Browsing by Author "Lermer, Anne"

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    Development of a LC-MS/MS method for the detection of snake venom toxins in human plasma
    (Stellenbosch : Stellenbosch University, 2022-12) Lermer, Anne; Kellermann, Tracy; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
    ENGLISH ABSTRACT: Background: Snakebite envenomation in sub-Saharan Africa significantly threatens human health. Species that was responsible for the envenomation are rarely positively identified. There are no rapid methods/diagnostics available for conclusive identification of the organism/species responsible. Venomous species can be categorised based on the composition and primary effect caused by their venom components as neurotoxic, cytotoxic and/or hemotoxic. Aim: To develop an LC-MS/MS method for the species-specific detection of snake venom toxins from human plasma. Methods: An epidemiological study on the incidence of snakebite in South Africa as reported to Poisons Information Helpline of Western Cape (PIHWC) was performed to provide rationale for the analytical study. Analytical methodology includes identification of species-specific venom toxins by high resolution (HR) LC-MS/MS, fractionation of Naja nivea venom by size exclusion chromatography and compositional analysis of the fractions by HR-LC-MS/MS. Venom fractions were screened for toxicity using a zebrafish model, utilising DanioVision for phenotypic screening and behavioural tracking of the larvae. Thereafter, a triple quadrupole LC-MS/MS method was developed containing species-specific toxins. Results: Analysis of PIHWC call data revealed that in 43.73% of the recorded snakebite cases the causative snake species were unidentified. The snake species Naja nivea, Bitis arietans and Bitis atropos species were identified as most medically significant in South Africa and consequently included in the study. Venom from N. nivea was chosen as the species for fractionation after common peptides from inter-region venom were identified as unique to the species. Behavioural changes in larvae were observed after exposure to sub-lethal concentrations of N. nivea venom fractions. Significant (p<0.0001) changes in larval behaviour were observed in two treatment groups compared to the control. Using transitions that was generated during HR-LC-MS/MS analysis, FASTA files were generated and converted into MRM’s onto the triple quadrupole LC-MS. Toxins were positively identified from human plasma by LC-MS/MS. Discussion: This study identified the species predominantly responsible for snakebites from PIHWC call data, and highlighted a need for a diagnostic to identify the species responsible for envenomation. Analysis of the venom proteomes by HR-LC-MS/MS revealed similarities and differences in the venom profiles of Naja nivea, Bitis arietans and Bitis atropos species. In-solution, HILIC assisted tryptic digestion produced the identification of more proteins from the N. nivea crude venom compared to in-gel digestion and while fractionation by size exclusion chromatography prior to MS analysis increased the detection of low molecular weight toxins. It was also shown that using a combination of conventional HR-MS (with database/library searches) and triple quadrupole MS, a method could be created that identified species specific venom peptides from human plasma to aid diagnosis. Conclusion: This is a proof of concept for future work that will include the development of a lateral flow assay to detect venom from envenomed plasma that is cost-effective to produce, aids in defining diagnosis and importantly serves victims of snakebite envenomation in rural communities.