metHb Formation Research Articles

Evaluation of Spontaneous Overtime Methemoglobin Formation in Post-Mortem Blood Samples from Real Cases in Critical Storage Conditions.

Nitrite/nitrate poisoning is an emerging problem, with an ongoing escalation of reported self-administration with suicidal intent in several countries. Nitrites toxicity mainly consists of their interaction with hemoglobin (Hb), causing its oxidization to methemoglobin (MetHb). In order to give support to the correct procedures for the analysis of these cases, this study aims to evaluate spontaneous sample degradation and consequent MetHb formation in the typical storage conditions of a forensic toxicology laboratory. Two different types of samples have been used in this study: the first stage of our study consisted of a retrospective analysis of blood samples obtained by judicial autopsies already stored in the toxicology laboratory, collected over four years (2018-2021), while the samples used for the second stage were appositely collected during judicial autopsies. The data obtained by the application of a derivative spectrophotometry method on these samples suggest that there seems not to be a maximum threshold for MetHb formation within which it is possible to state with a sufficient grade of certainty that the concentration of MetHb found is consistent with an ante-mortem formation and is not the result of an artifact due to sample degradation and storage conditions. On the other hand, the results suggest that MetHb formation depends on the time passed between sample collection and analysis, so that a tempestive sample processing, performed as soon as the samples are received in the laboratory, is crucial to obtain the maximum reliability and diagnostic values from the data when MetHb quantitation is necessary.

The impact of cannabinoids on methemoglobin formation and hemoglobin oxygen affinity: An ex-vivo study

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 (+1.4 mmHg and -0.9 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.

Mechanisms of Primaquine Induced Hemolysis in a Novel Humanized Murine Model of Mediterranean G6PD Deficiency

Background: Approximately half a billion individuals are genetically deficient in glucose 6-phosphate dehydrogenase (G6PD) due to amino acid variants generally thought to decrease enzyme half-life. Decreased G6PD activity limits the antioxidant capacity of red blood cells (RBCs). As such, certain drugs that induce oxidative stress can cause life-threatening hemolysis in G6PD deficient (G6DPd) individuals. Standard regiments of the only approved drugs that can cure liver phase Plasmodium vivax ( p. vivax) (i.e. primaquine and tafenoquine) are contraindicated in G6PDd patients due to risks of hemolysis. Effective treatment of G6PDd individuals infected with p. vivax requires a prolonged course of primaquine (8 weeks), leading to non-adherence, and subsequent reduced efficacy and increased risk of transmission. Despite decades of research and large drug screening programs, no non-hemolytic compound has been identified that can eradicate p. vivax. Lack of progress in this area has likely been hampered by an incomplete mechanistic understanding of primaquine induced hemolysis, in part due to the lack of an animal model that recapitulates G6PD instability. Methods: Recombinant non-deficient human hG6PD(ND) or the deficient Mediterranean variant (hG6PD(MED)) were expressed, purified, and subjected to thermal proteome profiling in combination with TMT10-assisted quantitative cross-linking proteomics. Mice were generated in which murine genomic G6PD is replaced with either genomic human hG6PD(ND) or hG6PD(MED). Hemolysis in humans is caused by primaquine metabolites (e.g. primaquine-5,6-orthoquinone (5,6-POQ)) and not primaquine itself. As such, RBCs from both strains were exposed to 5,6-POQ and then analyzed by 1) high resolution metabolomics and proteomics, 2) electron paramagnetic resonance (EPR) to measure superoxide, 3) methemoglobin (MetHb) formation, 4) light and electron microscopy, 5) in vitro hemolysis, and 6) in vivo hemolysis (clearance) after infusion into animals. In vivo pulse chase biotinylation allowed visualization and identification of younger (1-6 days) RBCs vs. older (7-55 day) RBCs. Results: Recombinant hG6PD(MED) enzyme had a 2.6-fold lower specific activity compared to hG6PD(ND). Thermal gradient crosslinking proteomics identified specific amino acid spacing (less than 26 angstroms) that was higher in hG6PD(ND) [82-95 and 91-432] vs. higher in hG6PD(MED) [89-205, 97-429, and 429-497]. Analysis of mice demonstrated that hG6PD(MED) RBCs had 5% of G6PD activity as compared to hG6PD(ND) RBCs, with an RBC age dependent decrease in G6PD activity in hG6PD(MED) RBCs. Exposure to 5,6-POQ caused increased superoxide generation, increased MetHb formation, alterations to morphology, in vitro hemolysis, and in vivo hemolysis of hG6PD(MED) RBCs compared to hG6PD(ND) RBCs. However, younger hG6PD(MED) RBCs with higher G6PD activity were highly resistant to 5,6-POQ induced hemolysis (both in vivo and in vitro). All experiments were carried out a minimum of 3 times and all stated findings were statistically significant (p<0.05). Proteomic and metabolomic analysis (with combined samples from multiple experiments) demonstrated oxidation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in a block in glycolysis that increases dihydroxyacetone phosphate (DHAP) leading to cytotoxic methylglyoxal that broadly oxidizes protein lysines to carboxymethyllysine (CML) - the first step in advanced glycation end products (AGEs) known to facilitate RBC clearance. Discussion: We identify specific conformational changes caused by the hG6PD(MED) variant resulting in decreased specific activity and half-life, and confirm that G6PD activity decreases with RBC age, in a humanized mouse model. The resistance of young hG6PD(MED) RBCs to 5,6-POQ has immediate practical ramifications, in that it supports the rationale of clinical studies currently proposing new dose escalation primaquine regimens to induce low level early hemolysis, leading to reticulocytosis, and decreasing the mean age of circulating RBCs such that increased parricidal doses of primaquine can then be tolerated in G6PDd patients. The elucidation of the metabolic and proteomic lesion (under conditions confirmed to cause 5,6-POQ induced hemolysis of hG6PD(MED) but not hG6PD(ND) RBCs) provides a mechanistic landscape to guide development of non-hemolytic drugs to treat p. vivax.

Rare Sighting of an Unstable Hemoglobin Variant [ Β11 (A8) Val → Asp]: Hemoglobin Windsor

Hemoglobin (Hb) is the major blood oxygen carrier where Hb subunits bind oxygen via the heme iron buried within a hydrophobic pocket that faces the outside of HbA tetramers. Iron must be in its reduced (ferrous, Fe2+) state for Hb to bind oxygen. Oxidized or metHb (ferric, Fe3+) cannot bind oxygen and is relatively unstable. Globin mutations that alter amino acids within the ligand pocket lead to changes in affinity for oxygen, and increased rates of metHb formation and heme loss. We present the case of a patient with a rare variant “Hb Windsor” which has only been described once in the literature. The patient is a 20-year-old Mexican male with a history of hepatitis B presenting with three days of fevers, chills, nausea and vomiting. The patient reported a diagnosis of hepatitis at the age of eight in Mexico and with jaundice all his life. He had no prior hospitalizations or blood transfusions. He denied any unusual food ingestions or recent foreign travel. In the emergency room, the patient was hypoxic with a SpO2 73% and tachypneic which improved with supplemental oxygen. On physical examination, the patient was jaundiced with scleral icterus and the spleen was easily palpable to the midline. CT scan confirmed splenomegaly measuring 20 cm. Labs showed a white blood count 21.6 nl, hemoglobin 4.2 g/dl, platelet count 33 nl, lactate dehydrogenase 13 unit/L and reticulocyte count 24%, total bilirubin 16 mg/dl, direct bilirubin 4 mg/dl and methemoglobin 20.9%. The patient was administered methylene blue and transfused two units of packed red blood cells with hemoglobin increasing to 7.1 g/dl. HIV, hepatitis B and C, direct antiglobulin test, and G6PD testing were negative. The peripheral smear showed numerous nucleated red cells, increased polychromasia with red cells showing numerous microspherocytes, microanthocytes, and bubbled cells with significant basophilic stippling. No fragmented cells were present. The clinical history and lab findings suggested a chronic hemolytic anemia, and the peripheral smear suggested a possible hemoglobinopathy. Hemoglobin electrophoresis by high performance liquid chromatography showed an unknown hemoglobin of 22.5%. Isoelectric focusing showed Hb A 71.9%, F, Hb A2 4.6%, Hb F 0.3%, and an abnormal band of 23% in the hemoglobin M range. Specimens were sent for DNA sequencing and a point mutation in the beta chain was found leading to a valine to aspartate substitution in the A8 position. Protein analysis showed a valine is substituted with aspartic acid in the ligand pocket which is the unstable varian Hb Windsor. This substitution in the base of the heme pocket results in instability of the hemoglobin molecule and an increase in oxygen affinity. The residues lining the heme pocket function as a hydrophobic basket from the heme iron, preventing its oxidation to the ferric state. Valine, which occupies position A8 is a hydrophobic amino acid. However, in Hb Windsor, the valine is substituted by aspartic acid, a polar molecule. The hydrophilic nature of aspartic acid, a polar molecule, may disrupt the hydrophobic nature of the heme pocket. The valine at position A8 also acts as a contact with the H helix which is lost in Hb Windsor leading to decreased Hb stability. These substitutions cause heme iron to auto-oxidize which results in methemoglobinemia. Hb M has iron in the ferric state (Fe3+) and is unable to carry oxygen which produces cyanosis. Our patient was found to be hypoxic requiring oxygen supplementation and found to have methemoglobinemia. We present a rare case of a hemoglobin variant Hb Windsor in a Mexican adult which has only been previously documented in a nine-month-old Anglo-Saxon infant. This variant seems to be a spontaneous de novo mutation as both patients had no family history of anemia. It is hypothesized that the hemolysis was precipitated by oxidative stress of an acute infection in the setting of an unstable hemoglobin. The patient was found to have stable hemoglobin of 12 g/dl and methemoglobin of 4.9% in follow up several years later.

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.

Mercury Chloride Affects Band 3 Protein-Mediated Anionic Transport in Red Blood Cells: Role of Oxidative Stress and Protective Effect of Olive Oil Polyphenols.

Mercury is a toxic heavy metal widely dispersed in the natural environment. Mercury exposure induces an increase in oxidative stress in red blood cells (RBCs) through the production of reactive species and alteration of the endogenous antioxidant defense system. Recently, among various natural antioxidants, the polyphenols from extra-virgin olive oil (EVOO), an important element of the Mediterranean diet, have generated growing interest. Here, we examined the potential protective effects of hydroxytyrosol (HT) and/or homovanillyl alcohol (HVA) on an oxidative stress model represented by human RBCs treated with HgCl2 (10 µM, 4 h of incubation). Morphological changes as well as markers of oxidative stress, including thiobarbituric acid reactive substance (TBARS) levels, the oxidation of protein sulfhydryl (-SH) groups, methemoglobin formation (% MetHb), apoptotic cells, a reduced glutathione/oxidized glutathione ratio, Band 3 protein (B3p) content, and anion exchange capability through B3p were analyzed in RBCs treated with HgCl2 with or without 10 μM HT and/or HVA pre-treatment for 15 min. Our data show that 10 µM HT and/or HVA pre-incubation impaired both acanthocytes formation, due to 10 µM HgCl2, and mercury-induced oxidative stress injury and, moreover, restored the endogenous antioxidant system. Interestingly, HgCl2 treatment was associated with a decrease in the rate constant for SO42- uptake through B3p as well as MetHb formation. Both alterations were attenuated by pre-treatment with HT and/or HVA. These findings provide mechanistic insights into benefits deriving from the use of naturally occurring polyphenols against oxidative stress induced by HgCl2 on RBCs. Thus, dietary supplementation with polyphenols might be useful in populations exposed to HgCl2 poisoning.

Alpha-Lipoic Acid and Its Enantiomers Prevent Methemoglobin Formation and DNA Damage Induced by Dapsone Hydroxylamine: Molecular Mechanism and Antioxidant Action

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.

Nitrite Toxicity: Chemical Analysis, Metabolism, and Health Effects

Nitrites can be formed from and away by the nitrate-nitrite-nitric oxide pathway. The properties of nitrite oxidation and metHb formation are carefully studied in mechanism, forming either N-binding or O-binding structures. Apart from nitrate and nitric oxide, nitrites can also form carcinogenic nitrosamines in acidic environments. MetHb can cause hypoxia and vasodilation, while symptoms are revealed in different degrees under recalled or present hypoxic conditions. The study thoroughly studied nitrite’s metabolic properties, chemical pathways, and dosage effects on health. The cancer risks of consuming dietary nitrite need more statistical support, while its metabolite N-nitrosodimethylamine and NDMA concentration are well considered with increasing cancer risks. ED50 of human vasodilation is identified, and lethal doses on juvenile pike-perch can be further utilized to predict related doses for humans. More studies should be done to investigate relative nitrite doses to boost utilization and studies about this chemical.

Effects of sodium chloride and sodium tripolyphosphate on the prooxidant properties of hemoglobin in washed turkey muscle system.

This study examined the effects of sodium chloride (NaCl) and sodium tripolyphosphate (STPP) on lipid oxidation induced by oxyhemoglobin (oxyHb) in washed turkey muscle (WTM) model. To explore the reasons for observed effects, the pro-oxidant abilities of Hb derivatives (e.g., metHb, oxyHb, hemin, Fe2+, and Fe3+), pH change, and antioxidation of Hb in the presence of NaCl or STPP were also analyzed. The observed lipid oxidation capacity in WTM followed the order metHb>hemin>oxyHb>Fe2+ > Fe3+. Added Fe2+ accelerated auto-oxidation of oxyHb and oxyHb-mediated lipid oxidation. Hb auto-oxidation to metHb increased as the pH decreased from 6.6 to 5.0. NaCl promoted oxyHb-mediated lipid oxidation due to NaCl causing decreased pH value and increased formation of metHb. STPP inhibited oxyHb-mediated lipid oxidation and weakened the pro-oxidative effect of NaCl. This could be attributed to STPP increasing the pH, inactivating free iron, and inhibiting formation of metHb.

Photoactivation of tDodSNO induces localized vasodilation in rats: Metabolically stable S-nitrosothiols can act as targeted nitric oxide donors in vivo

Nitric oxide (NO) is a key vasodilatory signalling molecule and NO releasing molecules (NO donors) are being examined as potential treatments for many pathologies. The photoresponsive NO donor tert-dodecane S-nitrosothiol (tDodSNO) has been designed to be highly resistant to metabolism; in principle photoactivation of tDodSNO should therefore enable the controlled release of NO in situ via light modulation. To investigate the therapeutic utility of tDodSNO, we tested drug efficacy in Sprague Dawley rats to assess systemic and localised hemodynamic responses under photoactivation, and to confirm drug safety. For comparison, drug action was evaluated alongside the existing NO donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Across a dosing range (0.1–3.0 mg/kg) tDodSNO exerted markedly reduced systemic hypotensive action compared to these standard NO donors, inducing a slight decrease in mean arterial pressure (maximum 14.2 ± 3.0%) without affecting heart rate. Target limb photoactivation of tDodSNO resulted in a substantial localized vasodilatory response, with increases to mean (26.0 ± 7.3%) and maximum (53.2 ± 10.4%) blood flow and decreases to vascular resistance (27.1 ± 3.9%) that were restricted to light exposed tissue. In comparison GSNO and SNP showed variable peripheral effects and were not responsive to photoactivation. tDodSNO did not induce met-Hb formation in blood, or display any signs of toxicity, and was rapidly cleared from the systemic circulation, with no hemodynamic effects detectable 5 min post administration. These data are the first demonstration that drugs based upon a metabolically stable S-nitrosothiol group can be photoactivated in vivo to release NO, and that such agents cause less systemic side effects than existing NO donors. Our data support the use of S-nitrosothiols to enable the spatiotemporal control of NO for therapeutic applications.

Antioxidant Activity of Quercetin in a H2O2-Induced Oxidative Stress Model in Red Blood Cells: Functional Role of Band 3 Protein.

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.

Rapid Formation of Methemoglobin via Nitric Oxide Delivery for Potential Use as an MRI Contrast Agent.

Contrast-enhanced magnetic resonance angiography is a vital tool for evaluating vascular pathology. However, concerns about the limitations and safety of gadolinium-based contrast agents have led to an interest in alternative agents. Methemoglobin (metHb) increases the T1-weighted signal intensity of the magnetic resonance image of blood and could provide a safe and effective alternative. MetHb can be produced by the reaction of nitric oxide (NO) gas with oxyhemoglobin followed by natural conversion back to hemoglobin by cytochrome b5 reductase. Since rapid production of metHb via NO has not been studied, the effectiveness of producing metHb via NO delivery to blood was evaluated using a hollow-fiber module. MetHb production began immediately and > 90% conversion was achieved within 10min. MetHb remained stable for at least 90min when NO delivery was removed following metHb formation. Comparison of experimental data for metHb formation with model predictions showed that only a fraction of the NO delivered was utilized for metHb production, suggesting an additional fast reaction of NO with other blood constituents. Directly delivering NO to blood for the rapid formation of metHb provides a potential platform for producing metHb as an alternative contrast agent.

HBOC-301 in Porcine Kidney Normothermic Machine Perfusion and the Effect of Vitamin C on Methemoglobin Formation.

Normothermic machine perfusion (NMP) of kidneys in combination with an optimized perfusate composition may increase donor organ preservation quality, especially in the case of marginal donor grafts. Optimization of currently employed perfusates is still a subject of present research. Due to the advantages of being cell-free, easy to store, and having minimal antigenicity, hemoglobin-based oxygen carriers, such as HBOC-301 (Oxyglobin®, Hemoglobin Oxygen Therapeutics LLC, Souderton, PA, USA), offer an alternative to the commonly used perfusates based on packed red blood cells (pRBC). As previously described, using HBOC results in formation of methemoglobin (metHb) as an adverse effect, inducing hypoxic conditions during the perfusion. As a potential counterpart to metHb formation, the application of the antioxidant ascorbic acid (VitC) is of high interest. Therefore, this study was conducted in four experimental groups, to compare the effect of NMP with (1) HBOC or (3) pRBC, and additionally examine a beneficial effect of VitC in both groups (2) HBOC + VitC and (4) pRBC + VitC. All groups were subjected to NMP for 6 h at a pressure of 75 mmHg. Kidneys in the HBOC groups had a significantly lower renal blood flow and increasing intrarenal resistance, with reduced renal function in comparison to the pRBC groups, as demonstrated by significantly lower creatinine clearance and higher fractional sodium excretion rates. Clinical chemistry markers for tissue damage (LDH, lactate) were higher in the HBOC groups, whereas no significant histological differences were observed. Although the application of VitC decreased oxidative stress levels, it was not able to significantly increase the outcome parameters mentioned above in either group. This study demonstrated that HBOC-301 is inferior to pRBCs in our porcine kidney NMP model, independent of additional VitC administration. Oxidative stress and fragmentation of the hemoglobin polymers could be detected as a possible reason for these results, hence further research, focusing on the use of cell-free oxygen carriers that do not exhibit this complex of issues, is required.

Pro-oxidative activity of trout and bovine hemoglobin during digestion using a static in vitro gastrointestinal model

The degradation of trout and bovine hemoglobin (Hb) and their pro-oxidant activities in washed cod muscle mince (WCM) were studied using simple pH-shifts to simulate gastrointestinal (GI) conditions (pH 7 → 6 → 3 → 7), as well as full static in vitro GI digestion. Following gastric acidification to pH 6, metHb formation increased, especially for trout Hb. Subsequent acidification to pH 3 promoted Hb unfolding and partial or complete heme group-loss. During full GI digestion, polypeptide/peptide analyses revealed more extensive Hb-degradation in the gastric than duodenal phase, without any species-differences. When digesting WCM +/-Hb, both Hbs strongly promoted malondialdehyde (MDA), 4-hydroxy-2-hexenal (HHE), and 4-hydroxy-2-nonenal (HNE) formation, peaking at the end of the gastric phase. Trout-Hb stimulated MDA and HHE more than bovine Hb in the first gastric phase. Altogether, partially degraded Hb, and/or free hemin -both mammal and fish-derived- stimulated oxidation of PUFA-rich lipids under GI-conditions, especially gastric ones.

Elevated methemoglobin levels in patients treated with hydroxocobalamin: a case series and in-vitro analysis

Background Historically, the first step in treating cyanide (CN-) toxicity utilized antidotes to induce methemoglobinemia. This is concerning in patients who are already hypoxemic or have elevated carboxyhemoglobin. Hydroxocobalamin (OHCbl) is now the first-line antidote for CN- toxicity and is not known to induce methemoglobinemia. We observed elevated methemoglobin (MetHb) levels in several patients treated with OHCbl and sought to investigate the incidence of MetHb formation following administration of OHCbl. Methods Chart review: A single-center, retrospective case series of patients who received 5 or 10 g of hydroxocobalamin from 01/01/2011 through 04/30/2019. Data was analyzed using descriptive statistics. In-vitro study: Discarded blood was separated into whole blood and plasma samples. OHCbl and normal saline was added to reach 0×, 1×, 2×, and 4× peak therapeutic concentrations and analyzed at times 0, 2, and 4 h after administration. Results Chart review: Twenty-seven cases of OHCbl administration were identified. The median age was 53 years (IQR 38 − 64) and 20 (74.1%) were male. Exposure to a house fire or smoke inhalation was the reason for OHCbl administration in 21 (77.8%) patients. Five (18.5%) patients received 10 g of OHCbl while the rest received 5 g. Six (22.2%) patients developed methemoglobinemia, all after 5 g OHCbl administration; four had been exposed to fire and smoke, two received the medication for severe acidosis of unknown etiology not related to fire or smoke. The median peak level was 7.1% (IQR 2.2 − 16.4%) at a median time of 11.4 h post-administration. Two patients received methylene blue (MB), neither responded. Death occurred in 17 (63%) cases. In-vitro study: We observed a dose dependent elevation in total hemoglobin but did not detect any increase in MetHb. Conclusion We observed a noteworthy temporal association between the formation of methemoglobinemia and the administration of hydroxocobalamin. This does not appear to be an artifact of the CO-oximeters. This could have profound implications for patients who are already hypoxemic or have impaired oxygen carrying capacity from carboxyhemoglobin.

The oxygen dissociation curve of blood in COVID-19.

COVID-19 hinders oxygen transport to the consuming tissues by at least two mechanisms: In the injured lung, saturation of hemoglobin is compromised, and in the tissues, an associated anemia reduces the volume of delivered oxygen. For the first problem, increased hemoglobin oxygen affinity [left shift of the oxygen dissociation curve (ODC)] is of advantage, for the second, however, the contrary is the case. Indeed a right shift of the ODC has been found in former studies for anemia caused by reduced cell production or hemolysis. This resulted from increased 2,3-bisphosphoglycerate (2,3-BPG) concentration. In three investigations in COVID-19, however, no change of hemoglobin affinity was detected in spite of probably high [2,3-BPG]. The most plausible cause for this finding is formation of methemoglobin (MetHb), which increases the oxygen affinity and thus apparently compensates for the 2,3-BPG effect. However, this “useful effect” is cancelled by the concomitant reduction of functional hemoglobin. In the largest study on COVID-19, even a clear left shift of the ODC was detected when calculated from measurements in fresh blood rather than after equilibration with gases outside the body. This additional “in vivo” left shift possibly results from various factors, e.g., concentration changes of Cl−, 2,3-BPG, ATP, lactate, nitrocompounds, glutathione, glutamate, because of time delay between blood sampling and end of equilibration, or enlarged distribution space including interstitial fluid and is useful for O2 uptake in the lungs. Under discussion for therapy are the affinity-increasing 5-hydroxymethyl-2-furfural (5-HMF), erythropoiesis-stimulating substances like erythropoietin, and methylene blue against MetHb formation.

Spectroscopic investigation on molecular aspects and structural and functional effects of tetraethyl pyrophosphate organophosphorus insecticide interaction with adult human hemoglobin

Organophosphates are extremely toxic compounds that use extensively in agriculture and household as insecticides. However, their binding mechanism to bio-macromolecules especially blood proteins is not clearly understood. In this research, various spectroscopic techniques utilized to analyze the effect of Tetraethyl Pyrophosphate (TEPP), as an organophosphorus insecticide, on the structure, function, stability, and aggregation of adult human hemoglobin and also hemolysis potential of the TEPP on red blood cells (RBCs) examined. Molecular docking was used for TEPP binding to human Hemoglobin (Hb), too. The results demonstrated that the TEPP insecticide has the potential for lysing RBCs. UV-Vis experiment indicated that globin part and heme group influenced by TEPP. Oxygen affinity measurements revealed the formation of deoxy-Hb and met-Hb, also decreased in oxygen affinity of Hb upon interaction with TEPP that is due to heme destruction. Fluorescence spectroscopy confirmed the production of heme degradation species after interaction of Hb with TEPP, which is inconsistent with oxygen affinity measurements. Thermal and aggregation studies indicated that TEPP induced aggregation of Hb in a concentration manner and Tm of protein reduced to lower temperatures. Docking's study also showed that TEPP interacts with Hb through hydrophobic interactions, which confirms UV-Vis results. ATR-FTIR study also revealed that TEPP can induce changes in the alpha helix element of Hb’s secondary structure. Totally, Experimental and theoretical results indicate that tetraethyl pyrophosphate has unfavorable effects on hemoglobin structure and function. Communicated by Ramaswamy H. Sarma

Risk assessment of nitrate and nitrite in feed.

The European Commission asked EFSA for a scientific opinion on the risks to animal health related to nitrite and nitrate in feed. For nitrate ion, the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) identified a BMDL 10 of 64 mg nitrate/kg body weight (bw) per day for adult cattle, based on methaemoglobin (MetHb) levels in animal's blood that would not induce clinical signs of hypoxia. The BMDL 10 is applicable to all bovines, except for pregnant cows in which reproductive effects were not clearly associated with MetHb formation. Since the data available suggested that ovines and caprines are not more sensitive than bovines, the BMDL 10 could also be applied to these species. Highest mean exposure estimates of 53 and 60 mg nitrate/kg bw per day in grass silage‐based diets for beef cattle and fattening goats, respectively, may raise a health concern for ruminants when compared with the BMDL 10 of 64 mg nitrate/kg bw per day. The concern may be higher because other forages might contain higher levels of nitrate. Highest mean exposure estimates of 2.0 mg nitrate/kg bw per day in pigs’ feeds indicate a low risk for adverse health effects, when compared with an identified no observed adverse effect level (NOAEL) of 410 mg nitrate/kg bw per day, although the levels of exposure might be underestimated due to the absence of data on certain key ingredients in the diets of this species. Due to the limitations of the data available, the CONTAM Panel could not characterise the health risk in species other than ruminants and pigs from nitrate and in all livestock and companion animals from nitrite. Based on a limited data set, both the transfer of nitrate and nitrite from feed to food products of animal origin and the nitrate‐ and nitrite‐mediated formation of N‐nitrosamines and their transfer into these products are likely to be negligible.

Disparity of selenourea and selenocystine on methaemoglobinemia in non-diabetics and diabetics.

Organoselenium drugs like selenourea (SeU) and selenocystine (SeC) are found to exhibit several medicinal properties and have reported roles in the field of cancer prevention. However, studies related to their interactions with the major erythroid protein, haemoglobin (HbA) are still in dearth despite being of prime importance. In view of this, it was considered essential to investigate the interaction of these two anticancer drugs with Hb. Both the drugs showed significant changes in absorption spectra of Hb at wavelength of maximum absorption (λmax) 630 nm. SeU itself had no effect on the absorbance value at 630 nm with respect to time even with 400 µM concentration. However, it was rapidly converted to nanoselenium in presence of nitrite and there was an increase in the absorbance rate at 630 nm from 3.39 × 10-3 min-1 (without nitrite) to 8.94 × 10-3 min-1 in presence of nitrite (200 µM) owing to the generation of reactive oxygen species in the medium. Although the generation and increase in peak intensity at 630 nm in Hb generally indicates the formation and rise in the levels of methaemoglobin (metHb), nanoselenium was observed to follow a different path. Instead of causing oxidation of Fe2+ to Fe3+ responsible for metHb formation, nanoselenium was found to interact with the protein part, thereby causing changes in its secondary structure which is reflected in the increasing absorbance at 630 nm. SeC, however, showed a different effect. It was shown to act as a novel agent to reduce nitrite-induced metHb formation in a dose-dependent manner. The efficiency of SeC was again found to be less in diabetic blood samples as compared to the non-diabetic ones. For similar ratio of metHb to SeC (1:8), % reduction of metHb was found to be 27.46 ± 0.82 and 16.1 ± 2.4 for non-diabetic and diabetic samples, respectively, with a two tailed P-value much <0.05 which implies that the data are highly significant.

D-Galactose Decreases Anion Exchange Capability through Band 3 Protein in Human Erythrocytes.

d-Galactose (d-Gal), when abnormally accumulated in the plasma, results in oxidative stress production, and may alter the homeostasis of erythrocytes, which are particularly exposed to oxidants driven by the blood stream. In the present investigation, the effect of d-Gal (0.1 and 10 mM, for 3 and 24 h incubation), known to induce oxidative stress, has been assayed on human erythrocytes by determining the rate constant of SO42− uptake through the anion exchanger Band 3 protein (B3p), essential to erythrocytes homeostasis. Moreover, lipid peroxidation, membrane sulfhydryl groups oxidation, glycated hemoglobin (% A1c), methemoglobin levels (% MetHb), and expression levels of B3p have been verified. Our results show that d-Gal reduces anion exchange capability of B3p, involving neither lipid peroxidation, nor oxidation of sulfhydryl membrane groups, nor MetHb formation, nor altered expression levels of B3p. d-Gal-induced %A1c, known to crosslink with B3p, could be responsible for rate of anion exchange alteration. The present findings confirm that erythrocytes are a suitable model to study the impact of high sugar concentrations on cell homeostasis; show the first in vitro effect of d-Gal on B3p, contributing to the understanding of mechanisms underlying an in vitro model of aging; demonstrate that the first impact of d-Gal on B3p is mediated by early Hb glycation, rather than by oxidative stress, which may be involved on a later stage, possibly adding more knowledge about the consequences of d-Gal accumulation.