Plasma renin activity by LC-MS/MS: development of a prototypical clinical assay reveals a subpopulation of human plasma samples with substantial peptidase activity.
BACKGROUND
For management and treatment of secondary hypertension, plasma renin activity (PRA) assay is considered an essential diagnostic tool. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to PRA offering improvements in laboratory workflow and throughput. During development, we observed a substantial number of clinical samples that have strong degradation activity toward angiotensin (Ang) I during generation. A preliminary characterization of this degradation activity was performed, and we provide here a method by which this degradation can be monitored via the addition of an isotope-labeled degradation standard.


METHODS
Automated online sample extraction coupled with HPLC was used to isolate Ang I and internal standard from plasma. The effluent from the analytical column was directed to a triple quadrupole MS operated in selected reaction monitoring mode, monitoring the a(5) and b(5) product ions from the [M+3H](+3) precursors. Routine analysis could be achieved with as little as 150 μL plasma.


RESULTS
We identified both C-terminal and N-terminal degradation products of Ang I using isotope-labeled peptides as controls and substrates. In 2%-5% of patient samples, the degradation essentially eliminated any Ang I produced during generation.


CONCLUSIONS
Our method requires reduced sample handling when compared with an RIA and eliminates the need for extended generation times for samples with low renin activity. Degradation of Ang I during generation appears to be a confounding variable in the interpretation of results from some clinical samples. Samples with profound degradation activity can be identified using a degradation standard that is added at the start of generation.
Bystrom
Cory E
CE
Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA 92694, USA.
Salameh
Wael
W
Reitz
Richard
R
Clarke
Nigel J
NJ
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000097:blood* / Q000592:standards; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D009842:Oligopeptides / Q000097:blood; D010447:Peptide Hydrolases / Q000097:blood*; D011863:Radioimmunoassay; D012015:Reference Standards; D012083:Renin / Q000097:blood*; D053719:Tandem Mass Spectrometry
D009842:Oligopeptides; D000803:Angiotensin I; D010447:Peptide Hydrolases; D012083:Renin