Arch. Toxicol.

33, 49--54 (1974) 9 by Springer-Verlag 1974

A Case of Fatal Poisoning by Gyromitra esculenta

G. V. Giusti a n d A. C a r n e v a l e Istituto di Medicina Legale e delle Assicurazioni Universits Cattolica dcl Sacro Cuore, Roma Received June 26, 1974 Abstract. A case of fatal poisoning by the mushroom Gyromitra esculenta (false morel) in a 53-year-old woman is reported. Clinical data were characterized initially by vomiting and diarrhea, and subsequently by hypotension, anuria, jaundice, hemiplegia, and coma. Death followed on the 3rd day. Prominent pathologic findings were brain edema, necrosis, fatty degeneration of the liver, nephrosis, scattered petechiae, and small hemorrhages. Gyromitrin was extracted in methanol, purified by thin layer chromatography, identified by I. R. spectrometry, and weighed. The relationship between O. D. at 277 m~ and the concentration (0.1--0.5 mg/ml in absolute ethyl alcohol) was established. Key words: Gyromitra esculenta (False Morel) -- Mushroom Poisoning. Zusammen/asssung. Es wird ein tSdlicher Vergiftungsfall mit Gyromitra esculenta (Friihjahrslorchel) bei einer 53-jahrigen Frau beschrieben. Initial kam es zu Erbrechen und Durchfall, danach zu Blutdrucksenkung, Anurie, Ikterus, Hemiplegie und Koma. Der Tod trat am 3. Tage ein. Der pathologische Befund zeigte Hirn5dem, Nekrosen und fettige Degeneration der Leber, Nephrose sowie Petechien und Hiimorrhagien. Gyromitrin wurde in Methanol extrahiert, diinnschichtchromatographisch gereinigt, mittels IR-Spektrometrie identifiziert und gewogen. Die Relationen zwischen Absorptions-Werten bei 277 mbt Wellenl~nge und Konzentration {0A--0.5 mg/ml in rcinem Athanol) werden angegeben. Schli~sselwSrter: Gyromitra esculenta (Friihjahrslorchel) -- Pilzvcrgiftung. Cases of f a t a l poisoning b y t h e m u s h r o o m Gyromitra esculenta are s e l d o m r e p o r t e d in t h e m e d i c a l l i t e r a t u r e because of t h e r a r i t y of t h i s e v e n t a n d t h e difficulty of a precise diagnosis. A n e x h a u s t i v e review on t h i s t o p i c was p u b l i s h e d in 1967 b y F r a n k e et al. ; since then, no o t h e r cases h a v e been described. This poisoning seems to be r e l a t i v e l y f r e q u e n t in E a s t e r n E u r o p e a n d G e r m a n y . A t least t w o f a t a l cases h a v e been p u b l i s h e d in t h e U n i t e d S t a t e s (Dearness, i924, H e n d r i c k s , 1940) b u t as far as is k n o w n no cases h a v e been d e s c r i b e d in s o u t h e r n E u r o p e . F a t a l i t y is r e p o r t e d t o v a r y b e t w e e n i 4 . 5 % (74 f a t a l cases o u t of 513 d e s c r i b e d in t h e m e d i c a l

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G. V. Giusti and A. Carnevale

l i t e r a t u r e from i782 to i965, after F r a n k e et al. 1967) a n d 34.5% (Orlov, t953). The t o x i n was first isolated i n i885 b y B o e h m a n d Kfilz a n d called helvellic acid. I t was synthesized a n d definitely identified, b y List a n d L u f t (1968), as t h e N - m e t h y l - N - f o r m i l h y d r a z o n e of acetaldehyde (gyromitrin). These authors describe g y r o m i t r i n as a colorless, volatile, oily substance, with a m e l t i n g p o i n t of i9.5 ~ C i n v a c u u m . The substance can be crystallized b y cooling a n d is soluble i n water, methanol, ethanol, acetone, ether, chloroform, benzene, m e t h y l e n e chloride, a n d carbon tetra~hloride. I t is t r a n s f o r m e d to a b r o w n oily substance when oxidized b y air. I t is a c o m m o n opinion t h a t cooking destroys the t o x i n : however fatalities have been reported after cooking of the m u s h r o o m a n d removal of the juice (Umber, 19i6; Herzog, 1917; Welsman, 1934). Case Report C. F., a 53-year-old, feeble-minded woman, ate some raw mushrooms that she had picked in the fields. The following day she vomited and had diarrhea. She was treated in a minor hospital with plasm~ infusions and cortieosteroids. As the symptoms still persisted, in the third day she was admitted to this University Hospital. On entering in the Intensive Care Unit, jaundice, hypotension, anuria, and a severe enlargement of the liver, together with a right hemiplegia were noted. Chemical tests yielded the following results: glucose 25 mg %; nitrogen 26 mg %; Na + t33 mEq/L; K + 3.4 mEq/L; Ca++ I1 mg %; total bilirubin 2.3 mg %; direct bilirubin 1.65mg %; SGOT> t000U.; SGPT> 1000U. No other tests could be made because the woman died a few hours after admittance. At autopsy, petechiae and interstitial hemorragiolae were noted in the mediastinum, mesentery, pleural surfaces, and renal pelvis; stomach and duodenum contained about 100 ml of blood. The brain showed a conspicuous edema, and the liver was enlarged, with the appearance of hepar variegatum. Histologiv sections, stained with hematoxylin and eosin, showed passive congestion of the viscera, massive edema of the brain, and slight edema of the lungs with desquamation of large round cells. The liver showed an actual necrosis and dissolution of the parenchyma. Some areas were characterized by marked fatty changes in the remaining cells and by intraparenchymatous hemorrhage. In the kidneys, swelling of the endothelial and epithelial cells was noted; proteinaceous material was observed in the capsular lumina and hyaline cylinders in the tubuli.

Toxilogic analyses were carried o u t with the aim of identifying the m u s h r o o m to which the d e a t h could be a t t r i b u t e d . The gastro-enteric tract, liver, a n d k i d n e y s were homogenized s e p a r a t e l y a n d covered with methanol. After a few days, the m e t h a n o l was decanted, centrifuged, a n d filtered, a n d the e x t r a c t was concent r a t e d o n a r o t a t i n g e v a p o r a t o r a t room t e m p e r a t u r e . The extract t h e n h a d a very distinct u n p l e a s a n t odour, similar to t h a t of mouse urine. This served as a basic i n d i c a t i o n for the diagnosis. However, the presence

Poisoning by Gyromitra esculenta Table i Rf values I00 iodine vapors 90 65~ 52a 34 t0 ninhydrin

51

Colour Reactions p-DMBA DPH vanillin-H2SO4 and heating yellow . . ----yellow blue-violet blue-violet blue-violet blue-violet blue-violet

yellow -. . . . . . yellow pink yellow --

These spots were much more distinct when the extract was chromatographed after standing for three days at room temperature. of a m a n i t a t o x i n s was e x c l u d e d b y a p p l y i n g t h e t h i n l a y e r c h r o m a t o g r a p h i c m e t h o d of P a l y z a a n d K u l h s (1970). To e x t r a c t g y r o m i t r i n , following t h e suggestions of L i s t a n d L u f t (1968), t h e s a m e v o l u m e of distilled w a t e r was a d d e d t o t h e m e t h a n o l i e residue, a n d t h e m e t h a n o l was distilled on a r o t a t i n g e v a p o r a t o r a t r o o m t e m p e r a t u r e . T h e w a t e r was t h e n e x t r a c t e d w i t h e t h y l ether, which was s u b s e q u e n t l y s e p a r a t e d a n d e v a p o r a t e d . A residue of 133 m g was obt a i n e d , which was t h e n dissolved in i.33 m l of a b s o l u t e e t h y l alcohol. P a r t of this m a t e r i a l was i n i t i a l l y c h r o m a t o g r a p h e d on a c t i v a t e d silica gel G F plates, using t h e m i x t u r e of m e t h y l e n e c h l o r i d e - m e t h a n o l 9 : i as a s o l v e n t s y s t e m (List a n d L u f t , i968). A b e t t e r r e s o l u t i o n was, however, o b t a i n e d using o n l y chloroform as a solvent. C h r o m a t o g r a p h i c results were f o u n d to be i d e n t i c a l for t h e t h r e e e x t r a c t s . T h e y are s u m m a r i z e d in Table t. I . R . a n d U.V. s p e c t r a were o b t a i n e d b y r e p e a t i n g t h e c h r o m a t o g r a p h i c r u n a n d s c r a p i n g off areas a t R f ' s 0.90, 0,24, a n d 0.10. T h e m a t e r i a l was t h e n e l u t e d in a few d r o p s of chloroform for I . R . spectrep h o t o m e t r y (NaC1 0 . i m m cells), a n d in a b s o l u t e e t h y l alcohol for U.V. spectrophotometry.

Fig. 1. I.R. spectrum of the spot at Rf 0.90

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G. V. Giusti and A. Carnevale Table 2 Rfvalues x 100 90 65 52 34 t0 max. 264, 274 (shoulders) 274, 266, 257 257, 264, 272 250, 257, 264, 270 272, 257, 266, 264 min. 269, 260, 253 269, 262, 254 267, 262, 254, 248 268, 265, 262, 255

Table 3 Rf value 0.90 0.65 0.52 0.34 0.10 mg (total) 24.73 1.60 t.60 27.26 16.62 % 18.61 1.20 1.20 20.51 12.50

I . R . s p e c t r u m of t h e s p o t a t R f 0.90 was f o u n d to be identical to t h e one r e p o r t e d b y L i s t a n d L u f t (1968) (Fig. i ) for g y r o m i t r i n , while spots a t R f 0.34 a n d 0.10 g a v e a s p e c t r u m lacking p e a k s below 1700 cm -2. U.V. s p e c t r a of these t h r e e spots were c h a r a c t e r i z e d b y a unique, clearly d i s t i n c t m a x i m u m a t 277 m~, a n d b y a m i n i m u m a t 254 m~. W i t h ageing (three days), s p e c t r a a p p e a r e d d e e p l y modified (Table 2). Quantitative determinations were m a d e on t h e intestine e x t r a c t b y r u n n i n g i 0 0 ~1 of t h e initial alcoholic solutions on TLC plates a n d weighing t h e m a t e r i a l e l u t e d a t c o r r e s p o n d i n g Rf's. (Table 3). T h e q u a n t i t y a p p e a r e d sufficient to establish a relationship b e t w e e n t h e o p t i c a l d e n s i t y a t 277 mf~ a n d t h e c o n c e n t r a t i o n (mg/ml of a n alcoholic solution) b y m e a s u r i n g t h e O.D. of k n o w n dilutions of t h e t h r e e m a i n c h r o m a t o g r a p h i c spots. This relationship was f o u n d to be linear for c o n c e n t r a t i o n s from 0.1 to 0.5 m g / m l a n d is r e p r e s e n t e d b y t h e following formulas : R f Values 0.90 y = 0 . 9 i x 0.04 0.34 y = 0.967 x - - 0.0283 0.t0 y = 1.t3 x + 0.030 where y = O.D. a t 277 m~, a n d x = C (mg/ml). Discussion Clinical d a t a a n d p a t h o l o g i c findings in this case are in a g r e e m e n t w i t h d a t a r e p o r t e d b y F r a n k e et al. (1967) in t h e i r review. On this basis, t h e

Poisoning by Gyromitra esculenta

53

case could be qualified as a mushroom poisoning, no real difference existing, from this point of view, between the poisoning from Gyromitra esculenta "and Amanita phalloides. A diagnosis therefore can be derived only from the results of the toxicologic analysis. A good indication for diagnosis is given by the odour of the extract, which seems to be typical. The method t h a t was followed, mainly based on the results obtained b y List and Luft (i968) from the study of the mushroom, has proved useful and rapid in practice in identifying the main poisonous component, gyromitrin. I t also permits other components to be revealed, although no hypothesis can be advanced about their nature, i.e. whether they represent metabolites of gyromitrin, or are compounds normally present in the mushroom, or even degradation products developed during the extraction procedures. I t is known t h a t the mushroom contains m a n y hydrazine derivatives (List and Luft, i969), and the possibility of a rapid degradation of the purified extracts is demonstrated here. The quantitative determination b y this U.V. spectrophotometric method allows an alternative procedure, t h a t seems specific providing it is carried out after chromatography, to the titrimetric technique of List and Luft (1969) employed on mushroom extracts. A method therefore can be proposed for qualitative and quantitative determination of gyromitrin in viscera. After extraction and chromatography, the spot at R f = 0.90 is eluted in a known quantity of ethyl alcohol and the U.V. spectrum registered. At the same time the O.D. of this solution is read and the concentration easily calculated. I f clinical and pathologic data are in agreement, this simple analysis seems sufficient for a really precise diagnosis. I n case of doubt, the registration of the I.R. spectrum should be made.
References

Boehm, R., Kiilz, R. Z. : ~ber den giftigen Bestandteil der ei~baren :Morchel (Helvella esculenta). Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 19, 43 (1885) Dearness, J.: Gyromitra poisoning. Mycologia 16, 199 (1924) Franke, S., Freimuth, U., List, P. H.: ]~ber die Giftigkeit der Friihjahrslorchel Gyromitra (Helvella) esculenta Fr. Arch. Toxikol. 22, 293 (1967) Hendricks, H. V. : Poisoning by false morel (Gyromitra esculenta). J. Amer. reed. Ass. 114, 1625 (1940) Herzog, G.: Miinch. med. Wschr. 44, 1366 (1917), (quoted by List and Luft, 1968) List, P. H., Luft, P. : Gyromitrin, das Gift der Friihjahrslorehel, Arch. Pharm. (Weinheim) 301, 294 (i968) List, P. H., Luft, P.: Naehweis und Gehaltsbestimmung yon Gyromitrin in frischen Lorcheln. Arch. Pharm. (Weinheim) 802, 143 (1969) Orlov, N. I.: Sjedobuye i jadovitye griby gribenye ostravlenjia i ich profilaktica, Megdiz, Moscow i953, p. 44 (quoted by List and Luft, 1968)

54

G. V. Giusti and A. Carnevale

Palyza, V., Kulh~nek, V. : i)ber die chromatographische Analyse yon Toxinen aus Amanita phalloides. J. Chromatog. 58~ 545 (1970) Umber, F. : Dtsch. med. Wschr. 42~ 627 (1916) (quoted by List and Luft, 1968) Welsman, L. : Pilzk. 18~ 119 (1934), (quoted by List and Luft, 1968) Dr. G. V. Giusti Mr. A. Carnevale Istituto di hfedieina Legale e delle Assicurazioni Universit~ Cattolica del Sacro Cuore Via della Pineta Saechetti, 644 1-00t 68 Roma, Italy