Analysis of α- and ß-amanitin in Human Plasma at Subnanogram per Milliliter Levels by Reversed Phase Ultra-High Performance Liquid Chromatography Coupled to Orbitrap Mass Spectrometry.

Amatoxins are known to be one of the main causes of serious to fatal mushroom intoxication. Thorough treatment, analytical confirmation, or exclusion of amatoxin intake is crucial in the case of any suspected mushroom poisoning. Urine is often the preferred matrix due to its higher concentrations compared to other body fluids. If urine is not available, analysis of human blood plasma is a valuable alternative for assessing the severity of intoxications. The aim of this study was to develop and validate a liquid chromatography (LC)-high resolution tandem mass spectrometry (HRMS/MS) method for confirmation and quantitation of α- and ß-amanitin in human plasma at subnanogram per milliliter levels. Plasma samples of humans after suspected intake of amatoxin-containing mushrooms should be analyzed and amounts of toxins compared with already published data as well as with matched urine samples. Sample preparation consisted of protein precipitation, aqueous liquid-liquid extraction, and solid-phase extraction. Full chromatographical separation of analytes was achieved using reversed-phase chromatography. Orbitrap-based MS allowed for sufficiently sensitive identification and quantification. Validation was successfully carried out, including analytical selectivity, carry-over, matrix effects, accuracy, precision, and dilution integrity. Limits of identification were 20 pg/mL and calibration ranged from 20 pg/mL to 2000 pg/mL. The method was applied to analyze nine human plasma samples that were submitted along with urine samples tested positive for amatoxins. α-Amanitin could be identified in each plasma sample at a range from 37-2890 pg/mL, and ß-amanitin was found in seven plasma samples ranging from <20-7520 pg/mL. A LC-HRMS/MS method for the quantitation of amatoxins in human blood plasma at subnanogram per milliliter levels was developed, validated, and used for the analysis of plasma samples. The method provides a valuable alternative to urine analysis, allowing thorough patient treatment but also further study the toxicokinetics of amatoxins.

Evaluation of complete blood count parameters, cell ratios, and cell volume indices in mushroom poisonings.

OBJECTIVE: Mushroom poisoning is still a significant health-care problem, with the potential to affect blood parameters. The present study analyses the effect of mushroom poisoning on complete blood cell parameters, cell ratios, and cell volume indices and the effect of the mentioned parameters on prognosis/hospital length of stay. METHODS: The study involved the retrospective assessment of 48 patients who became symptomatic after ingesting mushrooms. Patients who were 18 years and above and who spent at least one day in hospital were included in the study. The control group comprised individuals with similar characteristics to the patient group. The complete blood cell parameters, platelet indices (mean platelet volume, plateletcrit (PCT)), neutrophil to lymphocyte ratio (NLR), and platelet to lymphocyte ratio levels were compared between the two groups, and these ratios were analyzed for any correlation with length of hospital stay. RESULTS: NLR was significantly higher and PCT was significantly lower in the mushroom-poisoned group, while all other parameters were similar between the groups. NLR had a positive correlation and PCT had a negative correlation with length of hospital stay. CONCLUSION: NLR and PCT levels can provide information about prognosis and can aid in the prediction of hospital stay in mushroom-poisoned patients. Further investigations are needed to investigate effects of intoxication on hematological system elements.

A case study of Lepiota brunneoincarnata poisoning with endoscopic nasobiliary drainage in Shandong, China.

The most frequently reported fatal Lepiota ingestions are due to L. brunneoincarnata. We present a case of L. brunneoincarnata poisoning with endoscopic nasobiliary drainage known to be the first in China. The patient suffered gastrointestinal symptoms 9 h post ingestion of mushrooms. The patient was hospitalized 4 days after eating the mushrooms with jaundice. The peak ALT, AST, APTT, TBIL and DBIL values of the patient were as follow: ALT, 2980 U/L (day 4 post ingestion); AST, 1910 U/L (day 4 post ingestion); APTT, 92.8 seconds (day 8 post ingestion), TBIL, 136 µmol/L (day 10 post ingestion), DBIL 74 µmol/L (day 10 post ingestion). UPLC-ESI-MS/MS was used to detect the peptide toxins in the mushroom and biological samples from the patient. We calculated that the patient may have ingested a total of 29.05 mg amatoxin from 300 g mushrooms, consisting of 19.91 mg α-amanitin, 9.1 mg ß-amanitin, and 0.044 mg γ-amanitin. Amatoxins could be detected in bile even on day 6 after ingestion of L. brunneoincarnata. With rehydration, endoscopic nasobiliary drainage and intravenous infusion of Legalon SIL, the patient recovered after serious hepatotoxicity developed.

Serum Paraoxonase, Arylesterase, and Glutathione-S-Transferase Activities and Oxidative Stress Levels in Patients with Mushroom Poisoning.

OBJECTIVES: Consumption of toxic species of mushrooms may have detrimental effects and increase oxidative stress. Paraoxonase, arylesterase and glutathione-S-transferase are antioxidants that resist oxidative stress. In this study, we analyzed the changes in these enzymes during intoxication due to mushrooms. METHODS: The study enrolled 49 adult patients with a diagnosis of mushroom poisoning according to clinical findings and 49 healthy volunteers as the control group. The patients with mild clinical findings were hospitalized due to the possibility that the patient had also eaten the mushrooms and due to clinical findings in the late period, which could be fatal. Paraoxonase, arylesterase, and glutathione-S-transferase concentrations, as well as total antioxidant and oxidant status, were determined in the 49 patients and 49 healthy volunteers by taking blood samples in the emergency department. RESULTS: While paraoxonase, arylesterase, and total antioxidant status were significantly decreased in the patient group (p<0.05), glutathione-S-transferase, total oxidant status and the oxidative stress index were significantly higher (p<0.05). There was a positive correlation between the hospitalization time and the oxidative stress index (r=0.752, p<0.001), whereas a negative correlation was found with glutathione-S-transferase (r=-0.420, p=0.003). CONCLUSION: We observed a significant decrease in paraoxonase and arylesterase and an increase in glutathione-S-transferase and oxidative stress indexes in patients with mushroom poisoning, indicating that these patients had an oxidative status. In particular, a low total antioxidant status and high oxidative stress index may gain importance in terms of the assessment of hospitalization duration.

Serum paraoxonase, arylesterase, and glutathione-s-transferase activities and oxidative stress levels in patients with mushroom poisoning

OBJECTIVES: Consumption of toxic species of mushrooms may have detrimental effects and increase oxidative stress. Paraoxonase, arylesterase and glutathione-S-transferase are antioxidants that resist oxidative stress. In this study, we analyzed the changes in these enzymes during intoxication due to mushrooms. METHODS: The study enrolled 49 adult patients with a diagnosis of mushroom poisoning according to clinical findings and 49 healthy volunteers as the control group. The patients with mild clinical findings were hospitalized due to the possibility that the patient had also eaten the mushrooms and due to clinical findings in the late period, which could be fatal. Paraoxonase, arylesterase, and glutathione-S-transferase concentrations, as well as total antioxidant and oxidant status, were determined in the 49 patients and 49 healthy volunteers by taking blood samples in the emergency department. RESULTS: While paraoxonase, arylesterase, and total antioxidant status were significantly decreased in the patient group (p<0.05), glutathione-S-transferase, total oxidant status and the oxidative stress index were significantly higher (p<0.05). There was a positive correlation between the hospitalization time and the oxidative stress index (r=0.752, p<0.001), whereas a negative correlation was found with glutathione-S-transferase (r=-0.420, p=0.003). CONCLUSION: We observed a significant decrease in paraoxonase and arylesterase and an increase in glutathione-S-transferase and oxidative stress indexes in patients with mushroom poisoning, indicating that these patients had an oxidative status. In particular, a low total antioxidant status and high oxidative stress index may gain importance in terms of the assessment of hospitalization duration.

Acute Toxicity Study and the In Vitro Cytotoxicity of a Black Lingzhi Medicinal Mushroom, Amauroderma rugosum (Agaricomycetes), from Malaysia.

Amauroderma rugosum is a wild medicinal mushroom also known as budak cendawan sawan. Members of the indigenous Malaysian Temuan community wear the fresh stipes as a necklace to prevent epileptic seizure and unremitting crying by babies. In our previous studies, A. rugosum exhibited significant antioxidant and anti-inflammatory activities. The aim of this study was to determine the toxicity (in the event that a stipe is accidentally bitten) and cytotoxicity of this mushroom on Sprague-Dawley rats and selected cell lines. A. rugosum was orally administered to test chemicals according to Organisation for Economic and Co-operation and Development guidelines (TG 425, adopted October 3, 2008). Blood samples were hematologically and biochemically analyzed and multiple tissue sections from each organ were examined using light microscopy. Cytotoxicity of various A. rugosum extracts was also determined against MCF-7 and A-549 cell lines. Our results showed that oral administration of a single dose of mycelial powder (2000 mg/kg) had no adverse effect on the growth rate or hematological and clinical biochemical parameters. Histological studies showed that the treatments did not induce any pathological changes in the organs of the tested animals. All the treated rats survived beyond the 14-day observation period. Methanol and cold and hot water extracts of the freeze-dried mycelial culture of A. rugosum exhibited no or little cytotoxic effect against the MCF-7 and A-549 cell lines.

Early initiation of MARS® dialysis in Amanita phalloides-induced acute liver injury prevents liver transplantation.

 Amanita phalloides is the most relevant mushroom intoxication leading to acute liver failure. The two principal groups of toxins, the amatoxins and the phallotoxins, are small oligopeptides highly resistant to chemical and physical influences. The amatoxins inhibit eukaryotic RNA polymerase II causing transcription arrest affecting mainly metabolically highly active cells like hepatocytes and renal cells. The clinically most characteristic symptom is a 6-40 h lag phase before onset of gastrointestinal symptoms and the rapid progression of acute liver failure leading to multi-organ failure and death within a week if left untreated. Extracorporeal albumin dialysis (ECAD) was reported to improve patient's outcome or facilitate bridging to transplantation. In our tertiary center, out of nine intoxicated individuals from five non-related families six patients presented with acute liver injury; all of them were treated with ECAD using the MARS® system. Four of them were listed on admission for high urgency liver transplantation. In addition to standard medical treatment for Amanita intoxication we initiated ECAD once patients were admitted to our center. Overall 16 dialysis sessions were performed. All patients survived with full native liver recovery without the need for transplantation. ECAD was well tolerated; no severe adverse events were reported during treatment. Coagulopathy resolved within days in all patients, and acute kidney injury in all but one individual. In conclusion, ECAD is highly effective in treating intoxication with Amanita phalloides. Based on these experiences we suggest early initiation and repeated sessions depending on response to ECAD with the chance of avoiding liver transplantation.

Effectiveness of Fractionated Plasma Separation and Absorption as a Treatment for Amanita Phalloides Poisoning.

BACKGROUND Fractionated plasma separation and absorption (FPSA) is an extracorporeal liver support method that detoxifies accumulated toxins. There are limited data of its use in the treatment of Amanita phalloides intoxication. The objective of this study was to investigate whether FPSA before liver transplantation improves patients' short-term post liver transplantation survival in Amanita phalloides poisoning. MATERIAL AND METHODS The study population consisted of ten patients who had liver transplantation (LT) due to acute liver failure (ALF) caused by Amanita phalloides poisoning. Six patients were treated with FPSA before liver transplantation. All the patients who were started on FPSA were also placed on the liver transplantation list according to emergent liver transplantation criteria. RESULTS Patients treated with FPSA were in a more severe clinical condition presenting in higher mean MELD, total bilirubin, INR and ammonia along with more frequent hypoglycemia and hepatic encephalopathy grade 3/4. FPSA group had longer mean waiting time on the recipient list (3.5 vs. 1.25 days) but inferior thirty-day survival rate (16.5% vs. 100%). CONCLUSIONS When conservative medical modalities are ineffective, the only treatment for Amanita phalloides poisoning is a liver transplant. Although FPSA treated patients had inferior post-LT survival, FPSA was found to prolong the pre surgical waiting time for critically ill patients, consequently giving a chance of life-saving procedure.

A Simple and High-Throughput Analysis of Amatoxins and Phallotoxins in Human Plasma, Serum and Urine Using UPLC-MS/MS Combined with PRiME HLB µElution Platform.

Amatoxins and phallotoxins are toxic cyclopeptides found in the genus Amanita and are among the predominant causes of fatal food poisoning in China. In the treatment of Amanita mushroom poisoning, an early and definite diagnosis is necessary for a successful outcome, which has prompted the development of protocols for the fast and confirmatory determination of amatoxins and phallotoxins in human biological fluids. For this purpose, a simple, rapid and sensitive multiresidue UPLC-MS/MS method for the simultaneous determination of α-amanitin, ß-amanitin, γ-amanitin, phalloidin (PHD) and phallacidin (PCD) in human plasma, serum and urine was developed and validated. The diluted plasma, serum and urine samples were directly purified with a novel PRiME technique on a 96-well µElution plate platform, which allowed high-throughput sample processing and low reagent consumption. After purification, a UPLC-MS/MS analysis was performed using positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. This method fulfilled the requirements of a validation test, with good results for the limit of detection (LOD), lower limit of quantification (LLOQ), accuracy, intra- and inter-assay precision, recovery and matrix effects. All of the analytes were confirmed and quantified in authentic plasma, serum and urine samples obtained from cases of poisoning using this method. Using the PRiME µElution technique for quantification reduces labor and time costs and represents a suitable method for routine toxicological and clinical emergency analysis.

Treatment of Amanita phalloides intoxication by fractionated plasma separation and adsorption (Prometheus®).

OBJECTIVE: To investigate the effectiveness and safety of extracorporeal detoxification using the fractionated plasma separation and adsorption system (FPSA, Prometheus® 4008H, Fresenius Medical Care, Germany) in patients suffering from acute liver failure due to intoxication with Amanita phalloides (AP) toxin. METHODS: The study population consisted of 20 patients with proven AP intoxication (FPSA treatment group n=9, control group n=11). Urinary amanitin toxin concentration was measured by the Amanitin ELISA Kit (Bühlmann Laboratories, Germany, cut off level 1.5 ng/ml). All patients received standard medical treatment with activated charcoal, i.v. crystalloid fluids, silibinine and N-acetylcysteine. Additionally 9 patients underwent treatment with FPSA until undetectable amanitin levels. RESULTS: Mean urinary amanitin levels were significantly reduced by FPSA with 42.5 +/- 21.9 ng/ml before and 1.2 +/- 0.31 ng/ml after treatment (p=0.04). No hemodynamic, respiratory or hematological complications were observed. None of the patients had to undergo liver transplantation. All patients in the treatment group survived and were discharged fully recovered. One patient in the control group died due to shock and lactic acidosis; one patient remained dialysis dependent. Mean duration of hospital stay was 7.1 days in the treatment group and 11.7 days in the control group (p=0.30). CONCLUSIONS: Use of liver support therapy by fractionated plasma separation and adsorption (Prometheus®) offers a safe way for elimination of Amanita toxin with the potential to avoid the need for liver transplantation.

Hypoglycemia and death in mice following experimental exposure to an extract of Trogia venenata mushrooms.

BACKGROUND: Clusters of sudden unexplained death (SUD) in Yunnan Province, China, have been linked to eating Trogia venenata mushrooms. We evaluated the toxic effect of this mushroom on mice. METHODS: We prepared extracts of fresh T. venenata and Laccaria vinaceoavellanea mushrooms collected from the environs of a village that had SUD. We randomly allocated mice into treatment groups and administered mushroom extracts at doses ranging from 500 to 3500 mg/kg and water (control) via a gavage needle. We observed mice for mortality for 7 days after a 3500 mg/kg dose and for 24 hours after doses from 500 to 3000 mg/kg. We determined biochemical markers from serum two hours after a 2000 mg/kg dose. RESULTS: Ten mice fed T. venenata extract (3500 mg/kg) died by five hours whereas all control mice (L. vinaceoavellanea extract and water) survived the seven-day observation period. All mice died by five hours after exposure to single doses of T. venenata extract ranging from 1500 to 3000 mg/kg, while the four mice exposed to a 500 mg/kg dose all survived. Mice fed 2000 mg/kg of T. venenata extract developed profound hypoglycemia (median= 0.66 mmol/L) two hours after exposure. DISCUSSION: Hypoglycemia and death within hours of exposure, a pattern unique among mushroom toxicity, characterize T. venenata poisoning.

Acute and subchronic oral toxicity of Coriolus versicolor standardized water extract in Sprague-Dawley rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Coriolus versicolor, which is known as Yun Zhi, is one of the commonly used Chinese medicinal herbs. Recent studies have demonstrated its antitumor activities on cancer cells which led to its widespread use in cancer patient. However, little toxicological information is available regarding its safety. The present study evaluated the potential toxicity of Coriolus versicolor standardized water extract after acute and subchronic administration in rats. MATERIALS AND METHODS: In acute toxicity study, Coriolus versicolor water extract was administered by oral gavage to Sprague-Dawley (SD) rats (6 males, 6 females) at single doses of varying concentrations 1250, 2500 and 5000 mg/kg. In subchronic toxicity study, the extract was administered orally at doses of 1250, 2500 and 5000 mg/kg/day for 28 days to male and female SD rats respectively. General behavior, adverse effects and mortality were determined throughout the experimental period. Haematological and biochemical parameters, relative organ weights and histopathological were evaluated at the end of the experiment. RESULTS: There were no mortality and signs of toxicity in acute and subchronic toxicity studies. In the single dose acute toxicity and repeated dose 28-day subchronic toxicity studies, there were no significant difference in body weight, relative organ weight, haematological parameters, clinical chemistry, gross pathology and histopathology between treatment and control groups. CONCLUSIONS: Coriolus versicolor water extract did not cause remarkable adverse effect in SD rats. The oral lethal dose of Coriolus versicolor water extract is more than 5000 mg/kg and no-observed-adverse-effect level (NOAEL) of the extract for both male and female rats is 5000 mg/kg per day for 28 days.

[Lethal poisoning with the death-cap mushrooms].

We have analysed morphological manifestations in five cases of lethal poisoning with the Death Cap toxin and compared the relevant clinical and laboratory findings with those in 15 survived subjects. It was shown based on the results of autopsy that poisoning was associated with hepatopathy, extensive hepatic necrosis, and nephropathy. Elevated blood bilirubin levels in conjunction with lowered prothrombin and fibrin concentrations appear to be the main causative factors responsible for the unfavourable prognosis of this intoxication.

[Amanitin determination in mushroom poisoning diagnostics]. / Oznaczanie amanityny w diagnostyce zatruc grzybami.

There are some serious poisonings with toxic mushroom species in Poland every year. Good prognostics in the cases is correlated to short time from mushroom consumption to hospitalization, correct distinguish not specific gastrointestinal and Amanita phalloides syndrome and immediately specific treatment. The purpose of the paper was to make appraisal of usefulness of amanitin blood and urine determination and transaminases activity determination (ALT, AST) in diagnostics of mushroom poisoned patients up to three days after mushroom consumption. The material was twenty two retrospective histories of mushroom poisoned patients treated in the years 2007-2008. Amanitin blood and urine determinations were made by ELISA method. Urine amanitin results in samples collected within 40 h from mushroom consuming were positive in all Amanita phalloides syndrome cases. Serum amanitin determination was not useful for the diagnostics. Trans-aminases activity determinations let to distinguish Amanita phalloides syndrome on the second and the third day after mushroom consumption. In the first poisoning phase (within 24 h), the ALT and AST activities were in normal ranges and only amanitin urine determination let to confirm or exclude Amanita phalloides poisoning. Amanitin urine determinations were useful to take fast decision about specific treatment and avoid internal organs dysfunctions.

Acute poisoning with Tricholoma equestre.

Four cases, including three adults and one child, suffering from acute poisoning with Tricholoma equestre were described. The patients had eaten from 100 to 400 grams of the mushroom within a few consecutive meals. After consuming about 1000 grams of Tricholoma equestre for 3-4 days, the subjects developed fatigue, muscle weakness, myalgia, and in two cases acute respiratory failure with the need of respiratorotherapy. Maximal serum CK was 48136 U/L in the adults and 306 U/L in children. Maximal serum levels of AST and ALT were 802 U/L and 446 U/L in adults and 39 U/L, and 56 U/L in a child. All routine biochemical tests were within normal range. No other causes of rhabdomyolysis such as parasitic or viral infections, immune diseases, trauma or exposure to medications were found. Patient, aged 72 yrs., who developed acute respiratory failure, died in the second day of hospitalization. In other patients all the above mentioned symptoms and biochemical abnormalities disappeared from 2 to 3 weeks of hospitalization. Physicians should be aware of the possibility of appearance of rhabdo-myolysis after repeated consumption of large quantities of Tricholoma equestre.

Elevated serum hyaluronic acid and interleukin-6 levels in patients with mushroom poisoning admitted to the emergency department.

OBJECTIVE: To assess the possible use of hyaluronic acid (HA) and interleukin-6 (IL-6) together as a biochemical marker of liver damage in mushroom poisoning (MP). METHODS: We prospectively studied patients with MP who were admitted to the emergency service, between April 2005 and April 2007, Samsun, Turkey. Twenty-seven patients with MP were included in the study. Serum HA and IL-6 levels of the patients were determined using enzyme-linked immunosorbent assay daily for a total of 3 days. Ten healthy adults were included in the study to serve as controls. The patients were divided into survivors, and non-survivors. RESULTS: There was no significant difference between the patients and controls with respect to serum HA levels on admission (p>0.05). However, IL-6 levels on admission were significantly higher in the patients than the control group (p<0.01). Serum HA and IL-6 levels on admission, and the following days were significantly higher in non-surviving patients (n=5) than in surviving patients (n=22) (p<0.05). There was a significant correlation between HA and IL-6 (r=0.42, p<0.05) on admission. The HA concentration was also significantly correlated with aspartate aminotransferase, alanine aminotransferase, and creatinine levels during the observation period. Serum HA and IL-6 levels increased in non-surviving patients throughout the period of observation. CONCLUSION: Increased serum HA and IL-6 levels are associated with hepatic damage in acute MP. Hyaluronic acid may be a useful marker in the assessment of MP-induced acute liver failure in clinical practice.