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Presence of the neurotoxic amino acids β-N-methylamino-L-alanine(BMAA) and 2,4-diamino-butyric acid (DAB) in shallow springs from theGobi DesertDerek Craighead a; James S. Metcalf bcd; Sandra A. Banack b; Luvsanjamba Amgalan e; Harry V.Reynolds f; Mijiddorj Batmunkh g

a Beringia South, Kelly, Wyoming b Institute for Ethnomedicine, Jackson, Wyoming, USA c Division ofMolecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK d National Centerfor Natural Products Research, University of Mississippi, Oxford, Mississippi, USA e Institute ofBiology, Mongolian Academy of Sciences, Ulaanbaatar f Reynolds Alaska Wildlife Institute, Fairbanks,Alaska, USA g Great Gobi Special Protected Area, Bayantooroi, Gobi-Altai, Mongolia

Online publication date: 10 November 2009

To cite this Article Craighead, Derek, Metcalf, James S., Banack, Sandra A., Amgalan, Luvsanjamba, Reynolds, Harry V.and Batmunkh, Mijiddorj(2009) 'Presence of the neurotoxic amino acids β-N-methylamino-L-alanine (BMAA) and 2,4-diamino-butyric acid (DAB) in shallow springs from the Gobi Desert', Amyotrophic Lateral Sclerosis, 10: 1, 96 — 100To link to this Article: DOI: 10.3109/17482960903278469URL: http://dx.doi.org/10.3109/17482960903278469

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Amyotrophic Lateral Sclerosis, 2009; (Supplement 2): 96–100

Correspondence: D. Craighead Beringia South, PO Box 147, Kelly, Wyoming 83011, USA. E-mail: derek@bswy.us

(Received 13 August 2009; accepted 21 August 2009)

ISSN 1748-2968 print/ISSN 1471-180X online © 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)DOI: 10.3109/17482960903278469

ORIGINAL ARTICLE

Presence of the neurotoxic amino acids β -N-methylamino-L-alanine (BMAA) and 2,4-diamino-butyric acid (DAB) in shallow springs from the Gobi Desert

DEREK CRAIGHEAD1, JAMES S. METCALF2,3,4, SANDRA A. BANACK2, LUVSANJAMBAAMGALAN5, HARRY V. REYNOLDS7, & MIJIDDORJ BATMUNKH6

1Craighead Beringia South, Kelly, Wyoming, 2Institute for Ethnomedicine, Jackson, Wyoming, USA, 3Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK, 4National Center for Natural Products Research, University of Mississippi, Oxford, Mississippi, USA, 5Institute of Biology, Mongolian Academy of Sciences, Ulaanbaatar, 6Great Gobi Special Protected Area, Bayantooroi, Gobi-Altai, Mongolia, and 7Reynolds Alaska Wildlife Institute, Fairbanks, Alaska, USA

AbstractThe Gobi Desert in Mongolia, home to the critically endangered Gobi bear (Ursus arctos isabellinus), has few water resources for the animals that inhabit this environment. The majority of these water resources are shallow, small bodies of water, from approximately 30 cm to 3 m in diameter. Due to the harsh nature of the Gobi Desert environment, such pools of water are crucial resources for wildlife inhabiting the area and little information is currently available on the presence of organisms, including cyanobacteria, and the toxins they produce within these waters. Drinking water sources and small pools within the Gobi Desert were sampled on two separate occasions in October 2008 and April–May 2009. Samples were assessed for the presence of cyanobacteria; subsamples were taken for the analysis of β -N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB). According to LC-MS/MS analyses, both of these neurotoxic amino acids were present in both years and BMAA was present when cyanobacteria were major components of the pools. The results indicate that assessment of cyanotoxins to organisms that live in desert environments is warranted.

Key words: BMAA, DAB , Gobi Desert , cyanobacteria , water resources

Introduction

Since the fi nding that -N-methylamino-L-alanine (BMAA) production appears to be widespread in aquatic cyanobacteria from all fi ve morphological groupings ( 1) and in environmental samples ( 2), and the fact that this group of organisms is ubiquitous in environments worldwide, an understanding of the occurrence of this amino acid in other types of envi-ronments has become more pressing. This is in part due to the association of BMAA with a high inci-dence of amyotrophic lateral sclerosis/Parkinsonism-dementia complex among the Chamorro people of the island of Guam ( 3) and the positive identifi cation of this amino acid in the brains of North American ALS and Alzheimer’s patients ( 4). Cyanobacteria are important components of desert ecosystems where they stabilize the soil ( 5). In addition to their presence

in desert crusts, the Gobi Desert also supports small shallow pools that are important habitats and resources for a variety of organisms, including the Gobi bear (Ursus arctos isabellinus) and the wild Bactrian camel (Camellus bactrianus ferus), both of which are critically endangered. The Gobi bear pop-ulation in Mongolia is the last remnant of this sub-species of the brown bear adapted to living in the extreme environment of the Gobi Desert. Its distri-bution has declined substantially since the 1970s. Those that still exist are closely tied to aquatic hab-itats as a drinking water source in three oasis com-plexes in the Great Gobi Special Protected Area, a national park in south-western Mongolia that abuts the border with the People’s Republic of China. In addition to wild Bactrian camels, the springs serve as water sources for other large desert-dwelling

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Presence of BMAA and DAB in Gobi desert 97

precipitation in the region is about 100 mm, but during 1993–2007 drought conditions persisted and annual precipitation averaged approximately 50 mm. Years with no precipitation are not uncommon (Terbish, personal communication 2009).

Sample processing

Subsamples were removed and assessed by light and fl uorescence microscopy for the presence of cyanobac-teria using a Zeiss Axioplan fl uorescence microscope with excitation between 515 and 560 nm (green) and emission at 580 nm (red) using a 100 W mercury vapour lamp, with identifi cations performed accord-ing to Whitton ( 6). Further subsamples (1 ml) were removed and placed into pre-weighed microcentri-fuge tubes. These solutions were freeze-dried using a Labconco FreeZone 2.5 lyophilizer and, once dried, the tubes were re-weighed in order to determine the dry weight of the sample. Samples were hydrolyzed in 6.0 N HCl for 16 h at 110°C. Hydrolysates (50 μl) were centrifuge fi ltered (Ultrafree-MC, Millipore) at 13800 g for 3 min, with fi ltrates dried down (Thermo-Savant SC250DDA Speed Vac Plus, Waltham, MA) and then resuspended in 100 μl of 20 mM HCl. A 1/10 dilution was prepared with 20 mM HCl and the sample was derivatized with 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate, as described previously ( 7).

BMAA (Peter Nunn, Portsmouth University, UK) and DAB (Sigma, St. Louis, MO) were iden-tifi ed according to synthetic standards, prepared and derivatized as per hydrolyzed samples from the Gobi Desert. Separation was achieved by liq-uid chromatography (Waters AcQuity Ultra Perfor-mance LC, Milford, MA) using an a Waters AccQTag Ultra column ( 186003837) 2.1 × 100 mm at 0.65 ml/min using the eluents of 0.1% (v/v) formic acid (Thermo Scientifi c Formic Acid 99 � %, 46.02 g/mole, no. 28905) in Fisher Optima LC/MS (W6-4) water (eluent A) and 0.1% (v/v) formic acid in acetonitrile (eluent B) Honeywell, Burdick & Jackson LC/MS grade, LC441-2.5) with the following gradient: time 0� 99.1% A; 0.5 min � 99.1% A; 2.0 min � 95% A; 3.0 min � 95% A; 5.5 min � 90% A; 6.0 min � 15% A; 6.5 min � 15% A; 6.6 min � 99.1% A; 8.0 min � 99.1% A, all curve 6. Nitrogen gas (NitroFlow laboratory, Parker-Balston) was supplied to the Thermo Finnegan triple quadrupole MS H-ESI probe (heated electro-spray ionization) at a nebulizing pressure of 40 psi and a vaporizing temperature of 400°C. The triple quadrupole LC/MS/MS was operated under the fol-lowing conditions: the capillary temperature was set at 270°C, auxillary gas pressure of 35 psi, spray voltage of 3500 v, capillary offset of 35, tube lens offset of 110, source collision energy of 5, and multiplier voltage of �1789. The second quadrupole was pressurized to 1.5 mTorr with 100% argon. The derivatized molecule after ionization (MW 459) via HESI was selected as the precursor ion for analysis. Collision induced

mammals including wild ass (Equus hemionus), argali sheep (Ovis ammon), ibex (Capra sibirica), gazelle (Procapra gutturosa, and subgutturosa), snow leopard (Uncia uncial), and wolf (Canis lupus).

We have identifi ed three oasis complexes in the Great Gobi-A Specially Protected Area (GGSPTA-A). They are: 1) in the west, the Atas-Inges complex, comprising a minimum of four springs; 2) in the cen-ter, the Shar Khulsnii complex comprising a mini-mum of eight springs; and 3) in the east, the Tsagaan Bogd complex, comprising a minimum of fi ve springs. There are other springs around the periphery of our study area that may be isolated springs or associated with some other oasis complex. Most, if not all of the springs of the GGSPA, at one time or another are visited by all the large mammals of the region; Gobi bear, wild camel, ibex, argali sheep, gazelle, wild ass, wolf, snow leopard, and fox. These springs constitute the primary source of drinking water during the hot, dry summer months. During the fall and winter there is water available in the form of snow, and during the fall and spring, certainly snow melt and rain is avail-able in catchment basins. Following the infrequent rain showers, ephemeral, or intermittent streams fl ow. Depending on the amount and duration of the pre-cipitation these at times should be characterized as streams or small rivers.

Two expeditions to the Gobi Desert were carried out in October 2008 and April–May 2009 during which samples from shallow pools were obtained. The purpose of the study was to investigate these pools for the presence of cyanobacteria and two cyanotoxins, BMAA and its isomer DAB. The presence of cyanobacteria and their toxins within these resources potentially has important implica-tions for the health assessment of the organisms that use them and provides further evidence for the widespread occurrence of BMAA within the environment.

Materials and methods

Site selection and sampling

There are three oasis complexes in the Great Gobi Special Protected Area: complexes are separated from each other by 70–110 km ( Figure 1 ). Each complex supports 7–11 known springs, which vary in size from approximately 30 cm to 3 m in diameter. A few of the larger springs provide enough water to support small effl uent about 15-40 cm wide for distances of up to 400 m before they dissipate into the sand or other substrate. Most springs are confi ned to a single site and have negligible fl ow from their point of origin. Sample sites were selected from each complex.

In this region, daytime air temperatures average 38°C in summer and �27°C in winter months. Wind speeds in the spring, when bears are weakest after emerging from hibernation, average 16–18 m/s. The region is extremely arid: long-term annual

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98 D. Caighead et al.

Figure 1. A) Map of Mongolia showing Gobi Bear Special Protected Area A, gray shows previous range extension of Gobi bear whileblack area shows current restriction; B) Paw of tranquilized Gobi bear showing abrasion from harsh arid conditions; surface seeps and water in the summer in the Gobi Desert are rare; C) Gobi bear (Ursus arctos isabellinus) in profi le at Khutlin spring; D) Birge plankton net sampling of cyanobacteria from Khulsnii oasis; E) Aerial photo of Khulsnii oasis showing both algal and cyanobacterial blooms; F) Mongolian research scientists at Bogts Tsagaan Ders seep; G) Differential interference contrast (DIC) microphotography of cyanobacterial sample from Mukharzadgae seep showing cyanobacteria of the Oscillatoriales; H) DIC microphotograph of Anabaena fi laments sampled from Khulsnii seep; I) Mixed cyanobacterial taxa from Altan Tevsh seep using fl uorescence microscopy.

dissociation (CID) was achieved in the second quadrupole using the following parameters: 459 to 119 CE, 21; 459 to 171 CE, 38; and 459 to 289 CE, 17. The resultant three product ions of the deriva-

tized BMAA (MW 119, 289, 171) were scanned by the third quadrupole, subsequently detected, and their relative abundances quantifi ed. The ratios of these three product ions were compared to the ratios

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Presence of BMAA and DAB in Gobi desert 99

of the product ions created by injection of AQC-derivatized BMAA or DAB compared with Gobi Desert extracts.

Results

Microscopic analysis of samples collected from shal-low pools in the Gobi Desert showed fairly diverse groups of organisms that were present in the major-ity of the pools. The phytoplankton primarily com-prised green algae and diatoms, with cyanobacteria present in the majority of samples (8/9 samples). When cyanobacteria were present, they were pri-marily of the orders Oscillatoriales and Chroococ-cales, with Nostocales (Anabaena) present in one sample from 2009 (site 8, Figure 1 ). LC-MS/MS analyses showed the presence of both BMAA and DAB in samples ( Figure 2 ,Table I). DAB was more frequently found than BMAA, but the presence of BMAA was more indicative of a signifi cant pres-ence of cyanobacteria. Ratios and retention times from LC-MS/MS analyses for BMAA and DAB in samples were the same as for synthetic standards analyzed under the same conditions.

Discussion

LC-MS/MS analysis of water samples from the Gobi Desert showed the presence of cyanobacte-ria, BMAA and DAB. Water resources are limited in this area and are used by a number of species for drinking, including critically endangered Gobi bears and wild Bactrian camels. As there are only 20–50 Gobi bears remaining, the potential contribution of cyanobacterial toxins to their health is some-thing that requires urgent attention. Endangered species have been shown to be susceptible to the action of cyanobacterial toxins, including the Lesser Flamingo (Phoeniconaias minor) which is consid-ered ‘near-threatened’ by the International Union for Conservation of Nature (IUCN) in their red species list ( 8) and this species succumbs to the action of cyanobacterial toxins on a periodic basis ( 9). A further compounding factor that requires inves-tigation, not just in desert environments, but in all environments where cyanobacteria occur is the co-occurrence of BMAA and DAB and the potential synergistic or antagonistic effects that may result. BMAA has previously been shown to be coproduced

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NL: 7.24E6Base Peak m/z= 170.93-171.13 F: +c ESI SRM ms2 459.050[119.099-119.101, 171.099-171.101,289.099-289.101] MS ICIS

NL: 4.74E5Base Peak m/z= 288.96-289.16 F: +c ESI SRM ms2 459.050[119.099-119.101, 171.099-171.101,289.099-289.101] MS ICIS

NL: 3.70E5Base Peak m/z= 119.00-119.20 F: +c ESI SRM ms2 459.050[119.099-119.101, 171.099-171.101,289.099-289.101] MS ICIS

NL: 8.00E6TIC F: + c ESI SRM ms2 459.050[119.099-119.101, 171.099-171.101,289.099-289.101] MS ICIS

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Figure 2. LC-MS/MS analysis of an extract from a shallow pool in the Gobi desert showing the presence of BMAA and DAB. Daughterions were monitored at 171 (A), 289 (B) and 119 (C), with total ion counts at 459 (D).

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100 D. Caighead et al.

G. Dovchindorj (Great Gobi Special Protected Area Nayambayar, Great Gobi Special Protected Area).

Disclosure of interest: The authors report no con-fl icts of interest and alone are responsible for the content and writing of this paper.

References

Cox PA, Banack SA, Murch SJ, Rasmussen U, Tien G, 1. Bidigare RR, et al. Diverse taxa of cyanobacteria pro-duce -N-methylamino-L-alanine, a neurotoxic amino acid. PNAS. 2005;102:5074–8.Metcalf JS, Banack SA, Lindsay J, Morrison LF, Cox PA, 2. Codd GA. Co-occurrence of -N-methylamino-L-alanine, a neurotoxic amino acid with other cyanobacterial toxins in British waterbodies. Env Microbiol. 2008;10:702–8.Cox PA, Banack SA, Murch SJ. Biomagnifi cation of cyano-3. bacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. PNAS. 2003;100:13380–3. Pablo J, Banack SA, Cox PA, Johnson TE, Papapetropoulos S, 4. Bradley WG, et al. Cyanobacterial neurotoxin BMAA in ALS and Alzheimer’s disease. Acta Neurologica Scandinavica. 2009. (in press)Wynn-Williams DD. Cyanobacteria in deserts – life at 5. the limit? In: Whitton BA, Potts M, editors. The Ecology of Cyanobacteria. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2000. pp 341–66.Whitton BA. Phylum Cyanophyta (Cyanobacteria). In: 6. John DM, Whitton BA, Brook AJ, editors. The Freshwater Algal Flora of the British Isles . Cambridge, UK: Cambridge University Press ; 2002. pp 25–122.Banack SA, Cox PA. Biomagnifi cation of cycad neurotoxins 7. in fl ying foxes. Neurology. 2003;61:387–9.Childress B, Nagy S, Hughes B, compilers. International Single 8. Species Action Plan for the Conservation of the Lesser Flamingo (Phoeniconaias minor). CMS Technical Series No. 18, AEWA Technical Series No. 34. Bonn, Germany, 2008. Codd GA, Metcalf JS, Morrison LF, Krienitz L, 9. Ballot A, Pfl ugmacher S, et al. Susceptibility of fl amin-gos to cyanobacterial toxins via feeding. Vet Rec. 2003;152:722–3.Codd GA, Morrison LF, Metcalf JS. Cyanobacterial toxins: 10. risk management for health protection. Toxicol Appl Pharmacol. 2005;203:264–72.

with microcystins, nodularin, anatoxin-a and sax-itoxins in cyanobacterial blooms ( 2) and with cylin-drospermopsin in a cyanobacterial strain ( 1), along with LPS and suggests that toxicological assess-ment of cyanobacteria and the role of toxins in neurodegenerative disease requires urgent attention. Members of the Chroococcales, Oscillatoriales and Nostocales have been shown to produce microcys-tins and members of the Oscillatoriales and Nosto-cales can produce neurotoxins such as anatoxin-a and saxitoxins as examples ( 10). By studying a range of environments such as the Gobi Desert, a greater understanding of the occurrence and the potential for human exposure to BMAA may be elucidated.

Acknowledgements

The authors thank the following members of the Gobi Bear Project Team for their invaluable assistance with this study: Ts. Tuya (UNDP Great Gobi and Its Umbrella Species Project), M. Proctor (Birchdale Ecological), Jenny Ross (Jenny E. Ross Photography),

Table I. Summary of the occurrence of cyanobacteria, BMAA and DAB in samples from shallow pools in the Gobi Desert.

Spring location/year BMAA DAB Microscopy

2008Altan Tevsh � � GA, D, Osc, ChroSujiin Bulag � � D, GATsagaan Tokhoi � � D, GA, Osc, ChroKhukh Ders � � GA, OscMukhar Zadgai � � GA, Osc, Chro

2009Bogd Tsagaan Ders � � GA, D, Osc (minor)Shar Khulsnii � � GA, Osc (minor)Khukh Khuls � � D, ChrooKhotul Us � � GA, D, Osc, Nos

� : positive according to LC-MS/MS; –: not detected; GA: green algae; D: diatoms; Osc: Oscillatoriales; Chro: Chroococcales; Nos: Nostocales.

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