Elevated methemoglobin levels in patients treated with high-dose hydroxocobalamin.

OBJECTIVE: The aim of this study was to assess the impact of hydroxocobalamin (OHCbl) infusion on arterial blood gas and oximetry values in patients with vasoplegic syndrome. METHODS: Blood samples collected from 95 patients receiving OHCbl infusion were assayed using the ABL90 FLEX Plus blood gas analyzer for the concentration of methemoglobin (MetHb), total hemoglobin (tHb), carboxyhemoglobin (COHb), arterial oxygen saturation (SaO2), arterial oxygen partial pressure (PaO2), and arterial carbon dioxide partial pressure (PaCO2). Interference of OHCbl on these variables was evaluated using the measured difference between the preinfusion and postinfusion samples. RESULTS: Blood MetHb (%) measured after the infusion of OHCbl (5g) were significantly higher than the baseline levels, with a median of 4.8 (IQR, 3.0-6.5) versus 1.0 (IQR, 1.0-1.2) (P < .001). Blood COHb (%) increased from a median of 1.3 (IQR, 1.0-1.8) to 1.7 (IQR, 1.3-2.2) (P < .001) following the OHCbl infusion. No differences were seen in median levels of tHb, PaO2, PaCO2, and SaO2 between pre- and post-OHCbl treatment. CONCLUSION: The presence of OHCbl in blood clearly interfered with the oximetry measurements of the hemoglobin component fractions by falsely increasing the levels of MetHb and COHb. Blood levels of MetHb and COHb cannot be reliably determined by the co-oximetry when OHCbl is known or suspected.

A novel pre-clinical strategy to deliver antimicrobial doses of inhaled nitric oxide.

Effective treatment of respiratory infections continues to be a major challenge. In high doses (≥160 ppm), inhaled Nitric Oxide (iNO) has been shown to act as a broad-spectrum antimicrobial agent, including its efficacy in vitro for coronavirus family. However, the safety of prolonged in vivo implementation of high-dose iNO therapy has not been studied. Herein we aim to explore the feasibility and safety of delivering continuous high-dose iNO over an extended period of time using an in vivo animal model. Yorkshire pigs were randomized to one of the following two groups: group 1, standard ventilation; and group 2, standard ventilation + continuous iNO 160 ppm + methylene blue (MB) as intravenous bolus, whenever required, to maintain metHb <6%. Both groups were ventilated continuously for 6 hours, then the animals were weaned from sedation, mechanical ventilation and followed for 3 days. During treatment, and on the third post-operative day, physiologic assessments were performed to monitor lung function and other significative markers were assessed for potential pulmonary or systemic injury. No significant change in lung function, or inflammatory markers were observed during the study period. Both gas exchange function, lung tissue cytokine analysis and histology were similar between treated and control animals. During treatment, levels of metHb were maintained <6% by administration of MB, and NO2 remained <5 ppm. Additionally, considering extrapulmonary effects, no significant changes were observed in biochemistry markers. Our findings showed that high-dose iNO delivered continuously over 6 hours with adjuvant MB is clinically feasible and safe. These findings support the development of investigations of continuous high-dose iNO treatment of respiratory tract infections, including SARS-CoV-2.

Effects of encapsulated nitrate on growth performance, nitrate toxicity, and enteric methane emissions in beef steers: Backgrounding phase.

A long-term experiment was conducted to examine the effects of feeding encapsulated nitrate (EN) on growth, enteric methane production, and nitrate (NO) toxicity in beef cattle fed a backgrounding diet. A total of 108 crossbred steers (292 ± 18 kg) were blocked by BW and randomly assigned to 18 pens. The pens (experimental unit; 6 animals per pen) received 3 dietary treatments: Control, a backgrounding diet supplemented with urea; 1.25% EN, control diet supplemented with 1.25% encapsulated calcium ammonium NO (i.e., EN) in dietary DM, which partially replaced urea; or 2.5% EN, control diet supplemented with 2.5% EN (DM basis) fully replacing urea. Additionally, 24 steers were located in 4 pens and randomly assigned to 1 of the above 3 dietary treatments plus a fourth treatment: 2.3% UEN, control diet supplemented with 2.3% unencapsulated calcium ammonium NO (UEN) fully replacing urea. Animals in the additional 4 pens were used for methane measurement in respiratory chambers, and the pens (except UEN) were also part of the performance study (i.e., = 7 pens/treatment). The experiment was conducted for 91 d in a randomized complete block design. During the experiment, DMI was not affected by inclusion of EN in the diet. Feeding EN had no effect on BW, ADG, and G:F ( ≥ 0.57). Methane production (g/d) tended to decrease ( = 0.099) with EN and UEN, but yield (g/kg DMI) did not differ ( = 0.56) among treatments. Inclusion of EN in the diet increased ( ≤ 0.02) sorting of the diets in favor of large and medium particles and against small and fine particles, resulting in considerable increases in NO concentrations of orts without affecting DMI. Plasma NO-N and NO-N concentrations increased ( ≤ 0.05) for EN compared with Control in a dose response manner, but blood methemoglobin levels were below the detection limit. Nitrate concentration in fecal samples slightly increased (from 0.01% to 0.14% DM; < 0.01) with increasing levels of EN in the diet. In conclusion, EN can be used as a feed additive replacing urea in beef cattle during a backgrounding phase in the long term without NO intoxication or any negative effects on growth performance. In addition, the study confirmed that feeding EN tended to decrease enteric methane production in the long term.

[Evolution of the carboxyhemoglobinemia and methemoglobinemia during endoscopic resection: An observational study]. / Évolution de la carboxyhémoglobinémie et de la méthémoglobinémie lors de résections endoscopiques : une étude observationnelle.

AIM OF THE STUDY: Combustion of organic tissues due to endoscopic resection could induce methemoglobin (MetHb) and carboxyhemoglobin (COHb) formation. The aim of this study is to evaluate MetHb and COHb formation in patients undergoing prostatic or bladder endoscopic procedures. METHODS: COHb and MetHb measurements were performed in 44 patients at the beginning and end of the procedure. A third measurement was done in patients who stayed more than one hour in the recovery room. Means were compared using Student t-test, simple regressions were used for quantitative variables and ANOVA for categorical variables. Multiple linear regressions were used for multivariate analysis. RESULTS: COHb increased by 0.5±0.9 % (95 % CI: 0.2 to 0.7 % P=0.001). MetHb increase was 0.0±0.4 % (95 % CI: -0.1 to 0.2 % P=0.552). In univariate analysis, the variables associated with COHb increase are the length of surgery, the amount of irrigation fluid and location (prostate or bladder) of the procedure. In the multivariate model, COHb increase is associated with the amount of liquid and the location. CONCLUSION: MetHb did not increase during endoscopic surgery. In contrast, COHb increases, and can, in some patients, exceed 2-4 %. This could be responsible for a decreased angina threshold in patients with ischemic heart disease. LEVEL OF EVIDENCE: 4.

Interaction of recombinant octameric hemoglobin with endothelial cells.

Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbßG83C. We investigate the potential side effects of rHbßG83C on endothelial cells. The rHbßG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbßG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb ßG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.

Quantification of the chemical composition variations of tumors in photothermal therapy by photoacoustic spectroscopy: an in vitro study.

In this study, the change of tumors' chemical composition in the temperature range of 20 ~70 °C is quantified for photothermal tumor therapy by photoacoustic spectroscopy (PAS) with the wavelengths of 700~1000 nm. Based on the measured photoacoustic signals, two absorption peaks at the wavelengths of 750 nm and 950 nm are identified. It is also observed that high temperature (>55 °C) is able to induce the physical and chemical degeneration of tumors. According to the in vitro tests, a new chemical species, met-hemoglobin, which is absent in normal blood, is generated at high temperature with enhanced near-infrared absorption.

Antioxidant effect of zinc supplementation on both plasma and cellular red-ox status markers in a group of elderly Italian population.

OBJECTIVES: The aim of this work was to evaluate the effect of long term supplementation with two moderate dose of Zn on plasma and cellular red-ox status markers in elderly volunteers. DESIGN, SETTING AND SUBJECTS: In a double blind study 108 healthy volunteers, aged 70-85 years, were enrolled. They were randomly divided in 3 groups of treatment, receiving placebo, 15 mg/day and 30 mg/day of Zn for 6 months. Red-ox status markers were assessed at baseline and after 6 months evaluating carotenoids, vitamin A and E in plasma; glutathione (GSH), thiol groups (RSH), malondialdehyde (MDA), percentage of haemolysis and methemoglobin in erythrocytes. RESULTS: Zn supplementation had no significant effects on red-ox status markers except for vitamin A levels (from 1.94±0.44 to 2.18±0.48 µM in volunteers receiving 15 mg of Zn and from 1.95±0.46 to 2.26±0.56 µM in volunteers receiving 30 mg of Zn), which increased proportionally to zinc dose. CONCLUSIONS: It appears that, differently from unhealthy populations, long-term supplementation with two moderate doses of Zn in a healthy elderly population, with an adequate Zn nutritive status and macro and micronutrients intakes in the range of normality, is an inefficient way to increase antioxidant defences.

High-dose vitamin C management in dapsone-induced methemoglobinemia.

Methylene blue is the first-choice treatment of methemoglobinemia, but it is not readily available in most Korean emergency departments because of an import suspension. An 84-year-old woman with dapsone-induced massive methemoglobinemia visited our emergency department for unclear mentality and cyanosis. Because methylene blue was not available, we intravenously administrated vitamin C (VC) for symptomatic methemoglobinemia, although VC is not a universally accepted treatment. Vitamin C (10 g intravenously) administered 6 hourly successfully treated the dapsone-induced methemoglobinemia and did not adversely affect renal functions. Thus,we recommend that if methylene blue is unavailable, 6 hourly intravenous administrations of 10 g of VC should be considered for dapsone-induced methemoglobinemia.

Effect of chloride on nitrite-induced methaemoglobinemia in Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus (Mitchill).

We evaluated the effects of chloride concentration on the clinical pathology in juvenile Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus (Mitchill), following semi-static exposures to 1 mg L(-1) nitrite for 96 h. In spring water naturally low in chloride (5 mg L(-1)), plasma nitrite concentrated to more than 40× environmental levels resulting in a severe methaemoglobinemia characterized by torpid behaviour, 30-fold increase in methaemoglobin fraction, anaemia, leucopenia and hyperkalaemia. Loss of intracellular water and potassium to extracellular space may have resulted in hyperkalaemia and haemodilution. Fish survived nitrite exposure, but 60% of torpid fish died following capture and tissue sampling. Fish acclimated to 10-fold higher chloride content (55 mg L(-1)) did not concentrate nitrite in the plasma above environmental levels or develop methaemoglobinemia, but did exhibit similar haematology and plasma chemistry changes. Plasma nitrite returned to preexposure levels by 14 days following nitrite exposures, but severity of clinical pathology changes persisted or increased, suggesting that Atlantic sturgeon have reduced capacity to recover from methaemoglobinemia. Fish that survive methaemoglobinemia may be susceptible to mortality from the cumulative effects of intoxication, handling and other stresses for two or more weeks following nitrite remediation. Chloride buffering in aquaculture systems reduces the toxic effects of nitrite accumulation.

Potential behavioral and pro-oxidant effects of Petiveria alliacea L. extract in adult rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Petiveria alliacea (Phytolaccaceae) is a perennial shrub indigenous to the Amazon Rainforest and tropical areas of Central and South America, the Caribbean, and sub-Saharan Africa. In folk medicine, Petiveria alliacea has a broad range of therapeutic properties; however, it is also associated with toxic effects. AIM OF THE STUDY: The present study evaluated the putative effects of Petiveria alliacea on the central nervous system, including locomotor activity, anxiety, depression-like behavior, and memory, and oxidative stress. MATERIALS AND METHODS: Two-month-old male and female Wistar rats (n=7-10 rats/group) were administered with 900 mg/kg of hydroalcoholic extracts of Petiveria alliacea L. The behavioral assays included open-field, forced swimming, and elevated T-maze tests. The oxidative stress levels were measured in rat blood samples after behavioral assays and methemoglobin levels were measured in vitro. RESULTS: Consistent with previous reports, Petiveria alliacea increased locomotor activity. It also exerted previously unreported anxiolytic and antidepressant effects in behavioral tests. In the oxidative stress assays, the Petiveria alliacea extract decreased Trolox equivalent antioxidant capacity levels and increased methemoglobin levels, which was related to the toxic effects. CONCLUSIONS: The Petiveria alliacea extract exerted motor stimulatory and anxiolytic effects in the OF test, antidepressant effects in the FS test, and elicited memory improvement in ETM. Furthermore, the Petiveria alliacea extract also exerted pro-oxidant effects in vitro and in vivo, inhibiting the antioxidant status and increasing MetHb levels in human plasma, respectively.

Stabilization of erythrocytes against oxidative and hypotonic stress by tannins isolated from sumac leaves (Rhus typhina L.) and grape seeds (Vitis vinifera L.).

Erythrocytes are constantly exposed to ROS due to their function in the organism. High tension of oxygen, presence of hemoglobin iron and high concentration of polyunsaturated fatty acids in membrane make erythrocytes especially susceptible to oxidative stress. A comparison of the antioxidant activities of polyphenol-rich plant extracts containing hydrolysable tannins from sumac leaves (Rhus typhina L.) and condensed tannins from grape seeds (Vitis vinifera L.) showed that at the 5-50 µg/ml concentration range they reduced to the same extent hemolysis and glutathione, lipid and hemoglobin oxidation induced by erythrocyte treatment with 400 µM ONOO(-) or 1 mM HClO. However, extract (condensed tannins) from grape seeds in comparison with extract (hydrolysable tannins) from sumac leaves stabilized erythrocytes in hypotonic NaCl solutions weakly. Our data indicate that both hydrolysable and condensed tannins significantly decrease the fluidity of the surface of erythrocyte membranes but the effect of hydrolysable ones was more profound. In conclusion, our results indicate that extracts from sumac leaves (hydrolysable tannins) and grape seeds (condensed tannins) are very effective protectors against oxidative damage in erythrocytes.

Human erythrocytes as a system for evaluating the antioxidant capacity of vegetable extracts.

Free radicals are fairly unstable and highly reactive substances, able of causing oxidation and sometimes-irreversible damage to cells, compromising their function. The Brassicaceae family has many important species for the regular human diet as they provide several antioxidant constituents. In this study, the antioxidant potential of the hydroethanolic extracts prepared from the edible parts of kale, broccoli, and radish was investigated in vitro using human erythrocytes under oxidative stress imposed by phenylhydrazine as an experimental model, in which the methemoglobin levels were measured. When the results were compared with the antioxidant capacity shown by the traditional 2,2-diphenyl-2-picrylhydrazyl hydrate free radical and phosphomolybdenum complex methods, the extracts tested showed significant and correspondent antioxidant activity. Broccoli extract presented the highest antioxidant activity, followed closely by the kale, whereas the radish extract occupied the lowest position. The results derived from the human erythrocyte system have shown it as an alternative method for evaluating the antioxidant properties of vegetable extracts.

Pro-oxidative effects of Chinese herbal medicine on G6PD-deficient erythrocytes in vitro.

Glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects are susceptible to chemical-induced oxidative haemolysis. Little is known concerning the haemolytic properties of Chinese herbal medicine on G6PD-deficient subjects. Our objective was to investigate the pro-oxidative effect of 18 commonly used Chinese herbal medicine (CHM) on human G6PD-deficient red blood cells. G6PD-deficient (n=10) and normal (n=10) whole blood samples were incubated with water extracts of CHM. The resulting levels of reduced glutathione (GSH) and methaemoglobin (MetHb) were determined by biochemical assays. Rhizoma Coptidis significantly reduced GSH level by 48.9+/-5.4% (at 1 mg/mL) in the G6PD-deficient erythrocytes (P<0.001) compared with the respective control group without challenge. Similar dose-dependent responses were observed at higher concentrations of Cortex Moutan, Radix Rehmanniae, Radix Bupleuri, Rhizoma Polygoni Cuspidati and Flos Chimonanthi (P<0.01, 5-10 mg/mL). In addition, the levels of MetHb were elevated significantly when challenged with Rhizoma Coptidis (2.8 fold at 5 mg/mL) and Cortex Moutan (3.4 fold at 10 mg/mL). This is the first report on the pro-oxidative action of CHM on G6PD-deficient blood samples in vitro as demonstrated by the decrease of GSH and increase of MetHb. G6PD-deficient subjects should restrain from excessive consumption of these pro-oxidative herbs.

Antioxidant supplementation enhances erythrocyte antioxidant status and attenuates cyclosporine-induced vascular dysfunction.

The aim of this study was to determine the effects of dietary antioxidant supplementation with alpha-tocopherol and alpha-lipoic acid on cyclosporine-induced alterations to erythrocyte and plasma redox balance, and cyclosporine-induced endothelial and smooth muscle dysfunction. Rats were randomly assigned to either control, antioxidant, cyclosporine or cyclosporine + antioxidant treatments. Cyclosporine A was administered for 10 days after an 8-week feeding period. Plasma was analyzed for alpha-tocopherol, total antioxidant capacity, malondialdehyde and creatinine. Erythrocytes were analyzed for glutathione, methemoglobin, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, alpha-tocopherol and malondialdehye. Vascular endothelial and smooth muscle function was determined in vitro. Antioxidant supplementation resulted in significant increases in erythrocyte alpha-tocopherol concentration and glutathione peroxidase activity in both of the antioxidant-supplemented groups. Cyclosporine administration caused significant decreases in glutathione concentration, methemoglobin concentration and superoxide dismutase activity. Antioxidant supplementation attenuated the cyclosporine-induced decrease in superoxide dismutase activity. Cyclosporine therapy impaired both endothelium-independent and -dependent relaxation of the thoracic aorta, and this was attenuated by antioxidant supplementation. In summary, dietary supplementation with alpha-tocopherol and alpha-lipoic acid attenuated the cyclosporine-induced decrease in erythrocyte superoxide dismutase activity and attenuated cyclosporine-induced vascular dysfunction.

Effects of dietary antioxidant supplementation before and after oral acetaminophen challenge in cats.

OBJECTIVE: To determine effects of lipoic acid, vitamin E, and cysteine before and after oxidant challenge in cats. ANIMALS: 24 sexually intact adult cats. PROCEDURE: Cats were allocated into 4 equal groups. For 25 weeks, group A was fed a control dry diet and groups B, C, and D received this diet supplemented with vitamin E (2200 U/kg [dry matter basis {DMB}]) plus cysteine (9.5 g/kg [DMB]), lipoate (150 mg/kg [DMB]), or all 3 antioxidants together, respectively. Weights were measured every 3 days and venous blood obtained every 5 weeks for CBC; serum biochemical analyses; lymphocyte blastogenesis; thiobarbituric acid reactive substances concentration; and concentrations of plasma protein carbonyl, 8-OH d-guanosine, blood glutathione, plasma amino acid, lipoate, and dihydrolipoate. At 15 weeks, all cats received acetaminophen (9 mg/kg, PO, once), clinical effects were observed, and methemoglobin concentrations were measured. RESULTS: Lymphocyte blastogenesis increased transiently in group C and D cats. After acetaminophen administration, all groups had transient increases in methemoglobin within 4 hours and mild, brief facial edema; group C had decreased glutathione concentration and increased 8-OH d-guanosine concentration versus controls; and protein carbonyl concentration increased least for group B. Plasma lipoate and dihydrolipoate concentrations peaked by week 10 for groups C and D. Conclusions and Clinical Relevance-Lipoate, vitamin E, and cysteine did not have synergistic effects. Lipoate supplementation (150 mg/kg [DMB]) did not act as an antioxidant but appeared to enhance oxidant effects of acetaminophen. Vitamin E plus cysteine had protective effects.

Influence of laser wavelength and pulse duration on gas bubble formation in blood filled glass capillaries.

BACKGROUND AND OBJECTIVES: Hypervascular skin lesions (HVSL) are treated with medical lasers characterized by a variety of parameters such as wavelength lambda, pulse duration t(p), and radiant exposure E that can be adjusted for different pathology and blood vessel size. Treatment parameters have been optimized assuming constant optical properties of blood during laser photocoagulation. However, recent studies suggest that this assumption may not always be true. Our objective was to quantify thermally induced changes in blood that occur during irradiation using standard laser parameters. STUDY DESIGN/MATERIALS AND METHODS: Glass capillary tubes (diameter D = 100, 200, and 337 microm) filled with fresh or hemolyzed rabbit blood were irradiated once at lambda = 585, 595, or 600 nm, t(p) = 1.5 milliseconds; and also at lambda = 585 nm, t(p) = 0.45 milliseconds. E was increased until blood ablation caused formation of permanent gas bubbles. In a corroborative study, human blood was heated at 50 degrees C and absorbance spectra were measured as a function of time. RESULTS: Threshold radiant exposure, E(thresh), for gas bubble formation was found not to depend on lambda, which might be surprising in view of the 10-fold lower absorption coefficient at 600 nm as compared to 585 nm. The spectroscopic study revealed heat-induced changes in blood constituent composition of hemoglobins (Hb) from initially 100% oxyhemoglobin (HbO2) to deoxyhemoglobin (HHb) and, ultimately, methemoglobin (metHb) as the major constituent. Model calculations of E(thresh)(lambda,D) based on changing constituent blood composition during heating with milliseconds lasers were found to correlate with experimental results. CONCLUSIONS: For laser treatment of HVSL it appears that lambda is of secondary importance and that the choice of t(p) is a more important factor.

Antioxidant status of red blood cells and liver in hypercholesterolemic rats fed hypolipidemic spices.

Animal studies were carried out to examine the beneficial influence of known hypolipidemic spice principles--curcumin, capsaicin, and garlic--on the antioxidant status of red blood cells and liver under induced hypercholesterolemic conditions. Groups of experimental rats rendered hypercholesterolemic were maintained on curcumin (0.2%)/capsaicin (0.015%)/garlic (2.0% dry powder)-containing diets for eight weeks. Erythrocytes isolated at the end of the study were analyzed for intracellular antioxidant molecules and antioxidant enzymes. Intracellular thiols and glutathione content in red blood cells were significantly depleted (by about 35%) in hypercholesterolemic rats. This depletion in intracellular thiols and glutathione was effectively countered by dietary spice principles - curcumin, capsaicin, and garlic. Glutathione reductase activity that was lowered in hypercholesterolemic conditions (by 25%) was completely countered by dietary spice principles and garlic. Activities of glutathione transferase, glutathione peroxidase, and catalase in erythrocytes remained unchanged under hypercholesterolemic conditions. Although hemoglobin levels of erythrocytes were not affected, methemoglobin concentration was significantly increased in hypercholesterolemic rats. This alteration was partially countered by dietary spice principles. Significant fall in hepatic total thiols in the hypercholesterolemic situation was partially corrected by dietary spice treatment. Similarly, the lowered activities of hepatic antioxidant enzymes--glutathione reductase, glutathione-S-transferase, catalase, and superoxide dismutase--in hypercholesterolemic rats were effectively countered by the dietary spices treatment.

Comparison of methylene blue, riboflavin, and N-acetylcysteine for the reduction of nitric oxide-induced methemoglobinemia.

OBJECTIVE: To investigate the treatment of nitric oxide (NO)-induced methemoglobinemia by methylene blue (MB), riboflavin, and N-acetylcysteine (NAC) in vitro. DESIGN: Prospective, controlled in vitro study. SETTING: Research laboratory in a university hospital. PARTICIPANTS: Five healthy volunteers. INTERVENTIONS: Generation of 16% to 18% of methemoglobin in red blood cells by NO and subsequent addition of MB, riboflavin, or NAC. Simultaneous NO (32 ppm) and MB or riboflavin exposure of red blood cells. Induction of 14% to 18% of methemoglobin in red blood cells by NO, subsequent addition of MB or riboflavin, and further incubation with NO (80 ppm). MEASUREMENTS AND MAIN RESULTS: After discontinuation of NO, mean half-life for methemoglobin was significantly reduced by MB from 356 mins (controls) to 5 mins (10 microM) in a dose-dependent manner (p < .001). NAC did not alter the half-life for methemoglobin, and a reduction from 356 to 168 mins was seen for 120 microM riboflavin (p < .001). Methemoglobin formation after 3 hrs of NO exposure was 4.3%+/-0.7% in controls and 0.3%+/-0.1% with 10 microM MB (p < .001); 1 microM MB attenuated methemoglobin formation to 1.9%+/-0.1% (p < .01). With riboflavin (120 microM), methemoglobin was 2.2%+/-0.5% vs. 3.2%+/-0.6% in controls (p < .001). In the presence of high methemoglobin concentrations, further methemoglobin formation was inhibited by 1 and 10 microM MB (p < .001) and attenuated by 0.1 microM MB (p < .001) but not by riboflavin. CONCLUSIONS: In vitro, NO-induced methemoglobin formation is significantly decreased by medium (1 microM) and high (10 microM) concentrations of MB and partially by high riboflavin concentrations (120 microM). NAC and low concentrations of riboflavin do not alter methemoglobin formation.