Detection of Selenocyanate in Biological Samples by HPLC with Fluorescence Detection Using König Reaction.

We developed a simple and sensitive HPLC method for the determination of selenocyanate (SeCN-). The König reaction, which is generally used for the determination of cyanide and thiocyanate, was applied for the post-column detection, and using barbituric acid as a fluorogenic reagent made it possible to detect SeCN- with high sensitivity. The limits of detection (LOD) and quantification (LOQ) were 73.5 fmol and 245.1 fmol, respectively. Subsequently, the amounts of SeCN- in human blood and in cultured cell samples were analyzed, and no SeCN- was detected in human whole blood. Interestingly, we have found that some of the spiked SeCN- decomposed to cyanide in human whole blood. Ascorbic acid suppressed the decomposition of SeCN- to cyanide by reducing the ferric ion, which is typically involved in SeCN- decomposition. Then, SeCN- was detected in cultured HEK293 cells exposed to selenite. The established HPLC method with fluorescence detection of SeCN- is useful for investigating small amounts of SeCN- in biological samples.

Hemoglobin adducts in workers exposed to 1,6-hexamethylene diisocyanate.

We investigated the utility of 1,6-hexamethylene diamine (HDA) hemoglobin adducts as biomarkers of exposure to 1,6-hexamethylene diisocyanate (HDI) monomer. Blood samples from 15 spray painters applying HDI-containing paint were analyzed for hemoglobin HDA (HDA-Hb) and N-acetyl-1,6-hexamethylene diamine (monoacetyl-HDA-Hb) by GC-MS. HDA-Hb was detected in the majority of workers (≤1.2-37 ng/g Hb), whereas monoacetyl-HDA-Hb was detected in one worker (0.06 ng/g Hb). The stronger, positive association between HDA-Hb and cumulative HDI exposure (r(2) = 0.3, p < 0.06) than same day exposure (p ≥ 0.13) indicates long-term elimination kinetics for HDA-Hb adducts. This association demonstrates the suitability of HDA-Hb adducts for further validation as a biomarker of HDI exposure.

Red cell volume can be accurately determined in sheep using a nonradioactive biotin label.

The sheep has served as an informative animal model for investigation of human fetal and newborn erythropoiesis and red blood cell (RBC) kinetics. We previously validated the permanent label (14C)cyanate for measuring red cell volume (RCV) in sheep. Here, we validate biotin labeling of RBCs as a nonradioactive method for measuring RCV in sheep with the anticipation that it can be applied in studies of human infants. The RCV was determined simultaneously using two techniques for quantitation of the biotin label. The first one quantified total blood concentration of biotin label on biotin-labeled RBCs using (125I)streptavidin. The second one enumerated biotin-labeled RBCs by flow cytometry after incubation with fluorescein-conjugated avidin. RCV measurements made using the two biotin quantitation techniques were validated against both (14C)cyanate and 51Cr as reference methods. Both biotin techniques produced RCV values that agreed well with the reference methods and with each other, producing correlation coefficients averaging >or =0.93. Sequential repetitive measurements in the same animal also agreed with the (14C)cyanate method and each other (average difference <10%). These results establish biotin-labeled RBCs as an accurate method for performing RCV measurements in sheep. This biotin method can be applied in studies that model neonatal erythropoiesis.

Pseudolaminar necrosis in cyanide intoxication: a neuropathology case report.

We describe the gross and microscopic neuropathological changes in the brain of a 17-year-old male who died 4 days after being poisoned with cyanide. Previous reports indicate that following cyanide intoxication, the brain develops diffuse hypoxic/ischemic changes, predominantly of the basal ganglia. The case we describe here had similar features but in addition showed striking laminar necrosis of the cerebral cortex. This finding in cyanide poisoning has been previously demonstrated by neuroimaging, but not pathologically.

Red blood cell life span in the ovine fetus.

Red cell life span within the fetal circulation has not been reported, although erythrocyte life span has been studied in the adult and newborn. The present study quantified red cell life span in 12 chronically catheterized fetal sheep at 97-136 days gestation (term = 150 days) with the use of autologous red cells labeled with [(14)C]cyanate. Cyanate forms a permanent covalent bond with hemoglobin and acts as a permanent red cell label. In the fetuses, blood (14)C activity decreased in a curvilinear fashion with time and reached 50% of the initial activity at 16.4 +/- 1.6 (SE) days. In contrast, (14)C activity of autologous red cells in two adult ewes decreased linearly with time as expected, reached 50% of the initial (14)C activity in 59 days, and yielded life spans of 117 and 121 days. Computer modeling and parameter optimization taking into account growth and skewed life span distribution were used to analyze the (14)C disappearance curve in each fetus. The mean life span of all red cells in the fetal circulation was 63.6 +/- 5.8 days. Mean red cell life span increased linearly from 35 to 107 days as fetal age increased from 97 to 136 days (r = 0.83, P < 0.001). Life span of cells produced at the time of labeling was significantly greater than the mean life span. Fetal growth rate estimated from parameter optimization was 3.28 +/- 0.72%/day; this compared well with the rate of 3.40 +/- 0.14%/day calculated from fetal weights at autopsy. Mean corpuscular volume decreased as a function of gestational age, but the decrease was small compared with the large increase in red cell life span. We conclude the following: 1) red cell life span in the fetal circulation is short compared with the adult; 2) red cells in younger fetuses have shorter life spans than in near-term fetuses; 3) the curvilinear disappearance of labeled red cells in the fetus appears to be due primarily to an expanding blood volume with fetal growth; and 4) red blood cell life span in a growing organism will be significantly underestimated unless the expansion of blood volume with growth is taken into account.

Impaired biological activity of erythropoietin by cyanate carbamylation.

AIMS: During advanced renal failure, particularly in patients with end-stage renal disease (ESRD), proteins are carbamylated as a result of a reaction with cyanate. Some or all of the cyanate is derived from urea. If the carbamylation of proteins adversely alters their biologic activities, then urea must be viewed as an uremic toxin, rather than a surrogate. Therefore, we studied the effect of cyanate carbamylation on the erythropoietic activity of erythropoietin (EPO) in a rodent model. METHODS: EPO was carbamylated by incubation with cyanate at 37 degrees C. The extent of carbamylation was monitored using trinitrobenzenesulfonic acid. In Sprague-Dawley rats the erythrocyte count, hemoglobin concentration, and hematocrit were measured after the twice-weekly subcutaneous injection of either EPO or carbamylated EPO for 3 weeks. Two additional control groups received physiologic saline or 0.2 ml of 1 M cyanate. RESULTS: The level of carbamylated EPO was increased as the time of exposure to cyanate increased from 1 to 6 h, and as the cyanate concentration increased from 8 to 2,000 mM. EPO injections caused significantly large increases in all erythropoietic measures. Physiologic saline or 1 M cyanate-injected controls and the carbamylated EPO-injected animals demonstrated no change from baseline in erythropoietic parameters. CONCLUSION: These results support that EPO exposed to high levels of cyanate in vitro demonstrates diminished biologic activity in healthy Sprague-Dawley rats. This effect may be manifested by the carbamylation of EPO by the cyanate. Should this occur in ESRD patients, it may contribute to the suboptimal erythropoietic response to EPO therapy associated with high urea levels, especially related to inadequate dialysis. Targeting dialysis doses specifically to urea concentrations may be more important than previously considered.

Bioactivation of cyanide to cyanate in sulfur amino acid deficiency: relevance to neurological disease in humans subsisting on cassava.

Neurological disorders have been reported from parts of Africa with protein-deficient populations and attributed to cyanide (CN-) exposure from prolonged dietary use of cassava, a cyanophoric plant. Cyanide is normally metabolized to thiocyanate (SCN-) by the sulfur-dependent enzyme rhodanese. However, in protein-deficient subjects where sulfur amino acids (SAA) are low, CN may conceivably be converted to cyanate (OCN-), which is known to cause neurodegenerative disease in humans and animals. This study investigates the fate of potassium cyanide administered orally to rats maintained for up to 4 weeks on either a balanced diet (BD) or a diet lacking the SAAs, L-cystine and L-methionine. In both groups, there was a time-dependent increase in plasma cyanate, with exponential OCN- increases in SAA-deficient rats. A strongly positive linear relationship between blood CN- and plasma OCN- concentrations was observed in these animals. These data are consistent with the hypothesis that cyanate is an important mediator of chronic cyanide neurotoxicity during protein-calorie deficiency. The potential role of thiocyanate in cassava-associated konzo is discussed in relationship to the etiology of the comparable pattern of motor-system disease (spastic paraparesis) seen in lathyrism.

Modification of lipoproteins in uremia: oxidation, glycation and carbamoylation.

Lipoprotein modification occurs in uremic patients and in patients with end stage kidney disease under chronic renal replacement therapy. Forms of lipoprotein modification include lipid peroxidation, glycation, and carbamoylation. In this short review, we discuss the presence of these forms of lipoprotein modification and their association with various renal diseases. Methods to analyze lipoprotein modification are introduced, and functional consequences related to vascular and renal function are presented.

The search for the uremic toxin: the case for carbamoylation of amino acids and proteins.

Urea and cyanate, spontaneously transformed from urea, are increased with decreased renal function becoming potential toxins. Isocyanic acid, the active form of cyanate, carbamoylates proteins, amino acids and other molecules, changing molecular structure and function in vivo. Carbamoylation can occur at multiple sites with a cumulative effect over the the life-span of the molecule. Carbamoylation converts free amino acids to carbamoyl-amino acids (C-AA). C-AA interfere with protein synthesis and transamination reactions and contribute, in part, to protein-malnutrition. Insulin-sensitive glucose uptake is decreased by carbamoyl-asparagine. Cyanate inhibits superoxide release from neutrophils to an extent that interferes with microbiocidal activity. Antihomocitrulline antibodies identified homocitrulline (epsilon-amino-carbamoyl lysine) in situ in proteins in neutrophils in end stage renal disease. Also in uremic patients, homocitrulline was located in proteins in renal tissue but was not found in normal transplanted kidneys. Carbamoylated human low density lipoprotein interferes with human receptor binding, has decreased clearance, and is auto-immunogenic in animals. Carbamoylated insulin has decreased biological activity and changed immunological reactivity. Carbamoylation at a site of molecular activity can affect molecular function of enzymes, co-enzymes, antibodies, hormones and receptors. Carbamoyl-molecules can block, enhance, or be excluded from metabolic pathways, and can affect binding and trafficking, thereby influencing the fate of non-carbamoylated molecules. Normal renal function removes C-AA. In uremia, C-AA are removed by residual renal function or dialysis. Toxicity of cyanate is not an "all or none" phenomenon, but the actions of cyanate are a contributing factor in uremia. Removal of urea, cyanate and carbamoyl-molecules partially alleviates the morbidity and mortality of renal disease.

Quantification of carbamylated dehydroascorbate derivative produced from cyanate and dehydroascorbate.

We established a high-performance liquid chromatographic method for separating and quantifying carbamylated dehydroascorbate derivative (CDA), a reaction product of cyanate with dehydroascorbate. The separation of CDA from interfering substances was achieved by anion-exchange HPLC using a TSK gel SAX (250x4.6 mm I.D.) column and 0.12 M NaCl eluent. The detection of CDA was achieved through two steps: (1) degradation of CDA to cyanate and amino compounds in alkaline solution, and (2) detection of these products by an indophenol reaction. For the processing of plasma and urine samples, anion-exchange solid-phase extraction was used. The detection limit for quantitative determination was 0.1 microM CDA (S/N=3). The linear range found applying the optimized conditions was 0.2 to 200 microM. The intra- and inter-day assay precision (R.S.D.) of CDA (10 microM) were 4.8 and 7.2% for rat plasma, and 4.0 and 4.9% for rat urine, respectively. The usefulness of the present method was proved by the application to plasma and urine samples. The study of the biokinetics of CDA in rats revealed that the elimination of CDA is due to urinary excretion.

Circulating red cell volume and red cell survival can be accurately determined in sheep using the [14C]cyanate label.

The sheep commonly serves as an animal model for investigation of human fetal and newborn erythropoiesis and red blood cell kinetics. Measurement of red cell volume (RCV) and survival (RCS) in sheep would be useful for studying mechanisms of neonatal anemia. Unfortunately 51Cr, the standard method for RCV, is not suitable for RCS in sheep because 51Cr leaves the red cell too rapidly. We developed and validated the permanent label [14C]cyanate as a method for measuring both RCV and RCS in sheep. In 19 sheep, RCV was determined simultaneously using [14C]cyanate and 51Cr. RCV determined by [14C]cyanate agreed almost perfectly with RCV by 51Cr; correlation coefficient = 0.990. The line of regression had a slope of 0.94 and an intercept of 40; these parameters are not significantly different from a line of identity. In nine sheep, RCS was determined using [14C]cyanate. Survival after d 1 accurately fit a model containing two components: 1) an early exponential loss likely related to damage caused by labeling and handling and 2) a linear decrease that reflected normal survival of undamaged red cells. Mean potential life span (MPL) determined from the linear phase was 114 +/- 12 d (mean +/- 1 SD). These results agree with reported MPL values determined either by 59Fe or differential hemolysis. Together, these observations establish [14C]cyanate-labeled red cells as a tool for measuring both RCV and RCS in sheep and enhance the value of the ovine model for investigating neonatal anemia.

[14C]cyanate labeling of sheep red cells: covalent binding to hemoglobin continues in vivo for a day.

The sheep is a useful model to study fetal and newborn physiology including perinatal erythropoiesis and red cell kinetics. A practical, economical method for measuring red cell survival (RCS) in sheep would be very valuable. However, 51Cr is unsatisfactory, and suitable alternatives have not been published. In the course of investigating [14C]cyanate as a label for sheep red cells, we observed continued covalent labeling over 24 h in vivo that was great enough to introduce a substantial artifact into two commonly used parameters of RCS: posttransfusion recovery (PTR24) and time to 50% decrease (T50) when referenced to time zero. In a simulation of in vivo conditions, the amount of 14C bound to Hb increased 26 +/- 6% (mean +/- 1 SD, n = 11) over 24 h. To investigate the mechanism of the increasing 14C bound, acid-acetone extraction, molecular sieve chromatography, and density gradient separation were used separately or in combination to quantitate intracellular free 14C and 14C covalently bound to intracellular proteins. Free 14C decreased as protein-bound [14C]cyanate increased. These studies provide evidence that covalent binding of [14C]cyanate to intracellular Hb continues in vivo for the first 24 h and that the source of the increase is intracellular free [14C]cyanate. We conclude that 1) PTR24 cannot be accurately determined by [14C]cyanate unless labeled red cells are incubated before infusion to allow the cyanate reaction to approach completion and 2) RCS by [14C]cyanate should be referenced to blood concentrations at 24 h.

Fluorometric determination of cyanate in plasma by conversion to 2,4(1H,3H)-quinazolinedione and separation by high-performance liquid chromatography.

A fluorometric high-performance liquid-chromatographic method is described for the determination of cyanate in human plasma. The method is based on the derivatization of cyanate with 2-aminobenzoic acid (anthranilic acid), leading to a stable cyclic fluorescent product, 2,4(1H,3H)-quinazolinedione. The fluorescent product was extracted with ethyl acetate, passed through a cation-exchange resin to eliminate the excess of derivatization reagent, and then concentrated on a Sep-Pak C-18 cartridge before reversed-phase chromatography and fluorometric detection. The precision of the method was satisfactory (coefficient of variation 2.3%) and the analytical recovery was quantitative (103%). Detection limit was found to be 8 nmol/liter from a 1-ml plasma sample. Cyanate in plasma was stable for 3 months when stored in liquid nitrogen. The mean and SD cyanate level in human plasma from 17 healthy nonsmoking subjects was 45 +/- 21 nmol/liter.

Dicyanogold (I) is a common human metabolite of different gold drugs.

Gold based drugs and their metabolites have been characterized using reversed phase, ion pairing chromatography with an inductively coupled plasma mass spectrometer as an element specific detector. For a patient receiving gold sodium thiomalate the principal gold species in the urine is [Au(CN)2]-, which is also seen in a low molecular weight infiltrate of the blood. The same compound is also identified in the urine and blood of a patient taking auranofin and in patients taking solganol. This represents the first identification of a specific gold metabolite in biological fluids taken from patients undergoing gold therapy and the first evidence that different gold drugs have common metabolites.

Monitoring of haemoglobin-methyl isocyanate adduct by high-performance liquid chromatography with diode array detector.

Carbamylation of haemoglobin by methyl isocyanate (MIC) was detected by high-performance liquid chromatography (HPLC) using a photodiode array detector following cyclisation of the N-terminal valine into methyl isopropyl hydantoin (MIH). MIH was also synthesised by reaction of MIC with valine, the chromatographic conditions standardised and the spectrum derived by a photodiode array detector recorded for confirmation of the identity of MIH. This HPLC method is specific, sensitive and suitable for the detection of exposure of blood samples to methyl isocyanate.

GC-MS identification of MIC trimer: a constituent of tank residue in preserved autopsy blood of Bhopal gas victims.

Based on the external and internal findings of Bhopal gas disaster victims, it was apparent that the gases and particulate matter came out as an aerosol. This was possibly the pyrolysed, reformulated, reconjugated suspension of constituents of the tank E-610 of Union Carbide India Limited, Bhopal, while it was claimed to be methyl isocyanate (MIC) only. It was postulated by the manufacturer of MIC, that the material inhaled by the victims of the Bhopal gas disaster does not cross the lung barrier (UCC press conference on 14th December 1984). It was observed that the more the victims ran, the more aerosol they inhaled and the fatalities were observed in such victims. The tissues, which were preserved in the deep freeze, were randomly selected and analysed by GC coupled with MS (ITD) Finnigan MAT, UK. 14 out of 34 autopsy cases showed MIC trimer peak in extracts of blood. This was one of the constituents of the aerosol and was also located in the tank residue, thereby proving that the trimer had passed the lung barrier.

Stereospecific high-performance liquid chromatographic assay of sotalol in plasma.

A convenient high-performance liquid chromatographic (HPLC) assay was developed for determination of sotalol (STL) enantiomers in plasma. Following addition of the internal standard (IS; racemic atenolol), enantiomers of STL and IS were extracted using ethyl acetate. After evaporation of the organic layer, samples were derivatized with a solution of S-(+)-1-(1-naphthyl)ethyl isocyanate (NEIC). The resulting diastereomers were chromatographed with normal-phase HPLC with chloroform:hexane:methanol [65:33:2 (v/v)] as the mobile phase at a flow rate of 2 ml/min. The fluorescence detection wavelength was set at 220 nm for excitation with no emission filter. The suitability of the assay for pharmacokinetic studies was determined by measuring STL enantiomers in the plasma of a healthy subject after administration of a single 160-mg oral, racemic dose of STL.

State analysis of endogenous cyanate ion in human plasma.

Cyanate ion is continuously produced by spontaneous degradation of urea both in healthy human blood and in blood of patients with uremia on intermittent hemodialysis. The cyanate ion is trapped irreversibly by amino groups as N-carbamyl groups and reversibly by sulfhydryl groups as S-carbamyl groups. Consequently, these reactions keep the cyanate ion levels in plasma extremely low. Plasma proteins may play an important role in the detoxication of cyanate ion.