ACCIDENTAL COLCHICINE POISONING WITH FATAL OUTCOME AFTER INGESTION OF MEADOW SAFFRON (COLCHICUM AUTUMNALE L.)-REPORT OF AUTOPSY CASE ZADESNO TROVANJE KOLHICINOM SA SMRTNIM ISHODOM NAKON INGESTIJE BILJKE MRAZOVAC (COLCHICUM AUTUMNALE L.)-PRIKAZ

Introduction. Meadow saffron (Colchicum autumnale L.) is a perennial herbaceous plant belonging to the Lily family (Liliacea). It is similar to the edible wild garlic (Allium ursinum L.). Toxic substance in meadow saffron is alkaloid colchicine. Colchicine poisoning is a very dangerous condition which can lead to a fatal outcome. Case report. A 50-yearsold male was addmited to the hospital complaining of weakness, abdominal pain, nausea, vomiting and diarrhea without blood. The day before, the patient ate two plants thinking they were wild garlic and seven hours after ingestion he felt first symptoms. During the course of the hospital stay, he had gastroenterocolitis, acute renal faliure, hepatic lesions and cardiorespiratory insufficiency with a fatal outcome. Post-mortem examination revealed: brain oedema, lung oedema and congestion, heart weighing 700 g with ventricular hypertrophy, myocardial fibrosis, liver congestion and steatosis, spleen congestion, pancreatic fibrosis. Organs sections were taken for histopathological analysis. Body fluids and parts of organs were toxicologically analyzed. Histopathological findings were: brain oedema, diffuse perivascular and interstitial myocardial fibrosis, myocardial haemorrhage, lungs congestion and oedema, microvesicular and macrovesicular liver steatosis, centrilobular liver necrosis, lymphocytic inflammatory infiltrate in liver portions, red pulp congestion of the spleen, kidney congestion and interstitial bleeding, coagulation necrosis of the proximal tubules of the kidney. Toxicological analysis showed colchicine in the blood ? 0.011 mg/L, urine ? 0.051 mg/L, liver with gallbladder ? 0.007 mg/kg, kidney ? 0.008 mg/kg. Conclusion. Ingestion of meadow saffron can lead to poisoning with a fatal outcome due to the presence of the alkaloid colchicine. Colchicine intoxication should be suspected in patients with gastrointestinal symptoms after consuming wild plants.


Introduction
Meadow saffron (Colchicum autumnale L.) (Fig. 1) is a perennial herbaceous plant from the Lily family (Liliacea).It is commonly known as autumn crocus, wild saffron, naked lady, son-before-the-father.It's flower is very similar to the flower of saffron (Crocus sativus L.), it has no special smell and it is spread over mountain meadows and pastures.
The meadow saffron seems like the edible wild garlic (Allium ursinum L.) (Fig. 2), commonly called "sremush" in Serbian, which has specific garlic-like smell 1 .The toxic substance found in the meadow saffron is alkaloid colchicine.Colchicine poisoning is a very dangerous condition and fatal outcome is one of the possible consequences.The antidote for this substance does not exist yet, but there are possibility of colchicine specific monoclonal antibodies to be used in future 2 .All parts of the plant are poisonous and contain colchicine, but the highest concentration of this alkaloid is in seeds (0.2-0.8 %) and bulbs (0.4-0.6 %), while it is low in leaves.There are some other toxins present in the meadow saffron, but they are less dangerous to humans 1 .Colchicine is also used in medicine for the treatment of gout 3 , Mediterranean fever 4 , sarcoidosis 5 , scleroderma 6 , amyloidosis 7 , Behcet's disease 8 , Paget's disease 9 , psoriasis 10 , cutaneous vasculitis 10 , alcoholic cirrhosis of the liver 11 and primary biliary cirrhosis 12 .

Case report
A 50-year-old man was admitted to General Hospital in Loznica because of a suspicion of herbal poisoning.One day before the admission to hospital at 03:00 p.m. patient had eaten two whole herbs regarded as wild garlic.Seven hours after consumption the first symptoms appeared.He had diffuse abdominal pain, nausea, vomiting, and diarrhea without blood (at least ten watery stools).The patient had fifteen years history of arterial hypertension and diabetes mellitus type II treated with insulin.On examination he was conscious, well oriented to the place, time and person with slowed communication and euphoric.for histopathological analysis and body fluids (blood, urine), gastric contents and parts of organs (liver with a gallbladder, kidney, brain) for toxicological analysis.
Toxicological analysis: Toxicological analysis were done at the Military Medical Academy Belgrade, National Poison Control Center, the Institute of Toxicology and Pharmacology, the Department of Toxicological Chemistry.The presence of alkaloid colchicine was analyzed using method of liquid chromatography-mass spectrometry (LC-MS), comparing the mass spectra of examined compounds (blood, urine, liver with gallbladder, kidney and brain) with the spectra of standards (Table 2).The presence of ethanol has been established using method of gas chromatography (GC) with flame ionization detector (FID) -"head space" technique in blood-0.23 ‰ (4.9924 mmol/L), urine-0.58‰ (12.5895 mmol/L) and gastric contents-0.37 ‰ (8.0313 mmol/L).

Disccusion
A case of accidental fatal poisoning of a middle-aged male caused by colchicine from a meadow saffron was shown.One of the most common causes of meadow saffron ingestion and colchicine poisoning is its great similarity to the wild garlic, which is an edible plant and has a distinctive garlic odor.In addition to the accidental meadow saffron ingestion, suicide cases by meadow saffron ingestion were also reported 13 .Due to the extreme health risk, it is important to identify the symptoms and signs of colchicine poisoning and obtain anamnestic data in order to provide adequate immediate medical care, even though final confirmation of the poisoning is provided by toxicological analysis.Symptoms and signs of colchicine poisoning are most often manifested by gastrointestinal disorders such as nausea, vomiting, abdominal pain and diarrhea 14,15 as it was at the onset of the symptoms in our case.Clinical manifestations of poisoning, according to Stapczynski et al. develop through three phases 16 .The first phase of poisoning begins 4 to 12 hours after the ingestion and is characterized by vomiting and diarrhea.The second phase usually begins on the second day after ingestion and is followed by life-threatening conditions, such as arrhythmia, heart failure, kidney and liver failure, bone marrow damage and coagulopathy.
The third phase begins 5 to 7 days after ingestion, and it leads to leukocytosis and then very rapidly to pancytopenia (leukocytopenia and thrombocytopenia), which indicates bone marrow aplasia 17,18,19 .In the third stage poisoning may result in alopecia.This alopecia is transient, although cases in which there was no recurrence of hair have also been reported 20,21,22,23 .If patient survives poisoning, peripheral neurophathy may appear as longterm consequence 24 .Colchicine is a cytostatic that interrupts cell mitosis by interfering with the formation of microtubules and the mitosis spindle.It reversibly binds to tubulin and prevents its polymerization 25 .The half-life of this linked complex is 36 hours 1 .Colchicine primarily blocks cell mitosis in organs and tissues with high intensity of cell division.This is particularly expressed in the gastrointestinal system and bone marrow, which is manifested by a characteristic clinical presentation that was also seen in this case 26 .
Colchicine shows its toxicity to other organs, and in a number of cases occur arrhythmias, pancreatitis, acute liver failure 27,28,29 .Colchicine is accumulated in leukocytes and exhibits an inhibitory effect on leukocyte adhesion, mobility, mobilization, phagocytosis, degranulation of lysosomes and chemotaxis.Applied in the therapeutic dose, it blocks the release of chemotaxis factors by neutrophils and synoviocytes and thus reduces inflammation. 1Colchicin is predominantly absorbed in the small intestine.Due to its liposolubility it is largely bound to plasma proteins, primarily albumin, in blood.Colhicin is mostly metabolised in the liver, while one fifth remains unchanged and is eliminated by kidneys 30 .Colchicine is subjected to enterohepatic circulation.It is reported in the literature that when colchicine is administered in doses less than 0.5 mg/kg body weight, all poisoned survive, while when administered an amount higher than 0.8 mg/kg, all poisoned ones die.Toxic effects are rarely reported if the concentration is less than 3 ng/ml.
Consumption of 5 g of meadow saffron leads to fatal outcome 1,31,32 .Lethal concentration of colchicine in the blood is 0.009-0.024mg/L 33 .It is found in higher concentration in kidneys, liver and spleen than in heart, skeletal muscles and brain.Hepatic and biliary excretion are possible reasons for the onset of gastrointestinal symptoms 34 .Due to liver and kidney failure hepatic enzyme values alanine aminotransferase (ALT) and aspartate aminotransferase (AST), bilirubin and blood urea nitrogen are elevated 17 .Extreme damage of the central lobular regions of the liver is typical and can be explained by characteristic liver perfusion and by increasing the concentration of poisons in the liver and bile as a result of conspicuous enterohepatic circulation 35 .Myoglobinuria due to the effects of colchicine such as rhabdomyolysis and hypoxia may impair renal function and lead to renal failure 36 .Elevated blood creatine kinase (CK) and lactate dehydrogenase (LDH) levels are noted in colchicine poisoning.Increased blood CK concentration is associated with hypoxic damage of brain and heart.Brain damage (pericellular and perivascular edema) is probably due to the multiple systems organ failure.Autopsy findings in colchicine poisoning may include inflammation of gastrointestinal mucosa, lung and brain oedema, centrilobular necrosis of the liver, proximal tubule necrosis in kidney, petechial haemorrhage in fatty tissue 37,38 .Disseminated petechial bleeding in fatty tissue that some authors describe are caused by thrombocytopenia and liver failure 17 .There is still no antidote for colchicine poisoning, therefore treatment is symptomatic.In the future, monoclonal antibodies to colchicine will improve chance for survival 2 .
Although body fluids (blood, urine) and parts of the organs can be taken for toxicological analysis in fatal colchicine poisoning, liver with gallbladder is the best sample for analysis, because concentration level of substance is the highest 39 .In our case, samples of body fluids (blood and urine), gastric contents, liver with gallbladder, kidney and brain were taken for toxicological analysys during the autopsy.Presence of colchicine has been proven in blood, urine, liver with gallbladder and kidney.Blood concentration of colchicin was 0.011 mg/L, which is the lethal concentration 33 .

Conclusion
Ingestion of the meadow saffron plant is rare, but it is life threatening condition since it's content colchicine poisoning is commonly lethal.The significance of colchicine concentrations determination in body fluids and organs is in clinical toxicology for the more effective treatment of patients, as well as in forensic medicine in cases of accidental or rarely suicidal fatal poisoning.Colchicine poisoning is recommended to be treated supportively and symptomatically immediately until the finish of clinical trials for specific antidote, anti-colchicine antibody.Reference 37. Gilbert JD, Byard RW.Epithelial cell mitotic arrest -a useful postmortem histologic marker in cases of possible colchicine toxicity.Forensic Sci Int 2002; 126(2): 150-2.38.Sannohe S, Makino Y, Kita T, Kuroda N, Shinozuka T. Colchicine poisoning resulting from accidental ingestion of meadow saffron (colchicum autumnale).J Forensic Sci 2002; 47(6): 1391-6.39.Kintz P, Jamey C, Tracqui A, Mangin P. Colchicine poisoning: report of a fatal case and presentation of HPLC procedure for body fluid and tissue analyses, J Anal Toxicol 1997; 21: 70-2.

Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .
Fig. 1.Meadow saffron (Colchicum autumnale L.) The electrocardiogram showed sinus rhythm without change in ST and T segment.Abdomen was soft in the chest level, diffusely painfully sensitive to deep palpation, with no muscular defense.The extremities were with normal colour and without oedema.With the worsening health condition, the patient was shifted to referral tertiary health care institution, Military Medical Academy Belgrade, National Poison Control Center, Clinic for Emergency and Clinical Toxicology, same day at 03:45 p.m. On the arrival the patient was in the same condition as in the previous health care institution.The electrocardiogram showed sinus rhythm with occasional extrasystoles and no significant conduction and repolarization defects.The chest X-ray was normal.Laboratory data out of reference range are showen in (Table1), coagulation factors were: II 0.33; V 0.1; VII 0.13; Clinical findings: Auscultation of the heart and lungs showed no abnormalities.Patient had an oxygen saturation level of 96 %, a blood pressure of 140/90 mmHg and a heart rate of 103 counts/min.IX 0.61; APTT 117; INR 5.21; arterial blood gases were: pH 7.302; pCO2 28.3 mmHg; pO2 68.9 mmHg; BE 11.6, bicarbonates 15.9, lactates 5.4 mmol/L.Oxygen saturation level was 90 %.Colchicine was proven in urine by toxicological analysis.Patient developed gastroenterocolitis, acute renal faliure, hepatic leasion and cardiorespiratory insufficiency, and he was treated with rehydration and supportive therapy (oxygen, activated carbon, proton-pump inhibitors and diuretics).The next day, the patient went into cardiac arrest and cardiopulmonary resuscitation was unsuccessful.Death was pronounced at 10:55 a.m.Medicolegal autopsy was conducted at the Military Medical Academy Belgrade, Institute for Pathology and Forensic Medicine.Autopsy findings: The external examination of the body was unremarkable.Medicolegal autopsy revealed oedematous brain weighing 1500 g, congested and oedematous lungs with left and right lung weighing 650 g and 800 g respectively, the heart weighing 700 g with left and right ventricular hypertrophy (19 mm and 7 mm), cardiac muscle fibrosis, coronary arteries atherosclerosis, aortic atherosclerosis, liver congestion and steatosis, spleen congestion, and pancreatic fibrosis.During the autopsy, samples of the organs were taken