Gary A. Rockwood

The Analysis of Cyanide and its Breakdown Products in Biological Samples

Cyanide is a toxic chemical that may be introduced into living organisms as a result of natural processes and/or anthropogenic uses (legal or illicit). Exposure to cyanide can be verified by analysis of cyanide or one of its breakdown products from biological samples. This verification may be important for medical, law-enforcement, military, forensic, research, or veterinary purposes. This review will discuss current bioanalytical techniques used for the verification of cyanide exposure, identify common problems associated with the analysis of cyanide and its biological breakdown products, and briefly address the metabolism and toxicokinetics of cyanide and its breakdown products in biological systems.

Development of a Fluorescence-Based Sensor for Rapid Diagnosis of Cyanide Exposure

Although commonly known as a highly toxic chemical, cyanide is also an essential reagent for many industrial processes in areas such as mining, electroplating, and synthetic fiber production. The “heavy” use of cyanide in these industries, along with its necessary transportation, increases the possibility of human exposure. Because the onset of cyanide toxicity is fast, a rapid, sensitive, and accurate method for the diagnosis of cyanide exposure is necessary. Therefore, a field sensor for the diagnosis of cyanide exposure was developed based on the reaction of naphthalene dialdehyde, taurine, and cyanide, yielding a fluorescent β-isoindole. An integrated cyanide capture “apparatus”, consisting of sample and cyanide capture chambers, allowed rapid separation of cyanide from blood samples. Rabbit whole blood was added to the sample chamber, acidified, and the HCN gas evolved was actively transferred through a stainless steel channel to the capture chamber containing a basic solution of naphthalene dialdehyde (NDA) and taurine. The overall analysis time (including the addition of the sample) was <3 min, the linear range was 3.13–200 μM, and the limit of detection was 0.78 μM. None of the potential interferents investigated (NaHS, NH4OH, NaSCN, and human serum albumin) produced a signal that could be interpreted as a false positive or a false negative for cyanide exposure. Most importantly, the sensor was 100% accurate in diagnosing cyanide poisoning for acutely exposed rabbits.

In vitro and in vivo comparison of sulfur donors as antidotes to acute cyanide intoxication

Antidotes for cyanide (CN) intoxication include the use of sulfane sulfur donors (SSDs), such as thiosulfate, which increase the conversion of CN to thiocyanate by the enzyme rhodanese. To develop pretreatments that might be useful against CN, SSDs with greater lipophilicity than thiosulfate were synthesized and assessed. The ability of SSDs to protect mice against 2LD50 of sodium cyanide (NaCN) administered either 15 or 60 min following administration of an SSD was assessed. To study the mechanism of action of the SSD, the candidate compounds were examined in vitro for their effect on rhodanese and 3‐mercaptopyruvate sulfurtransferase (MST) activity under increasing SSD concentrations. Tests were conducted on nine candidate SSDs: ICD1021 (3‐hydroxypyridin‐2‐yl N‐[(N‐methyl‐3‐aminopropyl)]‐2‐aminoethyl disulfide dihydrochloride), ICD1022, (3‐hydroxypyridin‐2‐yl N‐[(N‐methyl‐3‐aminopropyl)]‐2‐aminoethyl disulfide trihydrochloride), ICD1584 (diethyl tetrasulfide), ICD1585 (diallyl tetrasulfide), ICD1587 (diisopropyl tetrasulfide); ICD1738 (N‐(3‐aminopropyl)‐2‐aminoethyl 2‐oxopropyl disulfide dihydrochloride), ICD1816 (3,3′‐tetrathiobis‐N‐acctyl‐l‐alanine), ICD2214 (2‐aminoethyl 4‐methoxyphenyl disulfide hydrochloride) and ICD2467 (bis(4‐methoxyphenyl) disulfide). These tests demonstrated that altering the chemical substituent of the longer chain sulfide modified the ability of the candidate SSD to protect against CN toxicity. At least two of the SSDs at selected doses provided 100% protection against 2LD50 of NaCN, normally an LD99. All compounds were evaluated using locomotor activity as a measure of potential adverse behavioral effects. Positive hypoactivity relationships were found with several disulfides but none was found with ICD1584, a tetrasulfide. Separate studies suggest that the chemical reaction of potassium cyanide (KCN) and cystine forms the toxic metabolite 2‐iminothiazolidine‐4‐carboxylic acid. An alternative detoxification pathway, one not primarily involving the sulfur transferases. may be important in pretreatment for CN intoxication. Although studies to elucidate the precise mechanisms are needed. it is clear that these newly synthesized compounds provide a new rationale for anti‐CN drugs, with fewer side‐effects than the methemoglobin formers. Copyright

Determination of cyanide exposure by gas chromatography–mass spectrometry analysis of cyanide-exposed plasma proteins

Exposure to cyanide can occur in a variety of ways, including exposure to smoke from cigarettes or fires, accidental exposure during industrial processes, and exposure from the use of cyanide as a poison or chemical warfare agent. Confirmation of cyanide exposure is difficult because, in vivo, cyanide quickly breaks down by a number of pathways, including the formation of both free and protein-bound thiocyanate. A simple method was developed to confirm cyanide exposure by extraction of protein-bound thiocyanate moieties from cyanide-exposed plasma proteins. Thiocyanate was successfully extracted and subsequently derivatized with pentafluorobenzyl bromide for GC-MS analysis. Thiocyanate levels as low as 2.5 ng mL(-1) and cyanide exposure levels as low as 175 μg kg(-1) were detected. Samples analyzed from smokers and non-smokers using this method showed significantly different levels of protein-bound thiocyanate (p<0.01). These results demonstrate the potential of this method to positively confirm chronic cyanide exposure through the analysis of protein-bound cyanide in human plasma.

The analysis of 2-amino-2-thiazoline-4-carboxylic acid in the plasma of smokers and non-smokers

ATCA (2-amino-2-thiazoline-4-carboxylic acid) is a promising marker to assess cyanide exposure because of several advantages of ATCA analysis over direct determination of cyanide and alternative cyanide biomarkers (i.e. stability in biological matrices, consistent recovery, and relatively small endogenous concentrations). Concentrations of ATCA in the plasma of smoking and non-smoking human volunteers were analyzed using gas-chromatography mass-spectrometry to establish the feasibility of using ATCA as a marker for cyanide exposure. The levels of ATCA in plasma of smoking volunteers, 17.2 ng/ml, were found to be significantly (p < 0.001) higher than that of non-smoking volunteers, 11.8 ng/ml. Comparison of ATCA concentrations of smokers relative to non-smokers in both urine and plasma yielded relatively similar results. The concentration ratio of ATCA for smokers versus non-smokers in plasma and urine was compared to similar literature studies of cyanide and thiocyanate, and correlations are discussed. This study supports previous evidence that ATCA can be used to determine past cyanide exposure and indicates that further studies should be pursued to validate the use of ATCA as a marker of cyanide exposure.

Neurotoxicological and Behavioral Effects of Cyanide and Its Potential Therapies

The use of the blood agent cyanide (CN) as a military threat agent has been recognized not only historically (Nero and Napoleon III) but also more currently in World War I, World War II, in the Iran–Iraq War in the 1980s, and elsewhere where terrorist activities have occurred. CN is easy and inexpensive to produce and can be obtained from normal commercial trade. CN can act very rapidly (within seconds) to prevent the normal utilization of oxygen by tissues. Excitable tissues, for example, heart and brain, are particularly affected. Hypoxia, convulsions, heart arrhythmias, and death can follow. If exposed, it is best to leave the affected area rapidly. Treatments such as nitrite, which forms methemoglobin that binds CN, and thiosulfate, which converts CN to thiocyanate, act within an appropriate time but produce central nervous system side effects. This article examines the literature on the neurotoxicological and behavioral effects of CN and its treatments. Sites and mechanisms of actions involved in these effects are evaluated. Factors that significantly alter the action of CN and may influence morbidity and mortality are discussed.

Comparison of cyanide exposure markers in the biofluids of smokers and non-smokers

Cyanide is highly toxic and is present in many foods, combustion products (e.g. cigarette smoke), industrial processes, and has been used as a terrorist weapon. In this study, cyanide and its major metabolites, thiocyanate and 2-amino-2-thiazoline-4-carboxylic acid (ATCA), were analyzed from various human biofluids of smokers (low-level chronic cyanide exposure group) and non-smokers to gain insight into the relationship of these biomarkers to cyanide exposure. The concentrations of each biomarker tested were elevated for smokers in each biofluid. Significant differences (p < 0.05) were found for thiocyanate in plasma and urine, and ATCA showed significant differences in plasma and saliva. Additionally, biomarker concentration ratios, correlations between markers of cyanide exposure, and other statistical methods were performed to better understand the relationship between cyanide and its metabolites. Of the markers studied, the results indicate plasma ATCA, in particular, showed excellent promise as a biomarker for chronic low-level cyanide exposure.

The Analysis of Protein-Bound Thiocyanate in Plasma of Smokers and Non-Smokers as a Marker of Cyanide Exposure

When cyanide is introduced into the body, it quickly transforms through a variety of chemical reactions, normally involving sulfur donors, to form more stable chemical species. Depending on the nature of the sulfur donor, cyanide may be transformed into free thiocyanate, the major metabolite of cyanide transformation, 2-amino-2-thiazoline-4-carboxylic acid or protein-bound thiocyanate (PB-SCN) adducts. Because protein adducts are generally stable in biological systems, it has been suggested that PB-SCN may have distinct advantages as a marker of cyanide exposure. In this study, plasma was analyzed from 25 smokers (chronic low-level cyanide exposure group) and 25 non-smokers for PB-SCN. The amount of PB-SCN found in the plasma of smokers, 1.35 µM, was significantly elevated (p < 0.0001) when compared to non-smokers, 0.66 µM. Differences in sub-groups of smokers and non-smokers were also evaluated. The results of this study indicate the effectiveness of analyzing PB-SCN in determining instances of chronic cyanide exposure with possible extension to confirmation of acute cyanide exposure.

Spectrophotometric Analysis of the Cyanide Metabolite 2-Aminothiazoline-4-Carboxylic Acid (ATCA).

Methods of directly evaluating cyanide levels are limited by the volatility of cyanide and by the difficulty of establishing steady-state cyanide levels with time. We investigated the measurement of a stable, toxic metabolite, 2-aminothiazoline-4-carboxylic acid (ATCA), in an attempt to circumvent the challenge of directly determining cyanide concentrations in aqueous media. This study was focused on the spectrophotometric ATCA determination in the presence of cyanide, thiocyanate (SCN−), cysteine, rhodanese, thiosulfate, and other sulfur donors. The method involves a thiazolidine ring opening in the presence of p-(hydroxy-mercuri)-benzoate, followed by the reaction with diphenylthiocarbazone (dithizone). The product is spectrophotometrically analyzed at 625 nm in carbon tetrachloride. The calibration curve was linear with a regression line of Y = 0.0022x (R2 = 0.9971). Interference of cyanide antidotes with the method was determined. Cyanide, thiosulfate, butanethiosulfonate (BTS), and rhodanese did not appreciably interfere with the analysis, but SCN− and cysteine significantly shifted the standard curve. This sensitive spectrophotometric method has shown promise as a substitute for the measurement of the less stable cyanide.

Dimethyl trisulfide A novel cyanide countermeasure

In the present studies, the in vitro and in vivo efficacies of a novel cyanide countermeasure, dimethyl trisulfide (DMTS), were evaluated. DMTS is a sulfur-based molecule found in garlic, onion, broccoli, and similar plants. DMTS was studied for effectiveness as a sulfur donor-type cyanide countermeasure. The sulfur donor reactivity of DMTS was determined by measuring the rate of the formation of the cyanide metabolite thiocyanate. In experiments carried out in vitro in the presence of the sulfurtransferase rhodanese (Rh) and at the experimental pH of 7.4, DMTS was observed to convert cyanide to thiocyanate with greater than 40 times higher efficacy than does thiosulfate, the sulfur donor component of the US Food and Drug Administration-approved cyanide countermeasure Nithiodote®. In the absence of Rh, DMTS was observed to be almost 80 times more efficient than sodium thiosulfate in vitro. The fact that DMTS converts cyanide to thiocyanate more efficiently than does thiosulfate both with and without Rh makes it a promising sulfur donor-type cyanide antidote (scavenger) with reduced enzyme dependence in vitro. The therapeutic cyanide antidotal efficacies for DMTS versus sodium thiosulfate were measured following intramuscular administration in a mouse model and expressed as antidotal potency ratios (APR = LD50 of cyanide with antidote/LD50 of cyanide without antidote). A dose of 100 mg/kg sodium thiosulfate given intramuscularly showed only slight therapeutic protection (APR = 1.1), whereas the antidotal protection from DMTS given intramuscularly at the same dose was substantial (APR = 3.3). Based on these data, DMTS will be studied further as a promising next-generation countermeasure for cyanide intoxication.