High-performance liquid chromatographic separation of renin-angiotensin system peptides and most of their metabolic fragments.
We describe here a gradient HPLC procedure for the separation, and quantification by UV absorption of renin tri- and tetradecapeptide substrates, angiotensins I, II, III, IV and V, angiotensin-derived peptides, and peptidase inhibitors including amastatin, bestatin, pepstatin, lisinopril, a renin peptide inhibitor, Z-Pro-prolinal, N-[1-(R,S)-carboxy-2-phenylethyl]-L-Ala-L-Ala-L-Phe-p-aminobenzoate, and phosphoramidon. Most peptides and peptidase inhibitors were baseline-resolved within 32 min. The overall intra- and inter-assay precisions ranged from 0.8 to 5.9 (n=6) and 2 to 13% (n=6), respectively. There was a linear relationship (correlation coefficients> or =0.9660) between peak height and peptide amount injected. In conclusion, the present method when combined with a peptidase-inhibitor paradigm can lead to the identification of renin-angiotensin system metabolizing enzymes, and when combined with radioimmunoassay can enhance the specificity of angiotensin measurement.
Pelegrini-da-Silva
Adriana
A
Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Av Bandeirantes 3900, 14049-900 Ribeirão Preto, São Paulo, Brazil.
Prado
Wiliam A
WA
Juliano
Maria A
MA
Wilk
Sherwin
S
Martins
Antonio R
AR
D000803:Angiotensin I / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D010446:Peptide Fragments / Q000302:isolation & purification*; D010455:Peptides / Q000302:isolation & purification* / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D012084:Renin-Angiotensin System; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet
D010446:Peptide Fragments; D010455:Peptides; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A