Snake Venom Variability and Methods Pt.1

Snake Venom Variability: Methods of study, results and interpretation- Chippaux et al. 1991

• Abstract: venom variability includes multiple factors such as: interfamily, intergenus, interspecies, intersubspecies and intraspecies, geographical variation, between individuals, seasonal variations, diet, habitat, age-dependent change, and sexual dimorphism.
• Introduction: Venom may aid in helping the snakes both immobilize their prey as well as digest it, which means they may be capable of a mixture of actions such as: neurotoxic (pre/post synaptic), cardiotoxic, myolytic, coagulant (anticoagulant), hemostatic (activating/inhibiting), hemorrhagic, as well as nephrotoxic (destroys kidneys) and hepatoxic (destroys pancreas) actions.
• Venom- Choice and Considerations
  1. Pooled venom: mainly used for commercial preparation, however individual variation cannot be studied via this manner.
  2. Individual venoms: much more accurate method of studying variation in venom with individual subjects, still should note where variation can occur like origin of the snakes, procedure of milking and storage. *check out the other papers mentioned in this part of the paper*
  3. Venom collection: Voluntary injection (when the snake bites onto a parafilm or rubber membrane into a receptacle), manual extraction (massaging venom glands), electrical stimulation (electrical shocks to improve venom flow). *check out papers mentioned here as well*
  4. Sample maintenance: fresh samples can be noted for colour and turbidity, samples can then be filtered or centrifuged, frosted, dried and lyophilized. Poor storage process can degrade venom components.
• Clinical observations and reports: The variation in venom is also seen in symptoms post-envenomation, especially species that are widespread. For example a single species of rattlesnake could produce strictly neurotoxic effects in one region and then produce neurotoxic as well as severe local toxic effects in another region. This disparity occurred to the point of some doctors thinking the bites were made by two different species. Also similar case for antivenom, patients with bites from southern brazil rattlesnakes found antivenom from the northern species to be ineffective.
• Toxicity studies: Back in the day they used to use a variety of models like crickets and pigeons, but nowadays toxicity is tested using LD₅₀ on mice as a way to compare different species. However this brings us to the problem of certain venoms working better on preferred prey types. Whole venom samples are used for this and can be injected intraperitoneal, intravenous, intramuscular, or subcutaneous.
• Measures of biological activity: most studies use a hemorrhagic zone in mice or other animals as an observation site for venom injections. Some studies have observed necrosis as well as the defibrinating capacity of venoms.
• Biochemical analysis: separated into 2 headings
  1. biochemical activities: can be categorized under 3 groups A) Pathophysiologically active substances like in vitro procoagulants, fibrinogenolytics and inhibitors B) Enzymatic like phospholipase, proteolytics, phosphodiesterase, 5′ nucleotidase, and L-amino oxidase C) Amidolytic which has more recently employed chromogenic substrates which can bind to different antibodies and proteins as a way to monitor activity levels. But biochemical activities cannot be used to compare the relativeness of species because it is possible that distantly related venomous snakes may seem very similar in pharmacologically may be produce by groups of non-homologus but functionally analgous proteins.
  2. Separation techniques: electrophoretic techniques and liquid chromatography. liquid chromatography is used to isolate fractions of venoms and elution profiles are used to compare composition by the absence or presence of known fractions. Gel filtration is the most employed method. However elution profiles should be compared with some caution because components could have “like-elution” where all the components aren’t visible. But it is also useful because by fractionation of venom via liquid chromatography means you can take the isolated components and compared them to other venoms, however should remember that certain components could be masked or grouped with another component because of like-elution profiles. In electrophoretic the most popular for comparing whole or individual components of venoms is polyacrylamide gel electrophoresis with to without SDS and in a native mercaptoethanol reduced state.