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Toxicology Letters 203 (2011) 142–146

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

he enterohepatic circulation of amanitin: Kinetics and therapeuticalmplications

hristian Thiela, Karolin Thiela, Wilfried Klingerta, Andreas Diewolda, Kathrin Scheuermanna,lmar Hawerkampa, Johannes Laubera, Johannes Scheppacha, Matthias H. Morgallab,lfred Königsrainera, Martin Schenka,∗

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

r t i c l e i n f o

rticle history:eceived 13 December 2010eceived in revised form 9 March 2011ccepted 10 March 2011vailable online 21 March 2011

eywords:manitin intoxicationnterohepatic circulation

a b s t r a c t

Background: Amatoxin poisoning induces a delayed onset of acute liver failure which might be explainedby the prolonged persistence of the toxin in the enterohepatic circulation. Aim of the study was todemonstrate 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 sampleswere measured.Results: Amanitin concentrations measured 5 h after intoxication of 219 ± 5 ng/mL (0.35 mg/kg) and

iliary excretion 64 ± 3 (0.15 mg/kg) in systemic plasma and 201 ± 8 ng/mL, 80 ± 13 ng/mL in portal plasma declined tobaseline levels within 24 h. Bile concentrations simultaneously recorded showed 153 ± 28 ng/mL and99 ± 58 ng/mL and decreased slightly delayed to baseline within 32 h. No difference between portal andsystemic amanitin concentration was detected after 24 h.Conclusions: Amanitin disappeared almost completely from systemic and enterohepatic circulationwithin 24 h. Systemic detoxification and/or interrupting the enterohepatic circulation at a later date

.

might be poorly effective

. Introduction

Over 95% of fatal mushroom poisoning in the world occurs afterngestion of Amanita species, primarily “death cap” (Amanita phal-oides). Amatoxins (Faulstich and Wieland, 1996; Karlson-Stibernd Persson, 2003), contained in these poisonous mushrooms, hasell-known effect on humans (Vetter, 1998) by binding to and

nhibiting nuclear RNA polymerase in eukaryotic cells. Lesions areound particularly in hepatocytes but also in kidney tubular cells.epatocytes incorporate the toxin fast and excrete them into theile, so that amatoxins could be detected in the gastrointestinaluid and faces (Jaeger et al., 1993), but toxin removal is mainlyased (>85%) on renal elimination.

The clinical course of amatoxin poisoning (Faulstich, 1979)s characterized by a asymptomatic incubation delay from 6 to2 h following gastrointestinal syndromes, such as vomiting, diar-hea, abdominal pain, hypoglycaemia and dehydratation after

∗ Corresponding author. Tel.: +49 7071 2986722; fax: +49 7071 29 4395.E-mail address: Martin.Schenk@med.uni-tuebingen.de (M. Schenk).

378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2011.03.016

© 2011 Elsevier Ireland Ltd. All rights reserved.

6–12 h. Hepatocellular damage will become evident clinically andbiochemically leading to progressive coagulopathy on day secondor third. In fatal cases patients develop acute liver failure includinghaemorrhages, encephalopathy and coma following renal and/ormultiorgan failure at around 6–8 days. Mortality ranges from about10%–20% in adults to 22%–50% in children (Enjalbert et al., 2002;Jander and Bischoff, 2000) reported by different authors. Amatoxinkinetics could yet not be clearly demonstrated in human poison-ing because of the delayed clinical presentation of most intoxicatedpatients.

It has first been postulated from results of animal studies withbeagle dogs that amanitin appears in the bile fluid after intravenousadministration (Faulstich and Fauser, 1973). They concluded thatthe biliary excretion of amanitin prolong their presence in systemicand enterohepatic circulation by intestinal reuptake. Therefore theclinical course of poisoning in humans and animals could be signifi-

cantly influenced by this mechanism. These results were confirmedby further experimental animal studies (Faulstich et al., 1980a;Faulstich et al., 1985; Faulstich and Fauser, 1973) and transferredinto clinical practise, although it has never been shown that aman-itin kinetics in portal plasma confirm the theory of relevant or

C. Thiel et al. / Toxicology Letters 203 (2011) 142–146 143

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ig. 1. Profile of prothrombin time (A), aspartate aminotransferase (B). (�) Indicateeriod over 120 min; black arrow indicates onset of ALF.

elayed intestinal reuptake. Based upon this theory, the attempt ofnterrupting the enterohepatic circulation through enteral admin-stration of activated charcoal or other medications has becomecommonly accepted substantial part of detoxification strategies.xtra-corporal blood purification through hemoperfusion as well aslocking the reuptake of amatoxins into hepatocytes using varioushemotherapies was introduced to detoxification management.ore recently, endoscopic nasobiliary drainage was performed to

emove bile fluid completely in the case of a 18 year old patientho ingested extremely high doses of amatoxin (Madhok et al.,

006). A total amatoxin concentration of 2.5 mg could be removedhrough bile sampling on day second after poisoning. But the clin-cal relevance remained unclear. Large animal models evaluatinghe significance of the enterohepatic amanitin reuptake still do notxist.

Aim of our study was to evaluate amanitin kinetics in the entero-epatic circulation representing the clinical relevance of enteraletoxification. Simultaneous measurements of amanitin concen-rations in systemic, portal, bile and urine samples have not beeneported previously in a pig model and the hypothesis of relevantnterohepatic circulation of amanitin via the bile could thereby beested.

. Methods

.1. Animals

After approval by the institutional review board for animal experiments,our female German landrace pigs weighing 34 ± 1 kg underwent �-amanitinAppliChem GmbH, Darmstadt, Germany) intoxication after overnight fasting. Allxperiments were performed according to the international principles governingesearch on animals and under the supervision of a veterinarian, who set the guide-ines for minimizing the suffering of the pigs. The aimed observation period in thistudy was 72 h. Pigs were euthanized by a single intravenous bolus of 10 mL T 61Intervet, Unterscheißheim, Germany).

.2. Anesthesia and surgical procedures

Intramuscular premedication consisted of atropine 0.1% (0.05 mg/kg), ketamine7 mg/kg), azaperone (10 mg/kg) and diazepam (1 mg/kg). Continuous infusion ofetamine (15 mg/kg/h), fentanyl 0.02 mg/kg/h and midazolam 0.9 mg/kg/h wasdministered to maintain anesthesia during the experiment. After oral intubation,nimals remained in deep general anesthesia receiving pressure-controlled ven-ilation (KION, Siemens Medical, Sweden) until conclusion of the study protocol.haracter of respiration, heart rate, eye movement and pain stimulus were used toonfirm the depth of anesthesia; if any of these parameters indicated a lessening of

nesthesia, infusion rates of anaesthetic agents were increased.

A stomach tube was placed for intestinal drainage. Adequate temperature8–39 ◦C was maintained with a warming mat. An antibiotic prophylaxis of 2 geftriaxon (Rocephin® , Hoffmann-La Roche, Basel, Switzerland) was given every4 h. The neck vessels were instrumented to measure arterial (Leadercath, Vygon,couen, France) and central venous pressure (Multi-Lumen Central Venous Catheter,

Time (h)

0.35 mg/kg group, (©) indicates 0.15 mg/kg group. Black bar indicates intoxication

Arrow International, Reading, PA, USA). Animals were laparotomized and a urinarycatheter was placed to measure urinary output. An 18-gauge catheter (Cavafix® ,Braun Melsungen, Germany) was inserted into the portal vein via cannulation of asmall mesenteric vein for amanitin intoxication and portal blood sampling. A 2.5 mmKehr T-tube (Ruesch GmbH, Kernen, Germany) was inserted into the common bileduct. The T-tube remained clamped so that no bile fluid was drained externally overthe observation period. The t-tube was only opened every 8 h for the sampling of500 �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 admin-istered intraportally over 120 min via the implanted portal vein catheter. Acute liverfailure was defined by a decline of prothrombin time below 30% confirmed by theclinical presence of hemodynamic changes and histology.

Monitoring included electrocardiogram, mean arterial, central venous andintracranial pressure, oxygen saturation and core body temperature. Urinary output,arterial blood gas analysis (ABL 800, Radiometer Copenhagen, Denmark) includinghaemoglobin, lactate, serum electrolytes, acid base balance and blood glucose levelswere monitored hourly and immediately corrected as required. Hydroxyethylstarch6% (Voluven® HES 130/0.4, Fresenius, Bad Homburg, Germany) and sodium chloridesolution 0.9% were used for fluid management to stabilize the hemodynamic param-eters such as mean arterial pressure within a range of 60–70 mmHg and centralvenous pressure within 6–12 mmHg. Norepinephrine was used to ensure hemo-dynamic stability in the end-stage of acute liver failure. Blood glucose levels weremaintained >100 mg/dL by glucose 20% solution, haemoglobin values remained sta-ble 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 uni-versity hospital Tuebingen (Zentrallabor, Innere Medizin IV, UniversitätsklinikumTuebingen, Germany). Prothrombin time, aspartate aminotransferase, total plasmaprotein, albumin, bilirubin, creatinine and ammonia were analyzed in systemicblood samples. Amanitin concentration measurements from systemic, portal, bileand urine samples were performed before starting surgical procedures and every8 h until conclusion of the study protocol. �-amanitin concentration measurementswere performed by an ELISA-kit (Buehlmann, Basel, Switzerland).

2.5. Statistical analysis

Results in the manuscript are reported as mean ± standard deviation (SD). Fig-ures 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 ven-tilation parameters could be stabilized in acute liver failure untilthe end of the observation period of 72 h. Relevant laboratoryparameters such as blood count, prothrombin time, aspartateaminotransferase, total plasma protein, albumin, ammonia, biliru-

144 C. Thiel et al. / Toxicology Letters 203 (2011) 142–146

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ig. 2. Haematoxylin–eosin staining of liver biopsies taken 48 h after amanitin intntoxicated animals.

in and creatinine were found to be within the normal range beforetart of intoxication. During the development of acute liver failurerothrombin time declined below 30% (Fig. 1A) paralleled by totallasma protein including albumine. Bilirubin slightly increased

n the further course of acute liver failure. Serum ammonia roseo levels of 137 ± 72 �mol/L (0.35 mg/kg) and 217 ± 241 �mol/L0.15 mg/kg) at the end of the observation period. Aspartateminotransferase increased to maximal values of 946 ± 717 U/L0.35 mg/kg) and 1381 ± 192 U/L (0.15 mg/kg) at 45 h after intox-cation and decreased in the further course (Fig. 1B). Creatinineemained stable during the observation period.

.2. Histological results

Light microscopic examination (haematoxylin–eosin staining)f liver biopsies taken 48 h after intoxication demonstrated typicalentrilobular necrosis confirming the clinical and laboratory onsetf acute liver failure as shown in Fig. 2.

.3. Amanitin kinetics

.3.1. Systemic and portal vein plasmaInitial amanitin concentration 5 h after intoxication produced

maximal level of 219 ± 5 ng/mL (0.35 mg/kg) and 64 ± 3 ng/mL0.15 mg/kg) in systemic plasma samples and 201 ± 8 ng/mL0.35 mg/kg); 80 ± 13 ng/mL (0.15 mg/kg) in portal plasma samples.manitin concentration rapidly decreased to baseline levels in sys-

emic and portal plasma within 24 h after intoxication as shown inig. 3A.

.3.2. BileAnalogue to systemic and portal vein plasma amanitin con-

entrations, maximal levels of 153 ± 28 ng/mL (0.35 mg/kg) and9 ± 58 ng/mL (0.15 mg/kg) could be detected in the bile fluid 5 hfter intoxication. The decline of amanitin concentration in theile fluid was slightly delayed respectively in the 0.15 mg/kg groupompared to systemic and portal amanitin plasma levels, so thatmanitin remained even at negligible levels detectable from base-ine up to 32 h after poisoning (Fig. 3B).

.3.3. Renal eliminationThe highest amanitin elimination was found in urine sam-

les with a maximal value of 1.4 ± 0.4 mg/h (0.35 mg/kg) and.5 ± 0.2 mg/h (0.15 mg/kg) in the low dose group, Fig. 3C. Kineticsemonstrated rapid renal elimination from plasma, but amanitinemained detectable in urine samples until the end of the observa-ion period.

ion. (A) 0.35 mg/kg body weight intoxicated animals; (B) 0.15 mg/kg body weight

3.3.4. �-Amanitin (portal - systemic plasma concentration)�-Amanitin was calculated as portal - systemic plasma

concentration to demonstrate the enterohepatic circulating aman-itin. Within the initial intoxication phase �-amanitin presentednegative values in the 0.35 mg/kg group 5 h after intoxication rep-resenting the systemic plasma excess of the high dose intoxicationgroup. In the further course values became slightly positive 16 hafter intoxication and trend to baseline levels within 24 h. In the lowdose group �-amanitin presented positive values 5 h after intoxica-tion verifying the rapid enterohepatic reuptake. Values decreasedto baseline levels within 16 h as shown in Fig. 3D.

4. Discussion

The optimal management of patients after ingestion ofamatoxin-containing mushrooms is still not determined. Becausethere is no specific antidote, treatment is only symptomatic andsupportive (Giannini et al., 2007) including gastric lavage, laxa-tives and enteral administration of activated charcoal as well asextra-corporal eliminations using haemodialysis, hemoperfusionand/or plasmapheresis. Liver assist devices to clear secondary tox-ins and support liver regeneration seemed to be beneficial (Hydziket al., 2005; Lionte et al., 2005; Sein et al., 2005). Blocking of theenterohepatic circulating amanitin by suction of the duodenal fluidthrough a nasoduodenal tube or complete external drainage ofthe bile through a nasobiliary tube placement as a more invasiveapproach for detoxification were discussed controversially becauseof the increased risk of haemorrhages through papilotomia or pan-creatitis (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 sup-port their clinical use but emergency liver transplantation remainsthe life saving therapy (Beckurts et al., 1997; Ganzert et al., 2005;Panaro et al., 2006) in fatal cases.

The clinical efficiency of these established therapeuticalapproaches is difficult to demonstrate because randomized, con-trolled clinical trials do not exist due to fortunately smallintoxication numbers and the delayed presentation of the intox-icated individual. Therefore large animal models are of primeimportance for the evaluation of the toxokinetics itself and to

standardize supportive therapy. Previous experimental studiesanalyzed amanitin uptake and kinetics in perfused pig livers. It wasdemonstrated that intraportally amanitin injection increased thehepatotoxic activity and the hepatic amanitin uptake correspondedto the portal perfusion (Faulstich et al., 1980b).

C. Thiel et al. / Toxicology Letters 203 (2011) 142–146 145

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ver 120 min; black arrow indicates onset of ALF.

Preliminary pig studies have been carried out to identify theose of 0.35 mg/kg which equivalents more than the double lethalose (LD50 = 0.15 mg/kg) in humans, to be the invariable lethalose for pigs. The presented experiment was aimed to demonstratemanitin kinetics in courses of high-dose and LD50 intoxications tovaluate the clinical significance of the enterohepatic circulation forpecific detoxification approaches. The hypothesis that amanitinlasma concentration could be significantly decreased by remov-

ng excreted amanitin in the bile fluid and thereby improving thelinical outcome has been verified neither experimentally nor clin-cally.

Overlooking the presented results, amanitin metabolism andlimination is a very rapid process lasting for approximately4 h after oral ingestion. Renal elimination kinetics demonstratedpproximately 75% �-amanitin elimination within the first 16 hfter start of intoxication.

The observed phenomenon in the low dose intoxication0.15 mg/kg) group that initial �-amanitin portal plasma exceededystemic plasma levels can be explained by a larger quantity ofiliary amanitin excretion within the early phase of poisoning.he nearly identical amanitin concentration kinetics in systemicnd portal plasma and �-amanitin disproved the theory of a clini-ally relevant delay of toxicity through biliary toxin excretion andeuptake in the enterohepatic circulation as previously reported.ntrahepatic and enterohepatic circulating amanitin might produce

delayed biliary excretion from hepatocytes into the intestine, butmanitin reuptake from the small intestine did not produce pro-onged appearance of the toxin in portal plasma samples. The facthat amanitin bile levels from 13 h after intoxication were lower inhe high dose intoxication group (0.35 mg/kg) than in the low dose

a (grey line) (A) and in the bile fluid (B). Renal elimination of �-amanitin (C) andg/kg group, (©) indicates 0.15 mg/kg group. Black bar indicates intoxication period

group may reflect more severe hepatocyte damage. The maximaltoxin levels exposed to hepatocytes could possibly affect the abilityof hepatocytes to excrete the toxin into the bile. Surprisingly serumparameters for amatoxin toxicity like aspartate aminotransferaseof the 0.15 mg/kg intoxication group exceeded the values of thehigh dose group. These findings could be explained by increased�-amanitin induced apoptosis of hepatocytes due to higher toxinlevels (Magdalan et al., 2010) which would result in a minor releaseof aspartate aminotransferase in systemic plasma samples. Never-theless it has been demonstrated (Escudie et al., 2007) that thisserum parameter can not be considered for predicting the clinicaloutcome.

We conclude that relevant toxin removal later than 16–24 hafter initial poisoning can not be gained by haemodialysis,hemoperfusion or albumin dialysis. Experimental liver transplantstudies done in pigs (Takada et al., 2001) demonstrated that no fur-ther liver injury could be observed in grafts after successful livertransplantation confirming our hypotheses. Interruption of theenterohepatic circulation might be poorly effective after 16–24 hbecause clinical relevant toxin concentration and/or intestinalreuptake do not longer exist at this time point. The delayed renalelimination of amanitin over 72 h offers the most sensitive methodidentifying this fatal intoxication.

Transferring our results into clinical practise, supportiveintensive care therapy including a rapid gastrointestinal decon-

tamination with initial enteric administration of charcoal couldbe effective especially in the early phase after ingestion. Additivehepatocyte protection with silymarin and antioxidants has beenreported to be useful in this life threatening condition. Nasobiliarydrain placement and hemoperfusion as more invasive therapeutic

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pproaches could be beneficial only by very early administrationithin 24 h after poisoning with extremely high doses of amanitin.ut it should be mentioned that the enterohepatic circulation – asemonstrated even in high dose poisoning – does not significantly

nfluence the total toxicity of amanitin.

onflict of interest statement

The authors declare that there are no conflicts of interest.

cknowledgments

The authors thank T.O. Greiner, A. Stolz and M. Seitzer for theirxcellent veterinarian and technical assistance.

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