01

Detection of allenic

norleucine from

Amanita smithiana

mushrooms

Dr. Ian Garber!

!

Medical Biochemistry Resident!

University of British Columbia!

Vancouver, BC

Clinical case

• A 63 year old man presented to our hospital with acute renal failure requiring hemodialysis.!

• Three days earlier, he had picked and eaten what he believed to be Pine mushrooms.!

• Soon after, he felt fatigued, and had vague flu-like symptoms. The next day, his symptoms progressed into nausea, vomiting, diarrhea, and abdominal pain.!

• When he finally came to medical attention the following day, he had not urinated for about 24 hours. Bloodwork on hospital admission confirmed that he was in severe renal failure, with serum creatinine of 1084 μmol/L.

A delicacy? Maybe not…

• Based on the patient’s description of the mushrooms, poisoning with Amanita smithana was suspected: 



Tricholoma magnivelare 


(Pine mushroom - edible)

Amanita smithiana 


(Nephrotoxic)

Amanita smithiana poisoning

• Stages of poisoning:!

1. Gastrointestinal:
Abdominal pain, nausea, vomiting, and diarrhea, starting 2-12 hours after ingestion.!

2. Delayed onset renal failure: 
Oliguria or anuria, starting anywhere from 1-5 days after ingestion.!

• Severe enough to require dialysis in all reported cases!

• However, kidney function spontaneously recovers within 1-5 weeks 



Related Amanita mushrooms in Europe

• Several European mushrooms cause a similar poisoning syndrome: 



Amanita ovoidea 


(edible)

Amanita proxima 


(Nephrotoxic)

Related Amanita mushrooms in Europe

• Several European mushrooms cause a similar poisoning syndrome: 



Amanita boudieri


(Nephrotoxic - case in Portugal)

Amanita solitaria (echinocephala) 


(Nephrotoxic - case in Germany)

Preliminary identification

• The patient had saved some leftovers of mushroom soup - these were submitted to the clinical laboratory.!

• Based on microscopic examination, a local mycologist was able to give preliminary identification of the cooked mushroom fragments as Amanita smithana.

Laboratory confirmation

• A sample of the soup was sent to a laboratory in Austria that had developed an assay for Amanita smithiana toxin. 


• Thin-layer chromatography confirmed that the toxin was present: 


(+)
Control

(-) 
Control

Mushroom fragment

Soup

The Toxin

• Allenic norleucine (2-amino-4,5-hexadienoic acid) is a modified amino acid produced by A. smithiana and several related mushroom species.!

• Generally accepted as the nephrotoxic component:!

• Animal study showing lethality in guinea pigs!

• Cytotoxic effect on cultured renal tubular cells!

• Potent insecticidal effect on Milkweed bug (70% lethality at 0.1%)!

• Co-migrates on TLC, and shows the same orange colour reaction with ninhydrin!

• Mechanism of toxicity unknown 









C6H9NO2

Monoisotopic mass: 127.06

Objective

• Could we develop an LC-MS/MS method for allenic norleucine? 


-The existing TLC method is laborious, and interpretation is subjective. 
 -Should also be able to greatly improve sensitivity.!

• Challenges: 


-Compound not commercially available to use as a standard 
 -No ESI spectrum published 


-Never detected by MS out of a complex matrix!

• Obtained reference material of A. smithiana, 
and set off into the unknown!

Method Development

Preparative TLC - Crude purification

• Extracted pulverized dried mushroom in 50% methanol!

• TLC method as developed by our Austrian colleague (Martin Kirchmair), but scaled up for preparative use!

✦ Silica G plate, 1000 µm thickness, 20x20 cm!

✦ Mobile phase:


Ethyl acetate : acetic acid : water : isopropanol : methanol

40:15:12:8:5!

• Spotted a range of volumes, to determine optimum loading (maximize yield, without smearing or distortion) 



xxxxxxxxxxxxxxxxxxx

HPLC

• Even though quite a polar analyte, decided to try on C18 column first!

• Found a method for separating standard amino acids on C18 column, using a

shallow gradient of H2O -> ACN. Used this as a starting point.!

• Final HPLC conditions:!

• Agilent Eclipse XDB-C18, 


4.6 x 150 mm, 5 µm!

• Mobile phase A = 


0.2% formic acid in water!

• Mobile phase B = 


0.2% formic acid in acetonitrile!

• Flow rate = 1 ml/min





Initial Q1 scan

• AB SCIEX QTRAP 5500, positive mode!

• One of the major peaks on the chromatogram showed a peak with the expected m/z for allenic norleucine!!

• Lower mass fragments (82 and 74) matched with previously published EI spectrum

Q1 scan

Product ion scan for 128

• Picked these 6 most intense fragments as transitions for an MRM method: 
 128 —> 82
 128 —> 80
 128 —> 74
 128 —> 67
 128 —> 65
 128 —> 55

CE = 20

MRM method

• All 6 transitions coelute

Solid Phase Extraction

• Could we skip the pTLC step and detect allenic norleucine directly out of a crude extract of the mushrooms?!

• SPE clean-up on Phenomenex Strata X-AW column.!✦ Load crude mushroom extract!

✦ Wash with 25 mM ammonium acetate!

✦ Wash with 100% MeOH!

✦ Elute in 5% formic acid in MeOH 




















MRM method with SPE

• No problem detecting out of complex matrix

Dilute and Shoot

• With SPE in 96-well plate format, we experienced problems with cross-contamination of adjacent wells!

✦ Dealing with huge range of concentrations (5 orders of magnitude), and need

to distinguish truly negative samples from positives with low concentration.!

• To minimize cross-contamination, attempted sample prep with simple “dilute-and-shoot” methodology!

✦ 100-fold dilution of raw extract into mobile phase A (0.2% formic acid in H2O)!

• Excellent results achieved, with no interferences apparent

High resolution confirmation

• Was able to use a Bruker Q-TOF at Simon Fraser University!

• Confirmed that molecular formula matched the observed exact mass for the putative allenic norleucine peak within 0.2 ppm!

• Using exact mass of the fragments, was able to rationalize the fragmentation scheme as follows:

CH2

O

OH

NH2

m/z 127.063329

C6H

9NO

2

M

CH2

O

OH

NH2

m/z 128.070605

C6H

10NO

2

M�+�H

+H+

CH2

CH+

NH2

m/z 82.065126

C5H

8N

M�-�CHO2

M�+�H

M�-�CH2O

2

CH2

NH2

+

m/z 82.065126

C5H

8N

M�-�CHO2

M�-�H2

CH2

C+

NH

m/z 80.049476

C5H

6N

M�-�CH3O

2

CH+

O

OH

NH2

m/z 74.023655

C2H

4NO

2

M�-�C4H

5

M�-�C4H

6

CH2

CH3

CH+

NH2

m/z 82.065126

C5H

8N

M�-�CHO2

C

CH2

NH2

+

m/z 67.041651

C4H

5N

M�-�C2H

4O

2

M�-�CH3

CH2

CH2

NH3

+

m/z 82.065126

C5H

8N

M�-�CHO2

CH2

C+

CH2

m/z 65.038577

C5H

5

M�-�CH4NO

2

M�-�NH3

CH2

O

OH

NH3

+

m/z 128.070605

C6H

10NO

2

M�+�HM�-�CH

2O

2

Summary of method development

• Because pure allenic norleucine was not available as a standard, instead started with mushrooms known to contain it!

• Used known migration position on TLC to crudely purify the compound!

• Q1 scan of scraped TLC spot —> looked for the known molecular mass!

• Product ion scan to determine fragmentation pattern —> set as MRM transitions!

• Demonstrated co-elution of these transitions, first from scraped TLC spot, then from crude mushroom extract with SPE cleanup only, and finally from crude mushroom extract with dilution only!

• Confirmed exact mass of parent ion and fragments using Q-TOF

Synthetic standard

• We later found a lab willing to custom-synthesize allenic norleucine for us to use as a standard!!

• Run on the existing method, the synthetic standard showed identical retention time and fragmentation pattern to our putative allenic norleucine peak.

Quantitation of allenic norleucine

• By building a standard curve of the pure compound, this allowed us to make the MS method quantitative.!

• Extraction also made quantitative:!

• Use of “Beadbug” homogenizer tubes!

• Precise weighing on analytical balance.!

• With these two modifications to the method, we are able to calculate the concentration by dry weight in the original mushroom specimen.

Key Results
with quantitative method

Key Results:

• The concentration of allenic norleucine in Amanita smithiana is very high: ~2.5% by dry weight!

• This is 25x higher than previously reported!

• Addresses previous concerns that the concentration in mushrooms was too low to cause poisoning based on the LD50 of allenic norleucine in animal studies.!

• Confirmed on duplicate extractions on each of two independent mushroom reference samples.

Key Results:

• Amanita proxima has been suspected to contain allenic norleucine, because of the similarity of its toxidrome, but this has never been proven.!

• We have now confirmed that Amanita proxima (most toxicity reports from France) does contain allenic norleucine, at a concentration of approximately 0.25 % (dry weight)!

• We also discovered that it contains a related amino acid called propargylglycine (2-amino-4-pentynoic acid) which is known to be hepatotoxic.

Key Results:

• Amanita boudieri and solitaria, causing poisonings leading to renal failure in Portugal and Germany respectively, were confirmed to contain allenic norleucine.!

• A. boudieri: ~1.2% allenic norleucine by dry wt.!

• A. solitaria (echinocephala): ~2.6% dry wt.

Ongoing Investigation

Clinical usage

• Can allenic norleucine be detected from urine or blood

specimens?!

• If so, what is the window of detection? This is important for

clinical utility, since the onset of renal failure is delayed by

up to several days.!

• No clinical samples from this poisoning case remaining.

Awaiting next case (local or referred in) to attempt this. 








Mycology/Chemotaxonomy

• Now collaborating with a world expert on Amanitas, RE Tulloss,

to answer some basic science questions with our assay.!

• Have screened 50+ different species of Amanita for allenic

norleucine. Preliminary results show that the distribution is

broader than previously thought). Confirmed that high levels of

ANL are present in other related Amanitas causing renal failure.!

• Have developed LC-MS/MS assays for other modified amino

acids found in these mushrooms. Comparing distribution of

these metabolites against existing taxonomic groupings, and

molecular phylogeny based on DNA sequencing.

Case Conclusion

Case: conclusion

• Patient continued hemodialysis over a two week hospital admission.!

• Discharged when his urine output had returned to normal and creatinine was consistently trending down!

• Kidney function remained normal at one year follow-up!

• Patient has given up mushroom picking

Acknowledgements

• Grace van der Gugten


Major work on assay development and optimization!

• Dan Holmes


Research supervisor!

• Rod Tulloss 
Herbarium Rooseveltensis Amanitarum, New Jersey, USA: 
Mycology expertise, herbarium sampling!

• Martin Kirchmair 


University of Innsbruck, Austria: 
Sharing of A. smithiana reference material, development of TLC method!

• Gang Chen


Simon Fraser University, Canada: Synthesis of allenic norleucine standard!

• Hongwen Chen


Simon Fraser University, Canada: Use of Q-TOF instrument

“There are old mushroom pickers and bold mushroom

pickers, but there are no old, bold mushroom pickers.”