Measurement of plasma angiotensin II: purification by cation-exchange chromatography.
Measurement of plasma angiotensin II (AII) by radioimmunoassay (RIA) usually requires prior purification of the plasma to remove substances that cross-react in the RIA, most notably angiotensin III (AIII). Purification of AII is generally accomplished by solid-phase extraction (SPE) followed by reverse-phase HPLC, with tedious evaporation and resuspension steps in between, and requires collection of many HPLC fractions per sample for RIA. In this report, we describe a rapid two-step SPE procedure for the purification of plasma AII, including an improved protease inhibitor cocktail for preventing the formation or degradation of AII in vitro. Plasma is first extracted on an S-Sepharose cation-exchange column, in which AII is separated from AIII by virtue of their difference in net charge, and then extracted on a C8 SPE column, without need for intermediate sample handling. The two-step SPE method is fast, results in only a single fraction for RIA per sample, and yields consistently high recoveries (77-86%) of AII, reducing the volume of plasma needed from 2 to 0.5 ml. Rat plasma was used in the present study, but the complete conservation of angiotensin peptide sequences (except angiotensinogen) in mammals suggests that this method will be applicable for other species including humans. In summary, the two-step SPE method offers the speed and simplicity of solid phase extraction while achieving a purity in AII (i.e., free of AIII) previously only obtained by laborious procedures involving HPLC.
Ledwith
B J
BJ
Department of Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania 19486.
Cahill
M K
MK
Losse
L S
LS
Satiritz
S M
SM
Eydelloth
R S
RS
Dallob
A L
AL
Tanaka
W K
WK
Galloway
S M
SM
Nichols
W W
WW
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000097:blood* / Q000302:isolation & purification*; D000809:Angiotensins / Q000097:blood; D000818:Animals; D002412:Cations; D002627:Chemistry, Physical / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D008969:Molecular Sequence Data; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D015203:Reproducibility of Results
D000809:Angiotensins; D002412:Cations; D000804:Angiotensin II