' biopsies, we found that GBM with a mesenchymal phenotype showed an up-regulation of several genes involved in antioxidant mechanisms as compared to proneural subtype. Thus, our results suggest that GBM subtypes differently respond to ferroptosis induction, emphasizing the significance of further molecular studies on GBM to better discriminate between various tumor subtypes and progressively move towards personalized therapy., (© 2024. The Author(s).)

Study Question: Is the determination of antioxidants, oxidative/nitrosative stress-related compounds, purines, pyrimidines and energy-related metabolites in human seminal plasma of utility to evidence biomarkers related to male infertility?, Summary Answer: The determination of 26 metabolites in seminal plasma allowed to evidence that 21/26 of them are biomarkers of male infertility, as well as to calculate a cumulative index, named Biomarker Score, that fully discriminates fertile controls from infertile patients and partially differentiates infertile without from infertile with spermiogram anomalies., What Is Known Already: Epidemiological studies indicated that a male factor is involved in ~50% of cases of pregnancy failure, with a significant percentage of infertile males having no alterations in the spermiogram. Further laboratory analyses of male infertility are mainly dedicated only to gross evaluations of oxidative stress or total antioxidant capacity., Study Design, Size, Duration: Seminal plasma of 48 fertile controls and 96 infertile patients (master group), were collected from September 2016 to February 2018. A second group of 44 infertile patients (validation group) was recruited in a second, independent centre from September 2017 to March 2018. Samples were analysed in blind using a 'Redox Energy Test' to determine various low-molecular weight compounds, with the aim of finding metabolic profiles and biomarkers related to male infertility., Participants/materials, Setting, Methods: In all seminal plasma, 26 water- and fat-soluble compounds (related to antioxidant defences, oxidative/nitrosative stress, purine, pyrimidine and energy metabolism) were analysed using high-performance liquid chromatographic methods. According to spermiogram, infertile patients of both groups were also categorized into normozoospermic (N, no anomalies in the spermiogram), or into the subgroup including all patients with anomalies in the spermiogram (asthenoteratooligozoospermic ATO + asthenozoospermic A + teratozoospermic T + oligozoospermic O)., Main Results and the Role of Chance: In the master group, results indicated that 21/26 compounds assayed in seminal plasma of infertile males were significantly different from corresponding values determined in fertile controls. These 21 compounds constituted the male infertility biomarkers. Similar results were recorded in patients of the validation group. Using an index cumulating the biochemical seminal plasma anomalies (Biomarker Score), we found that fertile controls had mean Biomarker Score values of 2.01 ± 1.42, whilst infertile patients of the master and of the validation group had mean values of 12.27 ± 3.15 and of 11.41 ± 4.09, respectively (P < 0.001 compared to controls). The lack of statistical differences between the master and the validation groups, in both the metabolic profiles and the Biomarker Score values, allowed to pool patients into a single cohort of infertile males. The Biomarker Score values showed that fertile controls and infertile males clustered into two distinct groups. Infertile patients without (N, n = 42) or with (ATO + A + T + O, n = 98) spermiogram anomalies differed in some biomarkers (ascorbic acid, all-trans retinol, α-tocopherol, cytidine, uridine, guanine). These differences were reinforced by distribution frequencies and posterior probability curves of the Biomarker Score in the three groups., Limitations, Reasons for Caution: Results were obtained in relatively limited number of human seminal plasma samples. Using the 'Redox Energy Test' it was possible to associate specific metabolic profiles and values of the Biomarker Score to fertile controls or infertile males. However, it was not possible to evaluate whether the different anomalies of the spermiogram are associated with specific metabolic profiles and values of the Biomarker Score., Wider Implications of the Findings: The 'Redox Energy Test', coupled with the Biomarker Score that cumulates the biochemical characteristics of seminal plasma into a single index, evidenced a set of low-molecular weight biomarkers potentially useful in the laboratory management of male infertility., Study Funding/competing Interest(s): The study was partly funded with research grants from the University of Catania. None of the authors have any conflicting interests to declare.

Fat-soluble vitamins and antioxidants are of relevance in health and disease. Current methods to extract these compounds from biological fluids mainly need use of multi-steps and multi organic solvents. They are time-consuming and difficult to apply to treat simultaneously large sample number. We here describe a single-step, one solvent extraction of fat-soluble vitamins and antioxidants from biological fluids, and the chromatographic separation of all-trans-retinoic acid, 25-hydroxycholecalciferol, all-trans-retinol, astaxanthin, lutein, zeaxanthin, trans-β-apo-8'-carotenal, γ-tocopherol, β-cryptoxanthin, α-tocopherol, phylloquinone, lycopene, α-carotene, β-carotene and coenzyme Q 10 . Extraction is obtained by adding one volume of biological fluid to two acetonitrile volumes, vortexing for 60s and incubating for 60min at 37°C under agitation. HPLC separation occurs in 30min using Hypersil C18, 100×4.6mm, 5μm particle size column, gradient from 70% methanol+30% H 2 O to 100% acetonitrile, flow rate of 1.0ml/min and 37°C column temperature. Compounds are revealed using highly sensitive UV-VIS diode array detector. The HPLC method suitability was assessed in terms of sensitivity, reproducibility and recovery. Using the present extraction and chromatographic conditions we obtained values of the fat-soluble vitamins and antioxidants in serum from 50 healthy controls similar to those found in literature. Additionally, the profile of these compounds was also measured in seminal plasma from 20 healthy fertile donors. Results indicate that this simple, rapid and low cost sample processing is suitable to extract fat-soluble vitamins and antioxidants from biological fluids and can be applied in clinical and nutritional studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neuroglobin is a neuron-specific hexacoordinated globin capable of binding various ligands, including O2, NO, and CO, the biological function of which is still uncertain. Various studies seem to indicate that neuroglobin is a neuroprotective agent when overexpressed, acting as a potent inhibitor of oxidative and nitrosative stress. In this study, we evaluated the pathophysiological response of the neuroglobin gene and protein expression in the cerebral tissue of rats sustaining traumatic brain injury of differing severity, while simultaneously measuring the oxidant/antioxidant balance. Two levels of trauma (mild and severe) were induced in anesthetized animals using the weight-drop model of diffuse axonal injury. Rats were then sacrificed at 6, 12, 24, 48, and 120 h after traumatic brain injury, and the gene and protein expression of neuroglobin and the concentrations of malondialdehyde (as a parameter representative of reactive oxygen species-mediated damage), nitrite + nitrate (indicative of NO metabolism), ascorbate, and glutathione (GSH) were determined in the brain tissue. Results indicated that mild traumatic brain injury, although causing a reversible increase in oxidative/nitrosative stress (increase in malondialdehyde and nitrite + nitrate) and an imbalance in antioxidants (decrease in ascorbate and GSH), did not induce any change in neuroglobin. Conversely, severe traumatic brain injury caused an over nine- and a fivefold increase in neuroglobin gene and protein expression, respectively, as well as a remarkable increase in oxidative/nitrosative stress and depletion of antioxidants. The results of this study, showing a lack of effect in mild traumatic brain injury as well as asynchronous time course changes in neuroglobin expression, oxidative/nitrosative stress, and antioxidants in severe traumatic brain injury, do not seem to support the role of neuroglobin as an endogenous neuroprotective antioxidant agent, at least under pathophysiological conditions., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

To date, little is known concerning the circulating levels of biochemically relevant metabolites (antioxidants, oxidative/nitrosative stress biomarkers, purines, and pyrimidines) in patients with primary myelofibrosis (PMF), a rare form of myeloproliferative tumor causing a dramatic decrease in erythropoiesis and angiogenesis. In this study, using a targeted metabolomic approach, serum samples of 22 PMF patients and of 22 control healthy donors were analyzed to quantify the circulating concentrations of hypoxanthine, xanthine, uric acid (as representative purines), uracil, β-pseudouridine, uridine (as representative pyrimidines), reduced glutathione (GSH), ascorbic acid (as two of the main water-soluble antioxidants), malondialdehyde, nitrite, nitrate (as oxidative/nitrosative stress biomarkers) and creatinine, using well-established HPLC method for their determination. Results showed that PMF patients have dramatic depletions of both ascorbic acid and GSH (37.3- and 3.81-times lower circulating concentrations, respectively, than those recorded in healthy controls, p < 0.0001), accompanied by significant increases in malondialdehyde (MDA) and nitrite + nitrate (4.73- and 1.66-times higher circulating concentrations, respectively, than those recorded in healthy controls, p < 0.0001). Additionally, PMF patients have remarkable alterations of circulating purines, pyrimidines, and creatinine, suggesting potential mitochondrial dysfunctions causing energy metabolism imbalance and consequent increases in these cell energy-related compounds. Overall, these results, besides evidencing previously unknown serum metabolic alterations in PMF patients, suggest that the determination of serum levels of the aforementioned compounds may be useful to evaluate PMF patients on hospital admission for adjunctive therapies aimed at recovering their correct antioxidant status, as well as to monitor patients' status and potential pharmacological treatments. [ABSTRACT FROM AUTHOR]

Nearly 40–50% of infertility problems are estimated to be of female origin. Previous studies dedicated to the analysis of metabolites in follicular fluid (FF) produced contrasting results, although some valuable indexes capable to discriminate control groups (CTRL) from infertile females (IF) and correlate with outcome measures of assisted reproduction techniques were in some instances found. In this study, we analyzed in blind FF of 35 control subjects (CTRL = patients in which inability to obtain pregnancy was exclusively due to a male factor) and 145 IF (affected by: endometriosis, n = 19; polycystic ovary syndrome, n = 14; age-related reduced ovarian reserve, n = 58; reduced ovarian reserve, n = 29; unexplained infertility, n = 14; genetic infertility, n = 11) to determine concentrations of 55 water- and fat-soluble low molecular weight compounds (antioxidants, oxidative/nitrosative stress-related compounds, purines, pyrimidines, energy-related metabolites, and amino acids). Results evidenced that 27/55 of them had significantly different values in IF with respect to those measured in CTRL. The metabolic pattern of these potential biomarkers of infertility was cumulated (in both CTRL and IF) into a Biomarker Score index (incorporating the metabolic anomalies of FF), that fully discriminated CTRL (mean Biomarker Score value = 4.00 ± 2.30) from IF (mean Biomarker Score value = 14.88 ± 3.09, p < 0.001). The Biomarker Score values were significantly higher than those of CTRL in each of the six subgroups of IF. Posterior probability curves and ROC curve indicated that values of the Biomarker Score clustered CTRL and IF into two distinct groups, based on the individual FF metabolic profile. Furthermore, Biomarker Score values correlated with outcome measures of ovarian stimulation, in vitro fertilization, number and quality of blastocysts, clinical pregnancy, and healthy offspring. These results strongly suggest that the biochemical quality of FF deeply influences not only the effectiveness of IVF procedures but also the following embryonic development up to healthy newborns. The targeted metabolomic analysis of FF (using empowered Redox Energy Test) and the subsequent calculation of the Biomarker Score evidenced a set of 27 low molecular weight infertility biomarkers potentially useful in the laboratory managing of female infertility and to predict the success of assisted reproduction techniques. [ABSTRACT FROM AUTHOR]

Oxidative/nitrosative stress and mitochondrial dysfunction is a hallmark of amyotrophic lateral sclerosis (ALS), an invariably fatal progressive neurodegenerative disease. Here, as an exploratory arm of a phase II clinical trial (EudraCT Number 2017-005065-47), we used high performance liquid chromatography(HPLC) to investigate changes in the metabolic profiles of serum from ALS patients treated weekly for 4 weeks with a repeated sub-cutaneous dose of 1 mg/kg of a proprietary low molecular weight dextran sulphate, called ILB®. A significant normalization of the serum levels of several key metabolites was observed over the treatment period, including N-acetylaspartate (NAA), oxypurines, biomarkers of oxidative/nitrosative stress and antioxidants. An improved serum metabolic profile was accompanied by significant amelioration of the patients' clinical conditions, indicating a response to ILB® treatment that appears to be mediated by improvement of tissue bioenergetics, decrease of oxidative/nitrosative stress and attenuation of (neuro)inflammatory processes. [ABSTRACT FROM AUTHOR]

Under physiological conditions, reactive oxygen species (ROS) play pivotal roles in various processes of human spermatozoa. Indeed, semen requires the intervention of ROS to accomplish different stages of its maturation. However, ROS overproduction is a well-documented phenomenon occurring in the semen of infertile males, potentially causing permanent oxidative damages to a vast number of biological molecules (proteins, nucleic acids, polyunsaturated fatty acids of biological membrane lipids), negatively affecting the functionality and vitality of spermatozoa. ROS overproduction may concomitantly occur to the excess generation of reactive nitrogen species (RNS), leading to oxidative/nitrosative stress and frequently encountered in various human pathologies. Under different conditions of male infertility, very frequently accompanied by morpho-functional anomalies in the sperm analysis, several studies have provided evidence for clear biochemical signs of damages to biomolecules caused by oxidative/nitrosative stress. In the last decades, various studies aimed to verify whether antioxidant-based therapies may be beneficial to treat male infertility have been carried out. This review analyzed the results of the studies published during the last ten years on the administration of low-molecular-weight antioxidants to treat male infertility in order to establish whether there is a sufficient number of data to justify antioxidant administration to infertile males. An analysis of the literature showed that only 30 clinical studies tested the effects of the administration of low-molecular-weight antioxidants (administered as a single antioxidant or as a combination of different antioxidants with the addition of vitamins and/or micronutrients) to infertile males. Of these studies, only 33.3% included pregnancy and/or live birth rates as an outcome measure to determine the effects of the therapy. Of these studies, only 4 were case–control studies, and only 2 of them found improvement of the pregnancy rate in the group of antioxidant-treated patients. Additionally, of the 30 studies considered in this review, only 43.3% were case–control studies, 66.7% enrolled a number of patients higher than 40, and 40% carried out the administration of a single antioxidant. Therefore, it appears that further studies are needed to clearly define the usefulness of antioxidant-based therapies to treat male infertility. [ABSTRACT FROM AUTHOR]

Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-γ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-γ (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism; (2) the amelioration of the macrophage energy state; (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde; (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat); (5) the increase of the transforming growth factor-β1 (TGF-β1) and the down-regulation of the expressions of interleukins 1β and 6 (IL-1β and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases). [ABSTRACT FROM AUTHOR]

Carnosine is an endogenous dipeptide composed of β-alanine and L-histidine. This naturally occurring molecule is present at high concentrations in several mammalian excitable tissues such as muscles and brain, while it can be found at low concentrations in a few invertebrates. Carnosine has been shown to be involved in different cellular defense mechanisms including the inhibition of protein cross-linking, reactive oxygen and nitrogen species detoxification as well as the counteraction of inflammation. As a part of the immune response, macrophages are the primary cell type that is activated. These cells play a crucial role in many diseases associated with oxidative stress and inflammation, including atherosclerosis, diabetes, and neurodegenerative diseases. In the present study, carnosine was first tested for its ability to counteract oxidative stress. In our experimental model, represented by RAW 264.7 macrophages challenged with phorbol 12-myristate 13-acetate (PMA) and superoxide dismutase (SOD) inhibitors, carnosine was able to decrease the intracellular concentration of superoxide anions (O2−•) as well as the expression of Nox1 and Nox2 enzyme genes. This carnosine antioxidant activity was accompanied by the attenuation of the PMA-induced Akt phosphorylation, the down-regulation of TNF-α and IL-6 mRNAs, and the up-regulation of the expression of the anti-inflammatory mediators IL-4, IL-10, and TGF-β1. Additionally, when carnosine was used at the highest dose (20 mM), there was a generalized amelioration of the macrophage energy state, evaluated through the increase both in the total nucleoside triphosphate concentrations and the sum of the pool of intracellular nicotinic coenzymes. Finally, carnosine was able to decrease the oxidized (NADP+)/reduced (NADPH) ratio of nicotinamide adenine dinucleotide phosphate in a concentration dependent manner, indicating a strong inhibitory effect of this molecule towards the main source of reactive oxygen species in macrophages. Our data suggest a multimodal mechanism of action of carnosine underlying its beneficial effects on macrophage cells under oxidative stress and inflammation conditions. [ABSTRACT FROM AUTHOR]