P185 SAMPLE MATRIX INTERFERENCES IN ANALYTICAL METHODS FOR DETECTION AND QUANTIFICATION OF BMAA
GLOVER B1, BROWN P2, MURCH S1
1University of British Columbia, Kelowna, British Columbia, Canada, 2British Columbia Institute of Technology, Burnaby, British Columbia, Canada
Email address for correspondence: [email protected]
Keywords: BMAA, mass spectrometry, matrix effects
BMAA is a non-protein amino acid found in samples of cyanobacteria, traditional foods and people of Guam. In in vitro studies, BMAA has been shown to be neurotoxic leading to interest in the prevalence of the amino acid in other parts of the world. However, difficulties in the measurement of BMAA in complex biological matrices have resulted in conflicting reports. Mass spectrometry (MS) is often seen as the “gold standard” of analytical techniques and analysis of BMAA by the fragmentation of the parent as m/z 119 - > 102 has been reported. This method of detection is prone to false negative error caused by chromatography interferences, voltage fluctuations, ion suppression and sample matrix effects. Only 0% - 28% of underivatized BMAA standard eluted from C18 columns using the published columns and eluents. MS issues were identified using a Time of Flight MS with mass accuracy 0.001. Optimal detection of the parent mass 119.028 is achieved at low inlet voltages (< 30) and MS response declined dramatically at higher inlet voltages to less than 20% of the standard. Regardless of voltages, all BMAA solutions tested contain a 102.055 signal at a steady 26% of the total ion count and corresponding to the daughter ion indicating spontaneous loss of the OH group in solution. Depending on voltages uses, between 3% and 66% of the BMAA standard was detected as the compound dimer with m/z 237.156. Interestingly, higher voltages increased the concentration of the dimer rather than increasing the daughter ion signal. Further, the measurement of BMAA in marine cyanobacteria is complicated by the formation of ion adducts in salt water. BMAA standards made in salt water solutions contain m/z signals of 143.525 (Mg-BMAA; 38%), 142.21 (Na-BMAA; 12%) and 159.183 (Ca-BMAA; 9%). Together, ion adducts account for more than 50% of the BMAA ion signal in underivatized standards. In contrast, ACQ-derivatized BMAA (m/z 459.17) forms a Na adduct (m/z 481.162) in marine samples that accounts for less than 4% of the total ion signal. These data indicate that, while MS can be a useful tool for analysis of BMAA, sample matrices and instrumental factors can lead to lost signal and false negative results.
P186 BMAA IS A CYANOBACTERIAL METABOLITE
DOWNING T, DOWNING S
Nelson Mandela Metropolitan University, Port Elizabeth, South Africa
Email address for correspondence: [email protected]
Keywords: BMAA, cyanobacteria, bioaccumulation
Background: β-N-methylamino-L-alanine (BMAA) has been associated with certain forms of progressive neurodegenerative disease, including sporadic Amyotrophic Lateral Sclerosis and Alzheimer's disease. The source of BMAA considered to have contributed to ALS/PDC on Guam was initially identified as Cycas micronesica. Subsequently, symbiotic Nostoc in the corraloid roots of cycads were shown to contain BMAA. Neurotoxic, non-protein amino acids have been intensively studied in eukaryotic phototrophs but have received less attention in cyanobacteria although BMAA in cyanobacterial cultures, and in cyanobacterial blooms continues to be reported by investigators from a variety of laboratories. However, it was recently suggested, based on reported instances of failure to detect BMAA in cyanobacteria, that BMAA may be confused with other compounds during analysis or that cyanobacteria may not be the source of the toxin.
Objectives: We sought to confirm the analytical validity of BMAA identification methods and to apply these methods to confirm the cyanobacterial origin of BMAA by stable isotope feeding of axenic and uni-algal cyanobacterial cultures. We also investigated the effect of biologically available nitrogen on cellular BMAA content so as to address the wide range of BMAA content observed in cyanobacteria. Additionally, we sought to confirm the presence of BMAA in organisms exposed to cyanobacterial blooms to support our data on uptake of BMAA by a range of organisms.
Methods: Two pre-column derivatization methods with either LC/MS or LC/MS/MS detection were used to distinguished BMAA from related compounds and to quantify BMAA in BMAA-exposed and unexposed samples. Nitrogen modulation of cyanobacterial culture media and 15N feeding experiments were used to evaluate BMAA production in axenic and uni-algal cultures, and the rate of assimilated nitrogen incorporation into BMAA.
Results: BMAA production by cyanobacteria was confirmed in axenic and uni-algal cultures of cyanobacteria based on chromatrographic retention time and by mass spectrometry using parent ion and product ions and ion ratios resulting from collision-induced dissociation. Nitrogen starvation of nutritionally replete cells resulted in an increase in free cellular BMAA. The addition of NO3- and NH4 + to the culture medium following starvation resulted in a decrease of free cellular BMAA, with ammonia resulting in a more rapid reduction. Addition of 15N ammonium chloride resulted in the appearance of 15N-containing amino acids, with no significant increase in 15N-containing BMAA. Subsequent starvation resulted in the appearance of 15N-containing BMAA.
Discussion and conclusion: BMAA is clearly distinguishable from common isomers and diamino acids in cyanobacteria. The production of BMAA by an axenic culture of cyanobacteria confirms the cyanobacterial origin of BMAA. This is further supported by the production of isotopically labeled BMAA by axenic cyanobacterial cultures from supplied raw materials, and by the presence of BMAA in aquatic organisms exposed to cyanobacterial blooms.
P187 CYANOBACTERIAL BLOOMS AND THE OCCURRENCE OF THE NEUROTOXIN BMAA IN FLORIDA AQUATIC FOOD WEBS
BRAND L, PABLO J, COMPTON A, HAMMERSCHLAG N, MASH D
University of Miami, Miami, Florida, USA
Email address for correspondence: [email protected]
Keywords: BMAA, cyanobacteria, seafood
Background: Recent papers have suggested that virtually all cyanobacteria species produce the neurotoxin beta-N-methylamino-L-alanine (BMAA). A large increase in the development of Amyotrophic Lateral Sclerosis (ALS)-Parkinsonism dementia complex in Guam in the middle of the 20th century has led to the hypothesis that the neurotoxin BMAA may be involved. High concentrations of BMAA have been found in humans who died of ALS or Alzheimer's disease, but little or no BMAA in the brains of those who died of other causes. These results suggest that BMAA from cyanobacteria could be involved in the development of neurodegenerative diseases.
Objectives: To determine if BMAA can biomagnify in aquatic food chains, BMAA concentrations were analyzed in animals collected from water bodies known to have blooms of cyanobacteria, primarily in Florida.
Methods: Cyanobacteria abundance was estimated by measuring concentrations of phycocyanin with a spectrofluorometer. BMAA was quantified by detection of the AQC fluorescent tag detected by reverse phase HPLC and verified by LC/MS/MS using product ion mode in a triple quadrupole system.
Results: Samples of pink shrimp in Florida Bay where a large bloom of cyanobacteria developed in central Florida Bay in the 1980s and persists have high concentrations of BMAA. Samples of fish and crustaceans in South Biscayne Bay-Eastern Florida Bay where a large bloom of cyanobacteria developed for 2 years show some species with no BMAA and others with very high concentrations. High concentrations were found in fish in the Caloosahatchee River where large blooms of cyanobacteria develop as a result of nutrient rich water from the Everglades Agricultural Area. High concentrations of BMAA were found in fish and blue crabs in Chesapeake Bay where blooms of cyanobacteria occur. Concentrations of BMAA similar to that of ALS and Alzheimer's patients were found in the brain tissue of bottlenose dolphins in the Indian River Lagoon where large blooms of cyanobacteria occur.
Discussion: These data suggest that BMAA could be found in high concentrations in aquatic animals in many areas of the world where cyanobacteria blooms occur.
Conclusions: A wide range of concentrations (ranging from undetectable to approximately 7000 ug/g) of the neurotoxin BMAA were found in various animals, from crustaceans to dolphin brains, in aquatic food webs. This suggests that BMAA can biomagnify up the food chain from cyanobacteria to seafood.
P188 IDENTIFYING ENVIRONMENTAL TRIGGERS OF AMYOTROPHIC LATERAL SCLEROSIS IN NORTHERN NEW ENGLAND
FIELD N, CALLER T, STOMMEL E
Dartmouth Hitchcock Medical Center, Lebanon, NH, USA
Email address for correspondence: [email protected]
Keywords: BMAA, cyanobacteria, epidemiology
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Sporadic ALS (sALS) represents the vast majority of cases, while approximately 5% of cases are familial and inherited (fALS). sALS likely results from a combination of genetic and environmental risk factors, however, few environmental factors have been identified as possible triggers for the disease. One proposed mechanism is exposure to the cyanobacteria produced neurotoxin β-methylamino-L-alanine (BMAA), which has been implicated as a cause of ALS/Parkinsons-Dementia Complex in Guam. Recent studies supporting this hypothesis found high levels of BMAA in the brain tissue of ALS patients and demonstrated a mechanism for bioaccumulation of BMAA within the food chain on Guam. Because cyanobacteria are found ubiquitously throughout the world, from the Baltic Sea to the desert sands of Iraq, our research aims to identify routes of exposure to BMAA and identify clusters of sALS in Northern New England, USA. Using 2010 Census and Landscan USA data, we constructed an expected model of sALS cases in Vermont, New Hampshire and Maine based on population density, age, and gender. Using geo-spatial analysis tools in ArcGIS we identified areas where the observed cases of ALS significantly exceeded the predicted number. A number of these clusters are located on water bodies known to harbor cyanobacterial blooms, potentially linking them to BMAA. In order to further examine the etiology of ALS and gather specific home address data for the previous analysis, an in-depth questionnaire was administered to over one-hundred and fifty ALS patients as well as to controls throughout the region. The questionnaire explored possible relationships between ALS and previously reported risk factors including smoking, military service, as well as potential exposure to BMAA which is produced by cyanobacteria found in blue-green algal blooms on lakes and rivers throughout New England. Sediment core, water, and bloom sample analysis has allowed for the detection of BMAA and historical identification of cyanobacteria in New England water bodies. Further geo-spatial analysis through the expansion of our study population will allow us to better determine if a correlation exists between proximity and exposure to cyanobacterial blooms and the development and onset of ALS.
P189 PROTEINS CONTAINING BMAA FORM AUTOFLUORESCENT AGGREGATES AND INDUCE CELL DEATH
DUNLOP R1, RODGERS K2
1The Heart Research Institute, Sydney, NSW, Australia, 2University of Technology, Sydney, NSW, Australia
Email address for correspondence: [email protected]
Keywords: BMAA, aggregation, apoptosis
Background: β-methylamino-L-alanine (L-BMAA), a non-protein amino acid produced by cyanobacteria (blue-green algae), has been linked to a neurodegenerative disease in the South Pacific (ALS/PDC) and more recently to sporadic Amyotrophic Lateral Sclerosis (sALS). Cyanobacteria are ubiquitously distributed in terrestrial, fresh water and marine environments thus human exposure to L-BMAA, possibly via bioconcentration through the food chain, is a likely route. Critically, the vast majority (> 90%) of L-BMAA present in extracts of cyanobacteria, cycad seeds, brain tissue from Chamorro ALS/PDC patients and from North American ALS and Alzheimer's patients is in a ‘protein-associated form’. Here we extend these studies to examine the toxicity of BMAA-containing proteins.
Objectives: To determine if proteins containing incorporated BMAA are toxic to cells.
Methods: Human fibroblasts (MRC-5) and neuroblastoma cells (SH-SY5Y) were incubated in amino acid-free media, supplemented with 500 μM L-BMAA in the presence and absence of the protein synthesis inhibitor cycloheximide (CHX) for 24 hrs. Cells were then examined by inverted fluorescent microscopy – for autofluorescent aggregates. Cell viability was assessed using acridine orange (AO) to indicate apoptosis and ethidium bromide (EtBr) to indicate necrosis.
Results: In cultures incubated with L-BMAA, perinuclear autofluorescence was observed under fluorescent light. Cells incubated with CHX (2 μg/mL) alone remained viable, while cells incubated with 500 μM BMAA showed an ∼4-fold increase in apoptosis/necrosis. In the presence of CHX, which reduces BMAA incorporation into protein by 75%, apoptosis/necrosis was significantly reduced. Further, co-incubation with amino acids to “out-compete” BMAA incorporation, also protected the cells from death.
Discussion: Post-mitotic cells, in particular neurons, are extremely sensitive to misfolded proteins, possibly because these potentially toxic species cannot be diluted out of the cell by normal cell division. As such, cellular inclusion bodies and aggregated mutant proteins, such as TDP-43 and alpha synuclein, have been implicated in the etiology of ALS. We have previously shown that the misincorporation of non-protein amino acids results in the formation of autofluorescent aggregates and cell death via apoptosis (1).
Conclusions: In cell culture, incubation with L-BMAA resulted in the formation of autoflourescent aggregates and the induction of apoptosis and necrosis. Thus, chronic exposure to BMAA may eventually result in the accumulation of misfolded/aggregated proteins which may induce cell death and thus, contribute to the pathology of sALS.
Reference
- Dunlop R A, Brunk U T, and Rodgers KJ. Biochem 2011; 435:207–16.
P190 EFFECTS OF THE CYANOBACTERIAL TOXIN, Β-METHYL-AMINO-L-ALANINE (BMAA), ON HUMAN NEURONS AND RAT GLIAL CELLS
GEHRINGER MM, CHIU A, WELCH JH, BRAIDY N, GUILLEMIN GJ, NEILAN BA
University of New South Wales, Sydney, NSW, Australia
Email address for correspondence: [email protected]
Keywords: BMAA, primary human neurons, glia
Background: The amino acid variant β-methyl-amino-L-alanine (BMAA) has long been associated with the increased incidence and progression of the amyotrophic lateral sclerosis/ Parkinson's disease complex (ALS/PDC) within the Chamorro people from Guam (1). BMAA has been reported to be produced by more than 90% of cyanobacterial species (2), and is therefore potentially ubiquitous in the environment. Even though BMAA has been detected in the brains of Chamorro people who died of ALS/PDC (3), as well as in the post-mortem brains of Alzheimer's disease (4) and ALS sufferers in North America, its effects on human primary neurons or neuron associated glial cells have not been determined.
Objectives: Here, we have challenged human primary neurons (HPN) and rat olfactory ensheathing cells (OECs) with exogenous BMAA, at concentrations from 100 μM to 1 mM, and measured its effects on cellular toxicity, apoptosis and generation of reactive oxygen species (ROS).
Methods: OECs and HPN were exposed to pure BMAA and media assayed for LDH release. DNA damage, generation of ROS and Ca2 + influx were measured. Neuronal nitric oxide synthase (nNOS) and caspase 3 cleavage were monitored microscopically in HPN. Mitochondrial activity of OECs was also measured. RNA was extracted from OECs and subjected to microarray analysis.
Results: We show that BMAA is cytotoxic, increases Ca2 + influx, enhances production of ROS and causes DNA damage in both cell types. Furthermore, BMAA disrupted mitochondrial activity in OECs. Caspase 3 cleavage and expression of nNOS were observed. Microarray expression data suggested a role of BMAA in the induction of apoptosis.
Discussion and conclusion: The results indicate that both cell types are significantly compromised after treatment and that the cyanobacterial toxin acts by direct excitotoxicity and via disturbance of mitochondrial activity. This is the first study investigating BMAA toxicity using human primary neurons and pure glial cells. The gliotoxicity of BMAA highlights its role as a key player in neurodegenerative disease as it can disturb neuronal homeostasis at several levels. The data presented align BMAA with the three proposed mechanisms of degeneration in ALS, those being non-cell autonomous death (5), excitotoxicity and mitochondrial dysfunction (6). The cytotoxicity of BMAA on different brain cells has important implications for the aetiology, progression and treatment of neurodegenerative disease.
References
- Spencer PS, et al. Science 1987;237:517–22.
- Cox PA, et al. Proc Natl Acad Sci USA 2005;102:5074–78.
- Murch SJ, Cox PA, Banack SA, Steele JC, Sacks OW. Acta Neurol Scand 2004;110:267–69.
- Ince PG, Codd GA. Neuropathol Appl Neurobiol 2005; 31:345–53.
- Barber SC, Shaw PJ. Free Radic Biol Med 2010; 48:629–41.
- Wang X, Michaelis EK. Front Aging Neurosci 2010;2:12.
P191 SYNERGISTIC TOXICITY OF THE ENVIRONMENTAL NEUROTOXINS METHYLMERCURY AND BETA-N-METHYLAMINO-L-ALANINE (BMAA)
LOBNER D, LIU X, RUSH T
Marquette University, Milwaukee, WI, USA
Email address for correspondence: [email protected]
Keywords: BMAA, methylmercury, glutathione
Background: Determination of the environmental factors involved in ALS has been elusive. Methylmercury and β-N-methylamino-L-alanine (BMAA) have both been implicated in this role. However, studying these factors in isolation probably does not accurately mimic the human condition. ALS often likely involves a complex interaction between genetic predisposition and multiple environmental factors. As a first step to assess how such factors may interact, we studied the interaction of BMAA and methylmercury. BMAA and methylmercury are widespread in the environment and exposure to both is likely common occurrence.
Objectives: The objective of the study was to test whether there is a synergistic effect between methylmercury and BMAA in causing neurotoxicity and then to determine the mechanism of the synergistic toxicity.
Methods: We used primary mixed neuronal and glial cortical cultures from embryonic mice to study the toxicity of BMAA and methylmercury. Neuronal death was assessed by release of the cytosolic enzyme lactate dehydrogenase. Since high concentrations of both methylmercury and BMAA have been shown to deplete cellular glutathione levels, we chose to analyze glutathione as a potential point of interaction between the two toxins. Cellular glutathione levels were assessed using an enzymatic assay that measures total glutathione.
Results: Exposure of cultures to methylmercury or BMAA independently induced concentration dependent neurotoxicity. The death caused by each toxin was selective to the neurons. Importantly, concentrations of BMAA (10 - 100 mM) that caused no toxicity by themselves potentiated methylmercury (3 mM) toxicity. BMAA plus methylmercury, at concentrations that had no effect by themselves on glutathione levels, together induced depletion of cellular glutathione. Furthermore, the combined toxicity of methylmercury and BMAA was attenuated by the cell permanent form of glutathione, glutathione monoethyl ester or the free radical scavenger, trolox. The combined toxicity was not blocked by the NMDA receptor antagonist MK-801. Toxicity of a high concentration of BMAA is mediated primarily by NMDA receptor activation, with significant protection provided by MK-801, and protection by trolox only occurring when NMDA receptors are blocked. Neurotoxicity of high concentration methylmercury in this culture system is attenuated by glutathione monoethyl ester, but not by trolox or MK-801. Therefore, the combined toxicity is somewhat different than that of each toxin alone. There is clearly a prominent role of oxidative stress in the neurotoxicity of the combined toxin treatment.
Discussion: There are two main findings of the current study. First, there is a synergistic toxicity induced by exposure to a combination of methylmercury and BMAA. Second, the mechanism of the synergistic toxicity appears to be at the level of cellular glutathione depletion.
Conclusions: Since glutathione depletion is known to occur in ALS, these results provide a potential mechanism for the involvement of methylmercury and BMAA in ALS.
P192 DISEASE PROGRESSION WITH EARLY TOXIN-INDUCED NEUROPATHOLOGY IN THE AGEING MUTANT SOD MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
LEE G1,2, SHAW CA1
1University of British Columbia, Vancouver, Canada, 2Vancouver Coastal Health, Vancouver, Canada
Email address for correspondence: [email protected]
Keywords: superoxide dismutase (SOD), motor axon, glia
Background: Familial adult onset amyotrophic lateral sclerosis (FALS) poses injuries to the central nervous system that lead to progressively debilitating and irreversible motor deficits. The loss of motor neurons results from a poorly understood, multifactorial neurodegenerative process. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are one cause of FALS. In contrast, sporadic ALS occurs with a vastly greater incidence and from unknown etiologies. Among suspected causes are various environmental toxins. An early report of environmental causes of neurodegenerative disease, including a form of ALS, pointed to a long latency neurotoxin in cycad seeds amongst the Chomorro people of Guam. Both washed cycad as well as isolated water insoluble steryl glucosides (SG) similar to that found in cycad seeds reproduced an ALS-PDC phenotype in an in vivo model.
Objectives: To determine whether environmental agents such as those from cycad accelerate disease onset in an otherwise late-onset condition, we combined two in vivo models of ALS testing dietary SGs for their potential synergistic properties in combination with genetic predisposition to adult onset ALS in the G37R mouse.
Methods: Male and female mice were treated with 42 mg of SG per kilogram of body weight daily in their diet. A cohort of animals harboured the SOD1G37R mutation for genetic predisposition to ALS.
Results: Results showed an additive effect of SG on spinal motor neuron loss and caused decreases in average soma diameter. While the presence of the transgene alone caused a leftward shift towards smaller diameter ventral root axons, SG exposure alone resulted in a bimodal distribution resembling a more immature state. The presence of the transgene alone markedly increased the amount of GFAP- and Iba1-positive cells in the spinal cord grey matter, with a heterogeneous expression of ramified (resting) and activated morphologies. The transgene in combination with SG did not significantly change glial numbers, but caused all glial cells to become extensively activated.
Discussion and conclusions: Although the mechanism of cycad toxin-induced neurodegeneration remains uncertain, the current results showed that dietary exposure to SGs alone was sufficient to produce a disease phenotype, but that when implemented in conjunction to a genetic predisposition to ALS was sufficient to produce a more severe disease phenotype. In conclusion, the environmental agent studied here has direct cytotoxic effects and contributes to disease progression in ALS. The mouse model of disease exploited in this study may be used further to understand the mechanisms of motor neuron death and CNS pathology in degenerative conditions exacerbated by environmental agents.
P193 CONCURRENT ELECTROPHYSIOLOGICAL AND LOCOMOTOR DEFECTS IN A FED DROSOPHILA MELANOGASTER MODEL OF ALS-PDC
GOTO JJ
California State University, Fresno, Fresno, California, USA
Email address for correspondence: [email protected]
Keywords: BMAA, environment, electrophysiology
Background: The L-form of the non-natural amino acid, beta-methylamino alanine (L-BMAA) is implicated in the ALS variant disease called amyotrophic lateral sclerosis-Parkinsonism dementia complex (ALS-PDC). While genetics and age can be effectors of MND, environmental factors like BMAA, produced by several or more species of cyanobacteria, contribute to neuronal dysfunction by affecting the glutamate NMDA and AMPA receptors. Drosophila melanogaster (fruit fly) is an example of an animal model that can be used to study the phenotypic effects of BMAA and the equivalent in-vivo effects at the neuronal level; glutamate is the major neutrotransmitter for fruit flies.
Objectives: The development of a fruit fly model to quantitatively characterize the locomotor defects of fed-flies, while simultaneously recording the dose-dependent and temporal development of defects at the post-synaptic neurons of flight muscles, using electrophysiology (EP).
Methods: The negative geotaxis or tapdown test was conducted to characterize gross locomotor defects over a period of 3-5 days continuous feeding of 12.5, 25 and 50 mM L-BMAA. Intracellular electrophysiological recordings were measured in individual Canton S flies that were anesthetized and immobilized, followed by insertion of the recording and stimulus electrodes for EP measurements. Flies were stimulated at pulses of 10 to 40 mHz. In all cases recording was done from the dorsal longitudinal flight muscles. This non-invasive method allows recording of the same fly over a period of several days.
Results: Three days after acute BMAA exposure, fed-flies showed a dose-dependent loss of locomotor climbing ability. The average percent of flies able to reach the top of the vial after tap down was 9.0% (50 mM), 33.6% (25 mM), 78.3% (12.5 mM) and 91.0% for the control female fly. The viability was also dose-dependent. Viability improved significantly with lysine and alanine; glutamate or histidine gave moderate protection when each amino acid was co-fed with equimolar BMAA. The EP showed spontaneous responses, decreases in the responses to low-level stimulation (1-10 Hz) and the inability to follow trains of stimulation at the postsynaptic DLM. These defects were progressive from days 1 to 3 during the feeding period. BMAA (4 mM) directly applied to the fly showed the same response effects and exogenous glutamate applied post-BMAA attenuated BMAA. Day 1 EP recordings showed defects at the DLM, prior to any significant locomotor disabilities.
Discussion and conclusions: The dose-dependent loss of motor ability correlates with the equivalent EP loss at the post-synapse level indicating BMAA acts at the neuronal level. The competition experiment with the chemically similar alanine and glutamate (equivalent to carbamylated-BMAA) showed a rescue in the viability and leads to developing a mechanism of where and how BMAA acts on the NMDA and AMPA glutamate fruit fly receptors.
P194 ZINC INHIBITION OF BMAA TOXICITY
LEVINE T1, HANK N1, SAPERSTEIN D1, BRADLEY W3, COX P2
1PNA Center for Neurological Research, Phoenix, AZ, USA, 2The Institute for Ethnomedicine, Jackson, WY, USA, 3University of Miami, Miami, FL, USA
Email address for correspondence: [email protected]
Keywords: zinc, BMAA, chelation
Background: Previous studies into the Guamian ALS-Parkinson's Dementia complex have identified β-methylamino-L-alanine (BMAA), as the potential neurotoxin responsible for this disease. BMAA is a non-essential amino acid produced by cyanobacteria, that are present in all ecosystems. The hypothesis has been that some individuals are vulnerable to BMAA deposition into their central nervous system where it is incorporated into proteins which can then serve as a reservoir for this neurotoxin. If BMAA is a potential neurotoxin responsible for ALS then methods directed at decreasing this toxin could treat the disease. It has been shown that BMAA binds strong to transition metal ions such as zinc, which may prevent BMAA from crossing into the brain and possibly clear BMAA from the CNS. In addition, zinc may serve as an antioxidant in the CNS and help protect the Blood Brain Barrier against oxidative stress and prevent BMAA from crossing into the brain. We hypothesize that by exposing patients to high levels of zinc, BMAA would be kept in a bound complex with zinc, which would increase its clearance.
Objective: To determine the safety and tolerability of zinc methionine (Optizinc) at high doses in patients with sporadic ALS and to measure levels of BMAA in blood and urine pre and post treatment.
Methods: Ten patients diagnosed with sporadic ALS on stable doses of riluzole were enrolled. Patients received Optizinc at 30 mg TID and copper 2 mg QD (to prevent copper depletion) for three months. Blood and urine were collected and shipped to the Institute for Ethnomedicine for BMAA analysis at baseline and at month 3. ALS-FRS-R, safety labs, and zinc and copper plasma levels were measured monthly. FVC and Quality of Life visual analogue scale (QOLVAS) were measured at baseline and at month 3.
Results: Eight patients completed the study. One patient went into a hospice and dropped out after one month of treatment. One patient was hospitalized for pneumonia early in the study. The majority of patients tolerated Optizinc at 90 mg/d. Only one patient couldn't tolerate the dose due to nausea, but completed the study on 60mg/d. Zinc levels were maintained between 80% and 125% of the upper limits of normal. Copper levels were maintained within the normal range in all patients. The rate of ALS progression, as measured by monthly change in ALS-FRS-R scores, did not improve or worsen in any of the patients over the course of treatment. All patients who completed the study (N = 8) opted to stay on treatment. Levels of BMAA from the blood and urine are being analyzed and will be presented.
Conclusion: Optizinc at 90mg/d was well tolerated in patients with ALS. No effect on the rate of disease progression was observed.