Hemoglobin Oxidation Research Articles

Anion exchanger1 (AE1/SLC4A1) function is impaired in red blood cells from prediabetic subjects: Potential benefits of finger lime (Citrus australasica, Faustrime cultivar) juice extract.

Prediabetes is a risk state that defines a high chance of developing diabetes and cardiovascular disease. Oxidative stress mediated by hyperglycemia-induced production of reactive species could play a crucial role in this context. In the present study, we investigated whether the anion exchange capability mediated by AE1 (SLC4A1), which is sensitive to oxidative stress, was altered in human red blood cells (RBCs) obtained from prediabetic volunteers. In addition, we assessed the precise composition of bioactive compounds and the potential benefits of finger lime juice extract (Citrus australasica, Faustrime cultivar) in counteracting oxidative stress-related functional alterations. Human RBCs from normal and prediabetic volunteers were incubated with 50 µg/mL juice extract for 2 h at 25°C. Juice extract restored alterations of the anion exchange capability mediated by AE1 and prevented the structural rearrangements of AE1 and α/β-spectrin in prediabetic RBCs. AE1 functional and structural alterations were not associated with an increase in lipid peroxidation or protein oxidation at the level of the plasma membrane. An increased production of intracellular ROS, which provoked the oxidation of hemoglobin to methemoglobin, both reverted by juice extract, was instead observed. Importantly, juice extract also induced a reduction in glycated hemoglobin levels in prediabetic RBCs. Finally, juice extract blunted the overactivation of the endogenous antioxidant enzymes catalase and superoxide dismutase and prevented glutathione depletion in prediabetic RBCs. These findings contribute to clarifying cellular and molecular mechanisms related to oxidative stress and glycation events that may influence RBC and systemic homeostasis in prediabetes, identify AE1 as a sensitive biomarker of RBC structural and function alterations in prediabetes and propose finger lime juice extract as a natural antioxidant for the treatment and/or prevention of the complications associated with the prediabetic condition.

Investigation of Antioxidant Mechanisms of Novel Peptides Derived from Asian Swamp Eel Hydrolysate in Chemical Systems and AAPH-Induced Human Erythrocytes.

Sixteen novel antioxidant peptides from Asian swamp eel (ASE) were identified in previous studies. However, their chemical and cellular antioxidant mechanisms remain unclear. Molecular docking of these peptides with ABTS and DPPH radicals revealed the critical role of hydrogen bonding and Pi-Pi stacking hydrophobic interactions between hydrophobic amino acid residues and free radicals. Residues, such as tryptophan, proline, leucine, and valine, played significant roles in these interactions. All these peptides exhibited notable erythrocyte morphoprotective effects in a model of AAPH-induced oxidative damage of human erythrocytes. Erythrocyte hemolysis was reduced primarily through the modulation of both non-enzymatic (GSH/GSSG) and enzymatic antioxidant systems (SOD, CAT, and GSH-Px) by these peptides. A decrease in levels of MDA, LDH release, and hemoglobin oxidation was observed. Among the peptides, VLYPW demonstrated superior chemical and cellular antioxidant activities, which may be attributed to its higher levels of tyrosine and tryptophan, as well as to its increased hydrophobic amino acid content.

Effects of Nitrosyl Iron Complexes with Thiol, Phosphate, and Thiosulfate Ligands on Hemoglobin.

Nitrosyl iron complexes are remarkably multifactorial pharmacological agents. These compounds have been proven to be particularly effective in treating cardiovascular and oncological diseases. We evaluated and compared the antioxidant activity of tetranitrosyl iron complexes (TNICs) with thiosulfate ligands and dinitrosyl iron complexes (DNICs) with glutathione (DNIC-GS) or phosphate (DNIC-PO4-) ligands in hemoglobin-containing systems. The studied effects included the production of free radical intermediates during hemoglobin (Hb) oxidation by tert-butyl hydroperoxide, oxidative modification of Hb, and antioxidant properties of nitrosyl iron complexes. Measuring luminol chemiluminescence revealed that the antioxidant effect of TNICs was higher compared to DNIC-PO4-. DNIC-GS either did not exhibit antioxidant activity or exerted prooxidant effects at certain concentrations, which might have resulted from thiyl radical formation. TNICs and DNIC-PO4- efficiently protected the Hb heme group from decomposition by organic hydroperoxides. DNIC-GS did not exert any protective effects on the heme group; however, it abolished oxoferrylHb generation. TNICs inhibited the formation of Hb multimeric forms more efficiently than DNICs. Thus, TNICs had more pronounced antioxidant activity than DNICs in Hb-containing systems.

Anaerobic glycolysis and oxidative stress interrelation in erythrocytes under administration of Cornus mas L. fruit extracts to rats with streptozotocin-induced diabetes mellitus

Background. In diabetes mellitus (DM), analysis of changes in the biochemical profile of erythrocytes is the important stage of complex scientific research to clarify the mechanism of action of medicinal products based on plant raw materials. The fruits of Cornus mas L. are widely known. The biologically active compounds of these fruits show multiple biological effects. However, the effect of the fruit extracts of cornelian cherry on the functional state of erythrocytes in diabetes has not been sufficiently studied. The high glucose concentration in erythrocytes induces various structural and functional changes, which lead to numerous disturbances in their metabolism. Glucose transported into erythrocytes by facilitated diffusion via GLUT2 undergoes catabolic breakdown in anaerobic glycolysis (90 % of all glucose) and pentose phosphate pathway (the rest 10 %). ATP and reduced coenzymes of NADH + H+ and NADPH + H+ formed due to metabolism participate in maintaining the structure of hemoglobin. Enzymes of the antioxidant defense system, which prevent hemoglobin oxidation into methemoglobin, are especially important. Hyperglycemia and the development of oxidative stress in diabetes are the cause of a decrease in the activity of antioxidant enzymes and the accumulation of ligand forms of hemoglobin (HbCO2, MetHb, HbA1c). Therefore, the work aimed to investigate the effect of extracts of red and yellow fruits of Cornus mas L. on the content of end products of the glycolytic breakdown of glucose in erythrocytes and biochemical markers of the antioxidant status of these blood cells in rats with streptozotocin-induced diabetes. Materials and Methods. DM 1 type in animals was induced by intraperitoneal injection of streptozotocin. Experiments were performed on male Wistar rats, who, from the 10th day after diabetes induction, were administered per os extracts of red and yellow fruits of the cornelian cherry and loganic acid obtained from yellow fruits at a dose of 20 mg/kg of body weight for 14 days. On the 24th day of the experiment, the rats were decapitated under ether anesthesia, and blood was taken. The content of pyruvate and lactate (as the end products of anaerobic glycolysis) and L-lactate dehydrogenase activity were determined in plasma and erythrocytes, as well as biochemical markers of the antioxidant status of erythrocytes (activity of superoxide dismutase, catalase and glutathione peroxidase, level of reduced glutathione, TBA-reactive substances, concentration of oxidative modifications of proteins and advanced oxidation protein products). Results. The activity of catalase, superoxide dismutase, glutathione peroxidase and the concentration of reduced glutathione significantly increased against the decrease in the content of oxidative modifications of proteins, advanced oxidation protein products, TBA-reactive substances, pyruvate, L-lactate, and lactate dehydrogenase in rats with DM after administration of the fruit extracts of the cornelian cherry. Noteworthy, these biochemical indicators made it possible to assess the intensity of anaerobic glycolysis and the antioxidant status of blood erythrocytes in streptozotocin diabetes. Conclusions. Extracts of Cornus mas L. fruits might be potential natural drugs for the treatment of metabolic disorders in diabetes, as they have a corrective effect on the catabolic breakdown of glucose and the antioxidant defense system of erythrocytes, preventing the development of oxidative stress. It should be pointed out that the extract of red fruits of cornelian cherry showed the best effect among the studied extracts in normalizing these indicators.

Glycated LDL generates reactive species that damage cell components, oxidize hemoglobin and alter surface morphology in human erythrocytes

Low-density lipoprotein (LDL) transports cholesterol to various tissues via the blood. Glycation of LDL occurs during hyperglycemic condition which is characterised by persistently high blood glucose level. Circulating erythrocytes can come in direct contact with glycated LDL (G-LDL). The objective of this study was to investigate the effect of G-LDL on human erythrocytes, specifically on hemoglobin, intracellular generation of reactive species and the antioxidant defence system. Isolated erythrocytes were incubated with G-LDL (3 and 6 mg/ml) and native LDL (6 mg/ml) at 37 °C for 24 h. Native LDL and G-LDL untreated erythrocytes were similarly incubated at 37 °C and served as control. G-LDL treatment increased hemolysis compared to control and native LDL-treated erythrocytes. Incubation of erythrocytes with G-LDL led to an increase in protein oxidation and lipid peroxidation while greatly decreasing the total sulfhydryl content. It also significantly enhanced hemoglobin oxidation, heme degradation, and the release of free iron moiety. Treatment with G-LDL led to an appreciable increase in the production of reactive oxygen and nitrogen species. The antioxidant power and activities of major antioxidant enzymes were drastically reduced, while critical membrane-bound enzymes were inhibited. The surface morphology of G-LDL-treated erythrocytes was altered leading to the formation of echinocytes. Importantly, treatment of erythrocytes with native LDL did not significantly affect the above-mentioned parameters and values were similar to the corresponding controls. Thus, G-LDL is cytotoxic to human erythrocytes and causes oxidative damage to cell components. This can reduce the oxygen-transporting ability of blood and also result in red cell senescence and anemia.

Effect of Garlic Extract on the Erythrocyte as a Simple Model Cell.

Garlic is known to have diverse effects on mammalian cells, being cytotoxic, especially to cancer cells, but also protect against oxidative stress. Mammalian erythrocyte is a simple cell devoid of intracellular organelles, protein synthesis ability, and most signaling pathways. Therefore, examination of the effects of garlic on erythrocytes allows for revealing primary events in the cellular action of garlic extract. In this study, human erythrocytes or erythrocyte membranes were exposed to garlic extract at various dilutions. Hemoglobin oxidation to methemoglobin, increased binding of hemoglobin to the membrane, and formation of Heinz bodies were observed. Garlic extract depleted acid-soluble thiols, especially glutathione, and induced a prooxidative shift in the cellular glutathione redox potential. The extract increased the osmotic fragility of erythrocytes, induced hemolysis, and inhibited hemolysis in isotonic ammonium chloride, indicative of decreased membrane permeability for Cl- and increased the membrane fluidity. Fluorescent probes indicated an increased level of reactive oxygen species and induction of lipid peroxidation, but these results should be interpreted with care since the extract alone induced oxidation of the probes (dichlorodihydrofluorescein diacetate and BODIPY C11). These results demonstrate that garlic extract induces oxidative changes in the erythrocyte, first of all, thiol and hemoglobin oxidation.

Síndrome de metahemoglobinemia felina induzida por fármacos

A Síndrome de Metahemoglobinemia (SM) é um quadro clínico resultante da concentração elevada de metahemoglobina no sangue e que, se não tratada, pode levar ao óbito. A formação de metahemoglobina é um processo metabólico normal ocasionado pela oxidação da hemoglobina (Hb), isto é, transformação do ferro presente, do seu estado ferroso (Fe2+) para o estado férrico (Fe3+), impedindo assim, a ligação do oxigênio à Hb e consequentemente seu transporte pelas hemácias. No entanto, o aumento excessivo de MHb ocasiona alterações respiratórias e hipóxia tecidual pela deficiência de oxigênio circulante. Os felinos são muito suscetíveis a SM, a qual decorre de alterações hereditárias ou de exposição a agentes químicos oxidantes. O objetivo desta revisão foi trazer informações atuais acerca da etiopatogenia, aspectos clínicos e de diagnóstico, além de conduta terapêutica frente à Síndrome de Metemoglobinemia Felina (SMF) provocada por fármacos. Foi realizada uma revisão de literatura com base nos últimos 10 anos de publicações sobre o tema, mas considerando os primeiros e clássicos relatos. O paracetamol foi o principal fármaco citado na literatura como agente químico etiológico; o diagnóstico presuntivo de SMF é considerado quando o paciente veterinário apresentar cianose e baixa saturação de oxigênio, confirmada pela co-oximetria de Tpulso e na ausência de doença cardiopulmonar; a investigação inicial como o spot teste e exames complementares podem auxiliar no diagnóstico; o tratamento básico da SMF consiste remoção precoce do agente causador, o uso de antídotos, oxigenioterapia e no controle dos sinais clínicos.

Processing of slaughterhouse blood for antianemic food products

Currently, rational processing of blood from slaughterhouses remains as a waste fluid in many regions. Traditional approaches to use the blood for food are significantly limited because of specific and non-favorable organoleptic characteristics. Present study provides a comparison of various methods for modifying the red blood cell (RBC) mass of animals and a more in-depth study of acid hemolysis. The solution of ascorbic acid has been proposed as a hemolyzing agent. There has been established experimentally that the addition of equal volumes of RBC and a solution of an ascorbic acid with a concentration of 0.75 mol/dm3 can effectively destroy the stroma more up 90% of red blood cells within 15 minutes. By this, the hemoglobin oxidation degree to methemoglobin is about 50%, which forms the desired color of the resulting hydrolysate. The dry semi-finished product has a neutral odor and brown color with high functional and technological properties. It also contains 0.9% organic iron with good biological value. Thus, the study shows that blood products can effectively use in various foods such as meat products, and also as a dietary supplement for various proposes. Consumption of these products has potent positive effect on hemoglobin levels and it is recommending for people with iron deficiency anemia.

New view on the compatibility of hemoglobin function in the erythrocytes.

Aim: To study the process of hemoglobin oxidation and the enzymatic reactions associated with it. Materials and Methods: Heparinized human blood (15 IU/ml) was obtained from the clinical department. The concentration of oxy- and methemoglobin, auto-oxidation of hemoglobin was determined spectrophotometrically spectrophotometrically. Autooxidation of hemoglobin was recorded spectrophotometrically, and protein concentration was determined by the Lowry method. Monooxygenase activity of hemoglobin was also measured by the method described by Lowry spectrophotometrically. The concentration of O2 and H2O2 in the reaction media was determined on a biomicroanalyzer OR 210/3 (Redelkis). Results: The obtained experimental data allow us to propose a mechanism of "spontaneous autooxidation" of oxyhemoglobin, which can be described by the following equations: Hb2+O2 → Hb3+ + O2 - (1) Hb2+O2 + 2e - + 2H+ →Hb3+ + H2O2 (2) Hb2+O2 + 2e - + 2H+ →Hb2+ + H2O2 (3) Hb2+ + O2 →Hb2+O2 (4) Spectral characteristics of the process of "spontaneous auto-oxidation" indicate the formation of a metform of hemoglobin, the depletion of oxygen by the system was established, at pH 5.6, an increase in the monooxygenase activity of hemoglobin is observed 3-4 times compared to the physiological level. Сonclusions: In addition to the main, previously known functions of hemoglobin (gas transport, peroxidase, monooxygenase), it catalyzes a two-electron oxidase reaction in which O2 is reduced to H2O2. This is confirmed by experimental data on the formation of one of the products of "spontaneous autoxidation" of oxyhemoglobin _ deoxyform at pH 5.6 _ 8.9.

Trehalose prevents hemoglobin oxidation during lyophilization of red blood cells

Trehalose prevents hemoglobin oxidation during lyophilization of red blood cells

New Insights in Sickle Cell Disease Provided By Intravascular Hemolysis and Total Hb Mass Parameters

New biomarkers may provide a better understanding of the complex pathophysiology of sickle cell disease. Anemia is defined as a reduction in the blood concentration of hemoglobin ([Hb]) to < 12 g/dL in females and < 13 g/dL in males. [Hb] will depend not just on the total circulating quantity of hemoglobin (tHbmass), but also on plasma volume (PV) in which it is suspended. Exercise capacity correlates poorly with [Hb], in contrast to tHbmass, as shown by studies comparing elite athletes and sedentary individuals. Similarly, classic parameters of hemolysis (LDH, bilirubin, ASAT) are not very specific for erythrocytes and do not always reflect the intravascular component of hemolysis. Plasma hemoglobin and heme, released by hemoglobin oxidation, are specific biomarkers of RBC destruction in the circulation and have their own deleterious effects on vascular walls, NO bioavailability, and the inflammatory state. In this study, we aimed to analyze the contribution of these biomarkers to cardiac and vascular parameters. Method: We carried out a preliminary analysis of patients included in the HEMOPROVE trial (NCT05199766) a prospective open-label study designed to better understand the effects of Voxelotor. Patients had to be more than one month from a vaso-occlusive crisis, 3 months from a transfusion and on a stable dose of hydroxyurea (HU) for at least 3 months to be included. We extracted and analyzed data from patients who had a pre-treatment Hb mass measurement. Hb mass, plasma volume were determined using the validated oCOR method using medical device Detalo Clinical TM - CO Rebreather (IEC 60601-1 and IEC 62304 for medical devices). Only patients with a HbCO of less than 3% benefited from this measurement; plasma hemoglobin and plasma heme were measured using the technique developed by our team (BET 18P3417). Blood pressure and heart rate were measured during echocardiography. All values were collected within the same week of inclusion in the protocol. Correlations were performed using a Spearman test, paired data comparisons were made with a Wilcoxon test (Graphpad9, PRISM). Results: Fourteen patients were included for blood mass measurement. Median age was 38 years [34.7-50.7], sex ratio F/M was 7/7, 10/14 patients were treated with HU. Median [Hb] was 7.4g/dL [6.6-7.7], tHbmass was 522 g [352-704] corresponding to 8.1 g/kg [6.1-11.7]. [Hb] and tHbmass were strongly correlated (p=0.008, r=0.68), whereas it was weaker with tHbmass/kg (p=0.046, r=0.54). Interestingly, some patients with approximately the same [Hb] had widely varying tHbmass. These results can be explained by significant variability in plasma volume, which was increased in half the patients, with a median increase of +135% [100-215] and a median volume of 5656 ml [4140-6931]. The tHbMass (p=0.01; r=0.64), tHbMass/kg (p=0.05, r=0.52) and above all PV (p=0.004; r=0.73) correlated with the E/E' used to evaluate the left ventricle filing pressure, while there was no correlation with [Hb] (p=0.5). Interestingly, the cardiac index was inversely correlated with plasma Hemoglobin (p=0.003, r=-0.74) with no correlation with [Hb] (p=0.4) or LDH (p=0.90), while mean and diastolic blood pressure were negatively correlated with plasma Heme (respectively p=0.01; r=-0.65 and p=0.001; r=-0.77) with no correlation with [Hb] and LDH (p>0.4). Conclusions: Measurements of total hemoglobin mass and intravascular hemolysis parameters provide a better understanding of the diversity of manifestations that lie behind sickle cell anemia, underpinned by complex mechanisms.

Changes in hemoglobin oxidation and band 3 during blood storage impact oxygen sensing and mitochondrial bioenergetic pathways in the human pulmonary arterial endothelial cell model.

Red blood cells (RBCs) undergo metabolic, oxidative, and physiological changes during storage, collectively described as the "storage lesion." The impact of storage on oxygen homeostasis, following transfusion, is not fully understood. We show that RBC storage induces changes in oxygen binding that were linked to changes in oxygen sensing (hypoxia-inducible factor, HIF-1α) mechanisms and mitochondrial respiration in human pulmonary arterial endothelial cells (HPAECs). A decrease in oxygen affinity (P50) to approximately 20 from 30mmHg was seen at the first week but remained unchanged for up to 42 days. This led to the suppression of HIF-1α in the first 3weeks due to limited oxygen supplies by RBCs. Furthermore, membrane oxidative damage, band 3 alterations, and subsequent microparticle (MP) formation were also noted. Mass spectrometric analysis revealed the upregulation of transitional endoplasmic reticulum ATPase, essential for clearing ROS-damaged membrane proteins and the protein DDI1 homolog, a proteasomal shuttle chaperone. Band 3 complex proteins and superoxide dismutase were among the downregulated proteins. Mitochondrial oxygen consumption rates measured in HPAECs incubated with RBC-derived MPs (14-day and 42-day) showed a rise in maximal respiration. Intervention strategies that target intracellular hemoglobin (Hb)'s redox transitions and membrane changes may lead to the reestablishment of oxygen homeostasis in old RBCs.

Free Radical Scavenging Activity of Boron and Vitamin C in Nitrite-Induced Hemoglobin Oxidation Model: In vitro and in vivo Studies

Objective: To investigate the dose-response relationship of the free radical-scavenging activities of boron and vitamin C in nitrite-induced hemoglobin oxidation in vitro and in vivo. Method: Different concentrations of boron and vitamin C were added to a hemolysate challenged with nitrite to induce methemoglobinemia (MetHb), and the most effective dose of boron and vitamin C was used before and after different intervals of inducing Hb oxidation, and the production of MetHb was monitored using a spectrophotometer. The effective doses of boron and vitamin C, alone and in combination, were administered to rats before challenging them with an oral dose of 100 mg/kg sodium nitrite. Results: In vitro results indicated that different concentrations of boron and vitamin C attenuated MetHb formation, with the maximum effect achieved at 0.08mg/L and 10mg/L, respectively. Moreover, when these doses were used at different time intervals, a maximum effect was achieved when added 10 min before nitrite. The in vivo results demonstrated a significant reduction in methemoglobin formation in rats treated with boron and vitamin C alone. The hematological markers were not changed except for the platelet levels, which increased in the boron-treated and combination groups. The monocyte-to-lymphocyte ratio decreased significantly in all treatment groups compared with the positive control group. Conclusion: Boron protects against Hb oxidation induced by nitrite, and a potentiated effect has been achieved with the combination of vitamin C.

An in-situ FTIR-LCR meter technique to study the sensing mechanism of MnO2@ZIF-8/CNPs and a direct relationship between the sensitivity of the sensors and the rate of surface reaction

Diethylamine vapor is harmful to people if inhaled or swallowed, as it results in the oxidation of hemoglobin in the body into unwanted methemoglobin, which is unable to transport oxygen in the blood, resulting in reduced blood oxygenation. Lack of blood oxygenation leads to hypoxemia. MnO2 nanorods, carbon soot, and MnO2@ZIF-8 are sensing materials used to prepare solid-state gas sensors that operate at room temperature. The prepared sensing materials were characterized by scanning electron microscopy, transmission electron microscopy, powder X-ray diffraction, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy. The performance of the MnO2@ZIF-8 based sensor improved significantly when the carbon soot was introduced into the composite. The effect of the amount of CNPs in the composite on the performance of the sensors was studied. The MnO2@ZIF-8/CNPs-based sensor with a 3:1 mass ratio was highly selective towards diethylamine vapor over acetone, methanol, ethanol, and 3-pentanone vapors. An in situ FTIR coupled with LCR meter was used to understand the sensing mechanism of diethylamine vapor and it was found that the sensing mechanism was by deep oxidation of diethylamine to CO2, H2O, and other molecules. The sensing mechanism was studied by monitored by CO2 band intensity which was produced from the reaction between the sensing materials and the analyte vapor. As the sensor's exposure time increased the intensity of the CO2 IR band increased. We observed the direct relationship between the surface reaction rate and the sensor's sensitivity.

Mechanisms and physiological function of daily haemoglobin oxidation rhythms in red blood cells.

Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox-sensitive covalent haem-Hb linkage that forms during SDS-mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in mouse and human RBCs cultured in vitro, or taken from humans in vivo, and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via nitric oxide (NO) signalling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.

Hemoglobin Derivatives in Beef Irradiated with Accelerated Electrons.

The efficiency of food irradiation depends on the accuracy of the irradiation dose range that is sufficient for inhibiting microbiological growth without causing an irreversible change to the physical and chemical properties of foods. This study suggests that the concentration of hemoglobin derivatives can be used as a criterion for establishing the limit for chilled beef irradiation at which irradiation-induced oxidation becomes irreversible. The express spectrophotometry method for estimating the hemoglobin derivative concentration shows a nonlinear increase in methemoglobin concentration from 15% to 50% in beef irradiated by accelerated electrons with the doses ranging from 250 Gy to 10,000 Gy. The monitoring of the hemoglobin derivative concentration for three days after irradiation shows nonmonotonous dependencies of methemoglobin concentration in beef in the storage time since the oxidation of hemoglobin occur as a result of irradiation and biochemical processes in beef during storage. The proposed method based on the quantitative analysis of the hemoglobin derivative concentration can be used to estimate the oxidation level for irradiation of foods containing red blood cells. The study proposes a model that describes the change in hemoglobin derivative concentration in beef after irradiation considering that oxidation of hemoglobin can be triggered by the direct ionization caused by accelerated electrons, biochemical processes as a result of bacterial activity, and reactive oxygen species appearing during irradiation and storage. This research throws light on the mechanisms behind food irradiation during storage that should be taken into account for selecting the optimal parameters of irradiation.

Mancozeb-induced cytotoxicity in human erythrocytes: enhanced generation of reactive species, hemoglobin oxidation, diminished antioxidant power, membrane damage and morphological changes

Mancozeb is an ethylene bis-dithiocarbamate fungicide extensively used in agriculture to safeguard crops from various fungal diseases. The general population is exposed to mancozeb through consumption of contaminated food or water. Here, we have investigated the effect of mancozeb on isolated human erythrocytes under in vitro conditions. Erythrocytes were treated with different concentrations of mancozeb (0, 5, 10, 25, 50, 100 μM) and incubated for 24 h at 37 °C. Analysis of biochemical parameters and cell morphology showed dose-dependent toxicity of mancozeb in human erythrocytes. Mancozeb treatment caused hemoglobin oxidation and heme degradation. Protein and lipid oxidation were enhanced, while a significant decrease was seen in reduced glutathione and total sulfhydryl content. A significant increase in the generation of reactive oxygen and nitrogen species was detected in mancozeb-treated erythrocytes. The antioxidant capacity and the activity of key antioxidant enzymes were greatly diminished, while crucial metabolic pathways were inhibited in erythrocytes. Damage to the erythrocyte membrane on mancozeb treatment was apparent from increased cell lysis and osmotic fragility, along with the impairment of the plasma membrane redox system. Mancozeb also caused morphological alterations and transformed the normal discoid-shaped erythrocytes into echinocytes and stomatocytes. Thus, mancozeb induces oxidative stress in human erythrocytes, impairs the antioxidant defense system, oxidizes cellular components, that will adversely affect erythrocyte structure and function.

Phytochemical Screening and In vitro Antioxidant, Antibacterial, and Antihemolytic Activities of Putoria calabrica Leaf Extracts

Background:: Putoria calabrica is a common Mediterranean plant used topically in folk medicine for medical purposes. The aim of this study is to evaluate the in vitro antioxidant, antibacterial, and antihemolytic properties of Putoria calabrica leaf extracts.Methods:Total phenol content (TPC), total flavonoid content (TFC), and condensed tannins content (CTC) were determined spectrophotometrically. The antioxidant activity was tested using TAC, DPPH, ABTS, and FRAP assays. Agar diffusion method was used to determine the antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. Anti-hemolytic activity was evaluated by the measurement of erythrocytes turbidity and extracellular hemoglobin concentration using AAPH method.Results:Methanol was the optimal solvent to extract the bioactive components with the highest extraction yield (30.11 ± 0.42%), TPC (142.11 ± 4.04 mg GAE/g dry extract) and TFC (29.16 ± 0.99 mg QrE/g dry extract). In terms of antioxidant activity, methanol extract was the most effective against ABTS free radical (IC50 = 5.02 ± 0.13 mg/ml) and FRAP (A0.5 = 2.91 ± 0.09 mg/ml). While, acetone extract showed the highest TAC (214.05 ± 8.74 mg GAE/g dry extract) and DPPH scavenging activity (IC50 = 1.94 ± 0.02 mg/ml). The strongest antimicrobial effect at a concentration of 10.24 mg/well was observed with acetone extract against P. aeruginosa (22.52 ± 0.60 mm) and C. albicans (17.33 ± 0.41 mm). While, methanol extract was the most active against S. aureus (17.31 ± 0.98 mm). Aqueous extract showed a good capacity to protect erythrocytes from oxidative damage by preventing their hemolysis and the oxidation of hemoglobin induced by AAPH.Conclusion:P. calabrica leaves could have a great importance as a source of therapeutic bioactive compounds.

New C8-substituted caffeine derivatives as promising antioxidants and cytoprotective agents in human erythrocytes

New structurally diverse groups of C8-substituted caffeine derivatives were synthesized and evaluated for their chemical and biological properties. Mass spectrometry, FT-IR, and NMR characterizations of these derivatives were performed. The cytotoxic activity of the derivatives was estimated in vitro using human red blood cells (RBC) and in silico pharmacokinetic studies. The antioxidant capacity of the compounds was analyzed using a ferrous ion chelating activity assay. The ability of the derivatives to protect RBC from oxidative damage, including the oxidation of hemoglobin to methemoglobin, was assessed using a water-soluble 2,2′-azobis(2-methyl-propionamidine) dihydrochloride (AAPH) as a standard inducer of peroxyl radicals. The level of intracellular oxidative stress was assessed using the fluorescent redox probe 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). The results indicate that all derivatives are biocompatible compounds with significant antioxidant and cytoprotective potential dependent on their chemical structure. In order to explain the antioxidant and cytoprotective activity of the derivatives, a mechanism of hydrogen atom transfer (HAT), radical adduct formation (RAF), or single electron transfer (SET), as well as the specific interactions of the derivatives with the lipid bilayer of RBC membrane, have been proposed. The results show that selected modifications of the caffeine molecule enhance its antioxidant properties, which expands our knowledge of the structure–activity relationship of caffeine-based cytoprotective compounds.

Sensitivity of High Conservation Value Birds to Para-Aminopropiophenone (PAPP) Determined by Sub-Lethal Dose-Response Assay.

Para-aminopropiophenone (PAPP) is a methaemoglobin (MetHb) forming compound used for the lethal control of invasive carnivores and mustelids. By measuring the dose-dependent inhibition of O2 transport arising from the oxidation of haemoglobin (HbFe2+) to MetHb (HbFe3+), we determined the sensitivity of nine bird species to PAPP. A methaemoglobinaemia absorbance index (MAI) was validated in five common bird species to determine thresholds associated with a 99% probability of survival (ST99) and a 50% probability of mortality (LT50). Dose-response trials in high conservation value birds sought MAI values below the ST99 threshold, projecting the LT50 value and avoiding the need for lethal outcomes. Black-backed gull (LT50 = 1784.7) and eastern rosella (LT50 = 1074 mg kg-1) were the most tolerant species, while brown kiwi (LT50 = 8.4 mg kg-1) and weka (LT50 = 9.3 mg kg-1) were the most sensitive. Takahē were of intermediate acute sensitivity (LT50 = 51 mg kg-1), although protracted impacts on haemoglobin were observed in takahē up to 72 h later and associated with PAPP doses as low as 25.6 mg kg-1. In pukeko (LT50 = 138.4 mg kg-1), protracted declines in haemoglobin 72 h later occurred at doses as low as 29.5 mg kg-1, while at higher doses (253 and 112 mg kg-1), deaths resulted after 4-6 days. Based upon PAPP doses that caused acute and protracted responses, we provide estimates for the lowest observable adverse effect level (LOAEL) and no observable effects level (NOEL) for nine bird species.