A Fatal Case of Acute Hydrogen Sulfide Poisoning Caused by Hydrogen Sulfide: Hydroxocobalamin Therapy for Acute Hydrogen Sulfide Poisoning

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1 A Fatal Case of Acute Hydrogen Sulfide Poisoning Caused by Hydrogen Sulfide: Hydroxocobalamin Therapy for Acute Hydrogen Sulfide Poisoning Case Report Yuji Fujita 1,2, *, Yasuhisa Fujino 3, Makoto Onodera 3, Satoshi Kikuchi 3, Tomohiro Kikkawa 3, Yoshihiro Inoue 3, Hisae Niitsu 4, Katsuo Takahashi 2, and Shigeatsu Endo 3 1 Poisoning and Drug Laboratory Division, Critical Care and Emergency Center, Iwate Medical University Hospital, Honchoudori, Morioka, Iwate , Japan; 2 Department of Pharmacy, Iwate Medical University Hospital, 19-1 Uchimaru, Morioka, Iwate , Japan; 3 Department of Emergency Medicine, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, Iwate , Japan; and 4 Department of Legal Medicine, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, Iwate , Japan Abstract A patient committed suicide with hydrogen sulfide (H 2 S) by combining. The patient was given hydroxocobalamin as an antidote in addition to treatment with cardiopulmonary resuscitation, but died approximately 42 min after his arrival at the hospital. The patient s cause of death was attributed to acute hydrogen sulfide poisoning. Serum concentrations of sulfide before and after administration of hydroxocobalamin were 0.22 and 0.11 μg/ml, respectively; serum concentrations of thiosulfate before and after hydroxocobalamin administration were 0.34 and 0.04 μmol/ml, respectively. Hydroxocobalamin is believed to form a complex with H 2 S in detoxification pathways of H 2 S. Although H 2 S is rapidly metabolized and excreted, the decreased sulfide concentration may be also associated with this complex formation. The decreased sulfide concentration suggests that hydroxocobalamin therapy may be effective for acute H 2 S poisoning. The decreased thiosulfate concentration seems to be associated with formation of a thiosulfate/hydroxocobalamin complex, because hydroxocobalamin can form a complex with thiosulfate. The thiosulfate concentration decreased to a greater extent than did sulfide, suggesting that hydroxocobalamin has a higher affinity for thiosulfate than for H 2 S. Therefore, prompt administration of hydroxocobalamin after H 2 S exposure may be effective for H 2 S poisoning. Introduction Hydrogen sulfide (H 2 S) gas is highly toxic, colorless, and flammable, with a characteristic rotten egg scent. It exists in raw petroleum and natural and volcanic gases, and is produced through leather tanning, processes for kraft and paper pulp, and decay of human and animal waste. Accidental H 2 S poisoning in industrial plants, dairy farms, and other locations has been reported (1 4). H 2 S is quickly absorbed through the lungs and the * Author to whom correspondence should be addressed: Yuji Fujita, Poisoning and Drug Laboratory Division, Critical Care and Emergency Center, Iwate Medical University Hospital, Honchoudori, Morioka, Iwate , Japan. yfujita@iwate-med.ac.jp. Figure 1. Main metabolic pathway of hydrogen sulfide. Reproduction (photocopying) of editorial content of this journal is prohibited without publisher s permission. 119

2 gastrointestinal tract; it is eliminated through the lungs or in feces, and its metabolites are passed in urine. H 2 S has three metabolic pathways: oxidation, alkylation, and reactions with metallo- or disulfide-containing proteins (5). Its main metabolic pathway is the oxidation pathway; H 2 S metabolizes to sulfate via thiosulfate (Figure 1) through non-enzymatic and enzymatic mechanisms. The oxidation and alkylation pathways are detoxification pathways, in which H 2 S s toxic mechanisms are reactions with essential proteins. In acute human H 2 S poisoning cases, thiosulfate usually cannot be detected in blood of survivors, but can be detected in their urine (6). In fatal cases, however, thiosulfate usually can be detected in blood, but not in urine (7 9). Therefore, detection of thiosulfate in urine for survivors and in blood for fatal cases is useful for diagnosing H 2 S poisoning (6). H 2 S binds cytochrome oxidase and inhibits its function, which is the conversion of molecular oxygen to water. It thus inhibits generation of adenosine triphosphate, which provides energy for many cellular functions. Most organ systems are susceptible to the effect of H 2 S, particularly mucous membranes and tissues that demand the most oxygen. H 2 S stops cellular respiration at high concentrations. The mechanism of toxicity of H 2 S is similar to that of cyanide. Increased exposure concentrations of H 2 S are associated with adverse effects of increased severity: conjunctival irritation occurs at about 50 ppm, irritation of the respiratory tract at ppm, loss of smell at ppm, pulmonary edema at ppm, and concentrations greater than 500 ppm often called the knock down concentration can cause respiratory arrest, collapse, and death within minutes (10). Although H 2 S has a characteristic rotten egg scent, its odor cannot be always identified, because the exposure to > 150 ppm paralyzes olfactory nerves. In acute H 2 S poisoning, victims should be immediately transferred to fresh air and cardiopulmonary resuscitation started for victims with no heartbeat. Therapy for acute H 2 S poisoning is supportive care. Although effective protocols for acute H 2 S poisoning are not established, nitrate therapy, such as inhaled amyl nitrate and intravenous sodium nitrate, and hyperbaric oxygen therapy may be useful for certain patients. Nitrates detoxify H 2 S poisoning by inducing the formation of methemoglobin, which has a higher affinity for H 2 S than does cytochrome oxidase; methemoglobin then binds H 2 S, decreasing its toxicity. Truong et al. (11) recently reported that hydroxocobalamin (Figure 2), which is part of the antidote kit for cyanide poisoning, is useful as an antidote against H 2 S poisoning in animal experiments; a cyanide antidote kit might be useful for emergency treatment of acute H 2 S poisoning. Hydroxocobalamin is thought to detoxify H 2 S poisoning by forming a complex with H 2 S, which metabolizes to thiosulfate and sulfate. To help establish an effective therapy for acute H 2 S poisoning, we report here on a fatal case of acute hydrogen sulfide poisoning, and serum concentrations of sulfide and thiosulfate before and after administration of hydroxocobalamin. This patient committed suicide using H 2 S made from two commercial products. Recently, H 2 S created from mixing two commercial products has been used as a tool for suicide in Japan (7 9,12). We think that case reports can offer useful information on therapy for acute intoxication, and an accumulation of case reports would be useful for the treatment of acute intoxications in the future. Case History In April 2008, a male in his early 20s committed suicide in a car by combining to make H 2 S gas. He was unconscious and not breathing when his family found him in the car (6:40 a.m.). His family confirmed him to have been alive at midnight (12:00 a.m.). He smelled noticeably of rotten eggs and had neither pulse nor spontaneous respirations when emergency workers arrived at the site (6:52 a.m.). On arrival at the hospital (7:19 a.m.), his eyes showed mydriasis, and an electrocardiogram showed asystole. On arterial blood gas analysis, his arterial carboxyhemoglobin was 1.5%, indicating that he had not developed carbon monoxide poisoning. He was treated with cardiopulmonary resuscitation and received an intravenous infusion of 2.5 g of hydroxocobalamin as an antidote for H 2 S poisoning. However, he died of H 2 S poisoning 42 min after arrival at the hospital (8:01 a.m.) in spite of the staff s efforts. Methods Analytical procedures for sulfide and thiosulfate Extraction of sulfide and thiosulfate from serum and derivatization of those were performed according to the method de- Figure 2. Structural formula of hydroxocobalamin. 120

3 Figure 3. Selected ion-monitoring chromatograms (SIM) of sulfide and thiosulfate in serum. Sulfide and thiosulfate were detected as bis(pentafluorobenzyl)sulfide and bis(pentafluorobenzyl)disulfide, respectively. The mass-to-charge ratios of the monitored ions of sulfide, thiosulfate, and internal standard were m/z 393.9, m/z 425.8, and m/z 313.7, respectively. SIM chromatogram of sulfide in spiked serum (0.29 μg/ml) (A); SIM chromatogram of sulfide in patient s serum before administration of hydroxocobalamin (B); SIM chromatogram of thiosulfate in spiked serum (0.50 μmol/ml) (C); and SIM chromatogram of thiosulfate in patient s serum before administration of hydroxocobalamin (D). Table I. Blood Concentrations of Sulfide and Thiosulfate in Fatal Cases of Acute Hydrogen Sulfide Poisoning Number SA Report of Sulfide Thiosulfate (Reference) Cases (µg/ml) (µmol/ml) Remarks Kage et al. (2) Accidental poisoning at a dye works Igawa et al. (7) Suicide, H 2 S produced from Kobayashi et al. (9) Suicide, H 2 S produced from Sasaki et al. (12) Suicide, H 2 S produced from Present case * 0.34* Suicide, H 2 S produced from * Before hydroxocobalamin administration. After hydroxocobalamin administration. scribed by Kage et al. (13,14). Sulfide was detected as bis(pentafluorobenzyl)sulfide. A serum sample (0.2 ml) was added to the mixture solution: 0.8 ml of 5 mm tetradecyl-dimethyl-benzyl ammonium chloride solution in oxygen-free water saturated with sodium tetraborate, 0.5 ml of 20 mm pentafluorobenzyl bromide (PFBBr) solution in ethyl acetate, and 2.0 ml of internal standard (IS) solution (10 μm 1,3,5-tribromobenzene (TBB) in ethyl acetate). The preparation was vortex mixed for 1 min, and about 0.1 g of potassium dihydrogen phosphate was added. The preparation was again vortex mixed for 10 s and centrifuged at 2500 rpm for 10 min. An aliquot of the organic phase was injected onto a gas chromatograph mass spectrometer (GC MS). Thiosulfate was detected as bis(pentafluorobenzyl)disulfide. A serum sample (0.2 ml) was added to the mixture solution: 0.05 ml of 200 mm L-ascorbic acid solution, 0.05 ml of 5% sodium chloride, and 0.5 ml of 20 mm PFBBr solution in acetone. The preparation was vortex mixed for 1 min; 2.0 ml of 25 mm iodine solution in ethyl acetate and 0.5 ml of IS solution (40 μm TBB in ethyl acetate) were added. The preparation was again vortex mixed for 30 s, centrifuged at 2500 rpm for 15 min, and left to stand for 1 h. An aliquot of the organic phase was injected onto a GC MS. The serum samples were stored at 80 C until analysis. Instrumentation and operating parameters A PerkinElmer AutoSystem XL GC and Turbomass MS (Waltham, MA) were used for GC MS analysis. GC was performed with an Agilent J&W DB-5MS column (30 m 0.25-mm i.d., 0.25-μm film thickness, Agilent Technologies, Santa Clara, CA); column oven temperature was maintained at 60 C for 4 min and then programmed to 300 C at 20 C/min. Carrier gas was helium (1 ml/min). Injection port and ion source temperatures were kept at 250 C and 200 C, respectively; MS was performed in electron impact ionization mode at ionization energy of 70 ev. Measurements were taken in selected ion monitoring mode. The mass-to-charge ratios of monitored ions of sulfide, thiosulfate, and IS were m/z 393.9, m/z 425.8, and m/z 313.7, respectively. 121

4 Calibration curves for sulfide and thiosulfate in serum In GC MS analysis, calibration curves for sulfide and thiosulfate in serum were obtained by plotting the peak-area ratio of each molecule relative to an internal standard. The calibration curve setting range for sulfide was μg/ml; for thiosulfate, the calibration curve range was μmol/ml. To examine the precision, three different concentrations of sulfide and thiosulfate were spiked in reference sera. Serum concentrations of sulfide were 0.072, 0.14, and 0.29 μg/ml; serum concentrations of thiosulfate were 0.05, 0.25, and 0.40 μmol/ml. These samples were analyzed five times in one day for intraday precision, and they were analyzed three times each on five separate days for interday precision. Results and Discussion This patient committed suicide using hydrogen sulfide. Similar H 2 S poisonings have been recently reported in Japan (7 9,12), combining, such as toilet bowl cleaner and liquid bath additive, to make H 2 S gas in sealed small spaces. The toilet cleaners and the liquid bath additives contain hydrochloric acid and polysulfide, respectively. Kobayashi and Fukushima (9) reported that mixing 120 ml of each of these products can produce approximately 1000 ppm of hydrogen sulfide, for example, in an ordinary motor vehicle with a volume of 3300 L. Because this reaction happens immediately after mixing, the production of lethal concentrations (over 500 ppm) of hydrogen sulfide is not very difficult in these cases. Sulfide and thiosulfate were detected in serum using GC MS analysis (Figure 3). Serum concentrations of sulfide before and after administration of hydroxocobalamin were 0.22 and 0.11 μg/ml, respectively; serum concentrations of thiosulfate before and after hydroxocobalamin administration were 0.34 and 0.04 μmol/ml, respectively (Table I). For the intraday and interday precision of the method, the relative standard deviations (RSDs) of each concentration of sulfide and thiosulfate in the intraday were below 10%, and the RSDs of those in the interday were below 15%. The overall precision of the method was acceptable. Reportedly, blood concentrations of sulfide and thiosulfate are lower than 0.05 μg/ml (15) and μmol/ml (14,16), respectively, in humans who are not exposed to H 2 S. On the other hand, in acute H 2 S poisoning fatalities, blood concentrations of sulfide and thiosulfate are μg/ml and μmol/ml, respectively (Table I). Serum concentrations of sulfide and thiosulfate in this case were similar to reported concentrations in fatal cases of acute H 2 S poisoning, and higher than concentrations of those in blood of humans who were not exposed to H 2 S. This result shows that the patient s cause of death was acute hydrogen sulfide poisoning. Serum concentrations of sulfide and thiosulfate in this case were less after administering hydroxocobalamin than before its administration. In the detoxification of H 2 S in the presence of hydroxocobalamin, hydroxocobalamin is thought to form a complex with H 2 S; H 2 S is then metabolized to thiosulfate and sulfate (11). Although H 2 S is rapidly metabolized and excreted, decreased serum concentration of sulfide may be also associated with this complex formation. Hydroxocobalamin has also sufficient potential for forming a complex with thiosulfate in vivo because hydroxocobalamin forms such a complex in vitro (17). Therefore, the formation of a thiosulfate/hydroxocobalamin complex seems associated with rapid decrease of serum concentration of thiosulfate. In acute H 2 S poisoning, thiosulfate can be detected in the urine of the survivors (6), but it cannot be detected in the urine of the fatal cases (7 9), indicating that thiosulfate is not excreted in urine in fatal cases; apparently thiosulfate cannot be excreted in urine in the short period between exposure and death. Therefore, it seems unlikely that the thiosulfate of this patient was excreted in urine. Serum concentration of thiosulfate decreased to a greater extent than did sulfide in this case. Given the findings, this result suggests that hydroxocobalamin has a higher affinity for thiosulfate than for H 2 S. In conclusion, this patient was given hydroxocobalamin as an antidote for H 2 S poisoning, but it was ineffective in this case. The patient would have succumbed regardless of treatment because he was in cardiopulmonary arrest upon arrival at the hospital. However, the decreased serum concentration of H 2 S after administration of hydroxocobalamin suggests that this therapy may be effective for acute H 2 S poisoning. Given the finding that hydroxocobalamin has a higher affinity for H 2 S s metabolite thiosulfate than for H 2 S itself, it seems likely that administration of hydroxocobalamin immediately after H 2 S exposure, when trace amounts of thiosulfate appear, is effective for H 2 S poisoning. Further case studies may elucidate this therapeutic effect of hydroxocobalamin against H 2 S poisoning. References 1. S. Kage, S. Kashimura, H. Ikeda, K. Kudo, and N. Ikeda. Fatal and nonfatal poisoning by hydrogen sulfide at an industrial waste site. J. Forensic Sci. 47: (2002). 2. S. Kage, H. Ikeda, N. Ikeda, A. Tsujita, and K. Kudo. Fatal hydrogen sulfide poisoning at a dye works. Leg. Med. (Tokyo) 6: (2004). 3. G. 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