A Single Laboratory Validation for the Analysis of Underivatized ß-N-Methylamino-L-Alanine (BMAA).

ß-N-Methylamino-L-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria that can accumulate in ecosystems and food webs. Human exposure to cyanobacterial and algal blooms may be a risk factor for neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Analytical chemists have struggled to find reliable methods for BMAA analysis in complex sample matrices. Analysis of BMAA is complicated by at least 3 naturally occurring isomers: N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB), and ß-aminomethyl-L-alanine (BAMA). More than 350 publications have reported detection and quantification of BMAA and its isomers, but varying results have led to controversy in the literature. The objective of this study was to perform a single laboratory validation (SLV) of a frequently published method for BMAA analysis using a ZIC-HILIC column. We investigated the selectivity, linearity, accuracy, precision, and sensitivity of the method and our data show that this HILIC method fails many of the criteria for a validated method. The method fails the criterion for selectivity as the chromatography does not separate BMAA from its isomer BAMA. Sensitivity of the method greatly decreased over the experimental period and it demonstrated a higher limit of detection (LOD) (7.5 pg on column) and a higher lower limit of quantification (LLOQ) (30 pg on column) than other published validated methods. The method demonstrated poor precision of repeated injections of standards of BMAA with % relative standard deviation (%RSD) values that ranged from 37 to 107% while HorRat values for BMAA had a fail rate of 80% and BAMA had a fail rate of 73%. No HorRat values between 0.5 and 2 were found for repeated injections of standards of AEG and DAB. Recovery of 13C3,15N2-BMAA in a cyanobacterial matrix was < 10% in experiments and we were also unable to accurately detect other protein amino acids including methionine, cysteine, or alanine, indicating matrix effects. The results of this study demonstrate that the ZIC-HILIC column is not fit for purpose for the analysis of BMAA in cyanobacterial matrices and further provides explanations for the high level of negative results reported by researchers using this method.

A virus-induced gene-silencing system for functional genetics in a betalainic species, Amaranthus tricolor (Amaranthaceae).

PREMISE OF THE STUDY: Research in Amaranthaceae could be accelerated by developing methods for targeted gene silencing. Most amaranths, including Amaranthus tricolor, produce betalains. However, the physiological and ecological roles of these pigments are uncertain. We sought to establish a virus-induced gene-silencing (VIGS) method for amaranths, using silencing of betalain pigments as a proof-of-principle. METHODS: We targeted AtriCYP76AD1, a putative cytochrome P450 component of the betalain biosynthetic pathway, using VIGS, and compared two different methods of introducing the VIGS construct into plants. We measured transcript abundance and concentrations of betalains and their l-DOPA precursor in VIGS-treated plants, and compared these to controls. RESULTS: We observed that when AtriCYP76AD1 was targeted by VIGS in normally red plants, AtriCYP76AD1 and the related genes AtriCYP76AD6 and AtriCYP76AD5 had diminished transcript abundance. Furthermore, newly emergent petioles and leaves of VIGS-treated plants appeared green, betacyanin accumulation was strongly reduced, and l-DOPA accumulation was increased. No betaxanthin could be detected in this variety of A. tricolor, either before or after VIGS treatment. DISCUSSION: These results help to establish the genetic basis of betalain synthesis in amaranths. Furthermore, this is the first report of VIGS in amaranths and demonstrates the potential of this technique for basic and applied research in these species.

The metabolic fate of dietary nicotine in the cabbage looper, Trichoplusia ni (Hübner).

Cabbage looper (Trichoplusia ni) larvae are generalist herbivores that feed on numerous cultivated plants and weeds including crucifers, other vegetables, flowers, and field crops. Consuming plant material from a wide range of plant species exposes these larvae to a considerable variety of plant secondary metabolites involved in chemical defense against herbivory. The ability of the cabbage looper larvae to detoxify plant secondary metabolites, such as nicotine, has been attributed to the rapid induction of excretion via the Malpighian tubules. However, the role of metabolism in the detoxification of plant secondary metabolites in cabbage looper larvae is not well studied. We investigated nicotine metabolism in 4th larval instar cabbage looper using UPLC-MS/MS analysis to resolve the time course of nicotine metabolism, the kinetic distribution of nicotine, and the presence or absence of major metabolites of nicotine in larval tissue and excretions. The major metabolite found in our analysis was cotinine, with trace amounts of cotinine N-oxide and nicotine N-oxide. The nicotine metabolites detected are similar to those of the nicotine-tolerant Lepidopteran tobacco hornworm (Manduca sexta). The results of our study demonstrate that the 5'C-oxidation of nicotine to cotinine is the primary pathway for nicotine metabolism in cabbage looper larvae. This study showed that metabolism of nicotine and subsequent excretion of nicotine and its metabolites occurs in the larvae of the cabbage looper. Our results suggest that 5'C-oxidation in lepidopteran insects is a conserved metabolic pathway for the detoxification of plant secondary metabolites.

Assessing Environmental Exposure to ß-N-Methylamino-L-Alanine (BMAA) in Complex Sample Matrices: a Comparison of the Three Most Popular LC-MS/MS Methods.

ß-N-Methylamino-L-alanine (BMAA) is a naturally occurring non-protein amino acid produced by cyanobacteria, accumulated through natural food webs, found in mammalian brain tissues. Recent evidence indicates an association between BMAA and neurological disease. The accurate detection and quantification of BMAA in food and environmental samples are critical to understanding BMAA metabolism and limiting human exposure. To date, there have been more than 78 reports on BMAA in cyanobacteria and human samples, but different methods give conflicting data and divergent interpretations in the literature. The current work was designed to determine whether orthogonal chromatography and mass spectrometry methods give consistent data interpretation from a single sample matrix using the three most common analytical methods. The methods were recreated as precisely as possible from the literature with optimization of the mass spectrometry parameters specific to the instrument. Four sample matrices, cyanobacteria, human brain, blue crab, and Spirulina, were analyzed as 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives, propyl chloroformate (PCF) derivatives separated by reverse phase chromatography, or underivatized extracts separated by HILIC chromatography. The three methods agreed on positive detection of BMAA in cyanobacteria and no detected BMAA in the sample of human brain matrix. Interpretation was less clear for a sample of blue crab which was strongly positive for BMAA by AQC and PCF but negative by HILIC and for four spirulina raw materials that were negative by PCF but positive by AQC and HILIC. Together, these data demonstrate that the methods gave different results and that the choices in interpretation of the methods determined whether BMAA was detected. Failure to detect BMAA cannot be considered proof of absence.

Plant signals during beetle (Scolytus multistriatus) feeding in American elm (Ulmus americana Planch).

American Elms were devastated by an outbreak of Dutch Elm Disease is caused by the fungus Ophiostoma novo-ulmi Brasier that originated in Asia and arrived in the early 1900s. In spite of decades of study, the specific mechanisms and disease resistance in some trees is not well understood. the fungus is spread by several species of bark beetles in the genus Scolytus, during their dispersal and feeding. Our objective was to understand elm responses to beetle feeding in the absence of the fungus to identify potential resistance mechanisms. A colony of Scolytus multistriatus was established from wild-caught beetles and beetles were co-incubated with susceptible or resistant American elm varieties in a controlled environment chamber. Beetles burrowed into the auxillary meristems of the young elm shoots. The trees responded to the beetle damage by a series of spikes in the concentration of plant growth regulating compounds, melatonin, serotonin, and jasmonic acid. Spikes in melatonin and serotonin represented a 7,000-fold increase over resting levels. Spikes in jasmonic acid were about 10-fold higher than resting levels with one very large spike observed. Differences were noted between susceptible and resistant elms that provide new understanding of plant defenses.