Human Plasma Renin Activity
Kheng Lim, Ph.D., principal scientist, and Daniel B. Kassel, Ph.D., vp of analytical sciences and DMPK at Takeda Pharmaceuticals, investigated renin inhibitors, a group of compounds employed for the treatment of hypertension, chronic renal disease, and congestive heart failure. Human plasma renin activity is reflective of the performance of the renin-angiotensin system and serves as a basis for the diagnosis of hypertension.
The renin-angiotensin system regulates blood pressure and fluid balance through a feedback mechanism in which the kidneys respond to low blood volume by secreting renin. This causes angiotensin levels to rise and blood vessels to constrict. So-called ACE inhibitors are important pharmacological agents used to block one of the critical steps in angiotensin II production. Typically, a radioimmunoassay (RIA) has been used to determine plasma levels of renin during the identification and investigation of inhibitory compounds.
In vitro human plasma renin activity assays incorporating radiolabeled angiotensinogen have been used routinely within the industry for identifying renin inhibitors. Compounds are spiked into human plasma, and the generation of angiotensin I from the enzymatic cleavage of angiotensinogen by renin is measured. “One of the major drawbacks to the RIA is that its linear dynamic range is not large,” Dr. Lim noted. “The standard curves level off rapidly, thus limiting the performance.”
There have been efforts to develop LC/MS/MS as a means for quantifying endogenous levels of angiotensin I; however the assays were time consuming and tedious, hardly representing an advance over the radioimmunoassay approach. Drs. Lim and Kassel have been working on an improved method that uses online solid-phase extraction. “Our online sampling approach replaces manual sampling and can save up to a full-time equivalent position,” Dr. Lim said.
Their system consists of an Applied Biosystems API 4000™ triple quadrupole mass spectrometer (Life Technologies), Shimadzu binary pumps, and a CTC PAL autosampler. In a series of experiments, Drs. Lim and Kassel optimized the procedure, evaluating different types of solid-phase extraction cartridges, analytical columns, and finally, the injection, wash, and elution steps.
“We found that the online SPE/LC/ MS/MS option for the human plasma renin assay is practical and robust,” Dr. Lim stated. “Indeed, we believe that the application of online solid-phase extraction could be extended to the analyses of small molecules, endogenous peptides, and other plasma biomarkers.”