Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 52) docx

6.2
6.2
© Springer-Verlag Berlin Heidelberg 2005
II.6.2 Mushroom toxins
by Kunio Gonmori and Naofumi Yoshioka
Introduction
As many as 5,000–6,000 mushroom species are growing in the world. Among them, only about
1,000 species are named; the majority of them are unnamed.  e number of species of edible
mushrooms in Japan is about 300; that of toxic mushrooms is said to be about 30. Various types
of toxic mushrooms exist; some show high toxicity, while others show hallucinogenic actions.
Morphological and chemical analyses for mushrooms are occasionally required in forensic sci-
ence practice. In this chapter, the characteristics of the representative toxic mushrooms and
some chemical methods for their toxins are presented.
Current situation of mushroom poisonings in Japan
According to “National Record of Food Poisoning Incidents” [1], the number of mushroom
poisoning incidents taking place in Japan in 1974–1997 was 1,068; it was 431 in 1988–1997
(10 years) with 1,842 poisoned people, including 20 fatal victims
a
. Among the 431 incidents,
the numbers of incidents according to causative mushrooms are: Rhodophyllus rhodopolius
plus Rhodophyllus sinuatus, 133; Lampteromyces japonicus, 127; Tricholoma ustale, 42; Amanita
virosa plus Amanita verna, 16; Amanita pantherina, 15; Clitocybe acromelalga, 15; Psilocybe
argentipes (a species of magic mushrooms), 12; other mushrooms, 36; not speci ed, 35
(
> Figure 2.1)
b
.
Toxic mushrooms can be classi ed into 6 groups according to their actions as follows.
•  ose which destroy cells, injure the liver and kidney and thus may cause death (latent
period, 6–10 h; Amanita virosa, Amanita verna and Amanita phalloides).
•  ose which act on the autonomic nervous system and provoke symptoms, such as sweat-

ing, lacrimation, vomiting and diarrhea (latent period, 20 min–2 h; Clitocybe gibba, Inocybe
species and others).
•  ose which inhibit the metabolism of acetaldehyde in blood (disul ram-like e ect), caus-
ing a  ushing phenomenon and palpitation upon drinking alcohol concomitantly (latent
period, 20 min–2 h; Clitocybe clavipes, Coprinus atramentarius and others).
•  ose which act on the central nervous system and provoke abnormal excitement and
hallucinations (latent period, 20 min–2 h; Amanita pantherina, Psilocybe argentipes and
others).
•  ose which irritate the gastrointestinal tract and provoke symptoms, such as abdominal
pain, vomiting and diarrhea (latent period, 30 min–3 h; Rhodophyllus rhodopolius, Lamptero-
myces japonicus and others).
• Others which cause swelling or necrosis of tips of extremities or sharp pain due to distur-
bances of the peripheral nerves (Clitocybe acromelalga and others).
470 Mushroom toxins
> Table 2.1 shows the outline of the mushroom poisoning analyses, which the authors had
undertaken in recent 9 years. As shown in this table, the number of the poisoning cases, in
which Amanita virosa had been (suspected to be) causative, was as many as 10. Amanita virosa
is highly toxic and sometimes causes fatalities.  e highest incidence of the Amanita virosa in
our laboratories is interpreted to mean that such fatal poisoning cases are selectively brought
to our Department for analysis. Two cases were suspected of poisoning by Rhodophyllus
rhodopolius (
> Table 2.1).
Representative mushrooms causing poisoning cases
Rhodophyllus rhodopolius ( > Figure 2.2)
 is mushroom shows the highest incidence of poisoning in Japan, because a very similar
edible species Rhodophyllus crassipes is available and grows at similar locations.  e poisoning
symptoms are vomiting, diarrhea and abdominal pain appearing 30 min–3 h a er ingestion.
 e stem of Rhodophyllus rhodopolius is easily crushed by pressure with the  nger, but that of
the edible Rhodophyllus crassipes is not.  e toxic compound being contained in the mush-
room is reported to be muscarine or choline.

Incidence ratio of mushroom poisonings according to species in Japan. It is calculated from the
data of “National Record of Food Poisoning Incidents”. The number of the mushroom poisoning
incidents was 431; the poisoned subjects involved were 1,842 people.
⊡ Figure 2.1
471
⊡ Table 2.1
Outline of mushroom poisoning analyses undertaken by Department of Legal Medicine, Akita University School of Medicine
No. Year Requesting institution Causative mushroom The patient
number
Outcome Specimen and detectability of the toxin
1 1993 H Univ. Dept. Legal Med. Amanita virosa 1 dead detected from the liver and the mushroom
2 1996 T Kyodo Hosp. Dept. Anaesth. Amanita virosa?
(mushroom not available)
1 dead not detected from blood, the liver or kidney
3 1996 Y Univ. Dept. Intern. Med Amanita virosa?
(mushroom not available)
1 dead not detected from blood
4 1997 D Univ. Emerg. Units Amanita virosa?
(mushroom available)
1 alive not detected from blood or the mushroom
5 1997 F Univ. Emerg. Units Amanita virosa?
(mushroom available)
1 alive not detected from blood stomach contents or
the mushroom
6 1998 A Pref. Hosp. Dept. Intern. Med. Agaricus blazei
(mushroom available)
2 alive not detected from blood or the mushroom
7 1998 O Pref. Hosp. Emerg. Units Amanita virosa
(mushroom available)
1 alive not detected from blood urine or the

mushroom
8 1998 J Med. Univ. Emerg. Units
and Dept. Nephrol
Amanita virosa 7 1 dead detected from blood of one patient
9 1998 J Med. Univ. Emerg. Units
and Dept. Nephrol.
Amanita virosa 5 alive not detected from blood or urine
10 1998 I Pref. Hosp. Emerg. Units Amanita virosa 1 alive not detected from blood or urine
11 1999 O Munic. Hosp. Dept. Urol. Amanita neoovoidea 1 alive not detected from blood
12 1999 J Med. Univ. Emerg. Units
and Dept. Nephrol.
not clear (Rhodophyllus
rhodopolius?) mushroom-
containing wheat-flour noodles
3 alive not detected from blood, urine or the
mushroom
13 1999 J Med. Univ. Emerg. Units
and Dept. Nephrol.
Lampteromyces japonicus
(mushroom available)
2 alive not detected from blood, urine or the
mushroom
14 1999 A Munic. Gen. Hosp. Dept. Intern.
Med.
not clear
(Rhodophyllus rhodopolius ?)
1 alive not detected from blood
15 2000 Y Publ. Health Center Amanita neoovoidea
(only mushroom available)
0 – not detected from the mushroom

16 2000 K Med. Univ. Emerg. Unit
and Dept. Pediat.
Amanita virosa
(mushroom available)
2 alive not detected from blood or urine, but
detected from the mushroom
17 2001 A Police H. Q. a magic mushroom (cultivated
with a culture medium)
1 dead detected from blood, urine and the
mushroom
Representative mushrooms causing poisoning cases
472 Mushroom toxins
Rhodophyllus rhodopolius.
⊡ Figure 2.2
Amanita virosa.
⊡ Figure 2.3
473
Amanita virosa ( > Figure 2.3)
It is a very beautiful white mushroom growing in mountain areas; it is thus being called “ de-
stroying”. Only with one mushroom of Amanita virosa, 2 or 3 adult subjects can be killed.  e
Amanita genus mushrooms should be watched most carefully also in the forensic toxicological
point of view.  e main toxin of this genus is considered to be amanitin (
> Figure 2.4) or
phalloidin (
> Figure 2.5).  e amanitin is subdivided into α-, β- and γ-amanitins. In Japan,
Amanita virosa and Amanita verna glow generally, while in Europe and America, Amanita
phalloides is responsible for poisoning.  ere is a report insisting that phalloidin does not exert
toxic e ect upon oral intake [2]. When chemical analysis was performed for 45 patients of
Structure of amanitin.
⊡ Figure 2.4

Structure of phalloidin.
⊡ Figure 2.5
Representative mushrooms causing poisoning cases
474 Mushroom toxins
Amanita verna poisoning in France, amanitin could be detected from plasma in only 11 of
43 patients, from urine in 23 of 35 patients, from the contents of the stomach and duodenum
in 4 of 12 patients and from feces in 10 of 12 patients [3].  e blood concentrations of amanitin
are highly dependent on the intervals a er ingestion; the concentrations in urine and the con-
tents of the stomach and duodenum are much higher than those in blood, and these specimens
are more suitable for analysis of amanitin [3].
Lampteromyces japonicus
 is is one of the most common toxic mushrooms with the highest incidence of poisoning,
like Rhodophyllus rhodopolius, in Japan. It is usually mistaken for the edible Lentinula edodes,
Pleurotus ostreatus, Panellus serotinus or others.  e shape of Lampteromyces Japonicus is
semicircular or kidney-like; the size is as large as 10–25 cm. When it matures, the color of its
cap part becomes purplish brown or dark brown.  e stem is as short as 1.5–2.5 cm and
located at a side part of the cap; there is a crater like protrusion in the reverse side of the cap
just around the stem. When this part of the cap including the stem is cut, dark coloration can
be observed there for the matured mushroom (
> Figure 2.6), and the folds and hyphae lumi-
nesce in a light yellow color in the dark; these are very useful for its discrimination. However,
it should be cautioned that the above dark coloration is absent or obscure in the immature
mushrooms. According to the growing circumstances, the Lampteromyces japonicus may show
a round cap like Lentinula edodes, and thus is confusing (
> Figure 2.7). Since the Lamptero-
myces mushrooms can grow in colonies on the dead beech or maple trees, a great number
of the mushrooms may be harvested at a single location.  e harvester distributes them to
neighbors and relatives, resulting in simultaneous occurrence of many poisoned patients.
Its toxin is lampterol ( illudin S), which causes vomiting and diarrea.  e fatality by the toxin is
very rare.

How to discriminate Lampteromyces japonicus.
⊡ Figure 2.6
475
Magic mushrooms (> Figure 2.8)
 e magic or hallucinogenic mushroom is a popular name for ones which exhibit hallucina-
tion (visual and auditory), mental derangement and muscle  accidness. In central and south
America, such mushrooms were being used in religious ceremonies since ancient times.  e
hallucinogenic e ects vary according to di erent individuals; they are similar to those ob-
tained with LSD, though they are much weaker than those of LSD.  ey were illegally sold, in
the forms of cultivation kits, dried pieces or tablets, on the streets and via the Internet before
2002. Various species of the Psilocybe genus are being used as magic mushrooms. Most magic
Lampteromyces japonicus mushrooms having circular umbrellas, which tend to be mistaken for
edible Lentinula edodes mushrooms.
⊡ Figure 2.7
Cultivation of “magic mushrooms” (Psilocybe cubensis).
⊡ Figure 2.8
Representative mushrooms causing poisoning cases
476 Mushroom toxins
mushrooms being circulated in Japan are Psilocybe cubensis and/or P. subcubensis and Cope-
landia genus.  e responsible toxins are psilocybin and psilocin.  e psilocybin is metabolized
into psilocin in human bodies (
> Figure 2.9).
From January 1997 to June 1999, 24 inquiries about magic mushrooms were received by
the o ce of Japan Poison Information Center [4]; the numbers of inquiries were 1 in 1997, 10
in 1998 and 13 in 1998 (6 months). An article entitled “Dangerous proliferation of hallucino-
genic mushrooms” appeared in the Asahi morning newspaper on July 18, 1999. It described a
case, in which a person had had a delusion of being capable of  ying in the air, had jumped
from a window of the 2nd  oor and had been severely injured, and also a case, in which a uni-
versity student had been mentally deranged on the campus; the article raised the alarm on such
dangers. In January, 2001, there was a case, in which a youngster ate a grown magic mushroom,

which had been purchased in the form of a cultivation kit via the Internet, and provoked hal-
lucinatory symptoms to result in his death due to cold inside a roadside gutter in the nude.
Accidents and incidents by ingestion of magic mushrooms are increasing recently; such
abuse should be controlled strictly. In the United States and Japan, the possession, cultivation
and intake of magic mushrooms have been completely prohibited recently.
Chemical analyses
For identi cation of a mushroom, in addition to the morphological method using the observa-
tions of its appearance and the form of its spores, chemical methods for analysis of toxins of
mushrooms are also important. In this section, some examples of such chemical methods are
described; especially, those for toxins of Amanita and Psilocybe mushrooms are presented.
Analysis of toxins of Amanita mushrooms
 e toxins of Amanita mushrooms are usually analyzed by HPLC.
As toxins, α-amanitin, β-amanitin, γ-amanitin and phalloidin are known.  eir authentic
standards can be purchased from Sigma (St. Louis, MO, USA).
Structures of psilocybin and psilocin.
⊡ Figure 2.9
477
i. HPLC conditions ( > Figures 2.10 and 2.11)
Column: Inertsil OD-3 (150 × 4 mm i.d., particle size 5 µm, GL Sciences, Tokyo, Japan); mobile
phase: 0.01 M ammonium acetate-acetic acid bu er solution (pH 5.0)/acetonitrile (84:16); its
 ow rate: 1.0 mL/min; detector: diode array detector (DAD); detector wavelengths: 302 nm for
amanitin and 292 nm for phalloidin.
ii. Extraction from a mushroom
A er a mushroom is minced into small pieces with a knife or scissors, they are extracted with
3 mL of methanol/ water/0.01 M HCl (5:4:1) by shaking the mixture at 4 °C for 24 h.
HPLC chromatograms for amanitins and phalloidin. A 0.25-µg aliquot each of the compounds
was injected into HPLC.
⊡ Figure 2.10
Tridimensional HPLC-DAD chromatograms for amanitins and phalloidin. 1: α-amanitin;
2: β-amanitin; 3: phalloidin. The amount of the compounds injected into HPLC was 0.25 µg each

in an injected volume.
⊡ Figure 2.11
Chemical analysis
478 Mushroom toxins
A er centrifugation, the supernatant solution is condensed under a stream of nitrogen and
injected into HPLC for analysis.
iii. Extraction from a body fluid
i. A 5-mL volume of serum is mixed with 10 mL acetonitrile, shaken for 10 min and centri-
fuged at 1,000 g for 10 min.
ii.  e supernatant solution is mixed with 30 mL dichloromethane, shaken for 20 min and
centrifuged at 1,000 g for 5 min.
iii.  e supernatant solution is condensed under a stream of nitrogen and injected into HPLC
for analysis.
Analysis of toxins of magic mushrooms (Psilocybe species)
For analysis of hallucinogenic toxins, such as psilocybin and psilocin, GC, GC/MS, LC and
LC/MS are being used.  e authentic standards of psilocybin and psilocin are not commer-
cially available in Japan; the solution vials of psilocin can be imported a er an appropriate
procedure from Sigma, USA.
i. HPLC
For HPLC, a spectrophotometric detector or an electrochemical detector (ECD)
c
can be used.
If LC/MS or LC/MS/MS is available, analysis with much higher sensitivity and reliability can
be realized. Here, an HPLC method with a relatively cheap and highly sensitive ECD detector
is described [5].
Column: Inertsil ODS-3 (150 × 4 mm i.d., particle size 5 µm, GL Sciences); mobile phase:
pH 3.8 bu er solution (300 mL of 0.1 M citric acid solution + 160 mL of 0.1 M sodium di-
hydrogenphosphate solution)/ethanol (9:1); its  ow rate: 1.0 mL/min; detector: ECD (+1.0 V).
ii. GC or GC/MS
A er ingestion of psilocybin, it is easily metabolized into psilocin in human bodies. In a recent

report [6], psilocin is said to exist in the glucuronide-conjugated form in human samples; they
have insisted that enzymatic hydrolysis with glucuronidase is required before analysis. Psilo-
cybin is dephosphorylated into psilocin in an injection chamber of GC at high temperature;
TMS derivatization is required for GC or GC/MS analysis.  e readers can refer to the refer-
ence [6] on the details of the method.
Scan range: m/z 50–550; retention index: 2,099; psilocin-di-TMS: m/z 290, 291 and 348.
iii. Extraction from a mushroom [5]
i. A 300-mg aliquot of a mushroom is mixed with 30 mL methanol and homogenized.
ii. A er shaking for 24 h, the homogenate is passed through a paper  lter.
iii.  e clear solution is evaporated to dryness under a stream of nitrogen; the residue is dis-
solved in 3.0 mL methanol and a 10-µL aliquot of it is injected into HPLC.
iv. Extraction from a dried mushroom [7]
i. A 100-mg aliquot of a dried mushroom is mixed with 9 mL methanol and extracted by
sonication for 120 min.
479
ii.  e volume of the mixture is adjusted to 10 mL and centrifuged at 1,000 g for 15 min. An
aliquot of the supernatant solution is injected into HPLC.
v. Extraction from cerebrospinal fluid (CSF) [5]
i. A 5-mL volume of CSF is mixed with 0.35 mL of 70 % perchloric acid solution, and centri-
fuged at 1,000 g for 30 min.
ii. A er decanting the supernatant solution, its pH is adjusted to 12 by adding 45 % KOH
solution with cooling, followed by centrifugation at 1,000 g for 5 min.
iii.  e supernatant solution is mixed with 1 g NaCl, extracted with 6 mL dichloromethane by
shaking for 15 min and centrifuged.  e organic phase is transferred to another tube, and
6 mL dichloromethane is again added to the aqueous phase; the same extraction procedure
is conducted.  e resulting organic phases are combined.
iv.  e combined extract is dehydrated with anhydrous Na
2
SO
4

and centrifuged at 1,000 g for
5 min.
v.  e organic extract is evaporated to dryness under a stream of nitrogen, and the residue is
dissolved in 200 µL methanol. An aliquot of the solution is injected into HPLC.
vi. Extraction from blood or urine [7]
i. A 1-mL volume of blood or urine is mixed with 10 µL of β-glucuronidase (E. coli origin,
Sigma) and incubated at 45 °C in a water bath with shaking for 1 h.
ii.  e mixture is diluted with 5 mL of 0.1 M potassium phosphate-NaOH bu er solution
(pH 8) and poured into a Bond Elut Certify LRC 300 mg column (Varian, Harbor City,
CA, USA).  e column had been activated by passing 2 mL methanol and 2 mL of 0.1 M
potassium phosphate-NaOH bu er solution (pH 8) in advance.
iii.  e above sample solution is poured into the column at a  ow rate of 1–2 mL/min.  ere-
a er, nitrogen gas is passed through the column to dry it.
iv.  e column is washed with 2 mL water, 2 mL of 0.2 M acetic acid-sodium acetate bu er
solution (pH 4) and 2 mL of 30 % methanol aqueous solution.
v. A er passing nitrogen gas through the column to dry it up, 2 mL of methanol/concen-
trated ammonia solution (98:2) and 1 mL of the same solution are passed for elution of
the target compound.
vi. A er both solutions are combined, they are evaporated to dryness under a stream of ni-
trogen with warming at 40 °C.
vii.
 e residue is mixed with 50 µL of N-methyl-N-trimethyl- silyltri uoroacetamide (MSTFA),
capped airtightly and heated at 80 °C for 15 min.
viii. A er cooling to room temperature, an aliquot of the solution is injected into GC/MS.
Toxic concentrations
Although there are great variation in concentrations among references, there is a report [3]
describing that the concentrations of α-amanitin and β-amanitin are 8–190 and 15.9–162 ng/mL
in blood plasma, respectively. Amanitin usually disappears from blood about 36 h a er in-
gestion.
A er oral ingestion of 10–20 mg (0.224 ± 0.02 mg/kg) of psilocin, its blood plasma con-

centrations were reported to be 8.2 ± 2.8 ng/mL [7].
Toxic concentrations
480 Mushroom toxins
Conclusion
 ere are some toxic mushrooms, the toxins of which are not clari ed; for such types of mush-
rooms, chemical analysis is useless. Especially for Amanita neoovoidea, which has been found
to be a toxic mushroom very recently, its toxin is only estimated to be a kind of peptides.  e
structure of the toxin remains to be clari ed.
Upon analysis of mushroom toxins, the causative foods, such as miso soup and sukiyaki,
even a er being cooked, and/or the corresponding mushrooms should be obtained together
with specimens of blood, urine and/or stomach contents. Raw mushrooms should not be
frozen, because their forms are destroyed upon thawing; they should be stored in a refrigerator
at 4 °C.  ey should be transported as soon as possible to reach a laboratory for analysis.  e
morphological  ndings of mushrooms themselves and their spores are very useful for interpre-
tation of the results obtained by chemical analysis.
Notes
a)  e numbers are based on only poisoning cases, which had been reported to a local public
health center; the unreported cases are not included in the numbers.
b) Upon totaling the numbers of each mushroom poisoning, the task may be undertaken by a
nonexpert for mushrooms.  erefore, it seems di cult to expect the exact classi cation of
mushrooms; the analogous species or genuses may be treated as a whole. For example, they
are probably treated as Rhodophyllus rhodopolius plus Rhodophyllus sinuatus and Amanita
virosa plus Amanita verna. In the Psilocybe argentipes mushrooms, other species of halluci-
nogenic mushrooms may be included.
c) HPLC-ECD is being widely used for sensitive analysis of catecholamines. Although it is not
a common detector, it enables the detection of trace levels of compounds, which cannot be
detected by the usual spectrophotometric detector. In addition, by using a CV stabilizer
(CV 1000 Denken), clean chromatograms without noises or dri can be obtained.
References
1) Life Hygiene Bureau of the Ministry of Health, Labour and Welfare (ed) (1974–1997) National Record of Food

Poisoning Incidents. Japanese Association of Food Hygiene, Tokyo (in Japanese)
2) Yamashita M, Furukawa H (1993) Mushroom poisonings. Kyoritsu-shuppan, Tokyo, p 110 (in Japanese)
3) Jaeger A, Jehl F, Flesch F et al. (1993) Kinetics of amatoxins in human poisoning. Therapeutic implications.
J Toxicol Clin Toxicol 31:63–80
4) Madono K, Sakai Y, Hatano Y et al. (1999) Hallucinogenic “magic mushroom” poisoning: a report by JPIC. Jpn J
Toxicol 12:443–447 (in Japanese)
5) Kysilka R, Wurst M, Pcakova V et al. (1985) High-performance liquid chromatographic determination of halluci-
nogenic indoleamines with simultaneous UV photometric and voltammetric detection. J Chromatogr 320:
414–422
6) Sticht G, Kaferstein H (2000) Detection of psilocin in body fluids. Forensic Sci Int 113:403–407
7) Musshoff F, Madea B, Beike J (2000) Hallucinogenic mushrooms on the German market. Simple instructions for
examination and identification. Forensic Sci Int 113:389–395