The enterohepatic circulation of amanitin: Kinetics and therapeutical implications

Toxicology Letters 203 (2011) 142–146

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The enterohepatic circulation of amanitin: Kinetics and therapeutical implications Christian Thiel a , Karolin Thiel a , Wilfried Klingert a , Andreas Diewold a , Kathrin Scheuermann a , Elmar Hawerkamp a , Johannes Lauber a , Johannes Scheppach a , Matthias H. Morgalla b , Alfred Königsrainer a , Martin Schenk a,∗ a b

Department of General, Visceral and Transplant Surgery, Tuebingen University Hospital, Hoppe-Seyler-Strasse 3, Tuebingen 72076, Germany Department of Neurosurgery, Tuebingen University Hospital, Hoppe-Seyler-Strasse 3, Tuebingen 72076, Germany

a r t i c l e

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Article history: Received 13 December 2010 Received in revised form 9 March 2011 Accepted 10 March 2011 Available online 21 March 2011 Keywords: Amanitin intoxication Enterohepatic circulation Biliary excretion

a b s t r a c t Background: Amatoxin poisoning induces a delayed onset of acute liver failure which might be explained by the prolonged persistence of the toxin in the enterohepatic circulation. Aim of the study was to demonstrate amanitin kinetics in the enterohepatic circulation. Methods: Four pigs underwent ␣-amanitin intoxication receiving 0.35 mg/kg (n = 2) or 0.15 mg/kg (n = 2) intraportally. All pigs remained under general anesthesia throughout the observation period of 72 h. Laboratory values and amanitin concentration in systemic and portal plasma, bile and urine samples were measured. Results: Amanitin concentrations measured 5 h after intoxication of 219 ± 5 ng/mL (0.35 mg/kg) and 64 ± 3 (0.15 mg/kg) in systemic plasma and 201 ± 8 ng/mL, 80 ± 13 ng/mL in portal plasma declined to baseline levels within 24 h. Bile concentrations simultaneously recorded showed 153 ± 28 ng/mL and 99 ± 58 ng/mL and decreased slightly delayed to baseline within 32 h. No difference between portal and systemic amanitin concentration was detected after 24 h. Conclusions: Amanitin disappeared almost completely from systemic and enterohepatic circulation within 24 h. Systemic detoxification and/or interrupting the enterohepatic circulation at a later date might be poorly effective. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Over 95% of fatal mushroom poisoning in the world occurs after ingestion of Amanita species, primarily “death cap” (Amanita phalloides). Amatoxins (Faulstich and Wieland, 1996; Karlson-Stiber and Persson, 2003), contained in these poisonous mushrooms, has well-known effect on humans (Vetter, 1998) by binding to and inhibiting nuclear RNA polymerase in eukaryotic cells. Lesions are found particularly in hepatocytes but also in kidney tubular cells. Hepatocytes incorporate the toxin fast and excrete them into the bile, so that amatoxins could be detected in the gastrointestinal fluid and faces (Jaeger et al., 1993), but toxin removal is mainly based (>85%) on renal elimination. The clinical course of amatoxin poisoning (Faulstich, 1979) is characterized by a asymptomatic incubation delay from 6 to 12 h following gastrointestinal syndromes, such as vomiting, diarrhea, abdominal pain, hypoglycaemia and dehydratation after

∗ Corresponding author. Tel.: +49 7071 2986722; fax: +49 7071 29 4395. E-mail address: [email protected] (M. Schenk). 0378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2011.03.016

6–12 h. Hepatocellular damage will become evident clinically and biochemically leading to progressive coagulopathy on day second or third. In fatal cases patients develop acute liver failure including haemorrhages, encephalopathy and coma following renal and/or multiorgan failure at around 6–8 days. Mortality ranges from about 10%–20% in adults to 22%–50% in children (Enjalbert et al., 2002; Jander and Bischoff, 2000) reported by different authors. Amatoxin kinetics could yet not be clearly demonstrated in human poisoning because of the delayed clinical presentation of most intoxicated patients. It has first been postulated from results of animal studies with beagle dogs that amanitin appears in the bile fluid after intravenous administration (Faulstich and Fauser, 1973). They concluded that the biliary excretion of amanitin prolong their presence in systemic and enterohepatic circulation by intestinal reuptake. Therefore the clinical course of poisoning in humans and animals could be significantly influenced by this mechanism. These results were confirmed by further experimental animal studies (Faulstich et al., 1980a; Faulstich et al., 1985; Faulstich and Fauser, 1973) and transferred into clinical practise, although it has never been shown that amanitin kinetics in portal plasma confirm the theory of relevant or

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delayed intestinal reuptake. Based upon this theory, the attempt of interrupting the enterohepatic circulation through enteral administration of activated charcoal or other medications has become a commonly accepted substantial part of detoxification strategies. Extra-corporal blood purification through hemoperfusion as well as blocking the reuptake of amatoxins into hepatocytes using various chemotherapies was introduced to detoxification management. More recently, endoscopic nasobiliary drainage was performed to remove bile fluid completely in the case of a 18 year old patient who ingested extremely high doses of amatoxin (Madhok et al., 2006). A total amatoxin concentration of 2.5 mg could be removed through bile sampling on day second after poisoning. But the clinical relevance remained unclear. Large animal models evaluating the significance of the enterohepatic amanitin reuptake still do not exist. Aim of our study was to evaluate amanitin kinetics in the enterohepatic circulation representing the clinical relevance of enteral detoxification. Simultaneous measurements of amanitin concentrations in systemic, portal, bile and urine samples have not been reported previously in a pig model and the hypothesis of relevant enterohepatic circulation of amanitin via the bile could thereby be tested. 2. Methods 2.1. Animals After approval by the institutional review board for animal experiments, four female German landrace pigs weighing 34 ± 1 kg underwent ␣-amanitin (AppliChem GmbH, Darmstadt, Germany) intoxication after overnight fasting. All experiments were performed according to the international principles governing research on animals and under the supervision of a veterinarian, who set the guidelines for minimizing the suffering of the pigs. The aimed observation period in this study was 72 h. Pigs were euthanized by a single intravenous bolus of 10 mL T 61 (Intervet, Unterscheißheim, Germany). 2.2. Anesthesia and surgical procedures Intramuscular premedication consisted of atropine 0.1% (0.05 mg/kg), ketamine (7 mg/kg), azaperone (10 mg/kg) and diazepam (1 mg/kg). Continuous infusion of ketamine (15 mg/kg/h), fentanyl 0.02 mg/kg/h and midazolam 0.9 mg/kg/h was administered to maintain anesthesia during the experiment. After oral intubation, animals remained in deep general anesthesia receiving pressure-controlled ventilation (KION, Siemens Medical, Sweden) until conclusion of the study protocol. Character of respiration, heart rate, eye movement and pain stimulus were used to confirm the depth of anesthesia; if any of these parameters indicated a lessening of anesthesia, infusion rates of anaesthetic agents were increased. A stomach tube was placed for intestinal drainage. Adequate temperature 38–39 ◦ C was maintained with a warming mat. An antibiotic prophylaxis of 2 g ceftriaxon (Rocephin® , Hoffmann-La Roche, Basel, Switzerland) was given every 24 h. The neck vessels were instrumented to measure arterial (Leadercath, Vygon, Écouen, France) and central venous pressure (Multi-Lumen Central Venous Catheter,

Arrow International, Reading, PA, USA). Animals were laparotomized and a urinary catheter was placed to measure urinary output. An 18-gauge catheter (Cavafix® , Braun Melsungen, Germany) was inserted into the portal vein via cannulation of a small mesenteric vein for amanitin intoxication and portal blood sampling. A 2.5 mm Kehr T-tube (Ruesch GmbH, Kernen, Germany) was inserted into the common bile duct. The T-tube remained clamped so that no bile fluid was drained externally over the observation period. The t-tube was only opened every 8 h for the sampling of 500 ␮L bile fluid aliquots. Liver biopsies were sampled every 24 h. 2.3. Amanitin intoxication, acute liver failure and intensive care monitoring 0.35 mg/kg or 0.15 mg/kg ␣-amanitin were dissolved in 25 mL saline and administered intraportally over 120 min via the implanted portal vein catheter. Acute liver failure was defined by a decline of prothrombin time below 30% confirmed by the clinical presence of hemodynamic changes and histology. Monitoring included electrocardiogram, mean arterial, central venous and intracranial pressure, oxygen saturation and core body temperature. Urinary output, arterial blood gas analysis (ABL 800, Radiometer Copenhagen, Denmark) including haemoglobin, lactate, serum electrolytes, acid base balance and blood glucose levels were monitored hourly and immediately corrected as required. Hydroxyethylstarch 6% (Voluven® HES 130/0.4, Fresenius, Bad Homburg, Germany) and sodium chloride solution 0.9% were used for fluid management to stabilize the hemodynamic parameters such as mean arterial pressure within a range of 60–70 mmHg and central venous pressure within 6–12 mmHg. Norepinephrine was used to ensure hemodynamic stability in the end-stage of acute liver failure. Blood glucose levels were maintained >100 mg/dL by glucose 20% solution, haemoglobin values remained stable within the range of 8.5–11.5 g/dL. 2.4. Biochemical analysis All blood samples were measured by the certified central laboratories of the university hospital Tuebingen (Zentrallabor, Innere Medizin IV, Universitätsklinikum Tuebingen, Germany). Prothrombin time, aspartate aminotransferase, total plasma protein, albumin, bilirubin, creatinine and ammonia were analyzed in systemic blood samples. Amanitin concentration measurements from systemic, portal, bile and urine samples were performed before starting surgical procedures and every 8 h until conclusion of the study protocol. ␣-amanitin concentration measurements were performed by an ELISA-kit (Buehlmann, Basel, Switzerland). 2.5. Statistical analysis Results in the manuscript are reported as mean ± standard deviation (SD). Figures are presented as mean ± standard error of mean (SEM).

3. Results 3.1. Clinical course and laboratory values All pigs developed ALF within 39 ± 10 h. Due to standardized intensive care therapy (Thiel et al., 2010) in pigs, vital and ventilation parameters could be stabilized in acute liver failure until the end of the observation period of 72 h. Relevant laboratory parameters such as blood count, prothrombin time, aspartate aminotransferase, total plasma protein, albumin, ammonia, biliru-

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Fig. 2. Haematoxylin–eosin staining of liver biopsies taken 48 h after amanitin intoxication. (A) 0.35 mg/kg body weight intoxicated animals; (B) 0.15 mg/kg body weight intoxicated animals.

bin and creatinine were found to be within the normal range before start of intoxication. During the development of acute liver failure prothrombin time declined below 30% (Fig. 1A) paralleled by total plasma protein including albumine. Bilirubin slightly increased in the further course of acute liver failure. Serum ammonia rose to levels of 137 ± 72 ␮mol/L (0.35 mg/kg) and 217 ± 241 ␮mol/L (0.15 mg/kg) at the end of the observation period. Aspartate aminotransferase increased to maximal values of 946 ± 717 U/L (0.35 mg/kg) and 1381 ± 192 U/L (0.15 mg/kg) at 45 h after intoxication and decreased in the further course (Fig. 1B). Creatinine remained stable during the observation period.

3.3.4. -Amanitin (portal - systemic plasma concentration) -Amanitin was calculated as portal - systemic plasma concentration to demonstrate the enterohepatic circulating amanitin. Within the initial intoxication phase -amanitin presented negative values in the 0.35 mg/kg group 5 h after intoxication representing the systemic plasma excess of the high dose intoxication group. In the further course values became slightly positive 16 h after intoxication and trend to baseline levels within 24 h. In the low dose group -amanitin presented positive values 5 h after intoxication verifying the rapid enterohepatic reuptake. Values decreased to baseline levels within 16 h as shown in Fig. 3D.

3.2. Histological results Light microscopic examination (haematoxylin–eosin staining) of liver biopsies taken 48 h after intoxication demonstrated typical centrilobular necrosis confirming the clinical and laboratory onset of acute liver failure as shown in Fig. 2. 3.3. Amanitin kinetics 3.3.1. Systemic and portal vein plasma Initial amanitin concentration 5 h after intoxication produced a maximal level of 219 ± 5 ng/mL (0.35 mg/kg) and 64 ± 3 ng/mL (0.15 mg/kg) in systemic plasma samples and 201 ± 8 ng/mL (0.35 mg/kg); 80 ± 13 ng/mL (0.15 mg/kg) in portal plasma samples. Amanitin concentration rapidly decreased to baseline levels in systemic and portal plasma within 24 h after intoxication as shown in Fig. 3A. 3.3.2. Bile Analogue to systemic and portal vein plasma amanitin concentrations, maximal levels of 153 ± 28 ng/mL (0.35 mg/kg) and 99 ± 58 ng/mL (0.15 mg/kg) could be detected in the bile fluid 5 h after intoxication. The decline of amanitin concentration in the bile fluid was slightly delayed respectively in the 0.15 mg/kg group compared to systemic and portal amanitin plasma levels, so that amanitin remained even at negligible levels detectable from baseline up to 32 h after poisoning (Fig. 3B). 3.3.3. Renal elimination The highest amanitin elimination was found in urine samples with a maximal value of 1.4 ± 0.4 mg/h (0.35 mg/kg) and 0.5 ± 0.2 mg/h (0.15 mg/kg) in the low dose group, Fig. 3C. Kinetics demonstrated rapid renal elimination from plasma, but amanitin remained detectable in urine samples until the end of the observation period.

4. Discussion The optimal management of patients after ingestion of amatoxin-containing mushrooms is still not determined. Because there is no specific antidote, treatment is only symptomatic and supportive (Giannini et al., 2007) including gastric lavage, laxatives and enteral administration of activated charcoal as well as extra-corporal eliminations using haemodialysis, hemoperfusion and/or plasmapheresis. Liver assist devices to clear secondary toxins and support liver regeneration seemed to be beneficial (Hydzik et al., 2005; Lionte et al., 2005; Sein et al., 2005). Blocking of the enterohepatic circulating amanitin by suction of the duodenal fluid through a nasoduodenal tube or complete external drainage of the bile through a nasobiliary tube placement as a more invasive approach for detoxification were discussed controversially because of the increased risk of haemorrhages through papilotomia or pancreatitis (Faulstich and Zilker, 1994). Penicillin-G (Broussard et al., 2001), silymarin (Abenavoli et al., 2010) and free radical scavengers (Enjalbert et al., 2002; Ganzert et al., 2008; Magdalan et al., 2011) were identified as hepatoprotective medication thatf strongly support their clinical use but emergency liver transplantation remains the life saving therapy (Beckurts et al., 1997; Ganzert et al., 2005; Panaro et al., 2006) in fatal cases. The clinical efficiency of these established therapeutical approaches is difficult to demonstrate because randomized, controlled clinical trials do not exist due to fortunately small intoxication numbers and the delayed presentation of the intoxicated individual. Therefore large animal models are of prime importance for the evaluation of the toxokinetics itself and to standardize supportive therapy. Previous experimental studies analyzed amanitin uptake and kinetics in perfused pig livers. It was demonstrated that intraportally amanitin injection increased the hepatotoxic activity and the hepatic amanitin uptake corresponded to the portal perfusion (Faulstich et al., 1980b).

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Fig. 3. ␣-Amanitin concentration and kinetics in systemic (black line) and portal plasma (grey line) (A) and in the bile fluid (B). Renal elimination of ␣-amanitin (C) and -amanitin (portal - systemic amanitin plasma concentration) (D). () indicates the .35 mg/kg group, () indicates 0.15 mg/kg group. Black bar indicates intoxication period over 120 min; black arrow indicates onset of ALF.

Preliminary pig studies have been carried out to identify the dose of 0.35 mg/kg which equivalents more than the double lethal dose (LD50 = 0.15 mg/kg) in humans, to be the invariable lethal dose for pigs. The presented experiment was aimed to demonstrate amanitin kinetics in courses of high-dose and LD50 intoxications to evaluate the clinical significance of the enterohepatic circulation for specific detoxification approaches. The hypothesis that amanitin plasma concentration could be significantly decreased by removing excreted amanitin in the bile fluid and thereby improving the clinical outcome has been verified neither experimentally nor clinically. Overlooking the presented results, amanitin metabolism and elimination is a very rapid process lasting for approximately 24 h after oral ingestion. Renal elimination kinetics demonstrated approximately 75% ␣-amanitin elimination within the first 16 h after start of intoxication. The observed phenomenon in the low dose intoxication (0.15 mg/kg) group that initial ␣-amanitin portal plasma exceeded systemic plasma levels can be explained by a larger quantity of biliary amanitin excretion within the early phase of poisoning. The nearly identical amanitin concentration kinetics in systemic and portal plasma and -amanitin disproved the theory of a clinically relevant delay of toxicity through biliary toxin excretion and reuptake in the enterohepatic circulation as previously reported. Intrahepatic and enterohepatic circulating amanitin might produce a delayed biliary excretion from hepatocytes into the intestine, but amanitin reuptake from the small intestine did not produce prolonged appearance of the toxin in portal plasma samples. The fact that amanitin bile levels from 13 h after intoxication were lower in the high dose intoxication group (0.35 mg/kg) than in the low dose

group may reflect more severe hepatocyte damage. The maximal toxin levels exposed to hepatocytes could possibly affect the ability of hepatocytes to excrete the toxin into the bile. Surprisingly serum parameters for amatoxin toxicity like aspartate aminotransferase of the 0.15 mg/kg intoxication group exceeded the values of the high dose group. These findings could be explained by increased ␣-amanitin induced apoptosis of hepatocytes due to higher toxin levels (Magdalan et al., 2010) which would result in a minor release of aspartate aminotransferase in systemic plasma samples. Nevertheless it has been demonstrated (Escudie et al., 2007) that this serum parameter can not be considered for predicting the clinical outcome. We conclude that relevant toxin removal later than 16–24 h after initial poisoning can not be gained by haemodialysis, hemoperfusion or albumin dialysis. Experimental liver transplant studies done in pigs (Takada et al., 2001) demonstrated that no further liver injury could be observed in grafts after successful liver transplantation confirming our hypotheses. Interruption of the enterohepatic circulation might be poorly effective after 16–24 h because clinical relevant toxin concentration and/or intestinal reuptake do not longer exist at this time point. The delayed renal elimination of amanitin over 72 h offers the most sensitive method identifying this fatal intoxication. Transferring our results into clinical practise, supportive intensive care therapy including a rapid gastrointestinal decontamination with initial enteric administration of charcoal could be effective especially in the early phase after ingestion. Additive hepatocyte protection with silymarin and antioxidants has been reported to be useful in this life threatening condition. Nasobiliary drain placement and hemoperfusion as more invasive therapeutic

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