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Bioaccumulation of β-methylamino-l-alanine (BMAA) through Cyanobacteria-contaminated food or water supplies has been suggested as a possible contributor to Parkinson-dementia complex (PDC) among the indigenous Chamorro of Guam as well as Alzheimer disease (AD) worldwide,1 a speculation that has been widely commented on in news and editorial sections of premier scientific and medical journals,2 and that has led to product recalls and government-sponsored assessments of public health in Europe. However, quantification of BMAA to date has been based on indirect high performance liquid chromatography methods. Given the potential global health significance, we developed a stable-isotope dilution assay for BMAA to more rigorously test its proposed bioaccumulation in cerebral cortex.
All studies were conducted following appropriate informed consent and approval by institutional review boards. We synthesized D3-BMAA by a published method and confirmed product as ≥98% pure by 1H and 13C NMR.3 Samples for gas chromatography–mass spectrometry were prepared with ethyl chloroformate; fragmentation patterns for both D3-BMAA and H3-BMAA were analyzed.4 Selected ion monitoring (SIM) focused on m/z 116 and 245 for H3-BMAA, and m/z 119 and 248 for D3-BMAA: m/z 116 and 119 for quantification, m/z 245 and 248 for qualifying ions. Frozen human cerebral cortex was analyzed in triplicate following established homogenization protocols.5 H3-BMAA spiked into cerebral cortex homogenates had limit of detection for free H3-BMAA = 0.10 μg/g tissue, limit of quantification = 0.25 μg/g tissue, and r2 = 1.00 for 0.25 to 10.0 μg/g tissue using six concentrations. D3-BMAA and H3-BMAA were stable to acid hydrolysis conditions; under these conditions to measure protein-associated H3-BMAA our limit of detection = 5 μg/g tissue, limit of quantification = 10 μg/g tissue, and r2 = 1 for 10 to 1,000 μg/g tissue using seven concentrations.
Frozen tissue from autopsies was obtained following appropriate informed consent as part of NIH-sponsored research programs. Samples were from Chamorro PDC patients on Guam (n = 8, 5 women, 64 to 89 years) who underwent a detailed clinical evaluation and were confirmed by neuropathologic examination; Chamorros on Guam who were free of neurologic symptoms during life and lacked neurofibrillary changes in brain (n = 2 women, 61 and 93 years); patients from the Seattle region who were diagnosed with definite AD (n = 5, 3 women, 74 to 83 years); and individuals from the Seattle region who were seen by a physician and had neuropsychological testing within the last 2 years of life with all results within normal ranges and who showed only age-related changes by neuropathologic examination (n = 5, 3 women, 82 to 91 years). All Seattle area residents in this study were Caucasian. Specific clinical and neuropathologic criteria for these cases have been published previously.6 On Guam, tissue from one half of the brain was fixed in formalin for histologic examination, while tissue from the other half was frozen and stored at −80°C; the postmortem interval ranged from 10 to 40 hours. In Seattle, all tissue was obtained by the Neuropathology Core of the AD Research Center at the University of Washington. Tissue from one half of the brain was dissected at the time of autopsy, flash frozen in liquid nitrogen, and then stored at −80°C. Tissue from the other half was fixed in formalin and used for histopathologic evaluation. The postmortem interval ranged from 3.5 to 9.0 hours.
No free or protein-associated H3-BMAA was detected in any autopsy sample of frontal or temporal cortex, despite our ability to detect other hydrolyzed amino acids (figure). Flash-frozen liver from mice fed BMAA was used as a positive control (figure e-1 on the Neurology® Web site at www.neurology.org). While these data cannot address the possibility of earlier exposure in life and clearance of BMAA from tissue, they do seriously challenge the proposal that BMAA, a nonproteinogenic amino acid, accumulates as a free form or is stored in a protein-associated form in human cerebral cortex. Although products of Cyanobacteria may have other health effects, our new data support the considered review of others which concluded that cycad products are unlikely contributors to PDC.7 Moreover, our findings do not support a global health concern for BMAA contributing to AD.
The authors thank Professor Gelb, Mr. Kruse, and the Mass Spectrometry Facility in the Department of Chemistry at the University of Washington.
Supplemental data at www.neurology.org
Supported by the NIH (AG14382 and AG05136) and the Nancy and Buster Alvord Endowment.
Disclosure: The authors report no disclosures. Christopher A. Shaw is the founder and chief operating officer of Neurodyn, a Canadian-based company examining early phase neurological disease.
Received October 21, 2008. Accepted in final form December 16, 2008.
Address correspondence and reprint requests to Laura R. Snyder, Department of Chemistry, Box 351700, University of Washington, Seattle, WA 98195-1700; ude.notgnihsaw.u@redynsrl