Diseño de investigación

5.2.5.1 Occurrence route or mechanism of formation

Psilocybin, 4-phosphoryloxy-N ,N -dimethyl-tryptamine, is a psychedelic pro-drug occurring inPsilocybe and several other mushroom genera together with a closely related psilocin,

N

N R R OR

H R⁴

1 2

3

Psilocybin, R¹ = PO3H2, R² = CH3, R³ = CH3, R⁴ = H Psilocin, R¹ = H, R² = CH3, R³ = CH3, R⁴ = H

Fig. 5.7 Structures of psilocybin and psilocin.

4-hydroxy-N ,N -dimethyl-tryptamine (see Fig. 5.7). In the body, psylocibin is enzymatically dephosphorylated by phosphatases yielding pharmacologically active psilocin. This reaction takes place also under strongly acidic conditions. Generally, psilocin is relatively unstable in solution due to its phenolic group on the benzene ring at the 4-position in the indole ring.

5.2.5.2 Methods of analysis

For qualitative analysis of psilocybin and psilocin, TLC can be used. The visualization of toxin spots is obtained either byN ,N dimethylaminobenzaldehyde (Ehlich reagent) or N ,N -dimethyl aminocinnamic aldehyde. For accurate GC/MS or HPLC employing fluorescence detector (FLD) or MS detection with electrospray ionization can be used (Bogusz, 2000;

Faulstich, 2005; Saitoet al., 2004, 2005). As an alternative, capillary-zone electrophoresis (CZE) and flow injection analysis (FIA) were reported as methods of choice for the determi-nation of these toxins in mushroom extracts (Anastoset al., 2005). Several methods such as HPLC with electrochemical detection (ECD) or DNA-based tests were applied for examina-tion of biological fluids human plasma and urine for the presence of pisloscybin and psilocin (the latter compound is excreted as glucuronide) (Faulstich, 2005).

5.2.5.3 Incidence and levels of occurrence

Mushrooms containing these hallucinogenic indole derivatives were known already in the l6th century in the Mayan culture of ancient Mexico. Species from generaPsilocybe, Conocybe, Panaeolus and Gymnopilus may contain psilocybin in amounts as high as 2000–16 000 mg kg⁻¹ dry weight, and a second psychoactive component, psilocin, in amounts ranging from 0 to 10 000 mg kg⁻¹ dry weight (Faulstich, 2005); the average is 5000 and 5000 mg kg⁻¹, respectively. The quantity of toxins in the mushrooms can vary widely, depending on species, phase of growth, mushroom size and drying conditions (both psilocybin and psilocin are temperature-sensitive). Anastoset al. (2005) found only small content of psylocin and psylocibin in dried mushroomsPsilocybe subaeruginosa and Hypholoma auranitiaca, also Saitoet al. (2005) reported levels of psylocibin in Psylocybe mushroom lower than 20 mg kg⁻¹dried samples. In dry form, the mushrooms are available in the black market in various countries (Faulstich, 2005).

5.2.5.4 Exposure assessment

Psilocybin mushrooms are commonly called ‘magic mushrooms’, or more simply ‘shrooms’, they are seldom mistaken for food fungi by innocent hunters of wild mushrooms. For recre-ational and entheogenic use, most common arePsilocybe semilanceata (liberty cap), and Psilocybe cubensis (golden tops). When ingested, psilocybin is absorbed through the lining of the mouth and stomach, then it is metabolized mostly in the liver where it becomes psilocin (it is broken down by the enzyme monoamine oxidase). The toxin affects CNS with the pro-duction of a syndrome, often described as pleasant, somewhat similar to alcohol intoxication, sometimes accompanied by hallucinations. Its intensity and duration depends on many fac-tors including dosage, individual physiology (metabolism), setting etc. Typically, following dosage 10–50 mg psilocybin corresponding to approximately 1–5 g dried mushroom or 10–

50 g wet mushrooms effect begins in 10–60 minutes and lasts 2–6 hours.

Poisonings by these mushrooms are rarely fatal in adults. The most severe cases of psilo-cybin poisoning occur in small children, where large doses may cause the hallucinations accompanied by fever, convulsions, coma and death.

It should be noted that psilocybin and psilocin are listed as Schedule I drugs under the United Nations 1971 Convention on Psychotropic Substances (Schedule I drugs are illicit drugs that are claimed to have no known therapeutic benefit). Parties to the treaty are re-quired to restrict use of the drug to medical and scientific research under strictly controlled conditions.

5.2.6 Coprine

5.2.6.1 Occurrence route or mechanism of formation

Coprine,N 5-1-hydroxycyclopropyl-L-glutamine is a poison occurring in the popular Inky Cap mushroom (Coprinus atramentarius) and several other species. This unusual amino acid, consisting of cyclopropanone and glutamine, is metabolized to 1-aminocyclopropanol and 1,1-cyclopropandiol (cyclopropanone hydrate). The latter compound reacts with essential SH-group of acetaldehyde dehydrogenase forming respective thio-hemiketal. Biologically active components exhibit similar metabolic effects as disulfiram (see Fig. 5.8).

5.2.6.2 Methods of analysis

No method of analysis for the determination of coprine in mushrooms has been published in the available literature, only analysis employing HPLC with electro-chemical detection for its and other disulfiram-like compounds in rat brain in vitro has been described (Nilsson and Tottmat, 1987).

HO N COOH

NH₂ O

H

N S S N

S

S

(CH2CH3)2

(CH3CH2)2 HO S-Enzyme

Fig. 5.8 Structures of coprine, disulfiram and thio-hemiketal.

5.2.6.3 Incidence and levels of occurrence

Coprinus atramentarius, or inky cap, has a delicious flavour when young. Like other inky caps, for instanceC. insignis, C. quadrifidus, C. variegates and C. atramentarius grows in tufts.

It is commonly associated with buried wood and is found in grassland, meadows, disturbed ground, and open terrain in autumn. It appears in spring, summer and autumn across the Northern Hemisphere, but has also been found in Australia. Traces of coprine were also found in other mushrooms likeClytocibe claviceps, Pholiota squarrosa and/or Boletus luridus.

5.2.6.4 Exposure assessment

Although generally considered edible, when consumed with alcohol,Coprinopsis atramen-taria and other related species are toxic. Coprine hydrolysis products originating in the human body interfere with the breakdown of alcohol at the intermediate stage, since they block ac-etaldehyde dehydrogenase. Consumption of alcoholic beverages within 72 hours after eating these mushrooms will cause headache, nausea and vomiting, flushing and cardiovascular disturbances that last for 2–3 hours. Although very unpleasant, the syndrome has not been associated with any fatalities. The symptoms are described as similar to those observed af-ter application of disulfiram (trade names for disulfiram in different countries are Antabuse or Antabus), that is a drug used to support the treatment of chronic alcoholism by produc-ing an acute sensitivity to alcohol. Coprine and disulfiram both caused an increase in the acetaldehyde/ethanol ratio, coprine being more potent than disulfiram.