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Case Report
A Fatal Case of Acute Hydrogen Sulfide Poisoning
Caused by Hydrogen Sulfide: Hydroxocobalamin
Therapy for Acute Hydrogen Sulfide Poisoning
Yuji Fujita1,2,*, Yasuhisa Fujino3, Makoto Onodera3, Satoshi Kikuchi3, Tomohiro Kikkawa3, Yoshihiro Inoue3,
Hisae Niitsu4, Katsuo Takahashi2, and Shigeatsu Endo3
1Poisoning
and Drug Laboratory Division, Critical Care and Emergency Center, Iwate Medical University Hospital,
3-16-1 Honchoudori, Morioka, Iwate 020-0015, Japan; 2Department of Pharmacy, Iwate Medical University Hospital,
19-1 Uchimaru, Morioka, Iwate 020-8505, Japan; 3Department of Emergency Medicine, Iwate Medical University School of
Medicine, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan; and 4Department of Legal Medicine, Iwate Medical University
School of Medicine, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan
Introduction
Abstract
A patient committed suicide with hydrogen sulfide (H2S) by
combining two commercial products. 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 H2S in detoxification pathways of H2S. Although H2S 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 H2S
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 H2S. Therefore, prompt
administration of hydroxocobalamin after H2S exposure may be
effective for H2S poisoning.
* Author to whom correspondence should be addressed: Yuji Fujita, Poisoning and Drug
Laboratory Division, Critical Care and Emergency Center, Iwate Medical University Hospital,
3-16-1 Honchoudori, Morioka, Iwate 020-0015, Japan. Email: [email protected].
Hydrogen sulfide (H2S) 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 H2S poisoning in
industrial plants, dairy farms, and other locations has been reported (1−4). H2S is quickly absorbed through the lungs and the
Figure 1. Main metabolic pathway of hydrogen sulfide.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
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gastrointestinal tract; it is eliminated through the lungs or in
feces, and its metabolites are passed in urine. H2S has three
metabolic pathways: oxidation, alkylation, and reactions with
metallo- or disulfide-containing proteins (5). Its main metabolic
pathway is the oxidation pathway; H2S metabolizes to sulfate via
thiosulfate (Figure 1) through non-enzymatic and enzymatic
mechanisms. The oxidation and alkylation pathways are detoxification pathways, in which H2S’s toxic mechanisms are reactions with essential proteins. In acute human H2S 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
H2S poisoning (6).
H2S 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 H2S, particularly mucous membranes and tissues that demand the most oxygen. H2S stops cellular respiration at high concentrations. The mechanism of
toxicity of H2S is similar to that of cyanide. Increased exposure
concentrations of H2S are associated with adverse effects of increased severity: conjunctival irritation occurs at about 50 ppm,
irritation of the respiratory tract at 50–100 ppm, loss of smell at
100–200 ppm, pulmonary edema at 250–500 ppm, and concentrations greater than 500 ppm—often called the “knock
down concentration”—can cause respiratory arrest, collapse,
and death within minutes (10). Although H2S has a characteristic “rotten egg” scent, its odor cannot be always identified, because the exposure to > 150 ppm paralyzes olfactory nerves.
In acute H2S poisoning, victims should be immediately transferred to fresh air and cardiopulmonary resuscitation started for
victims with no heartbeat. Therapy for acute H2S poisoning is
supportive care. Although effective protocols for acute H2S 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 H2S poisoning by inducing the formation of methemoglobin, which has a higher affinity for H2S than does cytochrome oxidase; methemoglobin then binds H2S, 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 H2S poisoning in animal experiments; a cyanide antidote kit might be
useful for emergency treatment of acute H2S poisoning. Hydroxocobalamin is thought to detoxify H2S poisoning by
forming a complex with H2S, which metabolizes to thiosulfate
and sulfate.
To help establish an effective therapy for acute H2S 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 H2S made from two commercial
products. Recently, H2S 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
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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 two commercial products to make H2S
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 H2S poisoning. However, he died
of H2S 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.
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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
of
Cases
Sulfide
(µg/mL)
SA
Thiosulfate
(µmol/mL)
Kage et al. (2)
4
0.32–9.36
0.11–0.23
Igawa et al. (7)
4
0.57–3.67
0.055–0.124
Suicide, H2S produced from
two commercial products
Kobayashi et al. (9)
1
0.66
0.14
Suicide, H2S produced from
two commercial products
13
0.06–14.13
0.05
Suicide, H2S produced from
two commercial products
1
0.22*
0.11†
0.34*
0.04†
Suicide, H2S produced from
two commercial products
Report
(Reference)
Sasaki et al. (12)
Present case
* Before hydroxocobalamin administration.
† After hydroxocobalamin administration.
Remarks
Accidental poisoning
at a dye works
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.
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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 0.036–0.36 μg/mL; for
thiosulfate, the calibration curve range was 0.025–0.50
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 H2S poisonings have been recently reported in Japan
(7−9,12), combining two commercial products, such as toilet
bowl cleaner and liquid bath additive, to make H2S 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 0.003
μmol/mL (14,16), respectively, in humans who are not exposed to H2S. On the other hand, in acute H2S poisoning fatalities, blood concentrations of sulfide and thiosulfate are
0.06–14.13 μg/mL and 0.05–3.02 μmol/mL, respectively (Table
I). Serum concentrations of sulfide and thiosulfate in this case
were similar to reported concentrations in fatal cases of acute
H2S poisoning, and higher than concentrations of those in
blood of humans who were not exposed to H2S. 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 H2S in the presence of
hydroxocobalamin, hydroxocobalamin is thought to form a
complex with H2S; H2S is then metabolized to thiosulfate and
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sulfate (11). Although H2S 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 H2S 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 H2S.
In conclusion, this patient was given hydroxocobalamin as
an antidote for H2S 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
H2S after administration of hydroxocobalamin suggests that
this therapy may be effective for acute H2S poisoning. Given
the finding that hydroxocobalamin has a higher affinity for
H2S’s metabolite thiosulfate than for H2S itself, it seems likely
that administration of hydroxocobalamin immediately after
H2S exposure, when trace amounts of thiosulfate appear, is
effective for H2S poisoning. Further case studies may elucidate
this therapeutic effect of hydroxocobalamin against H2S poisoning.
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Manuscript received May 1, 2010;
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