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Hemoglobin M is a rare hereditary disorder that causes methemoglobinemia and therefore cyanosis. The clinical manifestations of this condition differs considerably, so it should be considered as a differential diagnosis in a newborn with cyanosis, with no other apparent cause. The case of a 5 hours old newborn is presented below, with generalized cyanosis from birth with oxygen saturations between 60-70%, in whom, upon ruling out pathologies such as perinatal hypoxia, pulmonary disease, heart disease and sepsis, a high level of methemoglobin is documented, reported in 21,6%; the diagnosis of methemoglobinemia was established. The treatment administered was two doses of methylene blue with no response. For this reason, hemoglobin electrophoresis was performed, which was compatible with Hemoglobin M (Iwate or Kankakee), confirming its congenital cause., La hemoglobina M es un desorden hereditario infrecuente, causante de metahemoglobinemia y, por ende, cianosis. Las manifestaciones clínicas de esta enfermedad son amplias y variadas, por lo que se debe considerar como diagnóstico diferencial en un recién nacido con cianosis sin otra causa aparente. A continuación, se presenta el caso de un recién nacido de 5 horas de vida con cianosis generalizada desde el nacimiento y con saturaciones de oxígeno de entre 60-70%, en el que se descartaron patologías como hipoxia perinatal, patología pulmonar o cardíaca y sepsis y se documentó un nivel de metahemoglobina elevado, reportado en 21,6%, con lo cual se estableció el diagnóstico de metahemoglobinemia. El tratamiento administrado fueron dos dosis de azul de metileno, pero no hubo respuesta clínica. Por este motivo, se realizó electroforesis de hemoglobina, la cual fue compatible con hemoglobina M (Iwate o Kankakee), lo que se confirma su causa congénita de metahemoglobinemia.

Cyanosis is a pathological condition that is characterized by a bluish discoloration of the skin or mucous membranes. It may result from a number of medical conditions, including disorders of the respiratory system and central nervous system, cardiovascular diseases, peripheral vascular diseases, deep vein thrombosis, and regional ischemia. Cyanosis can also be elicited from methemoglobin. Therefore, a simple, rapid, and simultaneous monitoring of changes in oxygenated hemoglobin and deoxygenated hemoglobin is useful for protective strategies against organ ischemic injury. We previously developed a red-green-blue camera-based spectral imaging method for the measurements of melanin concentration, oxygenated hemoglobin concentration (C-HbO), deoxygenated hemoglobin concentration (C-Hb(R)), total hemoglobin concentration (C-HbT) and tissue oxygen saturation (StO(2)) in skin tissues. We leveraged this approach in this study and extended it to the simultaneous quantifications of methemoglobin concentration (C-metHb), C-HbO, C-Hb(R), and StO(2). The aim of the study was to confirm the feasibility of the method to monitor C-metHb, C-HbO, C-Hb(R), C-HbT, and StO(2). We performed in vivo experiments using rat dorsal skin during methemoglobinemia induced by the administration of sodium nitrite (NaNO2) and changing the fraction of inspired oxygen (FiO(2)), including normoxia, hypoxia, and anoxia. Spectral diffuse reflectance images were estimated from an RGB image by the Wiener estimation method. Multiple regression analysis based on Monte Carlo simulations of light transport was used to estimate C-HbO, C-HbR, C-metHb, C-HbT, and StO(2). C-metHb rapidly increased with a half-maximum time of less than 30 min and reached maximal values nearly 60 min after the administration of NaNO2, whereas StO(2) dramatically dropped after the administration of NaNO2, indicating the temporary production of methemoglobin and severe hypoxemia during methemoglobinemia. Time courses of C-HbT and S

IntroductionThough acetaminophen overdoses are common, acetaminophen induced methemoglobinemia is rare and it is thought to be due to oxidative stress from reactive metabolites. However, few prior cases of sulfhemoglobinemia in the setting of acetaminophen overdose have been reported. We report a case of mixed methemoglobinemia and sulfhemoglobinemia in the setting of a large, isolated acetaminophen ingestion.Case reportA 30-year-old African American male presented after intentionally ingesting 50 tablets of 500 mg acetaminophen two days prior. He was cyanotic and tachypneic. Peripheral oxygen saturation was 78 % on room air and minimally improved with high-flow oxygen. He was noted to have leukocytosis, thrombocytopenia, anion gap metabolic acidosis with lactic acidemia, acute kidney injury, transaminitis, hyperbilirubinemia, and coagulopathy. Arterial partial pressure of oxygen was normal. Methemoglobin and sulfhemoglobin concentrations were 8.5 % and 5.2 %, respectively. Along with intravenous N-acetylcysteine, methylene blue was administered without clinical improvement. Hemolytic anemia was subsequently noted. Glucose-6- phosphate dehydrogenase (G6PD) deficiency was then confirmed with a quantitative assay and genetic testing. He also received one dose of intravenous metoclopramide. The patient ultimately required eight units of packed red blood cells and several weeks of hemodialysis before discharge on hospital day 43.DiscussionAcetaminophen is structurally related to compounds known to cause methemoglobinemia and sulfhemoglobinemia. We hypothesize that these dyshemoglobinemias were triggered by acetaminophen-induced oxidative stress. The role of G6PD deficiency in the formation of sulfhemoglobinemia is unclear. Acetaminophen overdoses presenting with methemoglobinemia should prompt concern for underlying G6PD deficiency. Coincidental sulfhemoglobinemia should be considered if the clinical presentation is more severe than the methemoglobin

Oxidizing effects of ionizing radiation are well established and almost understood. However, exposure to low doses of widely used isotopes may result in minor and hidden oxidative stress in some forms of hemoglobin. This formation alteration regarding the legends of hemoglobin's stereochemical function may play a role in hemoglobin dysfunction. There are limited studies related to the effect of gallium isotope injections. The study intends to find the effect of gallium-68 isotope injection on male rabbits. It was conducted on thirty-two male rabbits (Orycytolagus cuniculus) divided into Group I: control and Group II: animals exposed to gallium-68 isotope at a similar dose commonly used in diagnostic protocols for humans. Blood samples were collected twice: the first was after two hours of injection with isotopes and the second was after twelve hours of injection. A linear, four-mathematical-equations matrix based on the Lamber-Beer law was used to measure the concentration of different hemoglobin derivatives. Results revealed a significant elevation (P<0.05) of methemoglobin, the oxidized form of hemoglobin, two hours after injection (Total hemoglobin = 4.463 ± 0.83), but this effect was completely reversed after twelve hours. This concluded that even low doses of isotopes result in oxidation of hemoglobin that recovers shortly. Furthermore, the outcome of the study supports the healthcare centres to understand the effect of gallium-68 injections on animals.

Cyanosis is a pathological condition that is characterized by a bluish discoloration of the skin or mucous membranes. It may result from a number of medical conditions, including disorders of the respiratory system and central nervous system, cardiovascular diseases, peripheral vascular diseases, deep vein thrombosis, and regional ischemia. Cyanosis can also be elicited from methemoglobin. Therefore, a simple, rapid, and simultaneous monitoring of changes in oxygenated hemoglobin and deoxygenated hemoglobin is useful for protective strategies against organ ischemic injury. We previously developed a red-green-blue camera-based spectral imaging method for the measurements of melanin concentration, oxygenated hemoglobin concentration (C-HbO), deoxygenated hemoglobin concentration (C-Hb(R)), total hemoglobin concentration (C-HbT) and tissue oxygen saturation (StO(2)) in skin tissues. We leveraged this approach in this study and extended it to the simultaneous quantifications of methemoglobin concentration (C-metHb), C-HbO, C-Hb(R), and StO(2). The aim of the study was to confirm the feasibility of the method to monitor C-metHb, C-HbO, C-Hb(R), C-HbT, and StO(2). We performed in vivo experiments using rat dorsal skin during methemoglobinemia induced by the administration of sodium nitrite (NaNO2) and changing the fraction of inspired oxygen (FiO(2)), including normoxia, hypoxia, and anoxia. Spectral diffuse reflectance images were estimated from an RGB image by the Wiener estimation method. Multiple regression analysis based on Monte Carlo simulations of light transport was used to estimate C-HbO, C-HbR, C-metHb, C-HbT, and StO(2). C-metHb rapidly increased with a half-maximum time of less than 30 min and reached maximal values nearly 60 min after the administration of NaNO2, whereas StO(2) dramatically dropped after the administration of NaNO2, indicating the temporary production of methemoglobin and severe hypoxemia during methemoglobinemia. Time courses of C-HbT and S

Background: Deliberate self-poisoning with sodium nitrite is uncommon. There is limited literature reporting survival after potentially fatal concentrations of methemoglobinemia. Method(s): A 25-year-old man researched suicide methods on the internet and found a recommendation for sodium nitrite as a painless and potentially reversible formof suicide. The patient purchased 100 g and ingested 15 g. He immediately had second thoughts about the attempt and called an ambulance within 10 minutes of ingestion. On paramedic arrival, he was alert, deeply cyanotic and admitted to sodium nitrite ingestion. In the emergency department, 40 minutes post-ingestion, GCS = 4, he had ashen gray skin, pulse-oximetry saturation 60% on mask high-flow oxygen, pulse 180 bpm, and BP 91/45 mmHg. He was emergently intubated to facilitate oxygen delivery. Peri-intubation hypotension was treated with intravenous crystalloids, metaraminol, and norepinephrine. Result(s): Initial VBG revealed a methemoglobin fraction of 88%, lactate 19mmol/L, and bicarbonate 14mmol/L. Three 70mg doses ofmethylene blue (1 mg/kg) were administered in the first hour post-presentation. Activated charcoal (50 g) was administered naso-gastrically. Methemoglobin fraction fell to 22%in the first hour and was 16%2 hours after methylene blue treatment. Hemodynamics also improved: pulse 120 bpm, BP 126/73 mmHg with successful cessation of norepinephrine infusion. Methemoglobin fraction fell to 0.9% over 30 hours. No further methylene blue was administered. He was extubated the next day without complication and discharged home after psychiatric treatment on day 4. Conclusion(s): Intentional ingestion of sodium nitrite is currently an uncommon method of deliberate self-poisoning. However, it is an easily accessible and unregulated salt, used in the food industry. Rapid development of methemoglobinemia can result in death without prompt medical care. In this case, early notification of the poisoning by the patient and timely

Background: Deliberate self-poisoning with sodium nitrite is uncommon. There is limited literature reporting survival after potentially fatal concentrations of methemoglobinemia. Method(s): A 25-year-old man researched suicide methods on the internet and found a recommendation for sodium nitrite as a painless and potentially reversible formof suicide. The patient purchased 100 g and ingested 15 g. He immediately had second thoughts about the attempt and called an ambulance within 10 minutes of ingestion. On paramedic arrival, he was alert, deeply cyanotic and admitted to sodium nitrite ingestion. In the emergency department, 40 minutes post-ingestion, GCS = 4, he had ashen gray skin, pulse-oximetry saturation 60% on mask high-flow oxygen, pulse 180 bpm, and BP 91/45 mmHg. He was emergently intubated to facilitate oxygen delivery. Peri-intubation hypotension was treated with intravenous crystalloids, metaraminol, and norepinephrine. Result(s): Initial VBG revealed a methemoglobin fraction of 88%, lactate 19mmol/L, and bicarbonate 14mmol/L. Three 70mg doses ofmethylene blue (1 mg/kg) were administered in the first hour post-presentation. Activated charcoal (50 g) was administered naso-gastrically. Methemoglobin fraction fell to 22%in the first hour and was 16%2 hours after methylene blue treatment. Hemodynamics also improved: pulse 120 bpm, BP 126/73 mmHg with successful cessation of norepinephrine infusion. Methemoglobin fraction fell to 0.9% over 30 hours. No further methylene blue was administered. He was extubated the next day without complication and discharged home after psychiatric treatment on day 4. Conclusion(s): Intentional ingestion of sodium nitrite is currently an uncommon method of deliberate self-poisoning. However, it is an easily accessible and unregulated salt, used in the food industry. Rapid development of methemoglobinemia can result in death without prompt medical care. In this case, early notification of the poisoning by the patient and timely

Burn wounds and wound healing invoke several biological processes that may complicate the interpretation of spectral imaging data. Through analysis of spatial frequency domain spectroscopy data (450 to 1000 nm) obtained from longitudinal investigations using a graded porcine burn wound healing model, we have identified features in the absorption spectrum that appear to suggest the presence of hemoglobin breakdown products, e.g., methemoglobin. Our results show that the calculated concentrations of methemoglobin directly correlate with burn severity, 24 h after the injury. In addition, tissue parameters such as oxygenation (StO2) and water fraction may be underestimated by 20% and 78%, respectively, if methemoglobin is not included in the spectral analysis.

Burn wounds and wound healing invoke several biological processes that may complicate the interpretation of spectral imaging data. Through analysis of spatial frequency domain spectroscopy data (450 to 1000 nm) obtained from longitudinal investigations using a graded porcine burn wound healing model, we have identified features in the absorption spectrum that appear to suggest the presence of hemoglobin breakdown products, e.g., methemoglobin. Our results show that the calculated concentrations of methemoglobin directly correlate with burn severity, 24 h after the injury. In addition, tissue parameters such as oxygenation (StO2) and water fraction may be underestimated by 20% and 78%, respectively, if methemoglobin is not included in the spectral analysis.

Pure tense (T) and relaxed (R) quaternary state polymerized human hemoglobins (PolyhHbs) were synthesized and their biophysical properties characterized, along with mixtures of T- and R-state PolyhHbs. It was observed that the oxygen affinity of PolyhHb mixtures varied linearly with T-state mole fraction. Computational analysis of PolyhHb facilitated oxygenation of a single fiber in a hepatic hollow fiber (HF) bioreactor was performed to evaluate the oxygenation potential of T- and R-state PolyhHb mixtures. PolyhHb mixtures with T-state mole fractions greater than 50% resulted in hypoxic and hyperoxic zones occupying less than 5% of the total extra capillary space (ECS). Under these conditions, the ratio of the pericentral volume to the perivenous volume in the ECS doubled as the T-state mole fraction increased from 50 to 100%. These results show the effect of varying the T/R-state PolyhHb mole fraction on oxygenation of tissue-engineered constructs and their potential to oxygenate tissues.

OBJECTIFS : Les résections endoscopiques entraînent la combustion de tissus organiques qui pourraient induire la formation de méthémoglobine (MetHb) et de carboxyhémoglobine (COHb). L’objectif de ce travail est d’évaluer en quelles proportions celle-ci sont formées chez les patients bénéficiant d’une chirurgie endoscopique de la prostate ou de la vessie. MÉTHODE : Un dosage de COHb et de MetHb a été réalisé chez 44 patients en début et en fin d’intervention. Un troisième prélèvement a été réalisé chez les patients ayant séjourné plus d’une heure en salle de réveil. Les moyennes ont été comparées avec des tests de Student. Des régressions simples ont été utilisées pour les variables quantitatives et des Anova pour les variables qualitatives. Des régressions linéaires multiples ont été utilisées pour les analyses multivariées. RÉSULTATS : L’élévation moyenne de COHb était de 0,5 ± 0,9 % (IC95 % : 0,2 à 0,7 % p = 0,001) tandis que celle de MetHb était de 0,0 ± 0,4 % (IC95 % : −0,1 à 0,2 % p = 0,552). En analyse univariée, les variables corrélées à l’élévation de COHb sont la durée de l’opération, la quantité de liquide d’irrigation et la localisation (prostate ou vessie). Dans le modèle multivarié, l’élévation de COHb est associée à la quantité de liquide d’irrigation et au site de la résection. CONCLUSION : Nous n’avons pas mis en évidence d’augmentation de MetHb au cours de la chirurgie endoscopique. En revanche, COHb augmente, et peut, chez certains patients, devenir supérieure à 2–4 %. Ceci pourrait être responsable d’une diminution du seuil angineux chez des patients souffrant d’une cardiopathie ischémique. NIVEAU DE PREUVE : 4., [Evolution of the carboxyhemoglobinemia and methemoglobinemia during endoscopic resection: An observational study] 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.

Pure tense (T) and relaxed (R) quaternary state polymerized human hemoglobins (PolyhHbs) were synthesized and their biophysical properties characterized, along with mixtures of T- and R-state PolyhHbs. It was observed that the oxygen affinity of PolyhHb mixtures varied linearly with T-state mole fraction. Computational analysis of PolyhHb facilitated oxygenation of a single fiber in a hepatic hollow fiber (HF) bioreactor was performed to evaluate the oxygenation potential of T- and R-state PolyhHb mixtures. PolyhHb mixtures with T-state mole fractions greater than 50% resulted in hypoxic and hyperoxic zones occupying less than 5% of the total extra capillary space (ECS). Under these conditions, the ratio of the pericentral volume to the perivenous volume in the ECS doubled as the T-state mole fraction increased from 50 to 100%. These results show the effect of varying the T/R-state PolyhHb mole fraction on oxygenation of tissue-engineered constructs and their potential to oxygenate tissues.

Two horses were referred for methemoglobinemia and hemolytic anemia following 5 acute deaths in their herd from an unidentified toxin source. Horses have a greater risk than other mammalian species of developing methemoglobinemia and hemolytic anemia following ingestion of oxidizing toxins, due to deficiencies in the mechanisms that protect against oxidative damage in erythrocytes. Their susceptibility to oxidative erythrocyte damage is evident in the numerous cases of red maple (Acer rubrum) toxicosis. The suspected toxins causing A. rubrum toxicosis are tannic acid, gallic acid, and a metabolite of gallic acid, pyrogallol. These compounds can be found in a variety of plants, posing a risk to equine health. In order to quickly identify toxin sources, 2 rapid in vitro assays were developed to screen plant extracts for the ability to induce methemoglobin formation or cause hemolysis in healthy equine donor erythrocytes. The plant extract screening focused on 3 species of the genus Pistacia: P. atlantica, P. terebinthus, and P. chinensis, which were located in the horse pasture. Extracts of the seeds and leaves of each species induced methemoglobin formation and resulted in hemolysis, with seed extracts having greater potency. The in vitro assays used in the current study provide a useful diagnostic method for the rapid identification of oxidizing agents from unidentified sources. There is no effective treatment for oxidative erythrocyte damage in horses, making rapid identification and removal of the source essential for the prevention of poisoning.

Tato bakalářská práce se v teoretické části zabývá problematikou dusičnanů v potravinách a v pitné vodě. Kromě koloběhu dusíku uvádí především účinky dusičnanů na lidské zdraví a dále pak přehled metod, které slouží k laboratornímu stanovení dusičnanů. Cílem této práce bylo stanovit dusičnany v různých balených pitných vodách různými metodami a tyto výsledky porovnat., The theoretical part presents the problem of nitrates in food and drinking water. The thesis presents nitrogen cycle, primarly nitrate effects on human healthy and further overview of the methods that are used for laboratory determination of nitrate. The aim of this thesis was to determine nitrate from various bottled water by using different methods and compare results., Ústav technologie potravin, obhájeno

The influence of galloyl residues on the antioxidant mechanism of polyphenols to prevent hemoglobin-promoted lipid oxidation was investigated by using polyphenolic fractions with different degrees of galloylation: nongalloylated structures from pine bark (IVP), medium-galloylated from grape pomace (IVG), and high-galloylated from witch hazel bark (IVH). Hemoglobin (Hb) from the pelagic fish horse mackerel (Trachurus trachurus) was employed as a Hb standard. In vitro experiments showed an important increase in the deoxygenation and autoxidation of horse mackerel Hb at acidic pH values. All polyphenolic fractions significantly reduced the redox stability of Hb in buffer solutions, showing a greater deoxygenation and methemoglobin (metHb) formation in the presence of IVH, followed in decreasing order by IVG and IVP. However, galloylated polyphenols (IVH and IVG) were efficient to inhibit the oxidation of the oxygenated Hb (OxyHb) and the formation of lipid oxidation products in chilled washed fish muscle. This antioxidant activity of galloylated proanthocyanidins showed a positive relationship with the phenolic concentration. Polyphenols devoid of galloyl groups (IVP) were less active to prevent either Hb oxidation or lipid oxidation in fish muscle. The results draw attention to the potential role of galloyl residues to lessen Hb-catalyzed lipid oxidation in muscle and to maintain Hb in reduced and oxygenated states, which exhibit lower pro-oxidant activity as compared to the metHb and deoxyHb species.

Human methemoglobin was crystallized in a unique unit cell and its structure was solved by molecular replacement. The hexagonal unit cell has unit-cell parameters a = b = 54.6, c = 677.4 Å, with symmetry consistent with space group P6₁22. The unit cell has the second highest aspect ratio of all unit cells contained in the PDB. The 12 molecules in the unit cell describe a right-handed helical filament having no polarity, which is different from the filament composed of HbS fibers, which is the only other well characterized fiber of human hemoglobin. The filaments reported here can be related to canonical sickle-cell hemoglobin filaments and to an alternative sickle-cell filament deduced from fiber diffraction by slight modifications of intermolecular contacts.

Human methemoglobin was crystallized in a unique unit cell and its structure was solved by molecular replacement. The hexagonal unit cell has unit-cell parameters a = b = 54.6, c = 677.4 Å, with symmetry consistent with space group P6₁22. The unit cell has the second highest aspect ratio of all unit cells contained in the PDB. The 12 molecules in the unit cell describe a right-handed helical filament having no polarity, which is different from the filament composed of HbS fibers, which is the only other well characterized fiber of human hemoglobin. The filaments reported here can be related to canonical sickle-cell hemoglobin filaments and to an alternative sickle-cell filament deduced from fiber diffraction by slight modifications of intermolecular contacts.

In order to evaluate the effects of nitrates on birds, we have exposed captive red-legged partridges to nitrates concentrations of 0 (control), 100 (dwell water in farming areas) or 500 mg/l (fertirrigation level). The cellular immune response, plasma biochemistry, methemoglobin concentration (metHb), and oxidative stress biomarkers in blood and tissues were studied after two weeks of exposure. Several blood parameters such as aspartate aminotransferase, creatinine phosphokinase and lactate dehydrogenase activities and magnesium level decreased with nitrate exposure, whereas alkaline phosphatase activity and creatinine level increased. The oxidant effect of nitrates was evidenced by the increase in blood metHb, accompanied by the lipid peroxidation of red blood cells, the increased levels of oxidized glutathione (GSH) in liver, and the generation of oxidative DNA damage in plasma lymphocytes. GSH in erythrocytes was negatively correlated with blood metHb. The cellular immune function was slightly lower at partridges exposed to nitrates. These results suggest that adverse effects of nitrates on birds occur at concentrations potentially present in the field.

Currently, no reliable noninvasive methods exist for monitoring the severity of in vivo cyanide (CN) toxicity, treatment, and resulting physiological changes. We developed a broadband diffuse optical spectroscopy (DOS) system to measure bulk tissue absorption and scattering. DOS was used to optically monitor CN toxicity and treatment with sodium nitrite (NaNO2). To perform experiments, the DOS probe was placed on the hind leg of rabbits. A sodium CN solution was infused intravenously. DOS and concurrent physiologic measurements were obtained. After completion of CN infusion, NaNO2 was infused to induce methemoglobinemia (MetHb). During infusion of CN, blood gas measurements showed an increase in venous partial pressure of oxygen (pO2), and following reversal, venous pO2 values decreased. DOS measurements demonstrated corresponding changes in hemoglobin oxygenation states and redox states of cytochrome-c oxidase (CcO) during CN infusion and NaNO2 treatment. Therefore, DOS enables detection and monitoring of CN toxicity and treatment with NaNO2.

Currently, no reliable noninvasive methods exist for monitoring the severity of in vivo cyanide (CN) toxicity, treatment, and resulting physiological changes. We developed a broadband diffuse optical spectroscopy (DOS) system to measure bulk tissue absorption and scattering. DOS was used to optically monitor CN toxicity and treatment with sodium nitrite (NaNO2). To perform experiments, the DOS probe was placed on the hind leg of rabbits. A sodium CN solution was infused intravenously. DOS and concurrent physiologic measurements were obtained. After completion of CN infusion, NaNO2 was infused to induce methemoglobinemia (MetHb). During infusion of CN, blood gas measurements showed an increase in venous partial pressure of oxygen (pO2), and following reversal, venous pO2 values decreased. DOS measurements demonstrated corresponding changes in hemoglobin oxygenation states and redox states of cytochrome-c oxidase (CcO) during CN infusion and NaNO2 treatment. Therefore, DOS enables detection and monitoring of CN toxicity and treatment with NaNO2.

The capacity of Atlantic pollock (Pollachius pollachius), seabass (Dicentrarchus labrax), and horse mackerel (Trachurus trachurus) hemoglobin (Hb) to promote lipid oxidation has been evaluated in liposomes and washed minced horse mackerel muscle. The pro-oxidant ability of fish Hbs was related with their vulnerability to suffer oxidation to metHb and release hemin either in spontaneous situation or in the presence of two representative lipid oxidation products, linolein hydroperoxides and trans-2-pentenal. The results indicated similar effectiveness to promote lipid oxidation in liposomes and washed fish muscle: pollock Hb > horse mackerel Hb > seabass Hb. Pollock Hb showed a more elevated autoxidation rate and spontaneous hemin loss and also faster oxidation to metHb in the presence of hydroperoxides and trans-2-pentenal. The autoxidation and spontaneous hemin loss rates were intermediate for horse mackerel Hb, whereas seabass Hb exhibited the highest stability. The isoelectrofocusing (IEF) pattern of pollock Hb revealed the presence of isoforms with elevated anionic character, which are known to have poor oxygen affinity at the pH values found in fish muscle (pH 7.0−5.5). In agreement with the IEF patterns, pollock Hb was less oxygenated at pH 6.8, and seabass Hb exhibited more oxygenation than did horse mackerel. MetHb forms were significantly more effective in catalyzing lipid oxidation than the corresponding reduced fish Hbs and free hemin. The present investigation highlights a direct correlation between the pro-oxidant capacity of fish Hbs and their susceptibility to undergo metHb formation and hemin loss and also suggests a potential role of lipid oxidation byproducts in activating the pro-oxidative action of hemoglobin

Many substances that form methemoglobin (MHb) effectively counter cyanide (CN) toxicity. Although MHb formers are generally applied as treatments for CN poisoning, it has been proposed that a stable, long-acting MHb former could serve as a CN pretreatment. Using this rationale, the 8-aminoquinoline WR242511, a potent long-lasting MHb former in rodents and beagle dogs, was studied in the rhesus monkey for advanced development as a potential CN pretreatment., The original document contains color images.

The authors investigated biochemically and histopathologically spontaneous and experimental methemoglobinemia in the Japanese eel (Anguilla japonica). 1. Spontaneous methemoglobinemia. The disease occurred in Japanese eels reared in culturing ponds using a water recirculating system which had high levels of nitrite (7.8～10.5ppm) in the winter of 1980, in Mie Prefecture. Twelve sampled fish had chocolate brown colored blood and liver characteristically. A few of them were accompanied by dropsy and jaundice. The blood of nine fish had high proportions of methemoglobin to total hemoglobin as 33～70%. Histopathological findings were varied with the levels of methemoglobin. In fish having 61～70% of methemoglobin levels, degeneration of erythrocytes in the blood, erythrophagocytosis in the spleen and thrombosis of gill lamellae were distincitive. In fish having 33～47% of methemoglobin levels, degeneration of erythrocytes was mild and hemosiderosis was intensive in the spleen and the kidney. 2. Experimental methemoglobinemia. Twelve eels were exposed to a nitrite concentration of 20, 30 and 40 ppm, using a flow-through system of aquaria, for 31 days. The fish exhibited methemoglobinemia and had 4～66% of methemoglobin to total hemoglobin. They manifested histopathological characteristics similar to those of fish suffering from spontaneous methemoglobinemia. Based on results from this study, it was judged that nitrite poisoning was a representative cause of methemoglobinemia.

We present noninvasive, quantitative in vivo measurements of methemoglobin formation and reduction in a rabbit model using broadband diffuse optical spectroscopy (DOS). Broadband DOS combines multifrequency frequency-domain photon migration (FDPM) with time-independent near infrared (NIR) spectroscopy to quantitatively measure bulk tissue absorption and scattering spectra between 600 nm and 1,000 nm. Tissue concentrations (denoted by brackets) of methemoglobin ([MetHb]), deoxyhemoglobin ([Hb-R]), and oxyhemoglobin ([HbO2]) were determined from absorption spectra acquired in "real time" during nitrite infusions in nine pathogen-free New Zealand White rabbits. As little as 30 nM [MetHb] changes were detected for levels of [MetHb] that ranged from 0.80 to 5.72 microM, representing 2.2 to 14.9% of the total hemoglobin content (%MetHb). These values agreed well with on-site ex vivo cooximetry data (r2= 0.902, P < 0.0001, n = 4). The reduction of MetHb to functional hemoglobins was also carried out with intravenous injections of methylene blue (MB). As little as 10 nM changes in [MB] were detectable at levels of up to 150 nM in tissue. Our results demonstrate, for the first time, the ability of broadband DOS to noninvasively quantify real-time changes in [MetHb] and four additional chromophore concentrations ([Hb-R], [HbO2], [H2O], and [MB]) despite significant overlapping spectral features. These techniques are expected to be useful in evaluating dynamics of drug delivery and therapeutic efficacy in blood chemistry, human, and preclinical animal models.

We present noninvasive, quantitative in vivo measurements of methemoglobin formation and reduction in a rabbit model using broadband diffuse optical spectroscopy (DOS). Broadband DOS combines multifrequency frequency-domain photon migration (FDPM) with time-independent near infrared (NIR) spectroscopy to quantitatively measure bulk tissue absorption and scattering spectra between 600 nm and 1,000 nm. Tissue concentrations (denoted by brackets) of methemoglobin ([MetHb]), deoxyhemoglobin ([Hb-R]), and oxyhemoglobin ([HbO2]) were determined from absorption spectra acquired in "real time" during nitrite infusions in nine pathogen-free New Zealand White rabbits. As little as 30 nM [MetHb] changes were detected for levels of [MetHb] that ranged from 0.80 to 5.72 microM, representing 2.2 to 14.9% of the total hemoglobin content (%MetHb). These values agreed well with on-site ex vivo cooximetry data (r2= 0.902, P < 0.0001, n = 4). The reduction of MetHb to functional hemoglobins was also carried out with intravenous injections of methylene blue (MB). As little as 10 nM changes in [MB] were detectable at levels of up to 150 nM in tissue. Our results demonstrate, for the first time, the ability of broadband DOS to noninvasively quantify real-time changes in [MetHb] and four additional chromophore concentrations ([Hb-R], [HbO2], [H2O], and [MB]) despite significant overlapping spectral features. These techniques are expected to be useful in evaluating dynamics of drug delivery and therapeutic efficacy in blood chemistry, human, and preclinical animal models.

Background and objectivesHypervascular 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 methodsGlass 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.ResultsThreshold 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.ConclusionsFor laser treatment of HVSL it appears that lambda is of secondary importance and that the choice of t(p) is a more important factor.

Background and objectivesHypervascular 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 methodsGlass 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.ResultsThreshold 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.ConclusionsFor laser treatment of HVSL it appears that lambda is of secondary importance and that the choice of t(p) is a more important factor.

During the investigation of aviation accidents, postmortem samples from accident victims are submitted to the FAA's Civil Aerospace Medical Institute for toxicological analysis. To determine if the accident victim was exposed to an in-flight/post crash fire or faulry heating/exhaust system, the analysis of carbon monoxide (CO) is conducted. While our laboratory predominantly uses a spectrophotometric method for the determination of carboxyhemoglobin (COHb), we consider it essential to confirm with a second technique based on a different analytical principle. Our laboratory encountered difficulties with many of our postmortem samples while employing a commonly used CC method. We believed these problems were due to elevated methemoglobin (MetHb) concentration in our specimens. MetHb does not bind CO, thus elevated MetHb levels will result in a loss of CO binding capacity. Since most commonly employed CC methods determine %COHb from a ratio of unsaturated blood to CO-saturated blood, a loss of CO binding capacity will result in an erroneously high %COHb value. Our laboratory has developed a new CC method for the determination of %COHb that incorporates sodium dithionite, which will reduce any MetHb present to Hb. Using numerous fresh human blood controls ranging from 1% to 67% COHb, we found no statistically significant differences between %COHb results from our new CC method and our spectrophotometric method. We then applied our new CC method to putrefied and non-putrefied postmortem samples. To validate the new CC method, postmortem samples were analyzed with our existing spectrophotometric method, a CC method commonly used without reducing agent, and our new CC method with the addition of sodium dithionite. As expected, we saw errors up to and exceeding 50% when comparing the unreduced CC results with our spectrophotometric method. With our new CC procedure, which incorporates a reducing agent, the error was virtually eliminated.

The high priority of monitoring workers exposed to nitrobenzene is a consequence of clear findings of experimental carcinogenicity of nitrobenzene and the associated evaluations by the International Agency for Research on Cancer. Eighty male employees of a nitrobenzene reduction plant, with potential skin contact with nitrobenzene and aniline, participated in a current medical surveillance programme. Blood samples were routinely taken and analysed for aniline, 4-aminodiphenyl (4-ADP) and benzidine adducts of haemoglobin (Hb) and human serum albumin (HSA). Also, levels of methaemoglobin (Met-Hb) and of carbon monoxide haemoglobin (CO-Hb) were monitored. Effects of smoking were straightforward. Using the rank sum test of Wilcoxon, we found that very clear-cut and statistically significant smoking effects (about 3-fold increases) were apparent on CO-Hb (P = 0.00085) and on the Hb adduct of 4-ADP (P = 0.0006). The mean aniline-Hb adduct level in smokers was 1.5 times higher than in non-smokers; the significance (P = 0.05375) was close to the 5% level. The strongest correlation was evident between the Hb and HSA adducts of aniline (rs = 0.846). Less pronounced correlations (but with P values < 0.02) appeared between aniline-Hb and 4-ADP-Hb adducts (rs = 0.388), between 4-ADP and 4-ADP-HSA adducts (rs = 0.373), and between 4-ADP-Hb and aniline-HSA adducts (rs = 0.275). In view of the proposal for additional use of the aniline-HSA adduct for biological monitoring, particularly in cases of acute overexposures or poisonings, the strong correlation of the Hb and HSA conjugates is noteworthy; the ratio aniline-HSA:aniline-Hb was 1:42 for the entire cohort.

Cyanide (CN) remains a viable threat as a chemical warfare agent. Methemoglobin (MHb) formation is one strategy used to counter CN toxicity. It has been suggested that blood levels of 5-12% MHb will protect a human against a 2X MLD (median lethal dose) of CN. Available MHb formers present certain drawbacks and limitations. To identify improved MHb formers, we characterized the locomotor activity effects and MHb formation capacity of three MHb formers with established efficacy against CN (p-aminopropiophenone PAPP, p-aminoheptanoylphenone PAHP and p-aminooctanoylphenone PAOP) as a function of time, dose (9.4-125.0 mg/kg) and route of administration (IM versus IP). Mice received a single injection of a test compound or its solvent, and were either placed in an automated activity monitoring chamber for 1 hr or monitored for MHb levels for 3 hr. Sodium nitrite (100 mg/kg) served as the positive control. Dose-related MHb formation was observed for each compound. A typical time-dependent decline in activity was exhibited in negative control groups and in drug-treated groups that exhibited greater than 20% MHb. A route of administration effect was also observed with PAHP and PAOP. These locomotor activity data, combined with other findings, support the idea that compounds producing low MHb levels can be effective against CN without debilitating side effects.

Many substances that form methemoglobin (MHb) counter cyanide (CN) toxicity. Although MHb formers can be applied as treatments for CN poisoning, it was reasoned that a long-acting MHb former could serve as a CN pretreatment. An 8- aminoquinoline drug, WR242511, was characterized as a long-lasting MHb former, producing sufficient MHb to protect against 2 X LD50 of CN. Transition for development of WR242511 was based on data from rodents and beagle dogs collected elsewhere. Advanced development testing of WR242511 at USAMRICD was conducted in the rhesus monkey. WR242511 was administered intravenously (IV) to two female and four male rhesus monkeys in doses of 3.5 and/or 7.0 mg/kg, with the objective of producing protective levels of MHb.

No escalation of curative doses of reference drugs (chloroquine and primaquine) in the simian radical curative models has been recorded over the last 14 years. A shorter 3- dose regimen of WR 238605 for anti-relapse activity has been established which would be operationally more acceptable. In addition this compound as exhibited significant blood-schizontocidal, prophylactic and gametocytocidal activities which can be exploited. A new anti-relapse regimen comprising of WR 238605 plus halofantrine or mefloquine as companion blood schizontocides has been developed for management of chloroquine resistant P. vivax in the field. Cyproheptadine has shown antimalarial action against multidrug resistant P. yoelii nigeriensis. Cyproheptadine also exerts mefloquine resistance reversal action against P. knowlesi. This finding will be useful for treatment of mefloquine resistant cases. In vitro bioassay for evaluation of antimalarials using synchronized P. knowlesi and in vitro model for screening of tissue schizontocidal drugs using P. cynomolgi have been established. An in vitro test for methemoglobin toxicity using mastomys erythrocytes to evaluate toxicity of 8 aminoquinoline agents has been developed.

Six Phase I clinical pharmacology studies were performed during this contract period under the Task Order numbers 90-01, 92-04, 92-05, 94-06, 94-07 and 94-09. Task Order 90-01 produced data suggesting that the Mark I auto-injector produced effective and desirable blood levels and effects of atropine and 2-PAM compared to other prototype auto-injectors. Task Order 92-04 demonstrated that WR 238, 605 was safe and well tolerated at single doses up to 600 mg. Task Order 92-05 compared the blood levels produced by a new dosing regimen for halofantrine in the fed compared to the fasting state. Task Order 94-06 demonstrated the lack of irritating potential for a topical skin protectant. Task Order 94-07 demonstrated the safety of weekly dosing with the antimalarial WR 238, 605 and documented the accumulation of non toxic methemoglobin levels at the higher doses. This side effect may be beneficial in prophylaxis against cyanide poisoning. Task Order 94-09 showed a lack of toxicity from multiple dose pyridostigmine bromide in healthy male and female subjects of different weights.

RIVM rapport:Dit rapport beschrijft de resultaten van een in vitro experiment naar het methemoglobine inducerende vermogen van nitriet in humaan bloed. Er werd gebruik gemaakt van verse, gehepariniseerde, veneuze bloedmonsters. Tien natriumnitriet doseringen, in een molaire verhouding, nitriet : hemoglobine, tussen 0.01 en 0.15, werden getest. Elk experiment werd in triplo uitgevoerd. De bloedmonsters werden op een temperatuur van 37 graden Celsius gehouden. De hemoglobineconcentratie, het percentage methemoglobine en de pH van het bloed werden gedurende 2 uur op vaste tijdstippen gemeten. Geen van de bloedmonsters toonde zichtbare hemolyse van het plasma na afloop van het experiment. Er werden methemoglobine percentages tot 24% geinduceerd. Er werd een lineaire relatie gevonden tussen de natriumnitriet doses en het maximale methemoglobine percentage dat werd bereikt. De lineaire regressie analyse toonde een gecorrigeerde R exp.2 > 0.99 (p=0.0001). De verhouding, natriumnitriet doses : maximaal geinduceerde hoeveelheid methemogobine in het bloedmonster, was ongeveer 0.7 voor elke dosering., This report describes the results of an in vitro experiment, investigating the methemoglobin inducing potential of nitrite in human blood. Fresh, heparinized, venous blood samples were used for the experiment. Ten sodium nitrite doses, with a molar ratio of nitrite to hemoglobin ranging between 0.01 and 0.15, were studied. Each sodium nitrite dose was tested in triplicate. During the experiment the blood was maintained at 37 degrees Celsius. The hemoglobin concentration, the percentage of methemoglobin and the pH of the blood were determined at timed intervals, for two hours following the start of the experiment. None of the blood samples showed visible hemolysis of the plasma after completing the experiment. Methemoglobin levels up to 24% were induced. A linear relationship was established between the nitrite dose and the maximum methemoglobin level that was induced in the blood. Linear regression analyses showed an adjusted R exp. 2 of > 0.99 (p=0.0001). The molar ratio of the nitrite dose to the maximum amount of methemoglobin that was induced at t-max, was approximately 0.7 at all dose levels.

Dit rapport beschrijft de resultaten van een in vitro experiment naar het methemoglobine inducerende vermogen van nitriet in humaan bloed. Er werd gebruik gemaakt van verse, gehepariniseerde, veneuze bloedmonsters. Tien natriumnitriet doseringen, in een molaire verhouding, nitriet : hemoglobine, tussen 0.01 en 0.15, werden getest. Elk experiment werd in triplo uitgevoerd. De bloedmonsters werden op een temperatuur van 37 graden Celsius gehouden. De hemoglobineconcentratie, het percentage methemoglobine en de pH van het bloed werden gedurende 2 uur op vaste tijdstippen gemeten. Geen van de bloedmonsters toonde zichtbare hemolyse van het plasma na afloop van het experiment. Er werden methemoglobine percentages tot 24% geinduceerd. Er werd een lineaire relatie gevonden tussen de natriumnitriet doses en het maximale methemoglobine percentage dat werd bereikt. De lineaire regressie analyse toonde een gecorrigeerde R exp.2 > 0.99 (p=0.0001). De verhouding, natriumnitriet doses : maximaal geinduceerde hoeveelheid methemogobine in het bloedmonster, was ongeveer 0.7 voor elke dosering., This report describes the results of an in vitro experiment, investigating the methemoglobin inducing potential of nitrite in human blood. Fresh, heparinized, venous blood samples were used for the experiment. Ten sodium nitrite doses, with a molar ratio of nitrite to hemoglobin ranging between 0.01 and 0.15, were studied. Each sodium nitrite dose was tested in triplicate. During the experiment the blood was maintained at 37 degrees Celsius. The hemoglobin concentration, the percentage of methemoglobin and the pH of the blood were determined at timed intervals, for two hours following the start of the experiment. None of the blood samples showed visible hemolysis of the plasma after completing the experiment. Methemoglobin levels up to 24% were induced. A linear relationship was established between the nitrite dose and the maximum methemoglobin level that was induced in the blood. Linear regression analyses showed an adjusted R exp. 2 of > 0.99 (p=0.0001). The molar ratio of the nitrite dose to the maximum amount of methemoglobin that was induced at t-max, was approximately 0.7 at all dose levels.

RIVM rapport:Dit rapport beschrijft de resultaten van een in vitro experiment naar het methemoglobine inducerende vermogen van nitriet in humaan bloed. Er werd gebruik gemaakt van verse, gehepariniseerde, veneuze bloedmonsters. Tien natriumnitriet doseringen, in een molaire verhouding, nitriet : hemoglobine, tussen 0.01 en 0.15, werden getest. Elk experiment werd in triplo uitgevoerd. De bloedmonsters werden op een temperatuur van 37 graden Celsius gehouden. De hemoglobineconcentratie, het percentage methemoglobine en de pH van het bloed werden gedurende 2 uur op vaste tijdstippen gemeten. Geen van de bloedmonsters toonde zichtbare hemolyse van het plasma na afloop van het experiment. Er werden methemoglobine percentages tot 24% geinduceerd. Er werd een lineaire relatie gevonden tussen de natriumnitriet doses en het maximale methemoglobine percentage dat werd bereikt. De lineaire regressie analyse toonde een gecorrigeerde R exp.2 > 0.99 (p=0.0001). De verhouding, natriumnitriet doses : maximaal geinduceerde hoeveelheid methemogobine in het bloedmonster, was ongeveer 0.7 voor elke dosering., This report describes the results of an in vitro experiment, investigating the methemoglobin inducing potential of nitrite in human blood. Fresh, heparinized, venous blood samples were used for the experiment. Ten sodium nitrite doses, with a molar ratio of nitrite to hemoglobin ranging between 0.01 and 0.15, were studied. Each sodium nitrite dose was tested in triplicate. During the experiment the blood was maintained at 37 degrees Celsius. The hemoglobin concentration, the percentage of methemoglobin and the pH of the blood were determined at timed intervals, for two hours following the start of the experiment. None of the blood samples showed visible hemolysis of the plasma after completing the experiment. Methemoglobin levels up to 24% were induced. A linear relationship was established between the nitrite dose and the maximum methemoglobin level that was induced in the blood. Linear regression analyses showed an adjusted R exp. 2 of > 0.99 (p=0.0001). The molar ratio of the nitrite dose to the maximum amount of methemoglobin that was induced at t-max, was approximately 0.7 at all dose levels.

RIVM rapport:Dit rapport beschrijft de resultaten van een in vitro experiment naar het methemoglobine inducerende vermogen van nitriet in humaan bloed. Er werd gebruik gemaakt van verse, gehepariniseerde, veneuze bloedmonsters. Tien natriumnitriet doseringen, in een molaire verhouding, nitriet : hemoglobine, tussen 0.01 en 0.15, werden getest. Elk experiment werd in triplo uitgevoerd. De bloedmonsters werden op een temperatuur van 37 graden Celsius gehouden. De hemoglobineconcentratie, het percentage methemoglobine en de pH van het bloed werden gedurende 2 uur op vaste tijdstippen gemeten. Geen van de bloedmonsters toonde zichtbare hemolyse van het plasma na afloop van het experiment. Er werden methemoglobine percentages tot 24% geinduceerd. Er werd een lineaire relatie gevonden tussen de natriumnitriet doses en het maximale methemoglobine percentage dat werd bereikt. De lineaire regressie analyse toonde een gecorrigeerde R exp.2 > 0.99 (p=0.0001). De verhouding, natriumnitriet doses : maximaal geinduceerde hoeveelheid methemogobine in het bloedmonster, was ongeveer 0.7 voor elke dosering., This report describes the results of an in vitro experiment, investigating the methemoglobin inducing potential of nitrite in human blood. Fresh, heparinized, venous blood samples were used for the experiment. Ten sodium nitrite doses, with a molar ratio of nitrite to hemoglobin ranging between 0.01 and 0.15, were studied. Each sodium nitrite dose was tested in triplicate. During the experiment the blood was maintained at 37 degrees Celsius. The hemoglobin concentration, the percentage of methemoglobin and the pH of the blood were determined at timed intervals, for two hours following the start of the experiment. None of the blood samples showed visible hemolysis of the plasma after completing the experiment. Methemoglobin levels up to 24% were induced. A linear relationship was established between the nitrite dose and the maximum methemoglobin level that was induced in the blood. Linear regression analyses showed an adjusted R exp. 2 of > 0.99 (p=0.0001). The molar ratio of the nitrite dose to the maximum amount of methemoglobin that was induced at t-max, was approximately 0.7 at all dose levels.

Task 92-28 was conducted to determine the protective efficacy provided by low methemoglobin (MHb) levels against a continuous sodium cyanide (NaCN) infusion, and to compare and quantitate the efficacy of long-acting and short-acting MHb-forming compounds in anesthetized canines. The time to respiratory arrest (TRA) was measured, after which the animals were revived with 10 mg/kg of hydroxylamine iv. The average estimated protective percent MHb was 5.4 percent with a 95 percent upper tolerance bound of 6.4 percent. Three compounds that induce the formation of MHb, p-aminopropiophenone (PAPP), p-aminoheptanophenone (PAHP), and an 8-aminoquinoline (WR242511), were effective in mitigating the effects of NaCN poisoning when compared to the controls. No statistically significant differences in efficacy were observed among the three compounds. Doses of two times the average concentration required to induce respiratory arrest (2xAvTRA) were tested in animals pretreated with PAPP. All of the PAPP pretreated anesthetized animals achieved approximately 6.5 percent MHb, did not cease breathing, and did not require any other medical intervention to recover from the NaCN challenge.

Chronic toxic effects of I ,3,5-trinitrobenzene (TNB) in male and female Fischer rats were evaluated by feeding certified powdered laboratory chow diet supplemented with varied concentrations of TNB (0, 5, 60 and 300 mg/kg diet). The study was designed to accommodate three interim sacrifices (10 rats/group/sex) at 90, 180 and 365 days. The final sacrifice was performed after two years. All data related to these interim sacrifices are presented independently in appendices J to L. The calculated average TNB consumption for females was 0.23, 2.68 and 13.31 mg/kg/day and was 0.22, 2.64 and 13.44 mg/kg/day for males. Terminal body weights were significantly decreased in both sexes in the 300 mg/kg group. Relative spleen weights were decreased in both sexes in the 300 mg/kg group while brain weights were increased in females in this same group. Methemoglobin was increased in both sexes in the 300 mg/kg group while other hematological effects noted at the interim sacrifice times were not evident at two years. Histopathological examinations suggested treatment related changes in both sexes involving the kidneys (cytoplasmic/hyaline droplets) in the 60 and 300 mg/kg groups and the spleen (erythroid cell hyperplasia and pigment deposition) in the 300 mg/kg group. The cytoplasmic/hyaline droplets were characterized by immunohistochemistry as alpha-2u-globulin. These renal droplets were also noted at the interim sacrifice times. A no observed adverse effect level (NOAEL) was established in this study at 2.68 mg/kg b.w./day for F-344 rats administered TNB for two years.

Methemoglobin (MHb) formation is one strategy to counter cyanide (CN) toxicity. Currently available MHb formers present certain drawbacks and limitations. The purpose of this study was to characterize, in mice, the hematologic effects of the MHb-forming compound p-aminopropiophenone (PAPP), and two structurally related phenones, p-aminoheptanoylphenone (PAHP) and p-aminooctanoylphenone (PAOP). Although these three phenones have been shown previously to be efficacious against CN, a more complete understanding of their hematologic effects is lacking. Using a hemoximeter, blood samples were obtained -2 to +180 min relative to intramuscular (im) or intraperitoneal (ip) injections. Sodium nitrite (NaNO2) and the appropriate solvents served as the positive and negative controls, respectively. Dose, time, route, and compound-related effects were observed. MHb and sulfhemoglobin levels tended to increase, whereas levels of those parameters related to oxygen carrying capacity of the blood, such as oxygen content, oxygen saturation, oxyhemoglobin and reduced hemoglobin were reduced. Small reductions or no measurable changes were observed in carooxyhemoglobin and oxygen capacity. In general, the effects of PAHP and PAOP tended to be longer lasting than those observed with PAPP and NaNO2. Furthermore, PAPP and NaNO2 were equally effective im or ip, whereas PAHP and PAOP showed larger effects when administered ip. Although additional work is needed, these data provide information which will be useful for the successful development of improved anti-CN MHb formers.

To study whether nitric oxide (NO) affects surfactant function, 36 young rats inhaled one of the following humidified environments for 24 h: 1) air; 2) 95% O2; 3) air and 100 parts/million (ppm) NO; and 4) 95% O2 and 100 ppm NO. The treatments did not change the recovery of phospholipid from bronchoalveolar lavage (BAL). Exposure to NO of animals that breathed either air or 95% O2 increased the minimum surface tension of surfactant from BAL at low (1.5 mumol/ml), but not at high (4 mumol/ml), phosphatidylcholine concentration. After inhaled NO, the nonsedimentable protein of BAL decreased the surface activity of surfactant (1 mumol phosphatidylcholine/ml) more than the protein from the controls. NO treatment of animals that breathed either air or 95% O2 affected neither the quantity nor the molecular weight distribution of nonsedimentable protein. Hyperoxia increased the amount of the nonsedimentable protein, whereas NO increased the iron saturation of transferrin. The surfactant fraction and the nonsedimentable protein from BAL were separately exposed to 80 ppm NO in vitro. NO exposure had no effect on the surface activity of surfactant fraction. NO exposure of nonsedimentable protein from the control animals (no NO) increased the inhibition of the surface activity and changed the adsorption spectrum of the protein, suggesting conversion of hemoglobin to methemoglobin. Nonsedimentable protein from NO-exposed animals contained methemoglobin. We propose that surfactant dysfunction caused by inhaled NO is in part due to alteration of protein(s) in epithelial lining fluid that in turn inactivates surfactant.

To study whether nitric oxide (NO) affects surfactant function, 36 young rats inhaled one of the following humidified environments for 24 h: 1) air; 2) 95% O2; 3) air and 100 parts/million (ppm) NO; and 4) 95% O2 and 100 ppm NO. The treatments did not change the recovery of phospholipid from bronchoalveolar lavage (BAL). Exposure to NO of animals that breathed either air or 95% O2 increased the minimum surface tension of surfactant from BAL at low (1.5 mumol/ml), but not at high (4 mumol/ml), phosphatidylcholine concentration. After inhaled NO, the nonsedimentable protein of BAL decreased the surface activity of surfactant (1 mumol phosphatidylcholine/ml) more than the protein from the controls. NO treatment of animals that breathed either air or 95% O2 affected neither the quantity nor the molecular weight distribution of nonsedimentable protein. Hyperoxia increased the amount of the nonsedimentable protein, whereas NO increased the iron saturation of transferrin. The surfactant fraction and the nonsedimentable protein from BAL were separately exposed to 80 ppm NO in vitro. NO exposure had no effect on the surface activity of surfactant fraction. NO exposure of nonsedimentable protein from the control animals (no NO) increased the inhibition of the surface activity and changed the adsorption spectrum of the protein, suggesting conversion of hemoglobin to methemoglobin. Nonsedimentable protein from NO-exposed animals contained methemoglobin. We propose that surfactant dysfunction caused by inhaled NO is in part due to alteration of protein(s) in epithelial lining fluid that in turn inactivates surfactant.

We have used infrared spectroscopy to evaluate the secondary structure of hemoglobin in red cells lyophilized by Dr. Spargo's most recent protocol. As noted for cells lyophilized by earlier methods, the hemoglobin is unfolded in the dried solid, but refolds during rehydration. Many studies with purified hemoglobin (including some of our preliminary work) have indicated that the rate of formation of methemoglobin is greatly increased if the protein is unfolded. If the same relationship is true for hemoglobin in the dried red cells, then generation of methemoglobin will most likely continue as the cells are stored in the dried solid. It may be possible to minimize this problem by developing methods to inhibit lyophilization-induced unfolding more effectively. We are investigating this approach with purified proteins. Dr. Spargo is attempting to improve stabilization of hemoglobin in the red cells by investigating methods to load cells with stabilizers that we have found to be more effective for purified proteins (e.g., trehalose). As another approach to help mitigate this problem, we will soon begin studying means by which to maximize the activity of methemoglobin reductase in rehydrated cells. If we can take advantage of this enzyme system in the red cells, then it may be possible to reverse oxidative damage to hemoglobin that occurs during lyophilization and storage.

An artillery propellant under development b the U.S. Army is a granular mixture of nitrocelinlose, nitroglycerin, nitroguanidine, ethylcentralite, cryolite, and graphite. The propellant, Modniar Artillery Charge System or MACS, consists of a single increment of propellant charge contained within a rigid combustible casing. Nitrocellulose, nitroglycerin, and nitroguanidine make up greater than 98% of the total propellant mixture. As part of the process to develop environmental and health effects criteria, a 90-day modified Screening Information Data Set reproductive assay is planned. In order to provide information on clinical signs, methemoglobin formation and possible target organs, as well as determine dose levels, a range- finding study was performed. Male and female Sprague-Dawley rats were treated with diet containing either O.O,0.2S, or 2.Og propellant/kg diet for a three-week period. No mortalities occurred and body weights were unaffected by treatment. Methemoglobin concentrations measured at the conclusion of the study indicated no differences between treated and control rats. Relative liver weights of the high-dose female rats were significantly (p<0. 1) greater than the control group. No gross lesions were noted at necropsy.

Subchronic toxic effects of 1,3,5-Trinitrobenzene (TNB) in male and female Fischer 344 rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of TNB (0, 66.67, 400 and 800 mg/kg diet) so as to achieve a final target dose of 0, 5, 30 and 60 mg/kg b.w. for ninety days. Food intake in the 400 and 800 mg TNB dose groups of both sexes was reduced throughout the study and resulted in a significant decrease in absolute body weights. The calculated average TNB dosage was 4, 25 and 49 mg/kg/ day for females and 4, 21 and 44 mg/kg/day for males. A decrease in testicular weight in males and increase in relative spleen weight of both sexes in the 400 and 800 mg TNB dose groups were noted. Also, the relative brain weight was increased in the male 400 and 800 mg TNB dose groups while the relative liver weight was increased in 800 mg TNB dose group of both sexes. Histopathological examinations suggested that the susceptible organs for TNB toxicity were kidney (hyaline droplets), spleen (extramedullary hemotopoiesis) and testes (seminiferous tubular degeneration). Hematology and clinical chemistry studies indicated a decrease in red blood cell count and hematocrit, a decrease in alkaline phosphatase, an increase in reticulocytes and increased methemoglobin concentration as compared to controls in both sexes

Subacute toxic effects of TNB in F344 male and female rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of TNB (0, 50, 200, 400, 800 and 1200 mg/kg diet) so as to achieve a final target dose of (0, 5, 20, 40, 80 and 120 mg TNB/kg body weight) for fourteen days. The calculated average daily TNB doses (50, 200, 400, 800 and 1200 mg/kg) for males was 4, 16, 34, 55 and 94 mg/kg b.w. and for females 4, 17, 34, 58 and 79 mg/kg b.w. Food intake by male and female rats consuming 1200 mg TNB/kg diet was reduced and resulted in a significant decrease in absolute body weights. A decrease in testicular weight in males and an increase in spleen weight of both sexes fed 800 or 1200 mg TNB/kg diet were noted. Histopathological changes for TNB toxicity (200-1200 mg/kg) were evident in the kidneys (hyaline droplets), spleen (extra-medullary hematopoiesis) and testes (seminiferous tubular degeneration). Hematology and clinical chemistry studies indicated a decrease in red blood cell count and hematocrit (400-1200 mg/kg), a decrease in alkaline phosphatase (400-1200 mg/kg) and an increase in Heinz bodies (800-1200 mg/kg) as compared to controls in both sexes. Methemoglobin levels were also significantly increased (400-1200 mg/kg). Subacute Oral Toxicity, Fischer 344 Rats, Hematology, Clinical Chemistry, 1,3,5- Trinitrobenzene

Liquid propellant XM46 is being considered as a replacement for solid propellants, both as part of a regenerative injection gun system as a working fluid in an electrothermal gun system. The XM46 formulation contains hydroxylammonium nitrate, triethanolammonium nitrate, and water. Male and female Sprague-Dawley rats received XM46 drinking water containing 2.0, 1.0, 0.2, or 0.0 g XM46/L throughout a 90-day study. Mating occurred following 14 days of treatment. One half the male rats per group were necropsied after 28 days of treatment, the remaining males and all dams were necropsied following 90 days. No mortality occurred in any of the parental animals during the study. The study did not produce any adverse effects on reproduction or litter parameters. Hemolytic anemia and methemoglobinemia were common findings in both sexes of rats. Splenomegaly was found in both sexes; in male rats, as early as 28 days. Exposure via drinking water containing XM46 for 90 days did not result in any decrease in reproductive performance in male or female rats, but it did result in clinical signs of hemolytic anemia at doses as low as 17 mg/kg/day.