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Biomagnification of cycad neurotoxins in flying foxes

Implications for ALS-PDC in Guam Sandra Anne Banack, PhD; and Paul Alan Cox, PhD

Abstract—!-Methylamino-L-alanine (BMAA) occurs in higher levels in museum specimens of the Guamanian flying fox than in the cycad seeds the flying foxes feed on, confirming the hypothesis that cycad neurotoxins are biomagnified within the Guam ecosystem. Consumption of a single flying fox may have resulted in an equivalent BMAA dose obtained from eating 174 to 1,014 kg of processed cycad flour. Traditional feasting on flying foxes may be related to the prevalence of neuropathologic disease in Guam. NEUROLOGY 2003;61:387–389

ALS–parkinsonism dementia complex (PDC), de- scribed in the Chamorro people of Guam, with as- pects similar to ALS, Parkinson’s disease (PD), and Alzheimer’s disease (AD), once occurred in Guam at 50 to 100 times the incidence of ALS elsewhere.1 Epidemiologic studies indicate that consumption of a Chamorro diet is the only variable significantly associ- ated with disease incidence.2 Seeds of cycad plants, Cycas micronesica Hill in the C. rumphii species com- plex, used by the Chamorro people as a source of torti- lla flour, have neurotoxins including !-methylamino-L- alanine (BMAA), a nonprotein amino acid that Spencer et al.3 and others have suggested may be associated with ALS-PDC. However, the concentrations of BMAA in processed cycad flour were believed to be too low to produce comparable disease states in animal models.4-6

Cox and Sacks7 suggested that high doses of cycad neurotoxins or their metabolites might be inadver- tently ingested by the Chamorros during their con- sumption of flying foxes during traditional feasts. The high rates and subsequent decline of ALS-PDC in Guam shadowed the rise and eventual decline in consumption of cycad-fed flying foxes by the Chamorro people.8

We analyzed three C. micronesica seeds obtained from Guam. Pteropus mariannus mariannus, an in- digenous flying fox of Guam, is now highly endan- gered. Therefore, we analyzed skin tissue from museum specimens of three flying foxes that were collected five decades ago in Guam, preserved as dried study skins, and deposited at the Museum of Vertebrate Zoology (MVZ) at the University of Cali-

fornia, Berkeley. We also analyzed three processed cycad flour specimens from Guam. We examined all samples for the presence of BMAA using high- performance liquid chromatography (HPLC). Results were confirmed with thin-layer chromatography (TLC) as well as gas chromatography–mass spectroscopy.

Methods. BMAA was quantified from free amino acid extracts of flying fox and cycad tissues. Samples were rehydrated for 30 minutes with water or trichloroacetic acid (mean tissue prepara- tion 80 " 32 [SD] mg/mL), macerated, and filtered. Extracts were derivatized with 6-aminoquinolyl-N-hydrozysuccinimidyl carbam- ate (ACQ) following standardized protocols.9 Free amino acids were separated by reverse-phase separation on a gradient HPLC system (Waters 717 Automated Injector, Waters 1525 Binary Sol- vent Delivery System, and Waters Nova-Pak C18 column, 300 # 3.9 mm; Waters, Milford, MA) at 37 °C. Individual compounds were eluted from the column with a gradient elution of 140 mM sodium acetate/5.6 mM triethylamine, pH 5.2 (mobile phase A) and 60% acetonitrile (mobile phase B) with a flow rate of 1.0 mL/min.9 Gradient conditions were as follows: initial $ 100% A, 2.0 minutes $ 90% A curve 11, 5.0 minutes $ 86% A curve 11, 10.0 minutes $ 86% A curve 6, 18.0 minutes $ 73% A curve 6, 30.0 minutes $ 60% A curve 10, 35.0 minutes $ 40% A curve 6, 39.0 minutes $ 10% A curve 6, followed by a wash with 100% B for 5 minutes and re-equilibration for 5 minutes at 100% A. BMAA peak identity was confirmed by comparison with a com- mercial standard (%94% pure; Sigma B-107, St. Louis, MO) and was reverified by modified gradient elution. The concentration of BMAA in samples was determined by fluorescence detection (Wa- ters 2487 Dual-l Fluorescence Detector) with excitation at 250 nm and emission at 395 nm. Detection of the ACQ-derivatized BMAA was dependent on concentration, and quantification was accom- plished with comparison of equal amounts of BMAA and a nor- leucine internal standard (representing a single midrange concentration), resulting in a mean response of 51.2%. These data express the average response of values for several experiments and depict the efficiency of the derivatization protocol and the

See also page 291

From the Institute for Ethnobotany (Drs. Banack and Cox), National Tropical Botanical Garden, Kalaheo, HI; and Department of Biological Science (Dr. Banack), California State University, Fullerton, CA. Supported by the ALS Association. Received December 23, 2002. Accepted in final form April 21, 2003. Address correspondence and reprint requests to Dr. P.A. Cox, Institute for Ethnobotany, National Tropical Botanical Garden, 3530 Papalina Rd., Kalaheo, Kauai, HI 96741; e-mail: [email protected]

Copyright © 2003 by AAN Enterprises, Inc. 387

relative ratio between BMAA and the internal standard. The re- sults may be indicative of internal quenching of the derivative but did not significantly affect sample quantification as the percent- age response was consistent across the quantifiable concentration range. The limits of detection (LOD; defined as the lowest concen- tration of an analyte in a sample that can be detected, though not necessarily quantitated) and limits of quantification (LOQ; the concentration within the linear range of the calibration curve re- lating absorbance to concentration) were determined by a concen- tration gradient of an authenticated standard (Sigma). The LOD was 0.00013 &mol/injection and the LOQ was 0.013 &mol/injection for all analyses. For data interpretations, all sample analyses were quantified within the range of the LOQ or were reported as not detected (table). Breeze software was used to control system operation and collect and analyze data.

TLC on glass-backed 250-&m analytical layer silica gel plates (20 # 20 cm) with a mobile phase of 60 mL of butanol/15 mL of glacial acetic acid/25 mL of 0.5 N NaCl confirmed the identity of BMAA-related compounds relative to standards (BMAA, Sigma B-107; methionine, Aldrich 15, 169-6, Milwaukee, WI). Collection of pooled 0.5-min HPLC fragments of derivatized standards and tissue samples was concentrated in a Savant speed-vac concentra- tor and spotted on TLC channels. After drying, the plates were visualized with a 365-nm ultraviolet light. Mass spectrometry of HPLC-identified peaks confirmed the presence of BMAA at 02.1 m/z for both the Sigma standard compound and a bat sample (MVZ 114607).

Results and Discussion. Flying fox skin tissue contained elevated quantities of BMAA (1,287 to 7,502 &g/g; see the table). This is in contrast to the sarcotesta of cycad seeds that they consume, with mean BMAA concentrations of 9 &g/g. We found it interesting that the outermost integument of the seed had extraordinarily high concentrations of BMAA, up to 2,657 &g/g, with a mean of 1,161 &g/g.

The abundance of BMAA in these 50-year old mu- seum specimens suggests that the Chamorro people who consumed this once-abundant flying fox species unwittingly ingested high doses of BMAA. For exam- ple, consumption of MVZ flying fox specimen no. 114607 (assuming a fresh weight of 500 g and uni- form distribution of BMAA throughout the specimen)

would result in the ingestion of 3,751 mg of BMAA, comparable with consuming 1,014 kg of processed cycad flour. We provide (see the table) comparative values of BMAA concentrations in cycad flour sam- ples from the literature.4,5 Differences in the values reflect distinct extraction methods, differences in the analytic methodology, and values unadjusted (as were ours) for BMAA recovery. It has been suggested that BMAA doses of %100 mg/kg/day would be neces- sary to further consider BMAA as a possible caus- ative factor.4 We suggest that such levels might be obtained by eating multiple flying foxes in their en- tirety, as is the Chamorro custom.8 Further ethnobo- tanical data on typical consumption patterns and chemical analysis of the effect on the levels of BMAA and BMAA-related compounds of boiling the flying fox during food preparation are necessary to under- stand the likely doses of neurotoxins consumed.

Other neurotoxic cycad molecules, including cyca- sin, sterol !-D-glucosides,10 or other unknown com- pounds, might be similarly biomagnified and should also be considered as possible environmental neuro- toxins related to ALS-PDC. Varying concentrations of BMAA in individual flying foxes, related to indi- vidual foraging patterns, might have resulted in dif- ferent cumulative doses of neurotoxins among Chamorros who consumed equivalent numbers of fly- ing foxes. This in turn might be tied to different clinical manifestations of ALS-PDC. Examination of the possible role of biomagnification of environmen- tal neurotoxins in ALS, PD, or AD in other areas of the world would be of interest.

Acknowledgment The authors thank C. Conroy, D. Janeke, J. Patton, and J. Steele for assistance in obtaining specimens; J. Douglass, K. Horak, G. Kisby, S. Murch, and D. Qualls for HPLC advice and assistance; G. Hiegel for TLC assistance; and J. Morre for gas chromatog-

Table BMAA in samples of cycads, cycad flour, and flying foxes

Species Sample Concentration,

&g/g

Cycas micronesica Gametophyte 240

Sarcotesta 9

Outer integument 2,657 Concentration,* &g/g of sarcotesta

Ref. 5 Ref. 4

Cycad seed flour Merizo village 3 18 73

Agat village 8 1 4

Yigo village ND 5 8

Equivalent mean dose in kg of cycad flour

Ref. 5 Ref. 4 Current article

Pteropus mariannus No. 114607 7,502 690 104 1,014

(dried skin) No. 114606 1,879 173 26 254

No. 114609 1,287 118 18 174

* Mean concentration reported based on published values4,5 with reported sample sizes ranging from 1 to 4.

388 NEUROLOGY 61 August (1 of 2) 2003

raphy–mass spectroscopy, and the Acacia Foundation for labora- tory support.

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3. Spencer PS, Nunn PB, Hugon J, et al. Guam amyotrophic lateral scle- rosis–parkinsonism–dementia linked to a plant excitant neurotoxin. Science 1987;237:517–522.

4. Duncan MW, Steele JC, Kopin IJ, et al. 2-Amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour: an unlikely cause of amyotrophic lateral sclerosis and parkinsonism–dementia of Guam. Neurology 1990;40:767–772.

5. Kisby GE, Ellison M, Spencer PS. Content of the neurotoxins cycasin (methylazoxymethanol !-D-glucoside) and BMAA (!-N-methylamino-L-

alanine) in cycad flour prepared by Guam Chamorros. Neurology 1992; 42:1336–1340.

6. Duncan MW, Kopin IJ, Garruto RM, et al. 2-Amino-3(methylamino)- propionic acid in cycad-derived foods is an unlikely cause of amyotro- phic lateral sclerosis/parkinsonism. Lancet 1988;II:631–632.

7. Cox PA, Sacks OW. Cycad neurotoxins, consumption of flying foxes, and ALS-PDC disease in Guam. Neurology 2002;58:956–959.

8. Monson CS, Banack SA, Cox PA. Conservation implications of Chamorro consumption of flying foxes as a possible cause of ALS-PDC in Guam. Conserv Biol 2003;17:678–686.

9. Cohen SA, Michaud DP. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 1993;211:279–287.

10. Khabazian I, Bains JS, Williams DE, et al. Isolation of various forms of sterol !-D-glucoside from the seed of Cycas circinalis: neurotoxicity and implications for ALS–parkinsonism dementia complex. J Neurochem 2002;82:516–528.

The potential value of ultrasonography in the evaluation of carpal tunnel syndrome

Henrich Kele, MD; Raphaela Verheggen, MD; Hans-Joachim Bittermann, MD; and Carl Detlev Reimers, MD

Abstract—The authors compared ultrasonography with electrophysiology for the diagnosis of carpal tunnel syndrome (CTS) on 110 clinically affected wrists. An increased cross sectional area in the proximal carpal tunnel larger than 0.11 cm2 in combination with compression signs on longitudinal scans proved to be highly predictive for CTS (sensitivity, 89.1%; specificity, 98.0%). Ultrasound was comparable to electrophysiology in the diagnosis of CTS, and in 35% of cases changes in morphology suggested a specific therapeutic strategy. NEUROLOGY 2003;61:389–391

The diagnosis of carpal tunnel syndrome (CTS) is usually based on clinical findings. Electrophysiology is helpful in confirming the diagnosis, and in less typical cases in differentiating other conditions, but it has a false negative rate with sensitivities ranging from 49% to 86%.1 Moreover, these methods provide no morphologic information regarding the median nerve and possible etiologic factors. The aim of this study was to determine the capability of ultrasonog- raphy as a basis for diagnosis, to correlate sono- graphic abnormalities with electrophysiologic and clinical findings, and to analyze the morphologic findings with regard to further therapy.

Patients and methods. A total of 77 consecutive patients (110 wrists) with clinical symptoms and signs of CTS (59 women, 18 men; mean age, 52 years; range, 22 to 84 years) and 33 asymptom- atic controls (55 wrists) (19 women, 14 men; mean age, 44 years; range, 27 to 80 years) were included in the study. The study group underwent clinical, electrophysiologic, and ultrasonographic ex- aminations. The control group, which consisted of employees of our department and their family members, did not undergo elec- trophysiologic examination. Exclusion criteria were the presence of polyneuropathy or wrist surgery.

Based on the clinical examination, which was considered gold standard, patients were classified as grade 1 CTS when intermit- tent paraesthesias and pain in the median nerve distribution with a normal motor and sensory examination were present. For grade 2, permanent symptoms and a decrease in fine touch sensitivity were present, and for grade 3, motor symptoms with weakness and wasting of the thenar muscles were found.

Electrophysiology was abnormal when the distal motor latency exceeded 4.2 msec, when the antidromic wrist-to-digit sensory nerve conduction velocity (SCV) to fingers with reported symp- toms was less than 47 m/sec, or when electromyographic signs of axonal lesion in the thenar muscles were found (acute or chronic). In patients older than 60 years, the normal SCV was !43 m/sec, and in patients older than 70 years, !40 m/sec.

Ultrasonography was performed by one examiner (H.K.), who was not informed about the patient’s history or clinical and elec- trophysiologic findings, using a real-time scanner (Logiq 500M, General Electric, WI) with an 11-MHz linear array probe. On longitudinal scans, qualitative nerve compression signs were sought. These were present when an abrupt reduction of the nerve diameter under the flexor retinaculum was seen (in contrast to the normally observed harmonic diminishing of the nerve diame- ter on the passage through the carpal tunnel). On axial scans, quantitative evaluation was performed measuring the nerve cross- sectional area (CSA) at the level of the radiocarpal joint, in the proximal (level of the pisiform bone) and in the distal carpal tunnel (level of the hamate bone). Nerve CSA were calculated automatically using integrated software. Quantitative measure-

From the Departments of Clinical Neurophysiology (Drs. Kele, Bittermann, and Reimers) and Neurosurgery (Dr. Verheggen), University of Göttingen, Germany. Received March 25, 2002. Accepted in final form March 22, 2003. Address correspondence and reprint requests to Dr. H. Kele, Dept. of Clinical Neurophysiology, University of Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany; e-mail: [email protected]

Copyright © 2003 by AAN Enterprises, Inc. 389