A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D₃BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D₃BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.     

Spermiogenesis and spermatozoon of the liver fluke Mediogonimus jourdanei (Microphalloidea: Prosthogonimidae), a parasite of Myodes glareolus (Rodentia: Cricetidae)

Spermatological characters of the liver fluke Mediogonimus jourdanei Mas-Coma et Rocamora, 1978 were studied by means of transmission and scanning electron microscopy. Spermiogenesis begins with the formation of the differentiation zone containing two centrioles associated with striated rootlets and an intercentriolar body. These two centrioles originate two free flagella that undergo a 90 degrees rotation before fusing with the median cytoplasmic process. Both nuclear and mitochondrial migrations toward the median cytoplasmic process occur before the proximodistal fusion of flagella. Finally, the constriction of the ring of arched membranes gives rise to the young spermatozoon. The mature sperm of M. jourdanei measures about 260 microm and presents two axonemes of different lengths with the typical pattern of the Trepaxonemata, two bundles of parallel cortical microtubules, one mitochondrion, a nucleus and granules of glycogen. An analysis of all the microphalloidean species studied to date emphasised some differences in certain characters found in Maritrema linguilla J?gerski?ld, 1908 and Ganeo tigrinum Mehra et Negi, 1928 in comparison to those in the remaining microphalloideans. The presence and variability of such ultrastructural characters according to family, superfamily or order have led several authors to propose their use in the analysis of trematode relationships and phylogeny. Therefore, apart from producing new data on the family Prosthogonimidae, the present study also compares the spermatological organization of M jourdanei with other available ultrastructural studies focusing on the Microphalloidea.     

Molecular mechanism of antioxidant synergism of tocotrienols and carotenoids in palm oil

During repeated deep-fat frying of potato slices at 163 degrees C in yellow or red palm olein of comparable fatty acid profiles, the oxidative stability (peroxide value and anisidine value) of the palm oleins was similar, and in yellow palm olein, the rate of antioxidant depletion decreased in the order gamma-T3 > alpha-T3 > delta-T3 (T3, tocotrienol). In red palm olein, which had a total tocopherol/tocotrienol content of 1260 vs 940 ppm in yellow palm olein and a corresponding longer induction period in the Rancimat stability test at 120 degrees C, only depletion of gamma-T3 was significant among the phenols during frying and slower as compared to that in yellow palm olein. The carotenes in the red palm olein were depleted linearly with the number of fryings, apparently yielding an overall protection of the phenols. In antioxidant-depleted palm olein and in phospholipid liposomes with added increasing concentrations of phenols, gamma-T3 was found to be a better antioxidant than alpha-T3. alpha-T3 and alpha-T (T, tocopherol) had a similar antioxidant effect in antioxidant-depleted palm olein in the Rancimat stability test, while in the liposomes the ordering as determined by induction period for the formation of conjugated dienes was gamma-T3 > alpha-T3 > alpha-T. The addition of 100-1000 ppm beta-carotene to antioxidant-depleted palm olein or liposomes (lycopene also tested) did not provide any protection against oxidation. In the liposomes, synergistic interactions were observed between beta-carotene or lycopene and alpha-T, alpha-T3, or gamma-T3 for carotene/phenol ratios of 1:10 and 1:2 but not for 1:1. In chloroform, carotenes were regenerated by tocopherols/tocotrienols from carotene radicals generated by laser flash photolysis as shown by transient absorption spectroscopy, suggesting that carotenes rather than phenols are the primary substrate for lipid-derived radicals in red palm olein, in effect depleting carotenes prior to phenols during frying. Regeneration of carotenes by the phenols also explains the synergism in liposomes. In the laser flash photolysis experiments, gamma-T3 was also found to be faster in regenerating carotenes than alpha-T3 and alpha-T.     

Identification of casein phosphopeptides released after simulated digestion of milk-based infant formulas

Adapted, follow-up, probiotic follow-up, toddler, and probiotic toddler infant formulas were subjected to an in vitro enzymatic procedure simulating physiological digestion. The formation and identification of casein phosphopeptides (CPPs) in the milk-based infant formulas were studied using reversed phase high-performance liquid chromatography coupled on line to an ion trap mass spectrometer. Most CPPs formed contained the cluster sequence SpSpSpEE, a mineral binding site. Phosphopeptide alpha(s2)-CN(1-19)4P was present in all formulas analyzed. Probiotic formulas released CPPs not detected in nonprobiotic formulas and probably formed by bifidobacteria action. These observations suggest that physiological digestion of these products promotes the formation of bioactive peptides with mineral carrier properties in the gastrointestinal tract, which resist further proteolysis.     

Mobile terrestrial laser scanner applications in precision fruticulture/horticulture and tools to extract information from canopy point clouds

LiDAR sensors are widely used in many areas and, in recent years, that includes agricultural tasks. In this work, a self-developed mobile terrestrial laser scanner based on a 2D light detection and ranging (LiDAR) sensor was used to scan an intensive olive orchard, and different algorithms were developed to estimate canopy volume. Canopy volume estimations derived from LiDAR sensor readings were compared to conventional estimations used in fruticulture/horticulture research and the results prove that they are equivalent with coefficients of correlation ranging from r = 0.56 to r = 0.82 depending on the algorithms used. Additionally, tools related to analysis of point cloud data from the LiDAR-based system are proposed to extract further geometrical and structural information from tree row crop canopies to be offered to farmers and technical advisors as digital raster maps. Having high spatial resolution information on canopy geometry (i.e., height, width and volume) and on canopy structure (i.e., light penetrability, leafiness and porosity) may result in better orchard management decisions. Easily obtainable, reliable information on canopy geometry and structure may favour the development of decision support systems either for irrigation, fertilization or canopy management, as well as for variable rate application of agricultural inputs in the framework of precision fruticulture/horticulture.     

Effects of ultra-high-pressure homogenization treatment on the lipolysis and lipid oxidation of milk during refrigerated storage

Free fatty acid (FFA) release and quantification and lipid oxidation extent of ultra-high-pressure homogenized (UHPH) milk samples were evaluated to assess the effect of UHPH on the susceptibility of milk lipids to lipolysis and oxidation. Milk was UHPH-treated at 200 and 300 MPa with inlet temperatures of 30 and 40 degrees C. UHPH-treated samples were compared to high-pasteurized milk (PA; 90 degrees C, 15 s). Results showed that all FFA increased significantly during storage only in 200 MPa samples. Lipid oxidation was measured as an accumulation of lipid hydroperoxides as the primary oxidation product and malondialdehyde and hexanal as the secondary oxidation products. Samples treated at 300 MPa presented higher malondialdehyde and hexanal content compared to 200 MPa treated-samples and to PA milk.     

Nitrogen assimilation studies using 15N in soybean plants treated with imazethapyr, an inhibitor of branched-chain amino acid biosynthesis

The pattern of nitrogen assimilation in soybean plants treated with a herbicide that inhibits branched-chain amino acid biosynthesis was evaluated by (15)N isotopic analysis. The herbicide imazethapyr caused a strong decrease in nitrate uptake by roots, partly due to a reduced stomatal conductance. The inhibition of (15)N uptake was accompanied by a decrease in the (15)N content in the plant and, concomitantly, an inhibition of translocation to the shoot. Imazethapyr inhibited nitrate reductase activity in leaves and roots. Among all parameters studied, "de novo" synthesis of proteins was the first parameter of the N assimilation metabolism affected by the herbicide. These results show that this class of herbicides totally damages N metabolism and indicates a regulatory effect on N uptake and translocation that would be mediated by the increase in free amino acid pool provoked by the inhibition of branched-chain amino acid biosynthesis.     

Ontogeny of digestive enzymatic capacities in juvenile seahorses Hippocampus guttulatus fed on different live diets

Differences in survival and growth rates in seahorse Hippocampus guttulatus juveniles feeding on Artemia sp. or copepods have been related to specific digestive capacities of seahorse newborn, which are capable of actively forage on available prey from the first day of live. Other seahorse species, such as H. abdominalis and H. hippocampus, show high success feeding on Artemia nauplii suggesting species-specific differences in the digestibility of prey among seahorses. In this study, the profiles of digestive enzyme activity during the initial 15 days after release (DAR) were very low for trypsin, chitinase and α-amylase. In contrast, higher activities towards any of the assayed substrates for lipase (butyrate, octanoate and oleate) were evident from 0 DAR onwards. From 15 DAR onwards, the effect of diet composition became evident in juveniles previously fed on a mixed diet (Artemia + copepods), which showed a clear increase in all the assayed enzymes when compared with juveniles fed on Artemia as a sole prey. As a practical applicability of this study, a feeding schedule ensuring an adequate digestibility of the prey is proposed based on ontogenetic enzymatic activities of seahorse juveniles fed on different prey.