ABSTRACT
Hemoglobin (Hb)-based oxygen carriers are investigated as a potential alternative or supplement to regular blood transfusions, particularly in critical and life-threatening scenarios. These include situations like severe trauma in remote areas, battlefield conditions, instances where blood transfusion is not feasible due to compatibility concerns, or when patients decline transfusions based on religious beliefs. This study introduces a novel method utilizing poly(ethylene glycol) (PEG) to entrap Hb within ZIF-8 nanoparticles (i.e., Hb@ZIF-8 NPs). Through meticulous screening, we achieved Hb@ZIF-8 NPs with a record-high Hb concentration of 34 mg mL-1. These NPs, sized at 168 nm, displayed exceptional properties: a remarkable 95 % oxyhemoglobin content, excellent encapsulation efficiency of 85 %, and resistance to Hb oxidation into methemoglobin (metHb). The addition of PEG emerged as a crucial factor amplifying Hb entrapment within ZIF-8, especially at higher Hb concentrations, reaching an unprecedented 34 mg mL-1. Importantly, PEG exhibited a protective effect, preventing metHb conversion in Hb@ZIF-8 NPs at elevated Hb concentrations.
Subject(s)
Hemoglobins , Nanoparticles , Polyethylene Glycols , Polyethylene Glycols/chemistry , Hemoglobins/chemistry , Nanoparticles/chemistry , Humans , Methemoglobin/chemistry , Methemoglobin/metabolism , Metal-Organic Frameworks/chemistryABSTRACT
Nitroxides are stable radicals consisting of a nitroxyl group, >N-Oâ¢, which carries an unpaired electron. This group is responsible for the paramagnetic and antioxidant properties of these compounds. A recent study evaluated the effects of pyrrolidine and pyrroline derivatives of nitroxides on the antioxidant system of human red blood cells (RBCs). It showed that nitroxides caused an increase in the activity of superoxide dismutase (SOD) and the level of methemoglobin (MetHb) in cells (in pyrroline derivatives) but had no effect on the activity of catalase and lactate dehydrogenase. Nitroxides also reduced the concentration of ascorbic acid (AA) in cells but did not cause any oxidation of proteins or lipids. Interestingly, nitroxides initiated an increase in thiols in the plasma membranes and hemolysate. However, the study also revealed that nitroxides may have pro-oxidant properties. The drop in the AA concentration and the increase in the MetHb level and in SOD activity may indicate the pro-oxidant properties of nitroxides in red blood cells.
Subject(s)
Antioxidants , Erythrocytes , Nitrogen Oxides , Superoxide Dismutase , Humans , Antioxidants/pharmacology , Antioxidants/chemistry , Ascorbic Acid/pharmacology , Ascorbic Acid/chemistry , Erythrocytes/metabolism , Erythrocytes/drug effects , Methemoglobin/metabolism , Nitrogen Oxides/chemistry , Oxidation-Reduction/drug effects , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Superoxide Dismutase/metabolismABSTRACT
The affinity of hemoglobin (Hb) to oxygen (O2) influences processes of oxygen delivery and extraction at the tissue level. Despite cannabinoids being utilized or ingested in various ways, their possible impact on Hb-O2 affinity has barely been studied. This is an experimental ex-vivo trial. Venous blood samples were drawn from 5 male and 6 female healthy volunteers and subsequently exposed to different cannabinoid types: (delta-9-tetrahydrocannabinol [Δ9-THC], delta-8-tetrahydrocannabinol [Δ8-THC], cannabidiol [CBD]) at different concentrations. Oxygen dissociation curves (ODC) were measured and blood gas analyses were performed for methemoglobin (MetHb) determination. The results revealed no MetHb formation. Besides two statistically significant changes (+1.4â¯mmHg and -0.9â¯mmHg) in the female cohort, following Δ9-THC and Δ8-THC exposure, no further P50 changes could be observed. The study demonstrated an in-vitro effect of selected cannabinoids and dosages on P50 values in female participants, with variations not observed at other dosages, leaving the underlying mechanisms open for debate. MetHb formation, as potential mechanism, was not detected in this study. The precise reasons why changes only occurred at specific dosages remain unclear, indicating a need for further in-vivo research to understand the interaction between cannabinoids and Hb-O2 affinity completely.
Subject(s)
Cannabidiol , Cannabinoids , Dronabinol , Hemoglobins , Methemoglobin , Oxygen , Humans , Female , Male , Adult , Methemoglobin/metabolism , Oxygen/metabolism , Dronabinol/pharmacology , Hemoglobins/metabolism , Young Adult , Cannabidiol/pharmacology , Dose-Response Relationship, Drug , Blood Gas AnalysisABSTRACT
The content of membrane-bound methemoglobin (MtHb) in nucleated erythrocytes was studied in the black scorpionfish Scorpaena porcus (Linnaeus, 1758) in vitro. Spectral characteristics were determined for a whole hemolysate, a hemolysate obtained by stroma precipitation (a clarified hemolysate), and a resuspended stroma. The MtHb proportion in the erythrocyte stroma was found to exceed 80% (6.20 ± 0.59 µM). Clarified hemolysates were nearly free of MtHb (0.5 ± 0.2 µM). Membrane-bound ferric hemoglobin did not affect the erythrocyte resistance to osmotic shock. The osmotic fragility range was determined using a LaSca-TM laser microparticle analyzer (BioMedSystems, Russia) to be 102-136 mOsm/kg, much the same as in other bony fish species. A nitrite load (10 mg/L) significantly increased the MtHb content in the blood. However, the membrane-bound ferric hemoglobin content did not change significantly, amounting to 6.34 ± 1.09 µM (approximately 95%). The finding suggested a functional importance for MtHb present in the plasma membrane of nucleated erythrocytes. Membrane-bound MtHb was assumed to neutralize the external oxidative load and the toxic effect of hydrogen sulfide in bottom water layers, where the species lives.
Subject(s)
Methemoglobin , Perciformes , Animals , Methemoglobin/metabolism , Perciformes/metabolism , Perciformes/blood , Hemoglobins/metabolism , Osmotic Fragility , Erythrocyte Membrane/metabolism , Erythrocyte Membrane/drug effects , Erythrocytes/metabolism , Erythrocytes/drug effects , Erythroblasts/metabolism , Fishes/metabolism , Fishes/bloodABSTRACT
Methemoglobinemia (MetHb) refers to the state of oxidation of the iron ion "ferrous" (Fe2+) to iron "ferric" (Fe3+) within the heme molecule that makes up hemoglobin (Hb). This state is physiological if its level remains controlled. The ferrous state of the heme molecule occurs in the event of significant oxidative stress. The pathophysiology of MetHb involves NADH, NADPH and glucose cycle enzymes such as cytochrome-b5-reductase. MetHb can be acquired or more rarely, congenital. Acquired causes include drug-induced effects such as topical anesthetics, or toxic effects such as nitrites. Primary causes are linked to enzyme deficiencies or constitutional Hb abnormalities. Excessively high MetHb causes symptoms of varying intensity, depending on the level of MetHb and associated comorbidities. Clinical signs are dominated by cyanosis, indicative of tissue hypoxia, which can be complicated by severe metabolic disorders leading to death. Diagnosis can be complex, as the resulting biological abnormalities may go undetected. Treatment is mainly based on identifying the etiology and restoring the heme molecule to its physiological state. Methylene blue is the main antidote in cases of elevated MetHb, but precautions must be taken in its use, and its physico-chemical effects must be understood. We provide an update on methemoglobinemia, summarizing its pathophysiology and clinical presentations, complementary tests and therapeutic principles.
Subject(s)
Methemoglobinemia , Methemoglobinemia/diagnosis , Methemoglobinemia/etiology , Humans , Methemoglobin/metabolism , Methemoglobin/analysis , Methylene Blue/therapeutic use , Oxidative Stress/physiology , Heme/metabolismABSTRACT
Methemoglobin (metHb), the oxidized form of hemoglobin, lacks the ability of reversible oxygen binding; however, it has a high binding affinity to toxic substances such as cyanide, hydrosulfide, and azide. This innate property of metHb offers the clinical option to treat patients poisoned with these toxins, by oxidizing the endogenous hemoglobin in the red blood cells (RBCs). The binding properties of naked metHb (isolated from RBC) with these toxins has been studied; however, the binding behaviors of metHb under the intracellular conditions of RBC are unclear because of the difficulty in detecting metHb status changes in RBC. This study aimed to elucidate the binding properties of metHb in RBC under physiological and poisoned conditions using artificial RBC, which was hemoglobin encapsulated in a liposome. The mimic-circumstances of metHb in RBC (metHb-V) was prepared by oxidizing the hemoglobin in artificial RBC. Spectroscopic analysis indicated that the metHb in metHb-V exhibited a binding behavior different from that of naked metHb, depending on the toxic substance: When the pH decreased, (i) the cyanide binding affinity of metHb-V remained unchanged, but that of naked metHb decreased (ii) the hydrosulfide binding affinity was increased in metHb-V but was decreased in naked metHb. (iii) Azide binding was increased in metHb-V, which was similar to that in naked metHb, irrespective of the pH change. Thus, the binding behavior of intracellular metHb in the RBC with cyanide, hydrosulfide, and azide under physiological and pathological conditions were partly elucidated using the oxidized artificial RBC.
Subject(s)
Azides , Methemoglobin , Humans , Methemoglobin/analysis , Methemoglobin/chemistry , Methemoglobin/metabolism , Azides/analysis , Azides/metabolism , Cyanides/toxicity , Cyanides/analysis , Cyanides/metabolism , Erythrocytes/metabolism , Hemoglobins/analysis , Hemoglobins/metabolismABSTRACT
OBJECTIVE: This study aimed to investigate the effect of diabetic plasma on human red blood cells (RBCs) in order to highlight the amplification mechanisms of oxidative stress (OS) in relation to methemoglobin (metHb) production, a potential bio-indicator that could be related to diabetes disease. RESEARCH DESIGN AND METHODS: Normal RBCs were co-incubated with the diabetic plasma of 24 patients at different HbA1c levels, for 0, 24, and 48 h in order to assess cell turbidity and hemoglobin (Hb) stability. Hb and metHb production were quantified inside and outside RBCs. Malonaldehyde (MDA) level and cell morphology were concomitantly evaluated. RESULTS: The cell turbidity was significantly decreased in the group co-incubated with diabetic plasma at high HbA1c levels (0.074 ± 0.010 AU) compared to the control group (0.446 ± 0.019 AU). A significant decrease in intracellular Hb (0.390 ± 0.075 AU) and its stability (0.600 ± 0.001 AU) were revealed. Also, we found an important increase of metHb levels inside RBCs (0.186 ± 0.017 AU) and in its supernatant (0.086 ± 0.020 AU) after 48 h. Consequently, MDA absorbance increased significantly (0.320 ± 0.040 AU) in RBCs exposed to diabetic plasma with high HbA1c. CONCLUSION: These findings suggest that poor glycemic control in diabetes leads to metHb generation which is the main factor of the OS amplification.
Subject(s)
Erythrocytes , Methemoglobin , Humans , Methemoglobin/metabolism , Methemoglobin/pharmacology , Oxidative Stress , Hemoglobins/metabolismABSTRACT
Sodium nitrite (NaNO2) is a universal antidote for patients with cyanide poisoning. However, its use has serious drawbacks in terms of efficacy and safety. Herein, we present a promising antidote: methemoglobin (metHb)-albumin clusters. The metHb-albumin cluster is made by a metHb core wrapped by covalently bound human serum albumin. Spectral analyses proved that the metHb-albumin clusters possessed cyanide-binding properties similar to those of naked metHb. In vitro cell experiments showed that metHb-albumin clusters prevented the cyanide-induced inhibition of cytochrome c oxidase activity, resulting in a strong cytoprotective effect. In mice subjected to cyanide poisoning, metHb-albumin clusters reduced mortality and alleviated metabolic acidosis, while maintaining the activity of cytochrome c oxidase in organs; their efficacy was better than that of NaNO2. Furthermore, the oxygen carrying capacity was maintained in poisoned mice treated with metHb-albumin clusters and was low in those treated with NaNO2. These results indicate that metHb-albumin clusters could be a more effective and safer antidote against cyanide poisoning than NaNO2.
Subject(s)
Cyanides , Methemoglobin , Humans , Mice , Animals , Methemoglobin/analysis , Methemoglobin/chemistry , Methemoglobin/metabolism , Cyanides/metabolism , Antidotes/pharmacology , Electron Transport Complex IV/metabolism , Albumins/metabolismABSTRACT
Intravascular hemolysis occurs in diverse pathological conditions. Extracellular hemoglobin and heme have strong pro-oxidative and pro-inflammatory potentials that can contribute to the pathology of hemolytic diseases. However, many of the effects of extracellular hemoglobin and heme in hemolytic diseases are still not well understood. Here we demonstrate that oxidized hemoglobin (methemoglobin) can modify the antigen-binding characteristics of human immunoglobulins. Thus, incubation of polyclonal or some monoclonal human IgG in the presence of methemoglobin results in an appearance of binding reactivities towards distinct unrelated self-proteins, including the protein constituent of hemoglobin i.e., globin. We demonstrate that a transfer of heme from methemoglobin to IgG is indispensable for this acquisition of antibody polyreactivity. Our data also show that only oxidized form of hemoglobin have the capacity to induce polyreactivity of antibodies. Site-directed mutagenesis of a heme-sensitive human monoclonal IgG1 reveals details about the mechanism of methemoglobin-induced antigen-binding polyreactivity. Further here we assess the kinetics and thermodynamics of interaction of a heme-induced polyreactive human antibody with hemoglobin and myoglobin. Taken together presented data contribute to a better understanding of the functions of extracellular hemoglobin in the context of hemolytic diseases.
Subject(s)
Heme , Methemoglobin , Humans , Heme/metabolism , Methemoglobin/metabolism , Hemoglobins/metabolism , Immunoglobulin G , Antibodies, Monoclonal , HemolysisABSTRACT
OBJECTIVE: To determine whether dogs with cytochrome b5 reductase (CYB5R) deficiency have a constitutive proinflammatory phenotype, characterize hematologic and serum chemistry results, and describe changes in methemoglobin (MetHb) levels and serum C-reactive protein (CRP) concentrations after long-term per os (PO) methylene blue (MB) therapy. ANIMALS: 21 client-owned dogs (CYB5R deficient, n = 10; healthy controls, 11). PROCEDURES: In this prospective, case-control study, methemoglobin levels were measured using a blood gas analyzer with co-oximetry. Plasma tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) concentrations were measured using a canine-specific multiplex bead-based assay. Serum CRP concentrations were measured with a canine-specific commercial ELISA kit. Serum CRP concentration and MetHb levels were measured in 6 dogs with CYB5R deficiency after ≥ 60 days of PO MB therapy. RESULTS: As expected, MetHb levels were higher in dogs with CYB5R deficiency compared to controls (P < .001). Plasma TNF-α, IL-6, IL-10, and serum CRP concentrations were no different between CYB5R-deficient and control dogs. Dogs with CYB5R deficiency had lower absolute lymphocyte (P = .005) and eosinophil counts (P = .04) and higher alanine transaminase (P = .04) and alkaline phosphatase activity (P = .02) than controls, but these changes were not clinically relevant. Methemoglobin levels decreased after PO MB therapy (P = .03). CLINICAL RELEVANCE: These results suggest that otherwise healthy dogs with CYB5R deficiency do not have a constitutive proinflammatory phenotype and clinically relevant abnormalities in hematologic and serum chemistry panels are not expected. Dogs with decreased quality of life attributed to methemoglobinemia from CYB5R deficiency might benefit from PO MB therapy.
Subject(s)
Dog Diseases , Methemoglobinemia , Dogs , Animals , Methemoglobinemia/veterinary , Methemoglobinemia/drug therapy , Methemoglobinemia/genetics , Methylene Blue/therapeutic use , Methemoglobin/genetics , Methemoglobin/metabolism , Methemoglobin/therapeutic use , Interleukin-10/genetics , Interleukin-10/therapeutic use , Cytochromes b5/genetics , Interleukin-6/genetics , Interleukin-6/therapeutic use , Tumor Necrosis Factor-alpha/genetics , Case-Control Studies , Prospective Studies , Quality of Life , Cytochrome-B(5) Reductase/genetics , Phenotype , Dog Diseases/drug therapy , Dog Diseases/geneticsABSTRACT
In this study, the effects of different oxidation intensities on the degradation of myofibrillar protein by endogenous enzymes in iron-catalyzed oxidizing (IOS) and metmyoglobin oxidizing system (MOS) were compared. The results showed that carbonyl content and endogenous enzyme activities (caspase-3, caspase-6 and calpain-1) increased significantly and the total sulfhydryl content decreased significantly with H2O2 concentration in both oxidation systems. Meanwhile, the rate of carbonyl formation and the inhibition of endogenous enzymes activities of IOS were significantly lower than MOS for the same oxidation intensity. In addition, IOS and MOS mainly produced myosin light chains degradation products of 20-25 kDa and 20-17 kDa. At the same oxidation intensity, MOS of myofibrillar protein significantly enhanced the degradation of troponin-T and desmin by caspase-3/-6 compared with IOS, while inhibiting the degradation of troponin-T by calpain-1. In conclusion, MOS inhibited endogenous enzyme degradation in vitro more than IOS during post-slaughter maturation of yak meat.
Subject(s)
Calpain , Myofibrils , Animals , Cattle , Proteolysis , Myofibrils/metabolism , Calpain/metabolism , Caspase 3/metabolism , Methemoglobin/metabolism , Troponin T/metabolism , Iron/metabolism , Hydrogen Peroxide/metabolism , Muscle, Skeletal/metabolism , Meat/analysis , Oxidation-Reduction , Metmyoglobin/metabolism , CatalysisABSTRACT
During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 µM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42- uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 µM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.
Subject(s)
Anion Exchange Protein 1, Erythrocyte , Antioxidants , Anion Exchange Protein 1, Erythrocyte/metabolism , Anion Exchange Protein 1, Erythrocyte/pharmacology , Antioxidants/metabolism , Antioxidants/pharmacology , CD47 Antigen/metabolism , Erythrocytes/metabolism , Humans , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Methemoglobin/metabolism , Oxidative Stress , Quercetin/metabolism , Quercetin/pharmacology , Reactive Oxygen Species/metabolism , Thiobarbituric Acid Reactive Substances/metabolismABSTRACT
Red blood cells (RBCs) carry large cholesterol fractions and imbalance in them leads to several vascular complications. RBCs band 3 protein plays an important role in maintaining membrane integrity and there are many reports on cholesterol and band 3 protein interaction. Yet, RBCs band 3 protein role in regulating cholesterol homeostasis needs to be investigated. In this study, we induced cholesterol-depletion and band 3 inhibition in RBCs; both of which cause stress by decreasing band 3 channel activity with an increase in RBCs adhesion to endothelial cells (EC) by elevating band 3 phosphorylation (Tyr21), methemoglobin level and decreasing nitric oxide level. We hypothesized that nitric oxide (NO), a prominent determinant for RBC structural stability, would protect RBCs from stressors. To estimate this, we used three NO donors (SpNO, Sildenafil citrate and 8-Bromo-cGMP) and found that all 3 NO donors were able to recover, with 8-Bromo-cGMP being the most effective as it not only increased band 3 channel activity but also decreased RBC-EC adhesiveness and methemoglobin level in both stressors. Whereas NO donor's treatment did not display an ameliorative impact when both stresses were combined. Overall, these findings may shed light on the role of 8-bromo-cGMP in regulating RBC cholesterol homeostasis by maintaining band 3 function. Further studies in this direction might help identify targets for the therapeutic use of NO donors in the treatment of blood disorders.
Subject(s)
Nitric Oxide Donors , Nitric Oxide , Anion Exchange Protein 1, Erythrocyte/metabolism , Cholesterol , Cyclic GMP/analogs & derivatives , Endothelial Cells/metabolism , Erythrocytes/metabolism , Methemoglobin/metabolism , Nitric Oxide/metabolism , Nitric Oxide Donors/pharmacologyABSTRACT
Hydrogen sulfide (H2S) has attracted significant attention as a seed in drug development. However, H2S is toxic and induces lethal acute intoxication. Here, we developed methemoglobin (metHb)-albumin clusters as detoxifying agents for H2S intoxication, which were designed based on the inherent binding property of metHb with H2S. The metHb-albumin clusters comprising an autoxidized ferric Hb center wrapped covalently with an average of three human serum albumins showed a similar H2S binding affinity to that of naked metHb. Owing to the H2S binding capability, metHb-albumin clusters suppressed cell death induced by H2S exposure while maintaining mitochondrial function in H9c2 cells. In addition, lethal H2S intoxication model mice were rescued by a single administration of metHb-albumin clusters, resulting from the recovery of cytochrome c oxidase activity. Furthermore, the metHb-albumin clusters possessed essential characteristics, such as adequate pharmacokinetic properties and biocompatibility, for their use as detoxifying agents against H2S intoxication. In conclusion, the results obtained in this study suggest that metHb-albumin clusters are promising detoxifying agents for H2S intoxication and that harnessing the inherent H2S binding properties of metHb is an innovative approach to develop detoxifying agents for H2S intoxication.
Subject(s)
Hydrogen Sulfide , Methemoglobin , Albumins/metabolism , Animals , Electron Transport Complex IV/metabolism , Humans , Hydrogen Sulfide/metabolism , Hydrogen Sulfide/toxicity , Iron/metabolism , Methemoglobin/metabolism , Methemoglobin/pharmacology , MiceABSTRACT
Hydrogen sulfide (H2S) induces acute and lethal toxicity at high concentrations. However, no specific antidotes for H2S poisoning have been approved. Liposomal methemoglobin (metHb@Lipo) was developed as an antidote for cyanide poisoning. As the toxic mechanism of H2S poisoning is the same as that of cyanide poisoning, metHb@Lipo could potentially be used as an antidote for H2S poisoning. In this study, we evaluated the antidotal efficacy of metHb@Lipo against H2S poisoning. Stopped-flow rapid-scan spectrophotometry clearly showed that metHb@Lipo scavenged H2S rapidly. Additionally, metHb@Lipo showed cytoprotective effects against H2S exposure in H9c2 cells by maintaining mitochondrial function. MetHb@Lipo treatment also improved the survival rate after H2S exposure in vivo, with the maintenance of cytochrome c oxidase activity and suppression of metabolic acidosis. Moreover, metHb@Lipo therapy maintained significant antidotal efficacy even after 1-year-storage at 4-37 °C. In conclusion, metHb@Lipo is a candidate antidote for H2S poisoning.
Subject(s)
Hydrogen Sulfide , Poisoning , Antidotes/pharmacology , Antidotes/therapeutic use , Cyanides , Humans , Hydrogen Sulfide/metabolism , Methemoglobin/metabolism , Methemoglobin/pharmacology , Poisoning/drug therapyABSTRACT
This aim of this study was to investigate whether macrophages protect endometriotic cells from oxidative injury and to elucidate the underlying mechanisms of any protection. Endometriotic cells cultured with or without differentiated macrophages (dTHP-1 cells) were treated with hydrogen peroxide (H2O2) or methemoglobin, a major component of hemoglobin species in endometriotic cyst fluid. Co-culture experiments, microarray analysis, screening and validation of differentially expressed genes (DEGs), cell proliferation and viability assays, and experiments using a specific inhibitor were conducted to investigate the functional cross-talk between endometriotic cells and macrophages. Microarray analysis revealed that endometriotic cells co-cultured with dTHP-1 differentially express several genes compared with monoculture. Quantitative enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis identified TGF-ß1 as a promising candidate gene expressed in endometriotic cells co-cultured with dTHP-1 cells. TGF-ß1 stimulated the expression of heme oxygenase-1 (HO-1) in dTHP-1 cells. HO-1 expression was increased in dTHP-1 cells co-cultured with endometriotic cells compared with the dTHP-1 monoculture. Both H2O2 and methemoglobin upregulated the expression of the HO-1 protein in the dTHP-1 monoculture; moreover, co-culture with endometriotic cells further enhanced HO-1 production. The co-culture with dTHP-1 protected endometriotic cells against oxidative injury. Blockade of HO-1 abolished the protective effects of macrophages. In an oxidative stress environment, TGF-ß1 produced by endometriotic cells may protect against oxidative injury through the upregulation of macrophage-derived HO-1. The cross-talk between endometriotic cells and macrophages may contribute to the progression and pathogenesis of endometriosis.
Subject(s)
Endometriosis , Transforming Growth Factor beta1 , Endometriosis/metabolism , Female , Heme Oxygenase-1/metabolism , Humans , Hydrogen Peroxide/pharmacology , Macrophages/metabolism , Methemoglobin/metabolism , Methemoglobin/pharmacology , Oxidative Stress , Transforming Growth Factor beta1/metabolismABSTRACT
Methemoglobin (metHb) arises from the oxidation of ferrous hemoglobin (HbFe2+, Hb) to ferric hemoglobin (HbFe3+, metHb), which is unable to bind gaseous ligands such as oxygen (O2) and carbon monoxide (CO), and binds to nitric oxide (NO) significantly slower compared to Hb. Therefore, metHb does not elicit vasoconstriction and systemic hypertension in vivo due to its extremely slow NO scavenging rate in comparison to cell-free Hb, but will induce oxidative tissue injury, demonstrating the potential of using metHb as a control material when studying the toxicity of cell-free Hb. Hence, the goal of this work was to develop a novel manufacturing strategy for production of metHb that is amenable to scale-up. In this study, small scale (e.g. 1 mL reaction volume) screening experiments were initially conducted to determine the optimal molar ratio of Hb to the oxidization agents hydrogen peroxide (H2O2) or sodium nitrite (NaNO2) to achieve the highest conversion of Hb into metHb. A spectral deconvolution program was employed to determine the molar fraction of various species (hemichrome, metHb, oxyHb, metHb-[Formula: see text], and NaNO2) in solution during the oxidation reaction. From this analysis, either a 1:1 or 1:5 molar ratio was identified as optimal molar ratios of Hb:NaNO2 (heme basis) that yielded the highest conversion of Hb into metHb with negligible amounts of side products. Hence in order to reduce the reaction time, a 1:5 molar ratio was chosen for large scale (i.e. 1.5 L reaction volume) synthesis of bovine metHb (metbHb) and human metHb (methHb). The biophysical properties of metHb were then characterized to elucidate the potential of using the synthesized metHb as a non-O2 carrying control material. The haptoglobin binding kinetics of metHb were found to be similar to Hb. Additionally, the synthesized metHb was stable in phosphate buffered saline (PBS, 50 mM, pH 7.4) at 4°C for approximately one week, indicating the high stability of the material.
Subject(s)
Hemoglobins/metabolism , Hydrogen Peroxide/chemistry , Methemoglobin/metabolism , Nitric Oxide/chemistry , Oxygen/chemistry , Sodium Nitrite/chemistry , Animals , Cattle , Hemoglobins/analysis , Humans , Methemoglobin/analysis , Oxidants/chemistry , Oxidation-ReductionABSTRACT
Dapsone (DDS) therapy can frequently lead to hematological side effects, such as methemoglobinemia and DNA damage. In this study, we aim to evaluate the protective effect of racemic alpha lipoic acid (ALA) and its enantiomers on methemoglobin induction. The pre- and post-treatment of erythrocytes with ALA, ALA isomers, or MB (methylene blue), and treatment with DDS-NOH (apsone hydroxylamine) was performed to assess the protective and inhibiting effect on methemoglobin (MetHb) formation. Methemoglobin percentage and DNA damage caused by dapsone and its metabolites were also determined by the comet assay. We also evaluated oxidative parameters such as SOD, GSH, TEAC (Trolox equivalent antioxidant capacity) and MDA (malondialdehyde). In pretreatment, ALA showed the best protector effect in 2.5 µg/mL of DDS-NOH. ALA (1000 µM) was able to inhibit the induced MetHb formation even at the highest concentrations of DDS-NOH. All ALA tested concentrations (100 and 1000 µM) were able to inhibit ROS and CAT activity, and induced increases in GSH production. ALA also showed an effect on DNA damage induced by DDS-NOH (2.5 µg/mL). Both isomers were able to inhibit MetHb formation and the S-ALA was able to elevate GSH levels by stimulating the production of this antioxidant. In post-treatment with the R-ALA, this enantiomer inhibited MetHb formation and increased GSH levels. The pretreatment with R-ALA or S-ALA prevented the increase in SOD and decrease in TEAC, while R-ALA decreased the levels of MDA; and this pretreatment with R-ALA or S-ALA showed the effect of ALA enantiomers on DNA damage. These data show that ALA can be used in future therapies in patients who use dapsone chronically, including leprosy patients.
Subject(s)
Methemoglobin , Thioctic Acid , Methemoglobin/metabolism , Antioxidants/pharmacology , Thioctic Acid/pharmacology , Dapsone/pharmacology , Superoxide Dismutase , DNA DamageABSTRACT
A patient with gunshots within inches of the skin developed intraoperative vasodilatory hypotension and methemoglobinemia, both recognized consequences of nitrite poisoning. A 1- mg/kg dose of methylene blue transiently and partially reversed methemoglobinemia, but the color of the methylene blue faded rapidly, consistent with bleaching of methylene blue by nitrite in vivo. Methylene blue did not raise blood pressure, consistent with inhibition of nitric oxide (NO) synthase. Because NO production from nitrite uses an NO synthase (NOS)-independent pathway, methylene blue is expected to have little effect on reversing hypotension from nitrite poisoning. Consider nitrite toxicity in gunshot patients with refractory vasodilatory hypotension and elevated methemoglobin.
Subject(s)
Hypotension , Methemoglobinemia , Wounds, Gunshot , Humans , Methemoglobinemia/chemically induced , Methemoglobinemia/drug therapy , Methylene Blue/therapeutic use , Nitrites/adverse effects , Methemoglobin/adverse effects , Methemoglobin/metabolism , Hypotension/chemically induced , Hypotension/drug therapyABSTRACT
INTRODUCTION: High-dose nitric oxide (NO) has been shown effective against a variety of micro-organisms in vitro, including common bacteria found in donor organs. However, clinical obstacles related to its implementation in vivo are the formation of methemoglobin and the accumulation of toxic nitrogen compounds. Ex vivo lung perfusion (EVLP) is a platform that allows for organ maintenance with an acellular perfusion solution, thus overcoming these limitations. The present study explores the safety of continuous high-dose inhaled (iNO) during EVLP for an extended period of 12 hours. METHODS: Lungs procured from Yorkshire pigs were randomized into control (standard ventilation) and treatment (standard ventilation + 200 ppm iNO) groups, then perfused with an acellular solution for 12 hours (n = 4/group). Lung physiology and biological markers were evaluated. RESULTS: After 12 hours of either standard EVLP or EVLP + 200 ppm iNO, we did not notice any significant physiologic difference between the groups: pulmonary oxygenation (P = .586), peak airway pressures (P = .998), and dynamic (P = .997) and static (P = .908) lung compliances. In addition, no significant differences were seen among proinflammatory cytokines measured in perfusate and lung tissue. Importantly, most common toxic compounds were kept at safe levels throughout the treatment course. CONCLUSIONS: High-dose inhaled NO delivered continuously over 12 hours appears to be safe without inducing any significant pulmonary inflammation or deterioration in lung function. These findings support further efficacy studies to explore the use of iNO for the treatment of infections in donor lungs during EVLP.