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

Th e number of species of edible mushrooms in Japan is about 300; that of toxic mushrooms is said to be about 30.. Among the 431 incidents, the numbers of incidents according to causativ

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II.6.2 Mushroom toxins

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 Th 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 victimsa 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 specifi ed, 35 (> Figure 2.1)b

Toxic mushrooms can be classifi ed into 6 groups according to their actions as follows

• Th 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).

• Th 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)

• Th ose which inhibit the metabolism of acetaldehyde in blood (disulfi ram-like eff ect), caus-ing a fl ushcaus-ing phenomenon and palpitation upon drinkcaus-ing alcohol concomitantly (latent

period, 20 min–2 h; Clitocybe clavipes, Coprinus atramentarius and others).

• Th 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)

• Th 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).

> 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 Th 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

Th 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 Th e poisoning symptoms are vomiting, diarrhea and abdominal pain appearing 30 min–3 h aft er ingestion

Th e stem of Rhodophyllus rhodopolius is easily crushed by pressure with the fi nger, but that of the edible Rhodophyllus crassipes is not Th 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

⊡

not clear (R hodoph

Rhodophyllus rhodopolius.

⊡ Figure 2.2

Amanita virosa.

⊡ Figure 2.3

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 Th e

Amanita genus mushrooms should be watched most carefully also in the forensic toxicological

point of view Th e main toxin of this genus is considered to be amanitin (> Figure 2.4) or

phalloidin (> Figure 2.5) Th 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 Th ere is a report insisting that phalloidin does not exert toxic eff 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

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] Th e blood concentrations of amanitin are highly dependent on the intervals aft 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

Th 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 Th 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 Th 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 Th 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 Th e fatality by the toxin is very rare

How to discriminate Lampteromyces japonicus.

⊡ Figure 2.6

Magic mushrooms ( > Figure 2.8)

Th e magic or hallucinogenic mushroom is a popular name for ones which exhibit hallucina-tion (visual and auditory), mental derangement and muscle fl accidness In central and south America, such mushrooms were being used in religious ceremonies since ancient times Th e hallucinogenic eff ects vary according to diff erent individuals; they are similar to those ob-tained with LSD, though they are much weaker than those of LSD Th 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

mushrooms being circulated in Japan are Psilocybe cubensis and/or P subcubensis and

Cope-landia genus Th e responsible toxins are psilocybin and psilocin Th 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 offi 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 fl ying in the air, had jumped from a window of the 2nd fl 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 identifi 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

Th e toxins of Amanita mushrooms are usually analyzed by HPLC.

As toxins, α-amanitin, β-amanitin, γ-amanitin and phalloidin are known Th eir authentic standards can be purchased from Sigma (St Louis, MO, USA)

Structures of psilocybin and psilocin.

⊡ Figure 2.9

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 buff er solution (pH 5.0)/acetonitrile (84:16); its

fl 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

Aft 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

Aft 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 Th e supernatant solution is mixed with 30 mL dichloromethane, shaken for 20 min and centrifuged at 1,000 g for 5 min

iii Th 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 Th e authentic standards of psilocybin and psilocin are not commer-cially available in Japan; the solution vials of psilocin can be imported aft 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 buff 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 fl ow rate: 1.0 mL/min; detector: ECD (+1.0 V)

ii GC or GC/MS

Aft 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 Th 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 Aft er shaking for 24 h, the homogenate is passed through a paper fi lter

iii Th 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

ii Th 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 Aft 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 Th e supernatant solution is mixed with 1 g NaCl, extracted with 6 mL dichloromethane by shaking for 15 min and centrifuged Th 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 Th e resulting organic phases are combined

iv Th e combined extract is dehydrated with anhydrous Na2SO4 and centrifuged at 1,000 g for

5 min

v Th 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 Th e mixture is diluted with 5 mL of 0.1 M potassium phosphate-NaOH buff er solution (pH 8) and poured into a Bond Elut Certify LRC 300 mg column (Varian, Harbor City,

CA, USA) Th e column had been activated by passing 2 mL methanol and 2 mL of 0.1 M potassium phosphate-NaOH buff er solution (pH 8) in advance

iii Th e above sample solution is poured into the column at a fl ow rate of 1–2 mL/min Th ere-aft er, nitrogen gas is passed through the column to dry it

iv Th e column is washed with 2 mL water, 2 mL of 0.2 M acetic acid-sodium acetate buff er solution (pH 4) and 2 mL of 30 % methanol aqueous solution

v Aft 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 Aft er both solutions are combined, they are evaporated to dryness under a stream of ni-trogen with warming at 40 °C

vii Th e residue is mixed with 50 µL of N-methyl-N-trimethyl- silyltrifl uoroacetamide (MSTFA),

capped airtightly and heated at 80 °C for 15 min

viii Aft 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 aft er in-gestion

Aft 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]