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.     

An unusual case of fatal accidental paraquat poisoning

Paraquat, a useful contact herbicide is now used in over 130 countries of the world, including Sri Lanka. The number of cases of accidental poisoning reported with paraquat is small, relative to instances of suicide. When a clear history is not available, accidental paraquat poisoning is sometimes difficult to diagnose. A 9-year-old boy was admitted to a peripheral hospital with a history of diarrhoea and vomiting. He later developed abdominal pain, subcutaneous emphysema and difficulty in breathing. Following transfer to a district hospital and then to a teaching hospital, poisoning with paraquat was suspected only on day 11 of the illness. On persistent questioning, on day 13 of the illness the child remembered that the day prior to the onset of illness, on his way from a shop, he felt thirsty and having found an empty bottle of Gramoxone (paraquat) on the wayside he used it to drink water from a water tank. The child died on day 17 and the histology of the lung showed typical changes of paraquat poisoning. This tragic episode emphasises the need for proper disposal of empty containers of all poisonous substances.     

大鼠百草枯中毒后体内的分布研究

目的建立生物检材中百草枯的超高效液相色谱-质谱联用检测方法,研究百草枯灌胃染毒致死的大鼠动物模型。方法大鼠以1/2 LD_(50)剂量灌胃染毒,分别于染毒后0.5h、2h、4h、8h、12h、24h、48h、72h处死解剖,采集心、肝、脾、肺、肾、脑、肌肉、膀胱和胃组织,UPLC-MS/MS法定量检测各组织中百草枯。结果试验中大鼠灌胃后,4h以内胃是主要分布器官,胃中含量最多,其他器官中含量相对较低。4h内除胃以外的脏器含量变化不大,4h后胃内百草枯含量有所下降,除胃以外的脏器含量均升高。各组织与脑组织比较具有显著性差异(P0.05)。结论百草枯在大鼠体内死后分布不均匀并且各组织含量随着时间变化有所改变。百草枯UPLC-MS/MS方法、口服染毒致死的动物模型、各组织分布规律可为甲百草枯中毒死亡案件提供检测依据。     

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.     

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.     

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.     

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.     

Agrochemical poisoning in Sri Lanka

Mortality resulting from agrochemicals met within the Office of the Judicial Medical Officer, Colombo, which is the premier Medico-legal Institute in Sri Lanka, are analysed over a 3-year period and the morbidity and mortality rates of the entire country are examined over a 10-year period. The number of patients admitted to hospitals in Sri Lanka during the period 1975-1983, stood at around 11,000-15,000 each year, with the year 1983 recording 16,649 admissions. The number of deaths during the same period varied from 900 to 1500 each year, while the year 1983 recording 1521 deaths. About 75% of such cases of poisoning were due to self ingestion while accidental and occupational poisoning formed the balance. Principal agricultural districts like Kurunegala, Jaffna, Vavuniya, Nuwara-Eliya and Badulla recorded the highest incidence of poisoning. The mortality figures of the Office of the J.M.O., Colombo, indicated that 4% of all bodies subjected to autopsy were those of agrochemical poisoning. The male/female ratio was 2:1. Seventy-five percent of deaths from agrochemical poisoning were recorded in the 15-39 year age group, while 33% of deaths belonged to the 20-24 age group. One third of cases of agrochemical poisoning were dead on being brought to hospital, while 50% were dead within 2 h and 60% dead within 24 h. Organophosphates accounted for 57.6% of all cases of agrochemical poisoning, while paraquat accounted for 21.2% of cases. Deaths were also reported from what are called safe chemicals like Carbamates and Pyrethrums due to their lethal additives.     

Multicenter evaluation of ultrafiltration, dialysis, and thermal coagulation as sample pretreatment methods for the colorimetric determination of paraquat in blood and tissues

Three methods of sample pretreatment for the rapid colorimetric determination of paraquat were compared: ultrafiltration, dialysis, and thermal coagulation. Spiked autopsy blood and tissue samples were examined in parallel in Groningen and Krakow and some samples were interchanged. All three methods gave recoveries between 87 to 102%; accuracy at the 20-mg/L level was within 10% of the target value and coefficients of variation in the 10 to 60-mg/L range were between 3 to 15%. Determinations in blood and liver in a fatal case of Gramoxone poisoning showed excellent agreement. Because of its reliability, speed, and simplicity, ultrafiltration is the method of choice.     

Identification and quantitation of xenobiotics by 1H NMR spectroscopy in poisoning cases

In order to analyse a wide range of xenobiotics and their metabolites present in biological fluids, NMR spectroscopy can be used. A large variety of xenobiotics (therapeutic agents, pesticides, solvents, alcohols) can be characterized and quantitated directly, without sample preparation. NMR investigations were applied to acute poisoning cases, involving drugs such as salicylates and valproic acid (VPA). In a salicylate poisoning case, the three major metabolites of acetylsalicylic acid have been detected in crude urine, and rapid identification of lysine revealed the origin of the intoxication, namely lysine acetylsalicylate (Aspegic). Valproic acid as its glucuronide was identified in urine samples from two poisoned patients. 1H NMR was also used to identify and quantitate paraquat (Gramoxone) in urine owing to its two aromatic signals at 8.49 and 9.02 ppm, in two acutely poisoned patients (183 and 93 mg/l). An intentional poisoning case with tetrahydrofuran (THF) was also investigated. Serum and urine samples were collected. THF was characterized by its resonances at 1.90 and 3.76 ppm, and quantified at 813 and 850 mg/l in the two biological fluids, respectively. Moreover, two other compounds were detected: lactate and gamma-hydroxybutyric acid (GHB). 1H NMR spectroscopic analysis of serum samples from three poisoned patients revealed methanol in one case and ethylene glycol in the two others. Moreover, in the same spectrum, the corresponding metabolites formate and glycolate were found. Compared with the reference chromatographic or spectrophotometric methods, requiring time-consuming extraction and/or derivatization steps, NMR spectroscopy allows the determination of many exogenous and endogenous compounds, without any pre-selection of the analytes.     

Development and application of immunoassay for paraquat: radioimmunoassay

A sensitive radioimmunoassay for paraquat is reported. Anti-paraquat antisera were produced by repeated immunization in rabbits with 1-methyl, 1'-hexanoic acid-4,4'-bipyridinium (MHBP) coupled to bovine serum albumin (BSA). Less than 0.5 ng of paraquat dichloride was detectable by this assay system. These antisera were strongly cross-reactive with the bipyridyl ring and methyl group in either the 1- or 1'-position of paraquat. The determination of paraquat in tissues of paraquat-poisoned cadavers was also carried out.     

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.     

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。     

Determination of paraquat in raw and formalin-fixed tissues by electron spin resonance (ESR) spectroscopy

ESR method was applied to determine paraquat levels in fresh and formalin-fixed tissues. Paraquat was converted to paraquat radical by adding sodium dithionite to tissue homogenates and detected by ESR. Paraquat levels of more than 0.2 micrograms/ml homogenate could be quantified with 0.1 ml of the homogenate. The use of manganese ions for standardization of paraquat signal enabled much more accurate ESR measurements because this ion was quite stable and its signal did not overlap that of paraquat. Even with tissues fixed in formalin, tissues paraquat levels were measureable after removing formalin from the tissue extract. This fact was verified by studying two cases; the tissues were kept in formalin for 1.5 years in case 1 and for 6.5 years in case 2. In both cases, the paraquat contents in tissues were 0.02-0.08 micrograms/g. In this way ESR is one of the most suitable methods in determining low levels of paraquat in tissues even after they were preserved in formalin for a long time.     

高效液相色谱法测定生物体液中百草枯

目的建立HPLC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,水溶性正相液相色谱分析,结合保留时间和紫外光谱对样品中百草枯进行定性。结果经该方法测得百草枯的最小检出限为10ng/m l血(S/N≥3),线性范围为0.1~10μg/ml。结论该方法快速、灵敏、准确,能有效祛除复杂生物基质中杂质干扰,使待测组分获得满意的峰形,适用于生物检材中百草枯的分析。     

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 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.     

气相色谱-串联质谱法分析尿和血中除草剂百草枯

目的建立尿和血中百草枯的离子交换固相萃取-气相色谱-串联质谱分析方法。方法尿样加内标乙基百草枯,用732阳离子交换树脂提取;血样加内标乙基百草枯,用三氯乙酸凝聚蛋白质后取上清液用732阳离子交换树脂提取。提取物用硼氢化钠在水溶液中碱性条件下还原,还原物用有机溶剂提取进行气相色谱-串联质谱法分析。结果尿和血中百草枯的提取率分别为76％和74％,检测限分别为2ng／mL和10ng／mL,尿添加百草枯100ng／mL和血添加百草枯500ng／mL水平的回收率分别为99．6±5．6％和99．3±7．6％（Mean±CV）。结论本文建立的分析方法灵敏度高,能够满足中毒致死案件检验及临床毒物检验的需要。     

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.     

Histological examination of the temporal bone in medicolegal cases of asphyxia

Histological findings of the temporal bone in 23 autopsy cases of various asphyxial fatalities were studied. The temporal bones of 12 cases who died of tumors including mammary cancer, gastric cancer, myxoma of heart and craniopharyngioma, the bones of 3 cases of heart attack and the bones of 17 cases who died of various poisoning (barbiturate, amphetamine, paraquat and alcohol) were used as controls. In drowning, the primary finding was hemorrhage in the mastoid air cells of the bilateral temporal bones. In cases of strangulation by ligature, hemorrhage and edema of the cochlear duct in the inner ear as well as hemorrhage in the mastoid air cells were demonstrated bilaterally. In contrast, congestion and edema in the mastoid air cells and inner ear were found in cases of manual strangulation but there was no hemorrhage. From these results, the histological examination of the temporal bone is useful as an adjunct procedure for diagnosing the cause of asphyxia. Differentiation between drowning, strangulation by ligature and manual strangulation may be possible by observing hemorrhages or their absence in the mastoid air cells and inner ear.