No abstract

Giant-celled Characeae (Chara australis Brown), grown for 4 months on 12/12 hr day/night cycle and summer/autumn temperatures, exhibited distinct concentration maxima in auxin (indole-3-acetic acid; IAA), melatonin and serotonin about 4 hr after subjective daybreak. These concentration peaks persisted after 3 day pretreatment in continuous darkness: confirming a circadian rhythm, rather than a response to "light on." The plants pretreated for 3 d in continuous light exhibited several large IAA concentration maxima throughout the 24 hr. The melatonin and serotonin concentrations decreased and were less synchronized with IAA. Chara plants grown on 9/15 hr day/night cycle for 4 months and winter/spring temperatures contained much smaller concentrations of IAA, melatonin and serotonin. The IAA concentration maxima were observed in subjective dark phase. Serotonin concentration peaks were weakly correlated with those of IAA. Melatonin concentration was low and mostly independent of circadian cycle. The "dark" IAA concentration peaks persisted in plants treated for 3 d in the dark. The plants pretreated for 3 d in the light again developed more IAA concentration peaks. In this case the concentration maxima in melatonin and serotonin became more synchronous with those in IAA. The abscisic acid (ABA) and jasmonic acid (JA) concentrations were also measured in plants on winter regime. The ABA concentration did not exhibit circadian pattern, while JA concentration peaks were out of phase with those of IAA. The data are discussed in terms of crosstalk between metabolic pathways.

A single-laboratory validation study was completed for the determination of β-N-methylamino-L-alanine (BMAA), N-(2-aminoethyl)glycine (AEG), and 2,4-diaminobutyric acid (DAB) in bulk natural health product supplements purchased from a health food store in Canada. BMAA and its isomers were extracted with acid hydrolysis to free analytes from protein association. Acid was removed with the residue evaporated to dryness and reconstituted with derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Fluor). Chromatographic separation and detection were achieved using RP ultra-performance LC coupled to a tandem mass spectrometer operated in multiple reaction monitoring mode. Data from biological samples were evaluated for precision and accuracy across different days to ensure repeatability. Accuracy was assessed by spike recovery of biological samples using varying amino acid concentrations, with an average recovery across all samples of 108.6%. The analytical range was found to be 764-0.746 ng/mL prior to derivatization, thereby providing a linear range compatible with potentially widely varying analyte concentrations in commercial health food products. Both the U. S. Food and Drug Administration (FDA) and U. S. Pharmacopeia definitions were evaluated for determining method limits, with the FDA approach found to be most suitable having an LOD of 0.187 ng/mL and LLOQ of 0.746 ng/mL. BMAA in the collected specimens was detected at concentrations lower than 1 μg/g, while AEG and DAB were found at concentrations as high as 100 μg/g. Finding these analytes, even at low concentrations, has potential public health significance and suggests a need to screen such products prior to distribution. The method described provides a rapid, accurate, and precise method to facilitate that screening process.

β-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.