Oxygen, Volume 3, Issue 1 (March 2023) – 11 articles

Over 2000 publications including more than 100 human studies seem to indicate that humans have only recently benefited from or known about the medical effects of H₂ within the past 15 years. However, we have unknowingly benefited from H₂ since the dawn of time, from H₂-producing bacteria to the use of naturally occurring hydrogen-rich waters. Moreover, the first writings on the therapeutic effects of H₂ date to around 1793. Since then, papers appeared sporadically in the literature every few decades but never exploded until Ohsawa et al. again demonstrated hydrogen’s therapeutic effects in 2007. This landmark paper appears to have been the spark that ignited the medical interest in hydrogen. Although H₂ was used in the 1880s to locate intestinal perforations, in the 1940s in deep sea diving, and in the 1960s to measure blood flow, H₂ was largely viewed as biologically inert. This review highlights the history of hydrogen in the genesis/evolution of life and its medicinal and non-medicinal use in humans. Although hydrogen medicine has a long and erratic history, perhaps future history will show that, this time around, these 15 years of ignited interest resulted in a self-sustaining explosion of its unique medical effects. Full article

Red spinach extract is high in inorganic nitrate/nitrite (NO₃/NO₂) which has been shown to enhance vascular function, cognition, and physical performance. To date, there have been no investigations as to whether red spinach extract serves as an effective strategy to improve repeated exercise performance, which is applicable to many sports and activities. The purpose of this study was to investigate the effect of acute red spinach extract ingestion on repeated sprint ability in female athletes. Eleven Division I NCAA female athletes (ages 18–24) were recruited. In a double-blinded, randomized, counterbalanced design, participants completed two separate visits each with a different treatment: placebo (placebo; tomato juice) or red spinach extract (~400 mg nitrate). For each trial, participants consumed their respective treatment two hours before exercise. Following a warm-up, participants completed 3 × 15 s Wingate Anaerobic Tests (WAnTs) separated by 2 min of recovery. A capillary blood sample was obtained pre-exercise to measure NO₂ concentrations. Performance outcomes, heart rate (heart rate), and rate of perceived exertion were measured following each WAnT. Blood lactate (La-) was obtained prior to exercise (PRE) and after the completion of the repeated sprints (POST). Each visit was separated by a minimal recovery period of 72 h. Mean power (p = 0.204), peak power (p = 0.067), heart rate (p = 0.151), and rate of perceived exertion (p = 0.379) were not significantly different between treatments. POST La- concentration was significantly higher with red spinach extract when compared to the placebo (p = 0.030). Furthermore, the fatigue index (p = 0.018) was significantly lower with red spinach extract. The results do not support the use of red spinach extract for the enhancement of power output during repeated anaerobic exercise. However, it may result in improved La-/H⁺ removal from the muscle, thereby combating physical fatigue. Full article

Flooding induces low-oxygen stress, which reduces plant growth. The activity of the γ-aminobutyric acid (GABA) shunt is crucial for plant stress adaptation, in which it acts by changing cytosolic pH, limiting reactive oxygen species production, regulating nitrogen metabolism, and bypassing steps in the tricarboxylic acid cycle. GABA accumulates under osmotic stress as well as flooding stress. To clarify the dynamic roles of GABA accumulation in wheat under flooding stress, biochemical and enzymatic analyses were performed using a plant-derived smoke solution (PDSS), which rescued wheat growth from flooding stress. Alcohol dehydrogenase abundance increased under flooding stress; however, under the same conditions, pyruvic acid content increased only following PDSS application. Glutamic acid content increased under flooding stress, but decreased following the application of PDSS after 2 days of flooding. Glutamate decarboxylase abundance and GABA content increased under flooding stress, and further increased after 1 day of application of PDSS. Succinyl semialdehyde dehydrogenase accumulated after 2 days of flooding. These results suggest that flooding stress increases GABA content along with the increase and decrease of glutamate decarboxylase and succinyl semialdehyde dehydrogenase, respectively. Additionally, PDSS increased GABA content along with the increase of glutamate decarboxylase abundance at the initial stage of application. Full article

In 2007 a paper in Nature Medicine sparked a new wave of interest in the use of molecular hydrogen (H₂) for medical treatments. Since then there has been a flurry of papers looking at a range of medical aspects, from neurodegenerative disease to sports injuries. Several methods of application have been developed, including breathing the gas, or making a hydrogen-rich solution that can be ingested, or administered as saline. H₂ treatments are deemed to be safe and can be used in agricultural practice as well as in the biomedical arena. However, the first studies to investigate the use of H₂ in medicine were carried out by those interested in the use of a range of gases and how this may affect respiration in humans. Beddoes was published in 1793 and Cavallo in 1798, with Davy following shortly after in 1800. With so many papers now appearing on H₂ in biological systems, it seems timely, and interesting, to revisit the early papers, to humbly remind us of what these pioneers of respiratory research did. Full article

Oxygen binds to hemoglobin cooperatively, and a correct description of this binding is relevant not only for understanding the mechanisms of involved molecular processes but also for clinical purposes. Hill’s equation, derived over a hundred years ago, is still the simplest and most efficient way to perform this description. However, in terms of accuracy, it is inferior to Adair´s equation, which contains more parameters. We proposed to use a modified Hill equation and showed that it allows a more accurate description of the binding of oxygen to hemoglobin than Adair’s equation. At the same time, unlike Adair’s equation, our model retains the physical meaning given to the original Hill equation. We considered and analyzed the influence of the equation parameters on the course of the oxygenation curve and presented the relationship between the fitting parameters and other parameters derived from them in the form of a diagram-graph, which, in our opinion, simplifies the perception of these estimates and can be useful in solving a number of problems for which the traditional way of analyzing the degree of cooperative interaction was via the Hill equation. We suggest that the newly proposed parameter h_max introduced in our model should be regarded as crucial for a better description of the oxygenation curve. Full article

Catalase is a key antioxidative enzyme, and a deficiency or malfunction of catalase is hypothesized to be related to various diseases. To investigate catalase activity, it is important to use reliable methods and experimental protocols enabling consistent fallouts. One major problem, however, is that the activity values obtained with different techniques and procedures can vary to a large extent. The aim of this work was to identify experimental conditions that provide similar catalase activity values with two different methods based on either spectrophotometry or chronoamperometry. The investigated parameters include the concentration of catalase and its substrate (H₂O₂), as well as the effect of deoxygenation of the catalase medium by nitrogen (N₂). Within the frame of investigated conditions, we show that spectrophotometry is strongly affected by the catalase concentration, whereas chronoamperometry is shown to be more dependent on the substrate concentration. Deoxygenation leads to elevated catalase activity values in the case of chronoamperometry, whereas it shows no influence on the results obtained with spectrophotometry. In particular, in the case of low substrate concentrations (i.e., low catalase reaction rates), higher and more accurate results are obtained with deoxygenation in the case of chronoamperometry measurements due to minimized oxygen escape. The effect of deoxygenation, giving rise to elevated catalase activity values, however, is not statistically significant at high substrate concentrations, implying that the protocol can be simplified by excluding this step as long as the other parameters are optimized. Finally, by comparing the two methods at different experimental conditions, we identified protocols resulting in similar results, i.e., 10 mM H₂O₂ and catalase activity of 4–5 U/mL. Based on this work, improved consistency of catalase activity data obtained with different methodologies and in different labs is expected. Full article

Low tissue oxygenation, termed hypoxia, is a characteristic of solid tumors with negative consequences. Tumor-associated macrophages (TAMs) accumulate in hypoxic tumor regions and correlate with worse outcomes in cancer patients across several tumor types. Thus, the molecular mechanism in which macrophages respond to low oxygen tension has been increasingly investigated in the last decade. Hypoxia stabilizes a group of hypoxia-inducible transcription factors (HIFs) reported to drive transcriptional programs involved in cell survival, metabolism, and angiogenesis. Though both tumor macrophage HIF-1α and HIF-2α correlate with unfavorable tumor microenvironments, most research focuses on HIF-1α as the master regulator of hypoxia signaling, because HIF-1α expression was originally identified in several cancer types and correlates with worse outcome in cancer patients. The relative contribution of each HIFα subunit to cell phenotypes is poorly understood especially in TAMs. Once thought to have overlapping roles, recent investigation of macrophage HIF-2α has demonstrated a diverse function from HIF-1α. Little work has been published on the differential role of hypoxia-dependent macrophage HIF-2α when compared to HIF-1α in the context of tumor biology. This review highlights cellular HIF-2α functions and emphasizes the gap in research investigating oxygen-dependent functions of tumor macrophage HIF-2α. Full article

Autism spectrum disorder (ASD) is a multifaceted neurodevelopmental disorder that comprises a complex aetiology, where a genetic component has been suggested, together with multiple environmental risk factors. Because of its increasing incidence in the paediatric population and the lack of successful curative therapies, ASD is one of the most puzzling disorders for medicine. In the last two decades and more, the relationship between oxidative stress (OS) and ASD has been recurrently documented. For this reason, the former hypothesis, according to which reactive oxygen and nitrogen species (ROS and RNS) play an important role in ASD, is now a certainty. Thus, in this review, we will discuss many aspects of the role of OS in ASD. In addition, we will describe, in the context of the most recent literature, the possibility that free radicals promote lipid peroxidation, as well as an increase in other OS biomarkers. Finally, we will outline the possibility of novel nutritional interventions aimed at counteracting ROS production in people with ASD. In fact, new strategies have investigated the possibility that ASD symptoms, as well behavioral anomalies, may be improved after interventions using antioxidants as supplements or included in foods. Full article

Synthetic CpG oligonucleotides are promising components of immunomodulatory drugs for the treatment and prophylaxis of infectious diseases, cancers, and allergies. Phosphorothioate modification stabilizes these compounds, contributing to the achievement of a clinical effect, but at the same time changes their immunomodulatory properties. We used the diffusible fluorescent dye dihydroethidium and the non-diffusible 6-carboxy-2′,7′dihydrochlorofluorescein diacetate and cytochrome c probes to demonstrate that it is the phosphorothioate backbones that determine the pronounced nonspecific pro-oxidant effect of CpG ODN on neutrophils. At the same time, as was shown using diaminofluorescein diacetate, the potentiation of nitric oxide synthesis in these leucocytes by CpG ODN class A strictly depends on the presence of CpG motifs and a palindromic “hairpin”. The results obtained will contribute to a more complete understanding of the physiological action of therapeutic agents based on synthetic CpG oligonucleotides. Full article

The development of novel biologically active nanopharmaceuticals is a topical problem of medicine. Water-soluble fullerene derivatives are of particular interest due to their ability to regulate intracellular metabolism of reactive oxygen species (ROS) by direct oxidation or effects on oxidative and signaling enzymes. Here, we studied the effect of a water-soluble chlorine-containing derivative of C₆₀ fullerene on human embryonic lung fibroblasts. MTT tests, intracellular ROS visualization, detection of the ROS-associated gene and protein expression, repair, cell proliferation and cell cycle regulation, and quantitation of oxidative DNA damage were used. Within the first three hours after exposure, antioxidant effects were revealed. Later, oxidative damage appeared. Thus, the studied compound had an ambiguous effect on ROS metabolism associated with a switch in the regulatory effect, which must be taken into account when assessing its biological activity and toxicity. Full article