Sample preparation method with ultrafiltration for whole blood thiosulfate measurement.

Quantitative analysis of thiosulfate is useful for diagnosing hydrogen sulfide poisoning. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables more rapid and sensitive measurements than previous methodologies. As simple measurements of blood thiosulfate concentration are affected by the blood matrix, blood is used as the solvent to prepare the standard solution for calibration curve generation. Thus, a large amount of blood devoid of thiosulfate is required. We developed a preparation method by incorporating an ultrafiltration step to overcome this limitation and generate a calibration curve using a standard solution prepared with pure water. We used this improved method to investigate the stability of thiosulfate in refrigerated samples. To compare the effects of refrigeration, blood samples were prepared using the following two methods: one sample was treated with a 50-kDa exclusion ultrafiltration membrane and the other was not treated. The samples were stored at 4 °C, and then measured at 0, 3, 6, 24, 48, and 96 h. The incorporation of the ultrafiltration step in the measurement procedure enabled the quantification of thiosulfate, by plotting a calibration curve using a standard of pure water; it did not require a blood standard. Additionally, the reduction in whole blood thiosulfate concentration was within 10% during 2 days of refrigeration. Thus, the need for a large amount of blood to prepare the standard solution was resolved by the ultrafiltration step in test sample preparation. This method is useful to measure thiosulfate concentration and is not hindered by sample refrigeration for a few days.

Phase 1 study to evaluate safety, tolerability and pharmacokinetics of a novel intra-tympanic administered thiosulfate to prevent cisplatin-induced hearing loss in cancer patients.

Cisplatin is a widely used chemotherapy for the treatment of certain solid tumors. Ototoxicity and subsequent permanent hearing loss remain a serious dose-limiting side effect associated with cisplatin treatment. To date, no therapies have been approved to prevent or treat cisplatin-induced hearing loss (CIHL). Sodium thiosulfate effectively inactivates cisplatin through covalent binding and may provide protection against cisplatin-induced ototoxicity. DB-020 is being developed as a novel formulation of sodium thiosulfate pentahydrate in 1% sodium hyaluronate for intratympanic injection (IT), enabling the delivery of high concentrations of thiosulfate into the cochlea prior to cisplatin administration. In the DB-020-002 phase 1a single-ascending dose study, healthy volunteers were enrolled into 5 cohorts to receive different doses of DB-020 via IT injection. Cohorts 1-4 received unilateral injections while Cohort 5 received bilateral injections. Plasma thiosulfate pharmacokinetics was measured, and safety and audiometric data were collected throughout the study. This study has demonstrated that intratympanic administration of DB-020 results in nominal systemic increases in thiosulfate levels, hence it should not compromise cisplatin anti-tumor efficacy. Furthermore, DB-020 was safe and well tolerated with most adverse events reported as transient, of mild-to-moderate severity and related to the IT administration procedure. These results support the design and execution of the ongoing proof-of-concept study, DB-020-002, to assess otoprotection using DB-020 in cancer patients receiving cisplatin without negatively impacting cisplatin anti-tumor efficacy.

Lethal Hydrogen Sulfide poisoning in open space: An atypical case of asphyxiation of two workers.

Hydrogen sulfide is one of the most dangerous toxic gases that has led to the deaths in confined spaces of many workers. We report an atypical case of a fatal accident of H2S poisoning in an open space when two workers died during the opening of a hatch on a tanker truck filled with leachate water. Despite being outdoors, the two workers, were suddenly and quickly overwhelmed by a lethal cloud of H2S, which escaped like a geyser from the hatch and hovered over the top of the tanker making it impossible for them to survive. The first operator was engulfed by the sudden flow of lethal gas near the hatch while the second worker, who came to his aid, immediately lost consciousness and fell off the tanker onto the ground. Environmental toxicological analyses were carried out on the air near the hatch and inside the tanker 2h, 20 days and 70 days after the accident. Toxicological analyses on the blood were also carried out but unfortunately, no urine sample was available. The thiosulfate, detected by GC/MS analysis after derivatization of PFBBr, was found to be 0.01 and 0.04mM/L. These values are included in the medium-low lethal values of occupational fatalities involving H2S reported in the literature.

Toxicological investigations in a fatal and non-fatal accident due to hydrogen sulphide (H2S) poisoning.

Hydrogen sulphide (H2S) is one of the most toxic natural gas and represents a not rare cause of fatal events in workplaces. We report here a serious accidental poisoning by hydrogen sulphide inhalation involving six sailors. Three of them died while the other three survived and were transported to the emergency room. No greenish discolouration of the body, that could be a feature of these type of deaths, was observed at autopsy. Given that blood and/or urine H2S detection does not allow to discriminate if it is related to inhalation or to putrefactive processes, the determination of thiosulphate, H2S main metabolite, is decisive. The succession of fatal events reported here can be rebuilt by toxicological data interpretation: the subject 1 died after a longer interval of time as demonstrated by the highest blood and urine thiosulfate concentrations; the subject 2 died after a short interval of time as showed by a lower blood and urine thiosulfate concentrations than subject 1; the subject 3 died almost immediately after H2S inhalation since he showed the lowest blood thiosulfate concentration, and no trace of sulphide and thiosulfate was found in the urine.

Fatal inhalation of volcanic gases in three tourists of a geothermal area.

The study reports the environmental, toxicological and histopathological forensic investigations applied on three victims of accidental death (father, mother and son), due to the fall in a volcanic pothole, during the touristic visit of the "Solfatara park", near Naples (Italy). At autopsy greenish skin discolouration was observed and all bodies showed the classical signs of asphyxial deaths, such as cyanosis and hemorrhagic pulmonary edema. Focal micro-hemorrhages were found in the brain at intracranial and subpial levels. The hemogasanalysis and spectrophotometric test on blood for Methemoglobin (MetHb), Carboxyhemoglobin (HbCO) and Sulfhemoglobin (SHb) showed pCO2, SHb and MetHb above the physiological levels. On biological specimens, toxicological analyses performed by GC/MS revealed high concentrations of hydrogen sulfide (H2S) and of thiosulfate (TS), its main metabolite. The monitoring of toxic gases on the death scene showed an unsafe environment, into the pothole, able to cause the sudden loss of consciousness of the victims with subsequent asphyxiation (knockdown effect). In particular, at the bottom of the hole, the maximum levels of H2S and carbon dioxide (CO2) were 2200 ppm and 98% respectively. For the family members, the cause of the death was assessed as acute poisoning by H2S and CO2. The fatalities, happened in quick succession as for a domino effect, were pretty similar to the asphyxial deaths by confined spaces, frequently observed in occupational setting. Fatalities secondary to accidental volcanic gases inhalation, such as H2S and CO2 in geothermal areas, have been already described but often without a forensic approach. To the best of our knowledge this is the first case that reports the accidental poisoning by volcanic gases involving three people, with different caracteristic of age and sex, allowing the correlation between toxicological and pathological results with the true levels of asphyxiating gas, measured on the death scene.

Liquid chromatography-tandem mass spectrometry method for the determination of thiosulfate in human blood and urine as an indicator of hydrogen sulfide poisoning.

Being a stable metabolite of hydrogen sulfide, thiosulfate has been utilized as an index for hydrogen sulfide poisoning (HSP). Thiosulfate analysis is mainly performed using gas chromatography/mass spectrometry (GC-MS) due to its high sensitivity and specificity. The GC-MS analysis requires two-step derivatizations of thiosulfate, and the derivative is not stable in solution as it has a disulfide moiety. To resolve this stability issue, we developed a novel analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for monitoring the pentafluorobenzyl derivative of thiosulfate (the first reaction product of the GC-MS method) in this study. The established method exhibited high reproducibility despite being a more simplified and rapid procedure compare to the GC-MS method. Phenyl 4-hydroxybenzoate was used as an internal standard because 1,3,5-tribromobenzene which had been used in the GC-MS method was not suitable compound for LC-MS/MS with Electrospray ionization (ESI) negative detection. The linear regression of the peak area ratios versus concentrations was fitted over the concentration ranges of 0.5-250µM and 0.25-250µM in blood and urine, respectively. The validation results satisfied the acceptance criteria for intra- and inter-day accuracy and precision. Blood and urine samples from 12 suspected HSP cases were tested using this method. The thiosulfate concentration detected in the sample coincided well with that determined at the scene of each HSP accident.

Development for the measurement of serum thiosulfate using LC-MS/MS in forensic diagnosis of H2S poisoning.

Thiosulfate measurement is crucial to diagnosis of hydrogen sulfide (H2S) poisoning in forensic toxicology. Although GC-MS method is currently regarded as a standard thiosulfate measurement, it requires complicated sample preparation prior to analysis. This study presents a simple, rapid, and highly sensitive method for the quantitative analysis of serum thiosulfate by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is based on selected reaction monitoring and has high sensitivity with a lower quantification limit of 0.5µM. Precision and accuracy of this method meet the basic requirements for quantitative analysis (intra- and inter-day tests have a relative standard deviation of ⩽10.4%; range of analytical recovery is 94.3-102.6%). On the measurements of serum thiosulfate by our developed method, a thiosulfate concentration as 57.5µM was detected clearly in the H2S poisoning case comparing to the non poisoning case in which only a trace amount of thiosulfate was observed.

Serum free sulfhydryl status is associated with patient and graft survival in renal transplant recipients.

Oxidative stress contributes significantly to graft failure, morbidity and mortality in renal transplant recipients (RTR). In cells, free sulfhydryl groups (reduced thiols, R-SH) are the transducers of redox-regulated events; their oxidation status is modulated by interaction with reactive oxygen and nitrogen oxide species and thought to be in equilibrium with the circulating pool. We hypothesized that high levels of serum free thiols are a reflection of a favorable redox status and therefore positively associated with cardiovascular risk parameters, patient and graft survival in RTR. To test this, reactive free thiol groups (R-SH; corrected for total protein) were quantified in serum of 695 RTR (57% male, 53±13yr, functioning graft ≥1yr) using Ellman's Reagent, and R-SH determinants were evaluated with multivariable linear regression models. Associations between R-SH and mortality or graft failure were assessed using multivariable Cox regression analyses. In multivariable models, male gender, estimated glomerular filtration rate and serum thiosulfate positively associated with R-SH while BMI, HbA1c, corrected calcium and NT-pro-BNP inversely associated with R-SH (model R2=0.26). During follow-up (3.1 [2.7-3.9] yrs), 79 (11%) patients died and 45 (7%) patients developed graft failure. R-SH correlated inversely with all-cause mortality (HR 0.58 [95% CI 0.45-0.75] per SD increase) and graft failure (HR 0.42 [0.30-0.59]; both P<0.001), independent of parameters with which R-SH significantly associated in the multivariable regression analyses, except for NT-pro-BNP. Serum R-SH are associated with a beneficial cardiovascular risk profile and better patient and graft survival in RTR, suggesting potential usefulness as low-cost, high-throughput screening tool for whole-body redox status in translational studies. Whether R-SH modification improves long-term outcome of RTR warrants further exploration.

Case studies of hydrogen sulphide occupational exposure incidents in the UK.

The UK Health and Safety Executive has investigated several incidents of workplace accidents involving hydrogen sulphide exposure in recent years. Biological monitoring has been used in some incidents to determine the cause of unconsciousness resulting from these incidents and as a supporting evidence in regulatory enforcement. This paper reports on three case incidents and discusses the use of biological monitoring in such cases. Biological monitoring has a role in identifying hydrogen sulphide exposure in incidents, whether these are occupational or in the wider environment. Sample type, time of collection and sample storage are important factors in the applicability of this technique. For non-fatal incidents, multiple urine samples are recommended at two or more time points between the incident and 15 h post-exposure. For routine occupational monitoring, post-shift samples should be adequate. Due to endogenous levels of urinary thiosulphate, it is likely that exposures in excess of 12 ppm for 30 min (or 360 ppm/min equivalent) would be detectable using biological monitoring. This is within the Acute Exposure Guideline Level 2 (the level of the chemical in air at or above which there may be irreversible or other serious long-lasting effects or impaired ability to escape) for hydrogen sulphide.

What diagnostic tools are available to document death by H2S exposure?

The Occupational Medicine Forum is prepared by the ACOEM Occupational and Environmental Medical Practice Committee and does not necessarily represent an official ACOEM position. The Forum is intended for health professionals and is not intended to provide medical or legal advice, including illness prevention, diagnosis or treatment, or regulatory compliance. Such advice should be obtained directly from a physician and/or attorney.

Suicide with hydrogen sulfide.

This presentation will address the recent rise of suicide deaths resulting from the asphyxiation by hydrogen sulfide (H2S) gas.Hydrogen sulfide poisoning has been an infrequently encountered cause of death in medical examiner practice. Most H2S deaths that have been reported occurred in association with industrial exposure.More recently, H2S has been seen in the commission of suicide, particularly in Japan. Scattered reports of this phenomenon have also appeared in the United States.We have recently observed 2 intentional asphyxial deaths in association with H2S. In both cases, the decedents committed suicide in their automobiles. They generated H2S by combining a sulfide-containing tree spray with toilet bowl cleaner (with an active ingredient of hydrogen chloride acid). Both death scenes prompted hazardous materials team responses because of notes attached to the victims' car windows indicating the presence of toxic gas. Autopsy findings included discoloration of lividity and an accentuation of the gray matter of the brain. Toxicology testing confirmed H2S exposure with the demonstration of high levels of thiosulfate in blood.In summary, suicide with H2S appears to be increasing in the United States.

Fatal and non-fatal cases of lime sulfide exposure and pathogenetic mechanisms underlying pancreatic injury: Case reports with an animal experiment.

Lime sulfide poisoning by the oral route is rarely encountered in the practice of forensic science, whereas hydrogen sulfide poisoning is seen frequently. We report here two cases of fatal lime sulfide poisoning with several related cases and in addition induced histological damage with acute inflammation in animal models under at similar concentrations. We also evaluated sulfide and thiosulfate concentrations and speculated as to the cause of pancreatic damage in these cases.

Effective AAV-mediated gene therapy in a mouse model of ethylmalonic encephalopathy.

Ethylmalonic encephalopathy (EE) is an invariably fatal disease, characterized by the accumulation of hydrogen sulfide (H(2)S), a highly toxic compound. ETHE1, encoding sulfur dioxygenase (SDO), which takes part in the mitochondrial pathway that converts sulfide into harmless sulfate, is mutated in EE. The main source of H(2)S is the anaerobic bacterial flora of the colon, although in trace amount it is also produced by tissues, where it acts as a 'gasotransmitter'. Here, we show that AAV2/8-mediated, ETHE1-gene transfer to the liver of a genetically, metabolically and clinically faithful EE mouse model resulted in full restoration of SDO activity, correction of plasma thiosulfate, a biomarker reflecting the accumulation of H(2)S, and spectacular clinical improvement. Most of treated animals were alive and well >6-8 months after birth, whereas untreated individuals live 26 ± 7 days. Our results provide proof of concept on the efficacy and safety of AAV2/8-mediated livergene therapy for EE, and alike conditions caused by the accumulation of harmful compounds in body fluids and tissues, which can directly be transferred to the clinic.

Sodium thiosulfate pharmacokinetics in hemodialysis patients and healthy volunteers.

BACKGROUND AND OBJECTIVES: Vascular calcification is a major cause of morbidity and mortality in dialysis patients. Human and animal studies indicate that sodium thiosulfate (STS) may prevent the progression of vascular calcifications. The pharmacokinetics of STS in hemodialysis patients has not been investigated yet. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: STS was given intravenously to 10 hemodialysis patients on- and off-hemodialysis. Additionally, STS was applied to 9 healthy volunteers once intravenously and once orally. Thiosulfate concentrations were measured by using a specific and sensitive HPLC method. RESULTS: In volunteers and patients, mean endogenous thiosulfate baseline concentrations were 5.5 ± 1.82 versus 7.1 ± 2.7 µmol/L. Renal clearance was high in volunteers (1.86 ± 0.45 ml/min per kg) and reflected GFR. Nonrenal clearance was slightly, but not significantly, higher in volunteers (2.25 ± 0.32 ml/min per kg) than in anuric patients (2.04 ± 0.72 ml/min per kg). Hemodialysis clearance of STS was 2.62 ± 1.01 ml/min per kg. On the basis of the nonrenal clearance and the thiosulfate steady-state serum concentrations, a mean endogenous thiosulfate generation rate of 14.6 nmol/min per kg was calculated in patients. After oral application, only 4% of STS was recovered in urine of volunteers, reflecting a low bioavailability of 7.6% (0.8% to 26%). CONCLUSIONS: Given the low and variable bioavailability of oral STS, only intravenous STS should be prescribed today. The biologic relevance of the high hemodialysis clearance for the optimal time point of STS dosing awaits clarification of the mechanisms of action of STS.

Prevention of cisplatin-induced hearing loss by administration of a thiosulfate-containing gel to the middle ear in a guinea pig model.

PURPOSE: Thiosulfate may reduce cisplatin-induced ototoxicity, most likely by relieving oxidative stress and by forming inactive platinum complexes. This study aimed to determine the concentration and protective effect of thiosulfate in the cochlea after application of a thiosulfate-containing high viscosity formulation of sodium hyaluronan (HYA gel) to the middle ear prior to i.v. injection of cisplatin in a guinea pig model. METHODS: The release of thiosulfate (0.1 M) from HYA gel (0.5% w/w) was explored in vitro. Thiosulfate in the scala tympani perilymph of the cochlea 1 and 3 h after application of thiosulfate in HYA gel to the middle ear was quantified with HPLC and fluorescence detection. Thiosulfate in blood and CSF was also explored. The potential otoprotective effect was evaluated by hair cell count after treatment with thiosulfate in HYA gel applied to the middle ear 3 h prior to cisplatin injection (8 mg/kg b.w.). RESULTS: HYA did not impede the release of thiosulfate. Middle ear administration of thiosulfate in HYA gel gave high concentrations in the scala tympani perilymph while maintaining low levels in blood, and it protected against cisplatin-induced hair cell loss. CONCLUSION: HYA gel is an effective vehicle for administration of thiosulfate to the middle ear. Local application of a thiosulfate-containing HYA gel reduces the ototoxicity of cisplatin most likely without compromising its antineoplastic effect. This provides a minimally invasive protective treatment that can easily be repeated if necessary.

Simulation-based sodium thiosulfate dosing strategies for the treatment of calciphylaxis.

BACKGROUND AND OBJECTIVES: Calciphylaxis remains a poorly understood life-threatening disorder with limited therapeutic options. Sodium thiosulfate (STS) has reported efficacy, thought to be because solubilizing calcium deposits promote clearance by hemodialysis (HD). Lack of rigorous pharmacokinetic studies makes it problematic for determining proper STS dosing given the expanding range of dialysis prescriptions and intensities. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The purpose of this study was to determine the dosing strategies for STS during different dialysis regimens. Given reported successes using an empiric 25 g, intravenous, 3 times per week after HD, simulations were performed to predict dosing guidelines for alternative, more or less intense dialysis to produce equivalent area under the curve drug exposure. The modeled prescriptions varied HD time from 12 to 40 h/wk over three to six sessions (Q(b) 200 to 400 ml/min, Q(d) 500 to 800 ml/min), and continuous venovenous hemodialysis at low flow rates (Q(b) 100 to 200 ml/min, Q(d) 35 to 50 ml/min), using high-flux polysulfone hemofilters. RESULTS: Simulations showed a marked variation in STS doses depending on HD frequency and duration. Blood and dialysate flows have a less prominent effect. Assuming no residual renal function, HD prescription permutations caused the dose to vary from 72 to 245 g/wk (70-kg adult), and the simulations provide specific guidelines for clinicians. CONCLUSIONS: Based on the success reported for one STS dosing regimen and assuming area under the curve exposure of STS is proportional to its effect, pharmacokinetic simulations can be used to calculate the dose for alternative, higher or lower intensity dialysis regimens. These strategies are imperative to assure adequate treatment for this mortal disease, as well as to avoid toxicity from excess dosing.

Deaths in Rotorua's geothermal hot pools: hydrogen sulphide poisoning.

In late 2007 and early 2008 two gentlemen were found dead in, or near to, enclosed hot pools fed with Rotorua's geothermal waters. Amidst much publicity the Coroner has ruled that the deaths were related to hydrogen sulphide poisoning. Following post mortem examinations, blood and urine samples were frozen and sent to the Toxicology Unit of ESR. These were then stored frozen until analysis. Hydrogen sulphide (H(2)S) is a potentially deadly gas at elevated levels, but is rapidly eliminated from the body and is unstable post mortem. Thiosulphate is a marker for the exposure to H(2)S, and as it is stable post mortem the samples were analysed to determine the thiosulphate levels present. The urine thiosulphate levels detected were above those seen in the urine samples measured from the only previous study of people exposed to the Rotorua thermal area and the blood levels were similar to literature values from fatalities exposed in workplaces such as sewage treatment plants.

Detection of exhaled hydrogen sulphide gas in healthy human volunteers during intravenous administration of sodium sulphide.

INTRODUCTION: Hydrogen sulphide (H(2)S) is an endogenous gaseous signaling molecule and potential therapeutic agent. Emerging studies indicate its therapeutic potential in a variety of cardiovascular diseases and in critical illness. Augmentation of endogenous sulphide concentrations by intravenous administration of sodium sulphide can be used for the delivery of H(2)S to the tissues. In the current study, we have measured H(2)S concentrations in the exhaled breath of healthy human volunteers subjected to increasing doses sodium sulphide in a human phase I safety and tolerability study. METHODS: We have measured reactive sulphide in the blood via ex vivo derivatization of sulphide with monobromobimane to form sulphide-dibimane and blood concentrations of thiosulfate (major oxidative metabolite of sulphide) via ion chromatography. We have measured exhaled H(2)S concentrations using a custom-made device based on a sulphide gas detector (Interscan). RESULTS: Administration of IK-1001, a parenteral formulation of Na(2)S (0.005-0.20 mg kg(-1), i.v., infused over 1 min) induced an elevation of blood sulphide and thiosulfate concentrations over baseline, which was observed within the first 1-5 min following administration of IK-1001 at 0.10 mg kg(-1) dose and higher. In all subjects, basal exhaled H(2)S was observed to be higher than the ambient concentration of H(2)S gas in room air, indicative of on-going endogenous H(2)S production in human subjects. Upon intravenous administration of Na(2)S, a rapid elevation of exhaled H(2)S concentrations was observed. The amount of exhaled H(2)S rapidly decreased after discontinuation of the infusion of Na(2)S. CONCLUSION: Exhaled H(2)S represents a detectable route of elimination after parenteral administration of Na(2)S.