RESUMEN
BACKGROUND: Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. RESULTS: A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for ß-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled ß-L-ODAP) allowing accurate quantification of ß-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any ß-L-ODAP in these species. The LCMS method was also used to quantify ß-L-ODAP accurately in different tissues of grass pea. CONCLUSIONS: The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and ß-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of ß-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for ß-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-ß-L-ODAP genotypes.
Asunto(s)
Aminoácidos Diaminos/análisis , Lathyrus/química , Neurotoxinas/análisis , Cromatografía Liquida/economía , Cromatografía Liquida/métodos , Costos y Análisis de Costo , Marcaje Isotópico , Lathyrus/genética , Espectrometría de Masas/economía , Espectrometría de Masas/métodos , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrofotometría/economía , Espectrofotometría/métodos , Factores de TiempoRESUMEN
Neurolathyrism is a neurodegenerative disorder characterized by spastic paraplegia resulting from the excessive consumption of Lathyrus sativus (Grass pea). ß-N-Oxalyl-L-α,ß-diaminopropionic acid (L-ODAP) is the primary neurotoxic component in this pea. The present study attempted to evaluate the proteome-wide alterations in chick brain 2 hr and 4 hr post L-ODAP treatment. Proteomic analysis of chick brain homogenates revealed several proteins involved in cytoskeletal structure, signaling, cellular metabolism, free radical scavenging, oxidative stress and neurodegenerative disorders were initially up-regulated at 2 hr and later recovered to normal levels by 4 hr. Since L-ODAP mediated neurotoxicity is mainly by excitotoxicity and oxidative stress related dysfunctions, this study further evaluated the role of L-ODAP in apoptosis in vitro using human neuroblastoma cell line, IMR-32. The in vitro studies carried out at 200 µM L-ODAP for 4 hr indicate minimal intracellular ROS generation and alteration of mitochondrial membrane potential though not leading to apoptotic cell death. L-ODAP at low concentrations can be explored as a stimulator of various reactive oxygen species (ROS) mediated cell signaling pathways not detrimental to cells. Insights from our study may provide a platform to explore the beneficial side of L-ODAP at lower concentrations. This study is of significance especially in view of the Government of India lifting the ban on cultivation of low toxin Lathyrus varieties and consumption of this lentil.
RESUMEN
BACKGROUND: ß-N-oxalyl-L-α,ß-diaminopropionic acid (L-ODAP) is a non-protein amino acid with haemostatic property present in Lathyrus sativus. It is considered to be the causative agent of neurolathyrism that occurs upon prolonged overconsumption of Lathyrus sativus seeds. L-ODAP is used as a haemostatic drug in surgical dressings. We previously reported that it can stabilize hypoxia inducible factor (HIF)-1α in normoxic conditions. HYPOTHESIS: We hypothesised that L-ODAP might affect wound healing by modulating cellular proliferation, migration and angiogenesis via HIF-1α stabilization. STUDY DESIGN: We performed in vitro assays to evaluate wound healing activity of L-ODAP. Further, we prepared pharmaceutical gel containing L-ODAP and checked its effect on healing of full thickness excision wounds using Wistar albino rats. METHODS: Effect of L-ODAP on HT1080 cell line proliferation, migration and invasion was investigated. Further, gel containing L-ODAP was applied on full thickness excision wounds of Wistar rats. Western blot and zymography were performed with wound tissue extracts obtained 2 days post-wounding and histological and immunohistochemical analysis with regenerated tissue obtained 10 days post-wounding. Evaluation was made based on wound contraction percentage, histological analysis and protein expression levels. RESULTS: L-ODAP significantly (Pâ¯<â¯0.05) affected wound healing both in vitro and in vivo. At non-toxic concentrations, it induced cell proliferation, migration, invasion and MMP-2 & -9 expressions. L-ODAP treated wounds healed faster than vehicle treated ones. Significantly higher expression level of HIF-1α, VEGF-A, PDGF-A and matrix metalloproteases were observed in L-ODAP treated wounds. CONCLUSION: The present investigation explores potential of L-ODAP as a wound healing agent. L-ODAP positively affected wound healing both in vitro and in vivo and thus could be considered a natural wound healing agent.