Release of tissue paraquat into formalin solution during fixation

Formalin-fixed tissues and formalin solutions are among the most frequently found materials in pathology and forensic science laboratories. However, these materials are seldom used for the identification of poisons for forensic toxicology purposes. In this study, the possibility that paraquat may be released from formalin-fixed tissues during the fixation process was investigated. However, because of the interference of formaldehyde on the reduction of paraquat with dithionite reagent, paraquat in formalin solutions was treated with ion-pair column chromatography and then determined by measuring the derivative spectrum of reduced paraquat. The results show that the interference of formalin on paraquat determination has been eliminated by the proposed method. Furthermore, a study on the formalin solutions of fixed organs in cases with suspected paraquat intoxication revealed that portions of tissue paraquat had been released into formalin during the fixation process. Moreover, the paraquat levels in formalin increased with increased storage time. Therefore, these data suggest that the combined concentrations of paraquat in the formalin-fixed tissues and formalin solutions might reflect more reliably the total paraquat in the postmortem tissues. This investigation could be of value to the forensic toxicologist, especially in cases in which no fresh tissue samples are available for analysis.     

Determination of diquat in biological materials by electron spin resonance spectroscopy

An electron spin resonance (ESR) method already in use for the quantitative analysis of paraquat was applied to the analysis of diquat in blood, serum, urine, tissue homogenates and several drinks without purification of the samples. The diquat radical produced with ascorbic acid at alkaline pH was much more stable than that produced with the commonly used sodium dithionite. Radical decay in solutions covered with n-hexane was less than 5% after 60 min over a wide range of ascorbic acid concentrations. In 0.2 N NaOH solution 85% of the radicals was present even after 24 h. The limit of detection was 0.3 micrograms/ml and the required amount of sample was 0.1 ml. When both diquat and paraquat were present in a sample the diquat was first extracted with 1-butanol prior to the ESR measurement, because both species were converted to the radicals.     

Acute and sub-acute paraquat poisoning in a pack of foxhounds

A case of paraquat poisoning in a pack of foxhounds is reported. The incident provided a unique opportunity to compare residues levels of paraquat in dogs showing symptoms of acute and sub-acute poisoning. Paraquat was detected in the urine up to six days after exposure. A high performance liquid chromatography method for determining paraquat in tissue is described.     

Determination of paraquat from formalin-fixed tissue

In this paper, a sulfuric acid digestion method and a clean-up technique by using cation exchange resin followed by XAD-2 resin has been developed for the determination of paraquat from formalin-fixed tissue at the submicrograms per gram level. Formalin-fixed tissue is dissolved by hot sulfuric acid, then paraquat is isolated and purified with cation exchange chromatography. The eluted paraquat forms an ion-pair with sodium dodecyl sulfate, it is then adsorbed on XAD-2 resin. Paraquat is eluted, extracted and reduced with solvent mixtures, NaCl solution and dithionite reagent, respectively. The calibration graphs of zero-order and second-derivative spectroscopy are linear in the range of 0.01-5.0 mg/kg. The relative standard deviation was less than 5% and the detection limit was 0.02 mg/kg based on 0.5-g samples. The sensitivity of the proposed method could be increased by using larger sample sizes. The method was precise and gave a quantitative recovery of paraquat spiked into formalin-fixed liver homogenates (78%). The proposed method has been satisfactorily applied to the determination of paraquat in the formalin-fixed tissues of suspected poisoned cases. It has been shown to be of great value in the field of forensic toxicology especially when formalin-fixed tissue only is available.     

HPLC法测定百草枯急性中毒大鼠的体内分布

目的 应用高效液相色谱法对口服百草枯急性中毒大鼠体内分布进行测定。方法以200mg／kg剂量百草枯给予Wister大鼠灌胃,4h后脱臼处死,解剖取脑、心、肝、脾、肺、肾、胃、盲肠、肌肉等组织,应用固相萃取法提取,液相色谱法测定各器官组织中百草枯含量。结果各组织经检验,均检出百草枯;组织间百草枯含量（μg／g）相差明显,其中最高为胃（231．47±129．10）,其次为盲肠（87．08±39．86）、肺（22．73±10．20）,最低为心（2．01±0．36）。结论百草枯口服给药后组织分布较为广泛,除胃、肠外各脏器中以肺浓度最高。     

Blowfly Larval Tissues as a Secondary Detector for Determining Paraquat-Related Death in Rabbit Carcass

Paraquat poisoning is commonly associated with suicide or homicide in Malaysia. In a case involving advanced body decomposition, pathological analysis regarding the cause of death may become difficult or almost impossible. Insects serve as common alternative matrix for poison detection in forensic analysis. Paraquat detection via secondary bioaccumulation in fly larvae tissue has never been reported. In this study, tissues from blowfly larvae collected from a rabbit carcass with paraquat poisoning were analyzed for secondary bioaccumulation. Larvae samples were collected and analyzed using liquid–liquid extraction. The detection was performed via reduction of quaternary ammonium presence in paraquat and analyzed using gas chromatography–mass spectrometry (GC-MS) with selected ion monitoring mode (SIM mode). GC-MS showed the elution of reduced paraquat was at retention time 12.8 min. Blowfly larvae tissue has proven useful as a secondary detector in paraquat-related deaths.     

Determination of paraquat in tissue using ion-pair chromatography in conjunction with spectrophotometry

Homogenized tissue was deproteinized with sulfuric acid. Paraquat in the supernatant was quantificated directly with the dithionite reagent (step 1) or concentrated by the XAD-2 column chromatographic technique before paraquat determination (step 2). Tissue paraquat levels in the range of 0.01-75 mg/kg could be quantificated by second-derivative or zero-order spectroscopy using 2.5 g of tissues. The sensitivity could be increased tenfold by using 25 g of tissue samples. The coefficients of variation of within-run and day-to-day precisions of spiked paraquat in tissue homogenates were below 5% at concentrations of 10.0, 1.0 and 0.1 mg/kg, respectively. The recoveries of the spiked paraquat in tissues ranging from 0.1-10 mg/kg were 91% by step 1 and 74% by step 2. Using these simple methods, steps 1 and 2, the paraquat concentrations in the psoas muscle, liver, lung and kidneys of a swine dosed with 0.16 g/kg of paraquat were investigated. The results were in close agreement with those of the TCA deproteinization method followed by cation-resin column chromatography. The proposed method offers the advantages of simplicity, rapidity, reasonable sensitivity and a wide range of concentrations.     

Paraquat poisoning

A fatal case of paraquat poisoning is described. Postmortem concentration of paraquat in different tissues reveals that treatment in this case could not prevent lethal tissue accumulation. Although accumulation was more pronounced in renal tissue, lung toxicity caused death. The formation of enormous fecaliths and the appearance of hypercalcemia are reported. Both were most likely connected with Füllers earth therapy. In spite of the fact that the exact nature of the equilibrium between plasma levels and tissue accumulation of paraquat (static or dynamic) is not understood, aggressive treatment must be recommended, even after the distribution phase and despite likely "fatal" plasma levels.     

Paraquat myopathy: report on two suicide cases.

We describe two suicide cases in which old paraquat was ingested. In conjunction with lung involvement a pronounced degeneration was observed in skeletal muscle of one who died on the 14th day after the ingestion. The following sarcoplasmic or endoplasmic reticulum Ca2+ ATPase (SERCA) monoclonal antibodies were used for skeletal muscle fiber typing by an immunohistochemical method: NCL-SERCA1, reactive with type 2 fiber (fast-twitch), and NCL-SERCA2, reactive with type 1 fiber (slow-twitch). The examination revealed that the remarkably degenerated fibers belonged to type 1 muscle fibers. This case showed an abrupt increase of plasma CK levels (1796 mU/ml) on the fifth day after the ingestion. The authors presume that the damage to the skeletal muscle had occurred in this period. The degeneration of the muscle seemed to be attributable to the long retention of paraquat in the tissue because these findings were not observed in the other case who died on the fifth day. Paraquat-induced myopathy may develop in prolonged paraquat poisoning. The examination of CK levels in plasma will be useful for diagnosis of damage of skeletal muscle.     

Tissue distribution of paraquat and diquat after oral administration in rats

The differential distribution of paraquat and diquat in liver, kidney, lung, brain and heart has been studied after oral administration of the LD50 or LD50/2 to rats. Paraquat concentrations were highest in all organs at 24 hours; at this time concentrations in kidney and lung were 2 to 3 times higher than at 2 hours. In contrast, with the exception of kidney, tissue diquat concentrations were highest at 2 hours. There was severe lung damage at 24 hours after paraquat; diquat aid not produce severe lung lesions, but caused intestinal distention and diarrhea. These findings suggest that the difference in the toxicity of paraquat and diquat is related to their difference in tissue distribution and excretion.     

A fatal case of parenteral paraquat poisoning.

The chief clinical and analytical aspects of the suicide of a 21-year-old male with psychiatric problems by parenteral administration of a 200 g/l aqueous solution of paraquat are described. Paraquat levels were determined in plasma, urine, kidney, liver and lung after autopsy. Tissue damage was studied by electron microscopy. The death ensued from pulmonary dysfunction 15 days after hospital admission.     

LC／MS／MS法测定生物组织中百草枯

目的建立LC／MS／MS检测生物体液中百草枯方法。方法弱阳离子交换固相萃取小柱提取剂，应用LC／MS／MS法对生物样品中百草枯进行定性定量分析。结果经该方法测得百草枯的最小检出限为10ng／ml血（S／N≥3），线性范围为0．02～20μg／ml。结论该方法快速、灵敏、准确，适用于生物检材中百草枯的分析。     

Identification of N,N-dimethylamphetamine formed by methylation of methamphetamine in formalin-fixed liver tissue by multistage mass spectrometry

Methamphetamine is methylated in the presence of unbuffered formalin solutions within hours at room temperature. The product, N,N-dimethylamphetamine, is also found in human liver exposed to methamphetamine followed by incubation with formalin. In the present study, a direct mass spectrometric method was developed to identify N,N-dimethylamphetamine in human liver before and after treatment with formalin. Human liver samples were obtained from four deaths that were investigated by the West Virginia Office of Chief Medical Examiner. Full toxicological analysis was conducted on samples from the decedents and methamphetamine was among the positive findings in each case. The method used to expose liver tissue to formaldehyde involved treating a small piece of liver from each case with formalin solution (20% v/v) for 24 h at room temperature. The formalin treated tissues were homogenized and the resulting suspension was sonicated for 5 min, and then centrifuged. Supernatant aliquots were directly analyzed by electrospray ionization (ESI) mass spectrometry without chromatographic isolation. Positive ion multistage mass spectra recorded in MS, MS/MS and MS/MS/MS (MS3) modes were used to confirm the presence of N,N-dimethylamphetamine and methamphetamine in the mixture. Liver tissue not treated with formalin did not contain a detectable level of N,N-dimethylamphetamine. Decreases in methamphetamine concentrations in liver tissue resulting from treatment with formalin were measured using deuterium-labeled methamphetamine as internal standard. The method can be completed in less than 2 h on thawed tissue. The results suggest that the process of fixing tissues with formalin may lead to false negative findings for methamphetamine.     

An enzyme-linked immunosorbent assay for plasma-paraquat levels of poisoned patients

To investigate the efficacy of medical treatments, plasma-paraquat levels were measured in patients who had ingested the liquid weedkiller, Gramoxon, containing paraquat. We determined the levels by an enzyme-linked immunosorbent assay (ELISA) using a murine paraquat-specific monoclonal antibody developed by Niewola et al. (Clin. Chim. Acta, 148 (1985) 149-156). Using this ELISA, concentrations of paraquat in the range 1.56-100 ng/ml could be measured. This assay method had good precision and was suitable for measurement of low levels of paraquat. The therapeutic treatments were most effective on the day of admission. The plasma-paraquat levels of poisoned patients decreased significantly. However, the levels tended to increase again during the pause period of the haemoperfusion and this increase was serious for fatal cases. It is clear that elimination of poison not only from blood but also from tissues is necessary to lower the mortality rate.     

The stability of tetramine, morphine and meperidine in formalin solution.

The stability of tetramine, morphine and meperidine in formalin solution is an important factor for drug analysis in forensic investigation. In this paper, the tissues (liver, kidney, lung and heart) from poisoned rabbits were immersed in 50 ml 10% formalin solutions for 4 months before examination. We compared the levels of tetramine, morphine, meperidine and the main metabolite normeperidine, measured by GC/NPD or GC-MS, in frozen rabbit tissues, formalin-fixed rabbit tissues, and formalin solution. There was a decrease in the levels of tetramine, morphine, meperidine in formalin-preserved tissues compared with the levels of these drugs in the frozen tissues. It is suggested that the formalin-fixed tissues and formalin solution should be analyzed at the same time to assure the accurate results.     

Paraquat (gramoxone) poisoning in south-west Hungary, 1977-1984. Toxicological and histopathological aspects of group intoxication cases

Fifty-six cases of paraquat poisoning were examined during the 8 years 1977-1984. The ratio of cases of accidental to intentional ingestion of the poison was 1:1; 23.2% of the total (13/56) survived several months or years. In 1983, 11 subjects accidentally drank 10-30 ml of gramoxone; eight of 11 died in 2 weeks. Toxicological investigations demonstrated rapid elimination of poison from the blood, as well as prolonged fixation of paraquat in the lung, kidney, liver, and spleen tissues. Histological examinations showed multiorgan failure from renal tubular necrosis and pulmonary hemorrhage with alveolar epithelial injury. Pulmonary proliferative changes were present in only two cases who survived 7 and 10 days and in which artificial ventilation was utilized.     

Extraction of diquat with 1-butanol from biological materials

Diquat can be extracted with 1-butanol from high pH solution in the presence of several moderate reductants. The red colored reduced compound of diquat in water turns to a purple compound in 1-butanol. The absorption of the purple compound is 0.105 at 383 nm and 0.119 at 520 nm in 1 microgram diquat/ml 1-butanol. The latter value is a little higher than that of the red compound at 495 nm in water. The purple compound is much more stable than the red compound in water. More than 80% of 10 ppm diquat added can be extracted from serum, blood, tissues, urine and some drinks. The extraction with 1-butanol is useful for concentration of diquat contained in large volume. The lower limit of detection is 0.1 microgram/ml 1-butanol. Paraquat is insoluble in 1-butanol under the same condition. Therefore, this method is applicable for the determination of diquat when paraquat is also contained in the solution.     

Pharmacokinetic profile of paraquat following intravenous administration to the rabbit

Pharmacokinetic studies of paraquat in rabbits were performed using [methyl-14C]-paraquat. Plasma concentration of paraquat following i.v. administration to the rabbit was fitted to a 3-exponential function of pharmacokinetic analysis. Distribution and elimination were discussed on the basis of the 3-compartment open model system, which has a central and two peripheral compartments. Computer simulations of paraquat levels in each compartment indicated that the slow-uptake peripheral compartment contained a greater amount of paraquat than the central or the fast-uptake peripheral compartment. On the basis of the present results of the computer simulations in company with tissue distributions of paraquat reported by the other investigators, it is likely that the slow-uptake peripheral compartment contains the lung. In cases of paraquat-induced renal failures, the paraquat levels of the slow-uptake peripheral compartment were remarkably higher than in cases of normal renal functions. Histology of the rabbit tissues 7 days after i.v. administration of paraquat revealed that marked changes were observed only in the kidney, suggesting some renal failures induced by paraquat. In spite of the high concentration of paraquat, which was presumed with the computer simulations in this study, the rabbit lung showed a remarkable resistance to paraquat toxicity. The histology studies suggested the complexities of paraquat toxicity to the rabbit. The lung toxicity in the rabbit would be caused by not only the paraquat concentration in the lung but also some biochemical parameters in the tissue related to the mechanisms of paraquat toxicity.     

Homicidal Paraquat Poisoning

Paraquat poisoning usually results from suicide, occupational, or accidental exposure. Herein, we report a rare fatal case of homicidal paraquat poisoning. A 58‐year‐old man was poisoned by taking paraquat‐mixed medicine and wearing paraquat‐soaked underwear. In the absence of a history of paraquat exposure, the patient was misdiagnosed with pulmonary infection and scrotal dermatitis and died of respiratory failure 24 days after the initial exposure to paraquat. Ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) was applied to detect and quantify paraquat in postmortem specimens. The concentration of paraquat in postmortem specimens from high to low is lung (0.49 μg/g), brain (0.32 μg/g), kidney (0.24 μg/g), liver (0.20 μg/g), cardiac blood (0.11 μg/mL), and stomach wall (<LOQ). Identification of homicidal paraquat poisoning is not easy for a clinician or a forensic pathologist, it is important to consider the possibility of paraquat poisoning when patients suffer from rapidly aggravating pneumonia of unknown origin.     

离子对SPE—HPLC法检测生物检材中的百草枯

目的建立生物检材中百草枯的简便、快速、灵敏、可靠的高效液相色谱分析方法。方法生物检材酶解后,用以十二烷基三甲基溴化铵和十二烷基硫酸钠预处理过的C18固相柱萃取,HPLC/DAD进行分析。结果回收率81%～94%,检出限为1ng·mL-1,线性范围50ng·mL-1～1mg·mL-1,结论此方法适用于中毒生物检材中百草枯的检测。