Radiolabel Uncovers Nonintuitive Metabolites of BIIB104: Novel Release of [14C]Cyanide from 2-Cyanothiophene and Subsequent Formation of [14C]Thiocyanate.

BIIB104 (formerly PF-04958242), N-((3S,4S)-4-(4-(5-cyanothiophen-2-yl)phenoxy)tetrahydrofuran-3-yl)propane-2-sulfonamide, is an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiator investigated for the treatment of cognitive impairment associated with schizophrenia. Preliminary in vitro metabolism studies with non-radiolabeled BIIB104 in rat, dog, and human liver microsomes (RLM, DLM, and HLM) showed O-dealkylation in all three species, tetrahydrofuran hydroxylation dominating in DLM and HLM, and thiophene hydroxylation prevalent in RLM. However, a subsequent rat mass balance study with [nitrile-14C]BIIB104 showed incomplete recovery of administered radioactivity (∼80%) from urine and feces over 7 days following an oral dose, and an exceptionally long plasma total radioactivity half-life. Radiochromatographic metabolite profiling and identification, including chemical derivation, revealed that [14C]cyanide was a major metabolite of [nitrile-14C]BIIB104 in RLM, but a minor and trace metabolite in DLM and HLM, respectively. Correspondingly in bile duct-cannulated rats, [14C]thiocyanate accounted for ∼53% of total radioactivity excreted over 48 hours postdose and it, as an endogenous substance, explained the exceptionally long plasma radioactivity half-life. The release of [14C]cyanide from the 2-cyanothiophene moiety is postulated to follow an epoxidation-initiated thiophene-opening based on the detection of non-radiolabeled counterpart metabolites in RLM. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate. Additionally, the potential cyanide metabolite of nitrile-containing drug molecules may be detected in liver microsomes with liquid chromatography-mass spectrometry following a chemical derivatization. SIGNIFICANCE STATEMENT: Using [nitrile-14C]BIIB104, non-intuitive metabolites of BIIB104 were discovered involving a novel cyanide release from the 2-cyanothiophene motif via a postulated epoxidation-initiated thiophene-opening. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate.

Microextraction with smartphone detection of thiocyanate in saliva of tobacco smokers using paper-based analytical method.

This study presents a novel, cost-effective approach involving spectrophotometric and smartphone paper-based (SPB) methods and a distinctive salting-out air-assisted dispersive microextraction procedure to quantify thiocyanate in saliva samples. The method relies on the inhibitory effect of thiocyanate on quinoneimine dye formation during the Emerson reaction with sodium hypochlorite. Spectrophotometry quantifies the extracted dye by monitoring quinoneimine color intensity reduction at 525 nm. In the SPB method, extracted dye is applied to a paper strip, a smartphone captures the colored paper, and an application analyzes red, green, and blue components. All analyte determination and extraction variables were explored. Both methods exhibit good linearity (10-100 µg/L) with a coefficient of determination of 0.9991 and a limit of detection of 7.5 µg/L for the spectrophotometric method, and a coefficient of determination of 0.9988 and a limit of detection of 8.8 µg/L for the SPB method. The calculated values for the enrichment factor and extraction recovery of the developed extraction methodology were 46% and 93%, respectively. The methods detect thiocyanate in saliva samples, producing results comparable to a validated method.

Validation of Urinary Thiocyanate as a Robust Biomarker of Active Tobacco Smoking in the Prospective Urban and Rural Epidemiological Study.

INTRODUCTION: Tobacco smoking is a leading preventable cause of premature death globally. Urinary thiocyanate is a biomarker of cyanide exposure from tobacco smoke; however, few studies have evaluated its utility in diverse populations of smokers. AIMS AND METHODS: We examined the associations between urinary thiocyanate and self-reported never and current smokers among 1000 participants from 14 countries in the Prospective Urban and Rural Epidemiological study. We analyzed urinary thiocyanate in light and heavy smokers as compared to never-smokers from high- (HICs), middle- (MICs), and low-income countries (LICs) using a validated capillary electrophoresis method in conjunction with standardized questionnaires. RESULTS: The median urinary thiocyanate concentration was 31 µM, which ranged from 8.6 µM to 52 µM for never-smokers (n = 335) and current smokers (n = 660), respectively. Urinary thiocyanate was correlated with daily cigarette consumption (r = 0.621) and total nicotine equivalents (r = 0.514). Thiocyanate also displayed a better dose-response than urinary cotinine. A moderate association of urinary thiocyanate was found in biochemically verified never-smokers (r ~0.38) because of intake of vegetables, fruits, and dairy. Receiver-operating characteristic curves established cutoff values for urinary thiocyanate to differentiate current from never-smokers with an optimal threshold of 23.9 µM (Area Under the Curve or AUC = 0.861), which lowered progressively from HICs, MICs, and LICs. CONCLUSIONS: Elevated thiocyanate was evident in current smokers from high-income countries likely reflecting differences in smoking topography and greater toxicant burden. Background urinary thiocyanate in never-smokers was associated with goitrogenic food intake that obscured detection of secondhand smoke exposure. IMPLICATIONS: Urinary thiocyanate is a sensitive biomarker of active tobacco smoking relative to cotinine that can be measured by an inexpensive capillary electrophoresis assay. Regional cutoff values are demonstrated to improve discrimination of smoking status in developing countries because of differences in smoking habits and cigarette products consumed, as well as intake of goitrogenic foods. Urinary thiocyanate may allow for more reliable estimates of the hazards of tobacco smoking between countries with varying socioeconomic development as compared to self-reports.

Rapid quantification of thiocyanate in milk samples using a universal paper-based SERS sensor.

Sodium thiocyanate (NaSCN) is a naturally antibacterial component in milk, but excessive consumption of thiocyanate may cause potential risks to human health. Currently available methods for the detection of thiocyanate have some disadvantages such as poor sensitivity and high price. Here, we report a robust and cost-effective method to detect NaSCN based on paper substrates deposited with in situ reduced Ag nanoparticles by surface-enhanced Raman spectroscopy (SERS). Densely packed multilayer AgNPs provide uniform narrow nanogaps, which exponentially enhance the Raman signals. Moreover, these homogeneous narrow hotspots ensure that this method has high sensitivity (the limit of detection is 10-12 mol L-1), a wide linear range (from 10-9 mol L-1 to 10-4 mol L-1), and remarkable reproducibility (the coefficient of variation within a SERS sensor is 6.5%). Spiked milk samples were detected and the recovery rates of NaSCN were in the range of 95.1%-108.0%. This paper-based SERS sensor offers great potential for sensitive NaSCN detection and milk analysis.

Effects of different levels of rapeseed cake containing high glucosinolates in steer ration on rumen fermentation, nutrient digestibility and the rumen microbial community.

This trial was conducted to study the effects of dietary rapeseed cake (RSC) containing high glucosinolates (GLS) on rumen fermentation, nutrient digestion and the rumen microbial community in steers. Eight growing steers and four rations containing RSC (GLS 226·1 µmol/g DM) at 0·00, 2·65, 5·35 and 8·00 % DM were assigned in a replicate 4 × 4 Latin square design. The results indicated that increasing RSC levels increased the ruminal concentration of thiocyanate (SCN) (P < 0·01), decreased the ruminal concentration of ammonia nitrogen (NH3-N) and the molar proportion of isovalerate (P < 0·05), did not affect the ruminal concentration of total volatile fatty acids (P > 0·05), decreased the crude protein (CP) digestibility (P < 0·05) and increased the ether extract (EE) digestibility (P < 0·01). Increasing RSC levels tended to decrease the abundances of ruminal Ruminobacter amylophilus (P = 0·055) and Ruminococcus albus (P = 0·086) but did not affect methanogens, protozoa, fungi and other bacteria (P > 0·05). Increasing RSC levels in the ration did not affect the ruminal bacterial diversity (P > 0·05), but it increased the operational taxonomic units and the bacterial richness (P < 0·05) and affected the relative abundances of some bacteria at the phylum level and genus level (P < 0·05). In conclusion, RSC decreased the ruminal concentration of NH3-N and the CP digestibility, increased the EE digestibility and partly affected the ruminal bacterial community. SCN, as the metabolite of GLS, could be a major factor affecting these indices.

Laser engraved microapillary pump paper-based microfluidic device for colorimetric and electrochemical detection of salivary thiocyanate.

A microcapillary grooved paper-based analytical device capable of dual-mode sensing (colorimetric and electrochemical detection) was demonstrated for analysis of viscous samples (e.g., human saliva). Herein, a hollow capillary channel was constructed via laser engraved micropatterning functions as a micropump to facilitate viscous fluidic transport, which would otherwise impede analysis on paper devices. Using salivary thiocyanate as a model analyte, the proposed device was found to exhibit a promising sensing ability on paper devices without the need for sample pretreatment or bulky instrumentation, as normally required in conventional methods used for saliva analysis. An extensive linear dynamic range covering detection of salivary thiocyanate for both high and trace level regimes (5 orders of magnitude working range) was collectively achieved using the dual-sensing modes. Under optimal conditions, the limit of detection was 6 µmol L-1 with a RSD of less than 5%. An excellent stability for the µpumpPAD was also observed for over 30 days. Real sample analysis using the proposed device was found to be in line with the standard chromatographic method. Benefitting from simple fabrication and operation, portability, disposability, low sample volume (20 µL), and low cost (< 1 USD), the µpumpPAD is an exceptional alternative tool for the detection of various biomarkers in saliva specimens.

Detecting Mercury (II) and Thiocyanate Using "Turn-on" Fluorescence of Graphene Quantum Dots.

In this work, 1.8 nm graphene quantum dots (GQDs), exhibiting bright blue fluorescence, were prepared using a bottom-up synthesis from citric acid. The fluorescence of the GQDs could be almost completely quenched (about 96%) by adding Hg2+. Quenching was far less efficient with other similar heavy metals, Tl+, Pb2+ and Bi3+. Fluorescence could be near quantitatively restored through the introduction of thiocyanate. This "turn-on" fluorescence can thus be used to detect both or either environmental and physiological contaminants mercury and thiocyanate and could prove useful for the development of simple point-of-care diagnostics in the future. Graphical Abstract.

Surface-enhanced Raman spectroscopy (SERS) investigations of saliva for oral cancer diagnosis.

Saliva could be an optimal sample for non-invasive cancer detection, as it contains plenty of proteins and metabolites which can reflect the health status of an individual. Moreover, pairing it with high-sensitivity, label-free detection techniques could prove successful for early cancer diagnosis. In this study, we explore the enhancement of salivary characteristic Raman bands by using label-free, ultrasensitive surface-enhanced Raman scattering (SERS) based on gold nanoparticles. SERS maps were acquired from dry samples of saliva supernatant mixed with Au colloidal nanoparticles, which was then pipetted on clean glass slides. The SERS spectra presented a high variability of signal intensities and frequency shifts. However, several reproducible SERS spectra showing well-resolved bands were obtained at certain locations on the maps, where Au nanoparticles clustered together during the air-drying. The healthy and oral cancer saliva could be differentiated using principal components analysis based on several SERS bands assigned mainly to amino acids and proteins. Moreover, thiocyanate Raman modes were detected in saliva samples of both smoking and non-smoking volunteers and cancer patients. The analysis indicated that the cancer group displayed an overall higher level of the 2126 cm-1 band area assigned to C-N stretching vibrations of thiocyanate.

Potentiometric Solid-Contact Ion-Selective Electrode for Determination of Thiocyanate in Human Saliva.

A new solid-contact potentiometric ion-selective electrode for the determination of SCN- (SCN-ISE) has been described. Synthesized phosphonium derivative of calix[4]arene was used as a charged ionophore. The research included selection of the ion-selective membrane composition, determination of the ISEs metrological parameters and SCN-ISE application for thiocyanate determination in human saliva. Preparation of the ISEs included selection of a plasticizer for the ion-selective membrane composition and type of the electrode material. The study was carried out using ISE with liquid internal electrolyte (LE-ISE) and solid-contact electrodes made of glassy carbon (GC-ISE) and gold rods (Au-ISE). The best parameters were found for GC sensors for which the ion-selective membrane contained chloroparaffin as a plasticizer (S = 59.9 mV/dec, LOD = 1.6 ´ 10-6 M). The study of potentiometric selectivity coefficients has shown that the thiocyanate-selective sensor could be applied in biomedical research for determination of SCN- concentration in human saliva. The accuracy of the SCN- determination was verified by testing 59 samples of volunteers' saliva by potentiometric sensors and UV-Vis spectrophotometry as a reference technique. Moreover, SCN- concentrations in the smokers' and non-smokers' saliva were compared. In order to investigate the influence of various factors (sex, health status, taken medications) on the thiocyanate level in the saliva, more extensive studies on a group of 100 volunteers were carried out. Additionally, for a group of 18 volunteers, individual profiles of SCN- concentration in saliva measured on a daily basis for over a month were collected.

Electrochemical Assays and Immunoassays of the Myeloperoxidase/SCN-/H2O2 System.

Strategies to detect and characterize myeloperoxidase (MPO) are needed, given that this "split personality" enzyme kills harmful microorganisms but also damages a host tissue. Here, we describe electrochemical approaches to measure MPO by using the pseudohalogenation (MPO/SCN-/H2O2) and catalase-like (MPO/H2O2) cycles. Their kinetics were determined by monitoring the consumption of H2O2 with a nitrogen-doped carbon nanotubes (N-CNT) electrode, which could detect 0.50 µM H2O2 at -0.20 V. The unique design of internally calibrated electrochemical continuous enzyme assay (ICECEA) and electrode stability allowed use of one N-CNT electrode for over half a year to reliably determine MPO. The kinetic measurements showed that (a) SCN- did not affect the affinity of MPO for H2O2, (b) catalase-like cycle was slower, and (c) MPO retained enzymatically active conformation after complexation with its antibody Ab both in a solution and on the surface of an antibody dipstick (d/Ab). The homogeneous assays could detect 5.2 µg L-1 MPO (35 pM) via a faster cycle. The heterogeneous immunoassays with the capture of MPO on d/Ab could detect 60 µg L-1, which was suitable for the accurate detection of MPO in human saliva (101% recovery). Replacing a detection antibody of ELISA with ICECEA as a signal transducer for immunoassays offers a rapid method for the selective determination of enzymes; for example, time of MPO quantification was cut from 3-4 h (sandwich ELISA) to ∼20 min (ICECEA-dipstick).

Ionic liquids as mobile phase additives for determination of thiocyanate and iodide by liquid chromatography.

An analytical method was developed for the simultaneous determination of thiocyanate and iodide by reversed-phase liquid chromatography with UV detection using imidazolium ionic liquids as mobile phase additives. The chromatographic behaviors of the two anions on a C18 column were studied and compared with four types of reagents including imidazolium ionic liquids, pyridinium ionic liquids, 4-aminophenol hydrochloride and tetrabutylammonium as mobile phase additives. The effects of the concentrations of imidazolium ionic liquids, organic solvents and detection wavelength on separation and detection of the anions were investigated. The role of ionic liquids, retention rules and mechanisms were discussed. The separation of the anions was performed on the C18 reserved-phase column using acetonitrile-0.3 mmol/L 1-amyl-3-methylimidazolium tetrafluoroborate (10:90, v/v) as mobile phase, with column temperature of 35°C, flow rate of 1 mL/min and detection wavelength of 210 nm. Under these conditions, the two anions can be completely separated within 6 min. The limits of detection were 0.05 mg/L. The method was applied for the determination of thiocyanate and iodide in ionic liquid samples and iodide drugs, and the spiked recoveries ranged from 97 to 101%. The method is simple, accurate and meets the requirements of quantitative analysis for thiocyanate and iodide.

Surface Enhanced Raman Spectroscopy Detection of Sodium Thiocyanate in Milk Based on the Aggregation of Ag Nanoparticles.

A method is developed for detecting the concentration of sodium thiocyanate (NaSCN) in milk based on surface-enhanced Raman scattering (SERS) technology. A trichloroacetic acid solution can be used to enhance the SERS signal because of its function in promoting the aggregation of Ag nanoparticles (Ag NPs). Meanwhile, the protein in milk would be precipitated as trichloroacetic acid added and the interference from protein could be reduced during the detection. In this work, the enhancement factor (EF) is 7. 56 × 105 for sodium thiocyanate in water and the limit of detection (LOD) is 0.002 mg/L. Meanwhile, this method can be used to detect the concentration of sodium thiocyanate in milk. Results show that SERS intensity increased as the concentration of sodium thiocyanate increase from 10 to 100 mg/L. The linear correlation coefficient is R² = 0.998 and the detection limit is 0.04 mg/L. It is observed that the concentration of sodium thiocyanate does not exceed the standard in the three kinds of milk. The confirmed credibility of SERS detection is compared with conventional methods.

Levels of the Thiocyanate in the Saliva of Tobacco Smokers in Comparison to e-Cigarette Smokers and Nonsmokers Measured by HPLC on a Phosphatidylcholine Column.

The aim of the study was to estimate the thiocyanate levels in saliva of cigarette smokers in comparison to e-cigarette smokers and nonsmokers. To improve our understanding of the influence of smoking on the oral level of thiocyanate, we conducted an assessment of human saliva, in 24 individuals (eight tobacco smokers, eight e-cigarette smokers, and eight nonsmokers). High-Performance Liquid Chromatography with ultraviolet detection (HPLC-UV) using a unique phosphatidylcholine column was applied in this assay. Thiocyanate ion was detected directly by its absorbance at 210 nm. The method presents a new application of the IAM (Immobilized Artificial Membrane) column for quantification of inorganic anions. The whole process meets the criteria of green chemistry because it was carried out without the use of organic solvents. For compensating matrix effects, an eight-point standard addition protocol was used to quantify the thiocyanate level in saliva samples. The calibration graphs were linear in the range of 5-100 mg L-1 with a correlation coefficient higher than 0.99. The thiocyanate concentrations in the saliva of tobacco smokers, e-cigarette smokers, and nonsmokers were found in the range of 121.25-187.54 mg L-1, 121.24-244.11 mg L-1, 33.03-79.49 mg L-1, respectively. The present study indicates an obvious statistically significant elevation in salivary thiocyanate level in tobacco smokers in comparison to nonsmokers. The phosphatidylcholine-based stationary phase proved to be suitable for the detection and quantification of the thiocyanate ion. The salivary thiocyanate levels in e-cigarette smokers were not significantly different in comparison to tobacco smokers but higher if compared to nonsmokers. The criterion for statistical significance was p < 0.05.

Specific and robust ion chromatographic determination of hypothiocyanite in saliva samples.

The enzymatic system in saliva, consisting of salivary peroxidase (SPO), hydrogen peroxide (H2O2), and thiocyanate (SCN-), produces hypothiocyanite (OSCN-) as a high effective antibacterial compound. OSCN- is of great importance for the natural non-specific antibacterial resistance in the oral cavity. However, no analytical method currently exists to selectively quantify OSCN- in saliva samples. A robust and specific analytical method for the determination of OSCN- was developed based on ion chromatography with combined UV and electrochemical detection. Calibration was achieved by calculating a derived calibration factor based on the known ratio of molar extinction coefficients of SCN- and OSCN-. Thus, the specific quantification of OSCN- in saliva samples is possible, as demonstrated here. The median value of 200 saliva samples was determined to be 0.56 mg L-1 (median), with a maximum of 3.9 mg L-1; the minimum value was below the detection limit (< 0.09 mg L-1). The recovery rate in individual saliva samples was 95 ± 8%.

Determination of thiocyanate as a biomarker of tobacco smoke constituents in selected biological materials of human origin.

In order to protect human health, it is necessary to biomonitor toxic substances originating from tobacco smoke in biological materials sampled from persons with different exposures to tobacco smoke constituents. Thiocyanate anion is a biomarker of exposure to tobacco smoke components which is characterized by a relatively long half-life in the human body, i.e. 6 days. In this work, we present the results of thiocyanate determinations performed on samples of placenta, meconium, saliva, breast milk, sweat and blood. The placenta samples were subjected to accelerated solvent extraction with water. The thiocyanate concentrations were determined using ion chromatography. The analyzed biological materials were compared with regard to their applicability for biomonitoring toxic substances originating from tobacco smoke. The highest mean concentrations of thiocyanate were observed in the samples of biological materials collected from active smokers.

Improvement in blood pressure after short-term inorganic nitrate supplementation is attenuated in cigarette smokers compared to non-smoking controls.

Dietary supplementation with inorganic nitrate (NO3-) has been reported to improve cardiovascular health indices in healthy adults. Cigarette smoking increases circulating thiocyanate (SCN-), which has been suggested to competitively inhibit salivary nitrate (NO3-) uptake, a rate-limiting step in dietary NO3- metabolism. Therefore, this study tested the hypothesis that dietary NO3- supplementation would be less effective at increasing the circulating plasma nitrite concentration ([NO2-]) and lowering blood pressure in smokers (S) compared to non-smokers (NS). Nine healthy smokers and eight healthy non-smoking controls reported to the laboratory at baseline (CON) and following six day supplementation periods with 140 mL day-1 NO3--rich (8.4 mmol NO3- day-1; NIT) and NO3--depleted (0.08 mmol NO3- day-1; PLA) beetroot juice in a cross-over experiment. Plasma and salivary [SCN-] were elevated in smokers compared to non-smokers in all experimental conditions (P < 0.05). Plasma and salivary [NO3-] and [NO2-] were elevated in the NIT condition compared to CON and PLA conditions in smokers and non-smokers (P < 0.05). However, the change in salivary [NO3-] (S: 3.5 ± 2.1 vs. NS: 7.5 ± 4.4 mM), plasma [NO3-] (S: 484 ± 198 vs. NS: 802 ± 199 µM) and plasma [NO2-] (S: 218 ± 128 vs. NS: 559 ± 419 nM) between the CON and NIT conditions was lower in the smokers compared to the non-smokers (P < 0.05). Salivary [NO2-] increased above CON to a similar extent with NIT in smokers and non-smokers (P > 0.05). Systolic blood pressure was lowered compared to PLA with NIT in non-smokers (P < 0.05), but not smokers (P > 0.05). These findings suggest that dietary NO3- metabolism is compromised in smokers leading to an attenuated blood pressure reduction compared to non-smokers after NO3- supplementation. These observations may provide novel insights into the cardiovascular risks associated with cigarette smoking and suggest that this population may be less likely to benefit from improved cardiovascular health if they increase dietary NO3- intake.

A dual-mode colorimetric and fluorometric "light on" sensor for thiocyanate based on fluorescent carbon dots and unmodified gold nanoparticles.

A novel, highly sensitive and selective dual-readout (colorimetric and fluorometric) sensor based on fluorescent carbon dots (CDs) and unmodified gold nanoparticles (AuNPs) for the detection of thiocyanate (SCN(-)) was proposed. Amino-functionalized CDs could be readily adsorbed onto the surface of citrate-stabilized AuNPs through Au-N interactions, leading to the aggregation of AuNPs and the nonfluorescent off-state of CDs arising from potential fluorescence resonance energy transfer (FRET). However, SCN(-) had a stronger affinity toward AuNPs and could compete with CDs to bind onto the surface of AuNPs in priority, which prevented the aggregation of AuNPs and fluorescence quenching of CDs. Correspondingly, both the colorimetric and fluorometric signals remained "light-on". The color of the sensing solution remained red and the fluorescence remained unquenched. A distinguishable change in the color was observed at a SCN(-) concentration of 1 µM by the naked eye and a detection limit as low as 0.036 µM was obtained by virtue of fluorescence spectroscopy. Both colorimetric and fluorometric sensors exhibited excellent selectivity toward SCN(-) over other common metallic ions and anions. In addition, such a sensing assay featured simplicity, rapidity, cost-effectiveness and ease of operation without further modification. The accuracy and precision were evaluated based on the quantitative detection of SCN(-) in tap water and saliva samples with satisfactory results.

Detection of thiocyanate through limiting growth of AuNPs with C-dots acting as reductant.

We have found that hydroxyl-rich carbon dots (C-dots) have the ability to reduce Au(3+) to form gold nanoparticles (AuNPs). Thiocyanate (SCN(-)) can be absorbed on the surface of the AuNPs due to its high affinity toward the AuNPs, which inhibits the growth of the AuNPs. Meanwhile, SCN(-) has the ability to etch the as-synthesized big AuNPs to small AuNPs, which can also cause the absorption peak of the AuNPs to decrease. Therefore, an optical sensor is developed for the detection of SCN(-) based on measuring the plasmon resonance absorption peak change of the AuNPs. Under optimal conditions, this method yields excellent sensitivity (the limit of detection is 0.16 µM) and selectivity toward SCN(-). This method can detect SCN(-) in raw milk with satisfactory results. This work gives new insight into monitoring the quality of milk.

Toxicokinetic aspects of thiocyanate after oral exposure to cyanide in female Wistar rats in different physiological states.

Cyanide (CN) is an ion that has been well studied in toxicology and has been associated with several intoxication episodes: the ingestion of contaminated foods and water, chemical war, suicides, homicides, occupational exposures and the use of certain medicines. The aim of the present study was to determine the toxicokinetic parameters of thiocyante (SCN), the main metabolite of CN, after oral administration of potassium cyanide (KCN) to female rats at diestrus, gestational and lactational periods. Female Wistar rats were divided into three equal groups: virgins in the diestrus phase of the estrus cycle, females at the 14th day of gestation and females at the 14th day of lactation. Each group of rats received 3.0 mg of potassium cyanide per kilogram (KCN/kg body weight) by gavage, and blood was collected at several time points. We also collected amniotic fluid from pregnant rats and milk from the nursing rats to analyze thiocyanate concentration. The results showed that SCN levels were significantly increased in serum, milk and amniotic fluid after administration of KCN. In conclusion, the results of the present study evidence that the metabolism of CN varies greatly considering the physiologic state of the female rat, being females at estrus probably more exposed by these substances than at gestation and lactation because in these states there are other compartments, fetus and milk, which may capture these substances, as demonstrated by the V(d) values.

Development of a colorimetric method for the dosage of OI- anions and I2 in aqueous media.

Lactoperoxidase catalyzes the oxidation of thiocyanate (SCN-) and iodide (I-) in presence of hydrogen peroxide in hypothiocyanite (OSCN-) ions and, depending on the pH, in hypoiodite (OI-) ions or in iode (I2). Oxidized SCN- and I- are part of the lactoperoxidase system, which is a natural biological protection in cow milk, and are described as having inhibitory properties against pathogenic human bacteria, fungi and viruses. We have developed an aqueous solution containing only OSCN- and OI- ions (without the enzyme) and we tested it successfully against plant pathogens. In order to characterize this new soft chemical control against plant pathogens we had to determine the concentration of OSCN- and OI- ions. The dosage of OSCN- consists in a well referenced colorimetric method but no procedure is described for the determination of OI- ions. We have thus developed an easy method, based on the oxidation of the amine moiety of 3,3',5,5'- tetramethylbenzidine (TMB) by OI- or I2 in a strongly absorbing blue product for the detection and dosage of both molecules. Interestingly the OSCN- ions are not able to oxidize TMB and render this method specific to enzymatic oxidized iodide. We have calculated its sensitivity, repeatability and linearity. This method could also be used for the determination of OCI- and OBr- ions produced during the enzymatic oxidation of chloride and bromide by mammalian's peroxidases.