1. Flying foxes in Guam were found to have significantly higher levels of the neurotoxin BMAA than the cycad seeds they eat, confirming that cycad neurotoxins biomagnify within the Guam ecosystem. 2. Consuming a single flying fox could result in a BMAA dose equivalent to eating over 1,000 kg of processed cycad flour. 3. Traditional feasting on flying foxes by the Chamorro people of Guam may be related to the high prevalence of neurodegenerative diseases like ALS-PDC in Guam.

1. 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

2. 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

3. 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),

4. 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.

5. 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

6. 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;

7. 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

8. 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.
References
1. Kurland LT, Mulder DW. Epidemiologic investigations of
amyotrophic
lateral sclerosis. Neurology 1954;4:355–378, 438 – 448.
2. Reed D, Labarthe D, Chen KM, et al. A cohort study of
amyotrophic
lateral sclerosis and parkinsonism– dementia on Guam and
Rota. Am J
Epidemiol 1987;125:92–100.
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

9. 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

10. 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.

11. 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

12. 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
Biology 112 Spring 2016
Scientific Journal Article Critique
Each time you evaluate an article, you will complete Part I and
Part II. You will submit on a typed hardcopy Part I., and Part II
on the due date listed in your syllabus.

13. Part I.
1. Skim the article (take light notes)
· Read the abstract. The abstract informs you of the major
findings of the study, and the importance.
· What is the big picture of the study (this is done as you read
the article)
· Record terms or techniques you are not familiar with.
· Include questions to parts of the article you do not understand.
· If you are unfamiliar with concepts discussed throughout the
article, then perform a Google search.
2. Re-read the article
· Go to the Materials and Methods and Results section, and ask
the following questions within each section
· Was the study repeated? (You should know why a study must
be repeated. If you do not know ask Prof. Olave or Dr. Bignami
ASAP)
· What was the sample size? Is this representative of a large
population?
· What were the variables? Controls?
· What factors might affect the outcome (according to the
investigators)
· Interpret the data within each figure without looking at the
text. Once you have done this, then read the text.
· Understand the purpose of the Materials and Methods
3. Preparing to summarize the article:
· Describe the article in your own words first. Can you explain
to a friend without looking at your notes? If not, then most
likely you do not understand. Go over your notes again.
· What was the purpose of the study?
· A reader who has not read your article must understand your
summary.
4. Write a draft of your summary:
· Begin to write the article without looking at your notes. If you

14. choose to look at your notes, then you may not understand the
article, and may unintentionally plagiarize.
· Ask yourself the following questions to write your summary
(without looking at your notes) in your own words:
· What was the purpose of the study?
· What questions were asked?
· How did the study address these questions?
· What assumptions did the author make?
· What were the major findings?
· What questions are still unanswered (according to the authors
of the article)
Part II. Critical Review and Assessment of the Article
· In your summary, include your own analysis and evaluation of
the article.
· Do not include personal opinions
· Use professional language. For example:
Common language: Dipodomys merriami is a kangaroo rat that
has a longer Loop of Henle, and this helps it survive better in
the desert by retaining more water.
Professional language: A longer Loop of Henle in Dipodomys
merriami allows for greater water absorption, an adaptation that
has led to survival in an arid environment.
· How did this study answer questions proposed in the
introduction section of the paper?
· Include the limitations of the study:
· Does the data support the conclusions of the study. Explain.
· What questions remain unanswered?
· How could future studies be improved?
Note: This scientific writing critique is based on Pechenik, Jan
A. “Writing Summaries and Critiques.” A Short Guide to
Writing about Biology. Ed. Rebecca Gilpin. 6th ed. New York:
Pearson, 2007. 130-138.