Table 2.

Summary of Methodological Limitations in RAAS Quantification: Considerations for Rigor and Reproducibility

Renin-angiotensin-aldosterone component	Weakness or limitation	Best practice consideration
Peptides	Improper collection practices Improperly validated ELISAs Naturally low concentrations (femtomole/milliliter) Ongoing metabolism Interfering substances Poor specificity and cross-reactivity Interference from sedatives and paralytics	Design experiments with appropriate quantification methods a priori Collect plasma in EDTA with appropriate protease inhibitor cocktail validated for intended assay Extract for purification Use mass spectrometry, high-performance liquid chromatography, or well-validated RIAs
Enzymes	Improper collection and processing Interfering substances	Use serum over plasma Use validated assays
Receptors	Poor specificity of antibodies for use in Western blots and immunohistochemistry	Use radiolabeled peptide binding Use proper positive and negative controls
mRNA or protein expression	Differential expression of mRNA vs protein, transcriptional regulation, turnover, and activity	Combine several methods
Interpretation	Difficult-to-interpret values without normative ranges in isolation	Ensure that values are biologically plausible Assess multiple components simultaneously Interpret in the context of both major pathways Consider tissue expression when available

RAAS indicates renin-angiotensin-aldosterone system; and RIA, radioimmunoassay.