The Renin Angiotensin System and other Cardiovascular Hormones
Prosearch can determine the levels of renin, angiotensins and other peptides in not only plasma but also tissues such as kidney, heart, brain etc. using our specific tissue extraction methods and inhibitor cocktails. Your lab can do the extractions or ours but it is important that tissues are collected, stored, transported and extracted appropriately. Special consideration must be given to the process at every stage or your science will quickly become invalid. We have considered many published methods and have picked the eyes out of each and developed our own methods with the best outcomes, yet still practical and economical.We are happy to adopt any method you might require but generally we prefer homogenization in mechanically driven glass homogenizers with PVC pestle. We believe this rather antiquated method is best as it is more consistent at cell disruption with different tissues varying enormously in their fibrous content. We include cell lysis steps by hypotonicity and freezing. This must all be done in the presence of an appropriate inhibitor cocktail for the analyte.
Inhibition of proteolytic activity is important for the prevention of unwanted degradation of proteins or peptides during their isolation and measurement. We have considered the inhibitor requirements in each cocktail we use specifically for the analyte we are aiming to measure. For peptides such as angiotensin-I, angiotensin-II, angiotensin 1-7 and insulin we use a cocktail mixture containing EDTA, AEBSF, Aprotinin, Bestatin, E-64 and Leupeptin. If ACE or ACE2 are to be tested it is important to omit the EDTA and any other metal chelating agents. Pepstatin A inhibits aspartic proteases such as renin and should be omitted for renin activity determination. N-ethylmaleimide is an alkylating agent that stoichiometrically binds sulphydryl groups and should not be present for many protein analytical methods such as enzymatic and immuno assay.
To measure tissue renin activity a renin substrate must be added. The substrate enhancement is essential as few tissues contain substrate.