Blood Cyanide Research Articles

Rapid and sensitive headspace gas chromatographic method for cyanide determination in whole blood

We reported a simple and rapid headspace (HS) gas chromatographic (GC) assay for the determination of cyanide in human whole blood obtained from both cadaveric and intoxicated subjects. The poison was extracted from whole blood samples by HS technique after 10 min incubation at 80°C with a HCl (1N) acidic solution supplemented with 30 g L−1 of sodium chloride and 10 mg L−1 of isopropanol as internal standard. A SGE WAX capillary column (30 m × 0.32 mm ID × 0.5 µm df) was used. The Flame Ionization Detector (FID) was set at 250°C. The detection and the quantification limits were 0.02 and 0.2 mg L−1, respectively. In the within-day study, the coefficients of variation for three different whole blood cyanide concentrations varied between 3.2 and 6.5%, whereas those in the day-to-day study varied between 4.8 and 6.3%. The assay was performed in less than 20 min per sample, which appears to be suitable for emergency toxicological tasks involved in diagnosis of cyanide poisoning.

Determination of cyanide in blood by reaction head-space gas chromatography

A method describing determination of cyanide in blood by head-space gas chromatography with electron capture detector was reported. The method involves transformation of cyanide into cyanogen chloride by reacting hydrogen cyanide with chloramine-T on a stick of filter paper in the space above the blood in the head-space vial. The recovery was 84-96% and the coefficient of variation was 3.3-7.2%. The limit of quantitation was about 0.01 mg cyanide/l.

Analysis of Cyanide in Blood by Headspace-Isotope-Dilution-GC-MS

An uncomplicated, rapid, automated procedure for the analysis of low cyanide concentrations in whole blood is reported. The analysis was performed by headspace gas chromatography and mass spectrometry in the (1H12C14N) and m/z 29 (1H13C15N). Carryover from cyanide adsorption onto the surface of the needle was prevented by developing a new method that enabled automated flushing of the needle in between each cyanide analysis. Results were compared of ordinary calibrations and those of isotope dilutions. The total time of analysis was 18 min for a single cyanide analysis.

Stability of Cyanide in Cadavers and in Postmortem Stored Tissue Specimens: A Review

The toxicological and postmortem analysis of fire victims' blood and tissue can disclose the type and quantity of toxic species, such as carbon monoxide or hydrogen cyanide, that they inhaled prior to death. For fire cases, these toxicological data can reveal objective data about the nature and circumstances of a fire, and thus assist both the Medical Examiner and the Fire Investigator in their investigations. Assigning a level of significance to cyanide concentrations found in the blood and tissue of fire victims is often hampered by the fact that cyanide is inherently unstable in cadavers and in stored tissue samples. Numerous researchers have provided insight into and characterized the stability of cyanide in the body and in collected biological specimens. Based on studies by these researchers, the rate of transformation of cyanide in blood and tissue specimens is dependent on the initial cyanide concentration in the sample at time of death, the length of time that a sample remains in the cadaver, the length of time that a sample remains in storage, and the preservation (e.g., addition of sodium fluoride to sample) and storage conditions (e.g., temperature) of the sample.

Determination of cyanide and volatile alkylnitriles in whole blood by headspace solid-phase microextraction and gas chromatography with nitrogen phosphorus detection

Simultaneous determination of cyanide and volatile alkylnitriles such as acetonitrile, cis- and trans-crotononitrile, allylnitrile and butyronitrile at low ppb concentration on whole blood (rat and mice) by headspace solid-phase microextraction (HS-SPME) followed by gas chromatography (GC) with nitrogen phosphorus detection has been achieved for the first time. SPME extraction time and temperature were optimized using a star experimental design. Optimum conditions for cyanide extraction were chosen to analyze unspiked blood samples containing alkylnitriles as that analyte occurs at the lowest concentrations. For all analytes, the developed methodology yielded good quality parameters. In all cases, good reproducibility (relative standard deviation ≤12%), detection limits (<3 ng mL −1) and quantification limits (<4 ng mL −1) were recorded.

新しい質量分析技術の法医学領域への応用

In the fields of legal medicine and forensic toxicology, numerous drugs and poisons obtained from diverse biological specimens such as whole blood, urine, and hair need to be identified and quantified. Therefore, it is necessary to develop new methods for analysis of drugs, especially mass spectrometric methods. In this paper, we introduce three methods—surface ionization organic mass spectrometry, a combination of mass spectrometry and cryogenic oven trapping (COT), and laser spray ionization (LSI)—and their application to legal medicine. Gas chromatography/surface ionization organic mass spectrometry is very sensitive in detecting drugs containing tertiary amine residues; for Phencyclidine (PCP), its sensitivity is 20 to 1,000 times higher than that of the conventional electron ionization method. Surface ionization organic mass spectrometry is suitable for compounds containing cyclic tertiary amino side chains. COT in combination with gas chromatography has enabled detection of many volatile organic compounds (VOCs) with high sensitivity. We have succeeded in quantifying several VOCs, such as chloroform, cyanide, thinner components, ethanol, and general anesthetics in whole blood by COT; COT affords a sensitivity that is 10 to 50 times higher than that of the conventional headspace gas chromatography method. Recently, we succeeded in identifying and quantifying 15 VOCs in human whole blood with the combination of COT and mass spectrometry. LSI is a new ionization method for liquid chromatography/mass spectrometry. We have succeeded in detecting aconitine alkaloids and psychopharmaceuticals using LSI. In the infusion mode, aconitine and haloperidol were ionized with higher efficiency compared to that in the conventional electrospray mode. However, since the signals in the LSI mode are not stable, it is necessary to improve the stability of signals in the LSI mode for applying LSI to forensic toxicology.

Blood Cyanide Determination in Two Cases of Fatal Intoxication: Comparison Between Headspace Gas Chromatography and a Spectrophotometric Method*

Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automated headspace gas chromatographic method with nitrogen-phosphorus detector (HS-GC/NPD). In the former, hydrogen cyanide (HCN) was quantitatively determined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS-GC/NPD method, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liberated as HCN. The spectrophotometric (VIS) and HS-GC/NPD methods were validated on postmortem blood samples fortified with potassium cyanide in the ranges 0.5-10 and 0.05-5 mug/mL, respectively. Detection limits were 0.2 mug/mL for VIS and 0.05 mug/mL for HS-GC/NPD. This work shows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable for rapid diagnosis of cyanide in postmortem cases.

Hydroxocobalamin for severe acute cyanide poisoning by ingestion or inhalation

This chart review was undertaken to assess efficacy and safety of hydroxocobalamin for acute cyanide poisoning. Hospital records of the Fernand Widal and Lariboisière Hospitals were reviewed for intensive care unit admissions with cyanide poisoning for which hydroxocobalamin was used as first-line treatment from 1988 to 2003. Smoke inhalation cases were excluded. Hydroxocobalamin (5-20 g) was administered to 14 consecutive patients beginning a median 2.1 hours after cyanide ingestion or inhalation. Ten patients (71%) survived and were discharged. Of the 11 patients with blood cyanide exceeding the typically lethal threshold of 100 μmol/L, 7 survived. The most common hydroxocobalamin-attributed adverse events were chromaturia and pink skin discoloration. Severe cyanide poisoning of the nature observed in most patients in this study is frequently fatal. That 71% of patients survived after treatment with hydroxocobalamin suggests that hydroxocobalamin as first-line antidotal therapy is effective and safe in acute cyanide poisoning.

Prospective Study of Hydroxocobalamin for Acute Cyanide Poisoning in Smoke Inhalation

To assess outcomes in patients treated with hydroxocobalamin at the fire scene or in the ICU for suspected smoke inhalation-associated cyanide poisoning. Adult smoke inhalation victims with suspected cyanide poisoning as determined by soot in the face, mouth, or nose or expectorations and neurologic impairment received an intravenous infusion of hydroxocobalamin 5 g (maximum 15 g) at the fire scene or in the ICU in this observational case series conducted from 1987 to 1994. Blood cyanide specimens were collected before administration of hydroxocobalamin. The threshold for cyanide toxicity was predefined as greater than or equal to 39 micromol/L. The sample included 69 patients (mean age 49.6 years; 33 men), of whom 39 were comatose. Out-of-hospital deaths were excluded. Fifty of the 69 patients (72%) admitted to the ICU survived after administration of hydroxocobalamin. In the group in which cyanide poisoning was confirmed a posteriori (n=42), 67% (28/42) survived after administration of hydroxocobalamin. The most common adverse events were chromaturia (n=6), pink or red skin discoloration (n=4), hypertension (n=3), erythema (n=2), and increased blood pressure (n=2). No serious adverse events were attributed to hydroxocobalamin. Laboratory tests revealed transient alterations in renal and hepatic function consistent with the critical condition of the patients and mild anemia consistent with progressive hemodilution. Empiric administration of hydroxocobalamin was associated with survival among 67% of patients confirmed a posteriori to have had cyanide poisoning. Hydroxocobalamin was well tolerated irrespective of the presence of cyanide poisoning. Hydroxocobalamin appears to be safe for the out-of-hospital treatment of presumptive cyanide poisoning from smoke inhalation.

Cyanide Adducts with Human Plasma Proteins: Albumin as a Potential Exposure Surrogate

Cyanide (CN) is a ubiquitous environmental toxicant. The measurement of CN in whole blood is a common exposure assay, but values are error prone because of CN's rapid metabolism and clearance (t1/2 < 1 h) from this compartment. This study was undertaken to determine whether CN forms covalent adduct(s) with plasma proteins that could serve as stable biomarker(s) and potential surrogate(s) of exposure. When added to human blood, plasma, or serum, CN formed covalent adducts with immunoglobulin G (IgG) and serum albumin (HSA) in the plasma fraction. Covalent adducts were not detected in the cellular, primarily erythrocyte, fraction. With human, mouse, and rabbit IgGs, the reaction with CN occurred at intra- and/or interchain disulfide linkages in the heavy and light chains. Digestion of CN-treated HSA with trypsin or the endoproteinase Lys-C at basic pH produced tautomeric 2-iminothiazoline-4-carboxylyl/2-aminothiazolidine-4-carboxylyl (itcCys) N-terminal peptides exclusively, consistent with prior model peptide/protein studies showing that under basic conditions internal S-cyanylated-Cys residues cyclize with concomitant release of the upstream peptide. The most readily detectable reaction of CN with purified HSA was at Cys34, the only Cys of the 35 present not connected as internal cystines. Because CN does not react with free sulfhydryl groups, it is probable that S-cyanylation at Cys34 occurs at those residues that carry GSH, Cys, or other small molecules as mixed disulfides. Relatively less detectable, modified Cys residues were also identified at positions 53, 124, 392, 477, and 487. When 14CN was added to human serum or whole blood at concentrations spanning a putative nontoxic to lethal range, stable adduct formation with HSA occurred in a linear, concentration-dependent reaction that was complete within 2 h. These attributes of the reaction, coupled with a plasma compartment location, suggest that quantitation of CN bound to HSA would provide a much more reliable assessment of exposure than does measurement of CN in blood.

Oxidative stress pathways in the potentiation of noise-induced hearing loss by acrylonitrile

We hypothesize that the disruption of antioxidant defenses is a key mechanism whereby chemical contaminants can potentiate noise-induced hearing loss (NIHL). This hypothesis was tested using acrylonitrile (ACN), a widely used industrial chemical whose metabolism is associated with glutathione (GSH) depletion and cyanide (CN) generation. CN, in turn, can inhibit Cu/Zn superoxide dismutase (SOD). We have shown previously that ACN potentiates NIHL, even with noise exposure approaching permissible occupational levels. However, the relative involvement of GSH depletion and/or CN production in this potentiation is still unknown. In this study, we altered these metabolic pathways pharmacologically in order to further delineate the role of specific antioxidants in the protection of the cochlea. We investigated the effects of sodium thiosulfate (STS), a CN inhibitor, 4-methylpyrazole (4MP), a drug that blocks CN generation by competing with CYP2E1, and l- N-acetylcysteine ( l-NAC), a pro-GSH drug, in order to distinguish between GSH depletion and CN production as the mechanism responsible for potentiation of NIHL by ACN. Long-Evans rats were exposed to an octave-band noise (97 dB SPL, 4 h/day, 5 days) and ACN (50 mg/kg). Separate pre-treatments with STS (150 mg/kg), 4MP (100 mg/kg) and l-NAC (4 × 400 mg/kg) all dramatically reduced blood CN levels, but only l-NAC significantly protected GSH levels in both the liver and the cochlea. Concurrently, only l-NAC treatment decreased the auditory loss and hair cell loss resulting from ACN + noise, suggesting that GSH is involved in the protection of the cochlea against reactive oxygen species generated by moderate noise levels. On the other hand, CN does not seem to be involved in this potentiation.

Determination of Cyanide in Blood by Isotope-Dilution Gas Chromatography–Mass Spectrometry

Determination of Cyanide in Blood by Isotope-Dilution Gas Chromatography–Mass Spectrometry

174: Plasma Lactate Concentrations Correlate with Physiological Responses in Cyanide Poisoning But Not Other Types of Acute Poisoning

Confirmation of cyanide poisoning (CNP) by laboratory measurement of blood cyanide concentrations generally takes hours to days, and is seldom available to aid in decisions regarding antidote use. Lactic acidosis may be a useful marker of CNP-induced cellular hypoxia. To understand the extent to which lactic acidosis is a direct result of cyanide effects on oxidative phosphorylation versus a secondary response to cardiovascular shock, we examined the correlation between blood lactate levels and physiological responses in patients with acute poisoning due to cyanide in comparison with other agents.

Determination of Cyanide in Blood by Isotope-Dilution Gas Chromatography–Mass Spectrometry

Cyanide (CN) is a lethal toxin. Quantification in blood is necessary to indicate exposure from many sources, including food, combustion byproducts, and terrorist activity. We describe an automated procedure based on isotope-dilution gas chromatography-mass spectrometry (ID GC/MS) for the accurate and rapid determination of CN in whole blood. A known amount of isotopically labeled potassium cyanide (K13C15N) was added to 0.5 g of whole blood in a headspace vial. Hydrogen cyanide was generated through the addition of phosphoric acid, and after a 5-min incubation, 0.5 mL of the headspace was injected into the GC/MS at an oven temperature of -15 degrees C. The peak areas from the sample, 1H12C14N+, at m/z 27, and the internal standard, 1H13C15N+, at m/z 29, were measured, and the CN concentration was quantified by ID. The analysis time was 15 min for a single injection. We demonstrated method accuracy by measuring the CN content of unfrozen whole blood samples fortified with a known amount of CN. Intermediate precision was demonstrated by periodic analyses over a 14-month span. Relative expanded uncertainties based on a 95% level of confidence with a coverage factor of 2 at CN concentrations of 0.06, 0.6, and 1.5 microg/g were 8.3%, 5.4%, and 5.3%, respectively. The mean deviation from the known value for all concentrations was <4%. The automated ID GC/MS method can accurately and rapidly quantify nanogram per gram to microgram per gram concentrations of CN in blood.

The development of an electrochemical sensor for the determination of cyanide in physiological solutions

Hydrogen cyanide gas produced during fires can cause poisoning from smoke inhalation. Blood cyanide concentrations in healthy subjects are sub-micromolar. Toxic or fatal blood concentrations are generally considered to be greater than 40 μmol dm −3 but concentrations in survivors can exceed 200 μmol dm −3, while values exceeding 400 μmol dm −3 have been recorded from fatalities. Cyanide in blood is either free in plasma or bound to ferric haemoglobin. Current analytical techniques require sample pre-treatment, generally with extraction of the cyanide from the sample. An amperometric test was developed which could determine free cyanide at physiological pH in a solution of albumin and other blood constituents without sample pre-treatment, with a calibration range exceeding 400 μmol dm −3 and a limit of detection (LOD; using three standard deviations) of 4 μmol dm −3 which is lower than would be treated clinically.

Effect of cytochrome P450 inhibitors and anticonvulsants on the acute toxicity of acrylonitrile

Some of the more striking expressions of toxicity are the tremors and seizures observed approximately 100 min after exposure of rats to an acutely toxic dose of acrylonitrile (AN). These early events are followed by a second wave of severe clonic convulsions that occur just prior to death at about 3-4 h. For AN, at least two chemical entities could produce these toxic effects, namely the parent AN molecule, the metabolically-released cyanide, or both. Which of these two agents is responsible for each of the symptoms of acute intoxication is not known. To help dissect the toxicity, it was anticipated that an effective inhibitor of the oxidative metabolism of AN to cyanide could help us to understand which toxic symptoms might be associated with each agent. Three inhibitors of oxidative metabolism were tested, namely SKF-525A, 1-benzylimidazole and metyrapone and one alternative substrate, ethanol. As compared to SKF-525A and metyrapone, both 1-benzylimidazole and ethanol were highly effective in reducing blood cyanide levels to insignificant levels in rats treated with an LD90 dose of AN. In addition, both agents abolished the early seizure activity, suggesting that this first phase of seizures is due to cyanide and not the parent molecule. 1-Benzylimidazole did not prevent the severe clonic convulsive phase preceding death, suggesting that these terminal convulsions are due to the toxic effects of the parent AN molecule. The CNS depressant ethanol was only partially effective in attenuating the terminal convulsions. None of these agents affected the incidence of AN-induced mortality, clearly establishing that, even in the absence of cyanide, the parent AN molecule is acutely toxic. The partial effectiveness of ethanol suggested that anticonvulsants might be of benefit. Both phenobarbital and phenytoin protected rats from both the early and terminal convulsions, while valproic acid was ineffective. These effects were not related to a reduction in blood cyanide levels but rather due to their inherent anticonvulsant activity.

Thiosulfate de sodium et intoxication cyanhydrique aiguë : étude chez le rat

Unlike practices in the United States where it is associated with other antidotes, sodium thiosulfate is not used for emergency therapy for cyanide poisoning in France. The purpose of this study was to develop a rat model using intraperitoneal injections of sodium thiosulfate at a dose of 225 mg/kg to test its therapeutic efficacy for acute cyanide poisoning. Efficacy was assessed directly by quantifying arterial blood cyanide and indirectly using markers of hypoxia: serum lactate and arteriolization of venous blood gases. Cyanide poisoning induced intense biological anomalies which were persistent (serum lactate) or transient (blood gases). Sodium thiosulfate was found to be an effective antidote in the rat enabling rapid normalization of hypoxia markers and clearing of cyanide from arterial blood.

Isotope Dilution-Mass Spectrometry Determination of Blood Cyanide by Headspace Gas Chromatography

A direct and sensitive method for the determination of blood cyanide by isotope dilution was developed. The blood is placed in a headspace vial, and K13C15N is added as internal standard. Addition of phosphoric acid liberates the cyanide as HCN. The detection is accomplished by mass spectrometry after a fine mass calibration tuning. The detection limit obtained is 0.3 micromol/L. The within- and inter-run coefficients of variation are 4.4% (for a concentration of 2.5 micromol/L) and 3.9% (for a concentration of 4.7 micromol/L), respectively. The observed recovery is 98%. A round-robin exercise was carried out to compare the performance of this method with others currently in use in other clinical laboratories.

Comparative metabolism of methacrylonitrile and acrylonitrile to cyanide using cytochrome P4502E1 and microsomal epoxide hydrolase-null mice

Methacrylonitrile (MAN) and acrylonitrile (AN) are metabolized via glutathione (GSH) conjugation or epoxide formation. We have recently shown that CYP2E1 is essential for AN epoxidation and subsequent cyanide liberation. Current studies were designed to compare the enzymatic basis of MAN vs. AN metabolism to cyanide using wild-type (WT), CYP2E1-, and mEH-null mice. Mice received a single gavage dose of 0.047, 0.095, 0.19, or 0.38 mmol/kg of MAN or AN, and blood cyanide was measured at 1 or 3 h later. Blood cyanide levels in WT mice treated with AN or MAN were dose and time dependent. At equimolar doses, significantly higher levels of cyanide were detected in the blood of MAN- vs. AN-treated mice. Further, while significant reduction in blood cyanide levels occurred in MAN-treated CYP2E1-null vs. WT mice, AN metabolism to cyanide was largely abolished in CYP2E1-null mice. Pretreatment of mice with 1-aminobenzotriazole (ABT, CYP inhibitor) demonstrated that CYPs other than CYP2E1 also contribute to MAN metabolism to cyanide. Blood cyanide levels in mEH-null mice treated with aliphatic nitriles are generally lower than levels in similarly treated WT mice. Western blot analysis showed that expression of sEH was greater in male vs. female mice. The role of various epoxide hydrolases (EHs) in the production of cyanide from aliphatic nitriles is apparently structure and dose dependent. Regardless of genotype, significantly higher levels of cyanide were measured in the blood of male vs. female mice treated with MAN or AN. In conclusion, these data showed that (1) at equimolar doses, higher blood cyanide levels were detected in mice treated with MAN vs. AN; (2) while CYP2E1 is the only enzyme responsible for AN metabolism to cyanide, other CYPs also contribute to MAN metabolism; and (3) significantly higher levels of cyanide were measured in the blood of male vs. female treated with either nitrile. Higher blood cyanide levels in male vs. female mice and in MAN- vs. AN-treated mice may explain the gender-related differences in the toxicity of these chemicals and the greater potency of MAN vs. AN.

Prunus Spp. Intoxication in Ruminants: A Case in a Goat and Diagnosis by Identification of Leaf Fragments in Rumen Contents

Prunus serotina Ehrh. (black cherry) intoxication was diagnosed on postmortem examination of a goat. The clinical signs were weakness, depression, seizure-like activity, and lateral recumbency. Natural cases of black cherry intoxication have not been reported in goats in the United States. In the absence of a history of access to black cherry or the ability to detect cyanide or cyanogenic glycosides in blood or tissues, black cherry intoxication may be diagnosed in ruminants by the identification of black cherry leaves in rumen contents. Three distinctive features facilitate identification of black cherry leaves or leaf fragments: 1) a pair of small glands that protrude from the sides of the petiole just below the base of the blade, 2) incurved, gland-tipped (callous) teeth along the margins of the leaf, and 3) a band of hairs to each side of the lower half of the midvein on the surface of the leaf. Shape of the marginal teeth, presence or absence of glands at the tips of these teeth, the morphology of these glands, and presence or absence of petiolar glands and their morphology may allow identification and differentiation of small fragments of leaves from the 6 most important cyanogenic Prunus spp. in eastern North America: black cherry, Carolina laurel cherry, peach, English laurel cherry, choke cherry, and fire cherry.