Intratracheal cobinamide (vitamin B12 analog) administration increases survivability in rabbits exposed to a lethal dose of inhaled hydrogen sulfide.

BACKGROUND: Hydrogen sulfide is a highly toxic, flammable, and colorless gas. Hydrogen sulfide has been identified as a potential terrorist chemical threat agent in mass-casualty events. Our previous studies showed that cobinamide, a vitamin B12 analog, effectively reverses the toxicity from hydrogen sulfide poisoning. In this study, we investigate the effectiveness of intratracheally administered cobinamide in treating a lethal dose hydrogen sulfide gas inhalation and compare its performance to saline control administration. METHODS: A total of 53 pathogen-free New Zealand White rabbits were used for this study. Four groups were compared: (i) received no saline solution or drug intratracheally (n = 15), (ii) slow drip saline intratracheally (n = 15), (iii) fast drip saline intratracheally (n = 15), and (iv) slow drip cobinamide intratracheally (n = 8). Blood pressure was continuously monitored, and deoxy- and oxyhemoglobin concentration changes were monitored in real-time in vivo using continuous wave near-infrared spectroscopy. RESULTS: The mean (± standard deviation) weight for all animals (n = 53) was 3.87 ± 0.10 kg. The survival rates of the slow cobinamide and the fast saline groups were 75 percent and 60 percent, respectively, while the survival rates in the slow saline and control groups were 26.7 percent and 20 percent, respectively. A log-rank (Mantel-Cox) test showed that survival in fast saline and slow cobinamide groups were significantly greater than those of no saline control and slow saline groups (P < 0.05). The slow and no saline control groups were not significantly different (P = 0.59). The slow cobinamide group did significantly better than the slow saline group (P = 0.021). DISCUSSION: The ability to use intratracheal cobinamide as an antidote to hydrogen sulfide poisoning is a novel approach to mass-casualty care. The major limitations of this study are that it was conducted in a single species at a single inhaled hydrogen sulfide concentration. Repeated investigations in other species and at varying levels of hydrogen sulfide exposure will be needed before any definitive recommendations can be made. CONCLUSIONS: We demonstrated that intratracheal cobinamide and fast saline drip improved survival for hydrogen sulfide gas inhalation in rabbit models. Although further study is required, our results suggest that intratracheal administration of cobinamide and fast saline may be useful in hydrogen sulfide mass-casualty events.

Intramuscular administration of glyoxylate rescues swine from lethal cyanide poisoning and ameliorates the biochemical sequalae of cyanide intoxication.

Cyanide-a fast-acting poison-is easy to obtain given its widespread use in manufacturing industries. It is a high-threat chemical agent that poses a risk of occupational exposure in addition to being a terrorist agent. FDA-approved cyanide antidotes must be given intravenously, which is not practical in a mass casualty setting due to the time and skill required to obtain intravenous access. Glyoxylate is an endogenous metabolite that binds cyanide and reverses cyanide-induced redox imbalances independent of chelation. Efficacy and biochemical mechanistic studies in an FDA-approved preclinical animal model have not been reported. Therefore, in a swine model of cyanide poisoning, we evaluated the efficacy of intramuscular glyoxylate on clinical, metabolic, and biochemical endpoints. Animals were instrumented for continuous hemodynamic monitoring and infused with potassium cyanide. Following cyanide-induced apnea, saline control or glyoxylate was administered intramuscularly. Throughout the study, serial blood samples were collected for pharmacokinetic, metabolite, and biochemical studies, in addition, vital signs, hemodynamic parameters, and laboratory values were measured. Survival in glyoxylate-treated animals was 83% compared with 12% in saline-treated control animals (p < .01). Glyoxylate treatment improved physiological parameters including pulse oximetry, arterial oxygenation, respiration, and pH. In addition, levels of citric acid cycle metabolites returned to baseline levels by the end of the study. Moreover, glyoxylate exerted distinct effects on redox balance as compared with a cyanide-chelating countermeasure. In our preclinical swine model of lethal cyanide poisoning, intramuscular administration of the endogenous metabolite glyoxylate improved survival and clinical outcomes, and ameliorated the biochemical effects of cyanide.

Identification of Platinum(II) Sulfide Complexes Suitable as Intramuscular Cyanide Countermeasures.

The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum-sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5-DMSO and Na (NH3)2PtCl-DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

Treatment of life-threatening H2S intoxication: Lessons from the trapping agent tetranitrocobinamide.

We sought to evaluate the efficacy of trapping free hydrogen sulfide (H2S) following severe H2S intoxication. Sodium hydrosulfide solution (NaHS, 20 mg/kg) was administered intraperitoneally in 69 freely moving rats. In a first group (protocol 1), 40 rats were randomly assigned to receive saline (n = 20) or the cobalt compound tetranitrocobinamide (TNCbi) (n = 20, 75 mg/kg iv), one minute into coma, when free H2S was still present in the blood. A second group of 27 rats received TNCbi or saline, following epinephrine, 5 min into coma, when the concentration of free H2S has drastically decreased in the blood. In protocol 1, TNCbi significantly increased immediate survival (65 vs 20 %, p < 0.01) while in protocol 2, administration of TNCbi led to the same outcome as untreated animals. We hypothesize that the decreased efficacy of TNCbi with time likely reflects the rapid spontaneous disappearance of the pool of free H2S in the blood following H2S exposure.

Glyoxylate protects against cyanide toxicity through metabolic modulation.

Although cyanide's biological effects are pleiotropic, its most obvious effects are as a metabolic poison. Cyanide potently inhibits cytochrome c oxidase and potentially other metabolic enzymes, thereby unleashing a cascade of metabolic perturbations that are believed to cause lethality. From systematic screens of human metabolites using a zebrafish model of cyanide toxicity, we have identified the TCA-derived small molecule glyoxylate as a potential cyanide countermeasure. Following cyanide exposure, treatment with glyoxylate in both mammalian and non-mammalian animal models confers resistance to cyanide toxicity with greater efficacy and faster kinetics than known cyanide scavengers. Glyoxylate-mediated cyanide resistance is accompanied by rapid pyruvate consumption without an accompanying increase in lactate concentration. Lactate dehydrogenase is required for this effect which distinguishes the mechanism of glyoxylate rescue as distinct from countermeasures based solely on chemical cyanide scavenging. Our metabolic data together support the hypothesis that glyoxylate confers survival at least in part by reversing the cyanide-induced redox imbalances in the cytosol and mitochondria. The data presented herein represent the identification of a potential cyanide countermeasure operating through a novel mechanism of metabolic modulation.

Methyl mercaptan gas: mechanisms of toxicity and demonstration of the effectiveness of cobinamide as an antidote in mice and rabbits.

CONTEXT: Methyl mercaptan (CH3SH) is a colorless, toxic gas with potential for occupational exposure and used as a weapon of mass destruction. Inhalation at high concentrations can result in dyspnea, hypoventilation, seizures, and death. No specific methyl mercaptan antidote exists, highlighting a critical need for such an agent. Here, we investigated the mechanism of CH3SH toxicity, and rescue from CH3SH poisoning by the vitamin B12 analog cobinamide, in mammalian cells. We also developed lethal CH3SH inhalation models in mice and rabbits, and tested the efficacy of intramuscular injection of cobinamide as a CH3SH antidote. RESULTS: We found that cobinamide binds to CH3SH (Kd = 84 µM), and improved growth of cells exposed to CH3SH. CH3SH reduced cellular oxygen consumption and intracellular ATP content and activated the stress protein c-Jun N-terminal kinase (JNK); cobinamide reversed these changes. A single intramuscular injection of cobinamide (20 mg/kg) rescued 6 of 6 mice exposed to a lethal dose of CH3SH gas, while all six saline-treated mice died (p = 0.0013). In rabbits exposed to CH3SH gas, 11 of 12 animals (92%) treated with two intramuscular injections of cobinamide (50 mg/kg each) survived, while only 2 of 12 animals (17%) treated with saline survived (p = 0.001). CONCLUSION: We conclude that cobinamide could potentially serve as a CH3SH antidote.

Development of sodium tetrathionate as a cyanide and methanethiol antidote.

CONTEXT: Hydrogen cyanide and methanethiol are two toxic gases that inhibit mitochondrial cytochrome c oxidase. Cyanide is generated in structural fires and methanethiol is released by decaying organic matter. Current treatments for cyanide exposure do not lend themselves to treatment in the field and no treatment exists for methanethiol poisoning. Sodium tetrathionate (tetrathionate), a product of thiosulfate oxidation, could potentially serve as a cyanide antidote, and, based on its chemical structure, we hypothesized it could react with methanethiol. RESULTS: We show that tetrathionate, unlike thiosulfate, reacts directly with cyanide in vitro under physiological conditions, and based on rabbit studies where we monitor cyanide poisoning in real-time, tetrathionate likely reacts directly with cyanide in vivo. We found that tetrathionate administered by intramuscular injection rescues >80% of juvenile, young adult, and old adult mice from exposure to inhaled hydrogen cyanide gas that is >80% lethal. Tetrathionate also rescued young adult rabbits from intravenously administered sodium cyanide. Tetrathionate was reasonably well-tolerated by mice and rats, yielding a therapeutic index of ∼5 in juvenile and young adult mice, and ∼3.3 in old adult mice; it was non-mutagenic in Chinese Hamster ovary cells and by the Ames bacterial test. We found by gas chromatography-mass spectrometry that both tetrathionate and thiosulfate react with methanethiol to generate dimethyldisulfide, but that tetrathionate was much more effective than thiosulfate at recovering intracellular ATP in COS-7 cells and rescuing mice from a lethal exposure to methanethiol gas. CONCLUSION: We conclude that tetrathionate has the potential to be an effective antidote against cyanide and methanethiol poisoning.

Efficacy of oral administration of sodium thiosulfate in a large, swine model of oral cyanide toxicity.

INTRODUCTION: Cyanide is a deadly poison, particularly with oral exposure where larger doses can occur before symptoms develop. Prior studies and multiple governmentagencies highlight oral cyanide as an agent with the potential for use in a terrorist attack. Currently, there are no FDA approved antidotes specific to oralcyanide. An oral countermeasure that can neutralize and prevent absorption of cyanide from the GI tract after oral exposure is needed. Our objective was toevaluate the efficacy of oral sodium thiosulfate on survival and clinical outcomes in a large, swine model of severe cyanide toxicity. METHODS: Swine (45-55kg) were instrumented, sedated, and stabilized. Potassium cyanide (8 mg/kg KCN) in saline was delivered as a one-time bolus via an orogastric tube. Three minutes after cyanide, animals randomized to the treatment group received sodium thiosulfate (510 mg/kg, 3.25 M solution) via orogastric tube. Our primary outcome was survival at 60 minutes after exposure. We compared survival between groups by log-rank, Mantel-Cox analysis and trended labs and vital signs. RESULTS: At baseline and time of treatment all animals had similar weights, vital signs, and laboratory values. Survival at 60 min was 100% in treated animals compared to 0% in the control group (p=0.0027). Animals in the control group became apneic and subsequently died by 35.0 min (20.2,48.5) after cyanide exposure. Mean arterial pressure was significantly higher in the treatment group compared to controls (p=0.008). Blood lactate (p=0.02) and oxygen saturation (p=0.02) were also significantly different between treatment and control groups at study end. CONCLUSION: Oral administration of sodium thiosulfate improved survival, blood pressure, respirations, and blood lactate concentrations in a large animal model of acute oral cyanide toxicity.

Intramuscular cobinamide as an antidote to methyl mercaptan poisoning.

BACKGROUND: Methyl mercaptan occurs naturally in the environment and is found in a variety of occupational settings, including the oil, paper, plastics, and pesticides industries. It is a toxic gas and deaths from methyl mercaptan exposure have occurred. The Department of Homeland Security considers it a high threat chemical agent that could be used by terrorists. Unfortunately, no specific treatment exists for methyl mercaptan poisoning. METHODS: We conducted a randomized trial in 12 swine comparing no treatment to intramuscular injection of the vitamin B12 analog cobinamide (2.0 mL, 12.5 mg/kg) following acute inhalation of methyl mercaptan gas. Physiological and laboratory parameters were similar in the control and cobinamide-treated groups at baseline and at the time of treatment. RESULTS: All six cobinamide-treated animals survived, whereas only one of six control animals lived (17% survival) (p = 0.0043). The cobinamide-treated animals returned to a normal breathing pattern by 3.8 ± 1.1 min after treatment (mean ± SD), while all but one animal in the control group had intermittent gasping, never regaining a normal breathing pattern. Blood pressure and arterial oxygen saturation returned to baseline values within 15 minutes of cobinamide-treatment. Plasma lactate concentration increased progressively until death (10.93 ± 6.02 mmol [mean ± SD]) in control animals, and decreased toward baseline (3.79 ± 2.93 mmol [mean ± SD]) by the end of the experiment in cobinamide-treated animals. CONCLUSION: We conclude that intramuscular administration of cobinamide improves survival and clinical outcomes in a large animal model of acute, high dose methyl mercaptan poisoning.

Protein tyrosine phosphatase-α amplifies transforming growth factor-ß-dependent profibrotic signaling in lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-ß (TGF-ß), are central to its pathogenesis. Protein tyrosine phosphatase-α (PTPα) has emerged as a key regulator of fibrogenic signaling in fibroblasts. We have reported that mice globally deficient in PTPα (Ptpra-/-) were protected from experimental pulmonary fibrosis, in part via alterations in TGF-ß signaling. The goal of this study was to determine the lung cell types and mechanisms by which PTPα controls fibrogenic pathways and whether these pathways are relevant to human disease. Immunohistochemical analysis of lungs from patients with IPF revealed that PTPα was highly expressed by mesenchymal cells in fibroblastic foci and by airway and alveolar epithelial cells. To determine whether PTPα promotes profibrotic signaling pathways in lung fibroblasts and/or epithelial cells, we generated mice with conditional (floxed) Ptpra alleles (Ptpraf/f). These mice were crossed with Dermo1-Cre or with Sftpc-CreERT2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1-Cre/Ptpraf/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc-CreERT2/Ptpraf/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-ß signaling and downstream TGF-ß-induced fibrogenic responses were attenuated in isolated Ptpra-/- compared with wild-type fibroblasts. Furthermore, TGF-ß-induced tyrosine phosphorylation of TGF-ß type II receptor and of PTPα were attenuated in Ptpra-/- compared with wild-type fibroblasts. The phenotype of cells genetically deficient in PTPα was recapitulated with the use of a Src inhibitor. These findings suggest that PTPα amplifies profibrotic TGF-ß-dependent pathway signaling in lung fibroblasts.

Intramuscular aminotetrazole cobinamide as a treatment for inhaled hydrogen sulfide poisoning in a large swine model.

Hydrogen sulfide (H2 S), a high-threat chemical agent, occurs naturally in a variety of settings. Despite multiple incidents of exposures and deaths, no FDA-approved antidote exists. A rapid-acting, easy to administer antidote is needed. We conducted a randomized control trial in swine comparing intramuscular administration of aminotetrazole cobinamide (2.9 mL, 18 mg/kg) to no treatment following inhalation of H2 S gas. We found that aminotetrazole cobinamide administered 2 min after the onset of respiratory depression-defined as a tidal volume of less than 3 mL/kg for 2 consecutive minutes-yielded 100% survival, while all control animals died. Respiratory depression resolved in the treatment group within 3.6 ± 1.5 min (mean ± SD) of cobinamide administration, whereas control animals had intermittent gasping until death. Blood pressure and arterial oxygen saturation (SO2 ) returned to baseline values within 5 and 10 min, respectively, of cobinamide treatment, and plasma lactate concentration decreased to less than 50% of the highest value by the end of the experiment. In control animals, plasma lactate rose continuously until death. We conclude that intramuscular aminotetrazole cobinamide is effective in a large animal, inhalational model of acute, severe H2 S poisoning.

Diagnosis of cyanide poisoning using an automated, field-portable sensor for rapid analysis of blood cyanide concentrations.

Cyanide (both HCN and CN- are represented by CN) has multiple industrial applications, is commonly found in some foods, and is a component of fire smoke. Upon exposure, CN blocks production of adenosine triphosphate, causing cellular hypoxia and cytotoxic anoxia, which can eventually result in death. Considering CN's quick onset of action and the long analysis times associated with current techniques, the objective of this study was to develop and validate a rapid and field-portable sensor to detect blood CN concentrations focusing on both concentration and diagnostic accuracy. The sensor takes advantage of the chemical properties of CN by converting it exclusively to HCN via acidification of whole blood. High-speed headspace transfer is used to deliver HCN to a capture solution where it is reacted with naphthalene dialdehyde and taurine to produce a fluorescent ß-isoindole product. Simple spectrofluorometric analysis of the product provides quantitative analysis of CN from whole blood in 60 s and requires only 25 µL of blood (obtainable via fingerstick). A limit of detection of 5 µM, a linear range of 10-200 µM (with ≥15 µM considered CN exposed), and excellent accuracy (100 ± 15%) and precision (≤15.2% relative standard deviation) were obtained. To evaluate the diagnostic accuracy of the sensor, rabbit blood samples (N = 190, including 24 blinded samples) were analyzed by both the sensor and a lab-based spectrophotometric method. An excellent positive correlation was obtained between the sensor and the lab-based method (R2 ˃ 0.995) confirming the concentration accuracy of the CN sensor. Moreover, the sensor produced no false positives or negatives when diagnosing CN poisoning.

Intramuscular sodium tetrathionate as an antidote in a clinically relevant swine model of acute cyanide toxicity.

Background: Cyanide is a metabolic poison used in multiple industries and is a high threat chemical agent. Current antidotes require intravenous administration, limiting their usefulness in a mass casualty scenario. Sodium tetrathionate reacts directly with cyanide yielding thiosulfate and the non-toxic compound thiocyanate. Thiosulfate, in turn, neutralizes a second molecule of cyanide, thus, per mole, sodium tetrathionate neutralizes two moles of cyanide. Historical studies examined its efficacy as a cyanide antidote, but it has not been evaluated in a clinically relevant, large animal model, nor has it previously been administered by intramuscular injection.Objective: The objective of this study is to evaluate the efficacy of intramuscular sodium tetrathionate on survival and clinical outcomes in a large, swine model of severe cyanide toxicity.Methods: Anesthetized swine were instrumented for continuous monitoring of hemodynamics, then acclimated and breathing spontaneously prior to potassium cyanide infusion (0.17 mg/kg/min). At 6-min post-apnea (no breaths for 20 s), the cyanide infusion was terminated, and animals were treated with sodium tetrathionate (∼18 mg/kg) or normal saline control. Clinical parameters and laboratory values were evaluated at various time points until death or termination of the experiment (90 min post-treatment).Results: Laboratory values, vital signs, and time to apnea were similar in both groups at baseline and treatment. Survival in the sodium tetrathionate treated group was 100% and 17% in controls (p = 0.0043). All animals treated with sodium tetrathionate returned to breathing at a mean time of 10.85 min after antidote, and all but one control remained apneic through end of the experiment. Animals treated with tetrathionate showed improvement in blood lactate (p ≤ 0.002) starting at 30 min post-treatment. The average time to death in the control group is 63.3 ± 23.2 min. No systemic or localized adverse effects of intramuscular administration of sodium tetrathionate were observed.Conclusion: Sodium tetrathionate significantly improves survival and clinical outcomes in a large, swine model of acute cyanide poisoning.

Endoscopic Optical Coherence Tomography for Assessing Inhalation Airway Injury: A Technical Review.

Diagnosis of inhalation injury has been clinically challenging. Currently, assessment of inhalation injury relies on subjective clinical exams and bronchoscopy, which provides little understanding of tissue conditions and results in limited prognostics. Endoscopic Optical coherence tomography (OCT) technology has been recently utilized in the airway for direct assessment of respiratory tract disorders and injuries. Endoscopic OCT is capable of capturing high-resolution images of tissue morphology 1-3 mm beneath the surface as well as the complex 3D anatomical shape. Previous studies indicate that changes in airway histopathology can be found in the OCT image almost immediately after inhalation of smoke and other toxic chemicals, which correlates well with histology and pulmonary function tests. This review summarizes the recent development of endoscopic OCT technology for airway imaging, current uses of OCT for inhalation injury, and possible future directions.

Automatic proximal airway volume segmentation using optical coherence tomography for assessment of inhalation injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury with a mortality rate of up to 40%. Early management of ARDS has been difficult due to the lack of sensitive imaging tools and robust analysis software. We previously designed an optical coherence tomography (OCT) system to evaluate mucosa thickness (MT) after smoke inhalation, but the analysis relied on manual segmentation. The aim of this study is to assess in vivo proximal airway volume (PAV) after inhalation injury using automated OCT segmentation and correlate the PAV to lung function for rapid indication of ARDS. METHODS: Anesthetized female Yorkshire pigs (n = 14) received smoke inhalation injury (SII) and 40% total body surface area thermal burns. Measurements of PaO2-to-FiO2 ratio (PFR), peak inspiratory pressure (PIP), dynamic compliance, airway resistance, and OCT bronchoscopy were performed at baseline, postinjury, 24 hours, 48 hours, 72 hours after injury. A tissue segmentation algorithm based on graph theory was used to reconstruct a three-dimensional (3D) model of lower respiratory tract and estimate PAV. Proximal airway volume was correlated with PFR, PIP, compliance, resistance, and MT measurement using a linear regression model. RESULTS: Proximal airway volume decreased after the SII: the group mean of proximal airway volume at baseline, postinjury, 24 hours, 48 hours, 72 hours were 20.86 cm (±1.39 cm), 17.61 cm (±0.99 cm), 14.83 cm (±1.20 cm), 14.88 cm (±1.21 cm), and 13.11 cm (±1.59 cm), respectively. The decrease in the PAV was more prominent in the animals that developed ARDS after 24 hours after the injury. PAV was significantly correlated with PIP (r = 0.48, p < 0.001), compliance (r = 0.55, p < 0.001), resistance (r = 0.35, p < 0.01), MT (r = 0.60, p < 0.001), and PFR (r = 0.34, p < 0.01). CONCLUSION: Optical coherence tomography is a useful tool to quantify changes in MT and PAV after SII and burns, which can be used as predictors of developing ARDS at an early stage. LEVEL OF EVIDENCE: Prognostic, level III.

Determination of free cyano-cobinamide in swine and rabbit plasma by liquid chromatography tandem mass spectrometry.

In recent years, Cobinamide (Cbi) has shown promise as a therapeutic for cyanide poisoning. There are several forms of Cbi based on the identity of the ligands bound to the cobalt in Cbi and these different forms of Cbi have divergent behavior (e.g., the aquo and hydroxo forms of Cbi readily bind to proteins, limiting their distribution significantly, whereas [Cbi(CN)2] does not). While current analysis techniques only measure total Cbi, methods to elucidate the behavior of 'available' Cbi versus cyanide-complexed Cbi would be valuable for biomedical and pharmacokinetic studies. Therefore, a method was developed for the analysis of cyanide-complexed Cbi in plasma via liquid chromatography tandem mass spectrometry (LC-MS-MS). Plasma samples were prepared by denaturing proteins with 10% ammonium hydroxide in acetonitrile. The resulting mixture was centrifuged, and the supernatant was removed, dried, and reconstituted. Cyanide-complexed Cbi was then analyzed via LC-MS-MS. The limit of detection was 0.2 µM, and the linear dynamic range was between 1 and 200 µM. The accuracy was 100 ±â€¯17% and the precision, measured by relative standard deviation (%RSD), was ≤18.5%. Carryover, a severe problem when analyzing Cbi via liquid chromatography was eliminated using a polymeric-based stationary phase (PLRP-S) and a controlled washing protocol. The method allowed evaluation of the cyanide-bound and 'available' Cbi from treated animals and, when paired with a method for total Cbi analysis, allows for estimation of Cbi utilization when treating cyanide poisoning.

Hydrogen Sulfide Toxicity: Mechanism of Action, Clinical Presentation, and Countermeasure Development.

INTRODUCTION: Hydrogen sulfide (H2S) is found in various settings. Reports of chemical suicide, where individuals have combined readily available household chemicals to produce lethal concentrations of H2S, have demonstrated that H2S is easily produced. Governmental agencies have warned of potential threats of use of H2S for a chemical attack, but currently there are no FDA-approved antidotes for H2S. An ideal antidote would be one that is effective in small volume, readily available, safe, and chemically stable. In this paper we performed a review of the available literature on the mechanism of toxicity, clinical presentation, and development of countermeasures for H2S toxicity. DISCUSSION: In vivo, H2S undergoes an incomplete oxidation after an exposure. The remaining non-oxidized H2S is found in dissolved and combined forms. Dissolved forms such as H2S gas and sulfhydryl anion can diffuse between blood and tissue. The combined non-soluble forms are found as acid-labile sulfides and sulfhydrated proteins, which play a role in toxicity. Recent countermeasure development takes into account the toxicokinetics of H2S. Some countermeasures focus on binding free hydrogen sulfide (hydroxocobalamin, cobinamide); some have direct effects on the mitochondria (methylene blue), while others work by mitigating end organ damage by generating other substances such as nitric oxide (NaNO2). CONCLUSION: H2S exists in two main pools in vivo after exposure. While several countermeasures are being studied for H2S intoxication, a need exists for a small-volume, safe, highly effective antidote with a long shelf life to treat acute toxicity as well as prevent long-term effects of exposure.

Efficacy of Oral Administration of Sodium Thiosulfate and Glycine in a Large, Swine Model of Oral Cyanide Toxicity.

STUDY OBJECTIVE: Cyanide is a deadly poison, particularly with oral exposure, in which larger doses can occur before any symptoms develop. Multiple governmental agencies highlight oral cyanide as an agent that can be used in a terrorist attack because it can be easily weaponized and is readily available. Currently, there are no Food and Drug Administration-approved antidotes specifically for oral cyanide. An oral countermeasure that can neutralize and prevent absorption of cyanide from the gastrointestinal tract after oral exposure is needed. The objective of this study is to determine if the combination of glycine and sodium thiosulfate administered orally is effective in reducing mortality in a large, swine model of oral cyanide toxicity. METHODS: Nine swine (45 to 55 kg) were instrumented, sedated, and stabilized. Potassium cyanide (at 8 mg/kg) in saline solution was delivered as a onetime bolus through an orogastric tube. Three minutes after cyanide administration, animals that were randomized to the treatment group received sodium thiosulfate (508.2 mg/kg, 3.25-M solution) and glycine (30 mg/kg, 3.5-M solution) through an orogastric tube. Survival at 60 minutes was the primary outcome. We compared survival between groups by log-rank Mantel-Cox analysis and trended laboratory results and vital signs. RESULTS: At baseline and treatment, all animals were similar. Survival at 60 minutes was 100% in treated animals compared with 0% in the control group (P=.003). By the study end, defined as death or 60 minutes after cyanide administration, there was a significant difference in the lactate concentration between the treatment and control groups (control 9.43 mmol/L [SD 4.08]; treatment 1.66 mmol/L [SD 0.82]; difference between means 7.69 mmol/L [SD 2.07]; 95% confidence interval difference -14.05 to -1.32). Mean arterial pressure was significantly different between the treatment and control groups at study end (control 26 mm Hg [SD 6.7]; treatment 81 mm Hg [SD 14]; difference between means 55.2 mm Hg [SD 7.1]; 95% confidence interval difference 37.8 to 72.6). pH and oxygen saturation were also significantly different between the treatment and control groups at study end. CONCLUSION: The combination of oral sodium thiosulfate and glycine significantly improved survival and physiologic parameters in a large-animal model of oral cyanide toxicity.

Metabolism of Cyanide by Glutathione To Produce the Novel Cyanide Metabolite 2-Aminothiazoline-4-oxoaminoethanoic Acid.

The direct analysis of cyanide (HCN or CN- inclusively symbolized as CN) to confirm exposure has major limitations due to cyanide's volatility, reactivity, and short half-life in biological fluids. These limitations have led to the exploration of cyanide detoxification products for indirect verification of cyanide exposure. Although cyanide interacts strongly with sulfur-containing molecules, to date, biomarkers resulting from the interaction of cyanide with glutathione (GSH; i.e., a biologically abundant sulfur-donating biomolecule) have yet to be discovered. In this study, we studied the interaction of CN and GSH to produce 2-aminothiazoline-4-oxoaminoethanioc acid (ATOEA). An LC-MS/MS method was developed and validated to analyze ATOEA from plasma, producing a linear range of 0.5-50 µM, a limit of detection of 200 nM, and excellent precision and accuracy. ATOEA concentrations were significantly elevated in the plasma of animals following cyanide exposure. Moreover, the production of ATOEA from cyanide exposure was confirmed by detection of both ATOEA and ATOEA-13C15N in rabbit plasma ( N = 11 animals) following administration of NaCN:K13C15N (1:1), with a similar amount of ATOEA and ATOEA-13C15N formed ( R2 = 0.9924, p < 0.05). The concentration of ATOEA increased with cyanide dose and then decreased rapidly when an antidote was administrated. This study definitively showed that ATOEA is produced from interaction of CN and GSH and can serve as a biomarker of cyanide exposure.

Correction to: Monitoring Dose Response of Cyanide Antidote Dimethyl Trisulfide in Rabbits Using Diffuse Optical Spectroscopy.

The online version of the original article can be found at.