Chromatography and radioimmunoassay of angiotensin II and metabolites in blood

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BBA 33171

Chromatography and radioimmunoassay of angiotensin II and metabolites in blood Several procedures for the radioimmunoassay of angiotensin I I in plasma or blood have been recently described with mean peripheral levels 1-5 ng/Ioo mP -s. Such assays have not yet been completely validated in regard to specificity against interference by immunoreactive metabolic fragments 7-9 of angiotensin II. To examine this point, ethanolic extractsZ, 4 of normal and reconstituted plasma, shed blood and circulating blood were subjected to descending paper chromatography and radioimmunoassay. The solvent systems were n-butanol-acetic acid-water-pyridine (5 : 1:4: I, by vol. ; pH 4.9) and n-butanol-acetic acid-water-ethyl acetate (IO : 1:5 : io, by vol. ; pH 3.4). Eluates of 2-cm sections along the dried chromatogram were radioimmunoassayed in a system employing ~z31I]angiotensin II tracer and an antiserum to angiotensin I I with similar immunoreactivity against the 3-8 hexapeptide. When extracts of a reconstituted human plasma protein solution (Commonwealth Serum Laboratories, Melbourne) containing [llS~angiotensin I I were subjected to chromatography and radioimmunoassay, the peaks of immunoreactive material and radioactive tracer were'of identical mobility to the angiotensin I I markers run on adjacent chromatogram strips (Fig. I). Trace amounts of activity had the mobility of the 3-8 hexapeptide used as marker for one of the angiotensin II metabolites. Addition of both unlabelled and labelled angiotensin I I to reconstituted human plasma protein solution before extraction again have coincident peaks of peptide mass and radioactivity (Fig. i). The absence of immunoreactive fragments of angiotensin is consistent with the lack of angiotensinase activity in such solutions ~°. =i

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Fig. I. Chromatography of extracts of human reconstituted plasma and reconstituted plasma to which synthetic angiotensin II standard was added before extraction. The [l*sI]angiotensin II (counts/min) added in both cases before extraction was localised (cm) in relation to position of marker angiotensin II (A), the 3-8 hexapeptide (H) and the mass of peptide in terms of angiotensin II (ng). Biochim. Biophys. Acta, 199 (1969) 322-324

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To investigate the stability of angiotensin II in blood treated with EDTA, [l~q]angiotensin II was added to blood and plasma after collection into 0.03 M EDTA, then stored 15 min at 4 °. Identical aliquots of blood were similarly treated, with the additional step of initial acidification to pH 4.5. Chromatography showed a single tracer peak, with mobility of the octapeptide marker, in reconstituted plasma or blood taken into EDTA and adjusted to pH 4.5 before standing in the cold (Fig. 2). However, blood and plasma treated with EDTA alone showed a second major peak of radioactivity with the mobility of the hexapeptide. The distribution of unlabelled immunoreactive material was similar to that shown by the labelled tracer peptide. These observations are extremely relevant to the critical evaluation of assay procedures in which EDTA alone is used to inhibit angiotensinase activityS,5,11 and of tracer preparation methods involving exposure of labelled angiotensin to high concentrations of antiserum which may contain angiotensinase activity n. With direct assay of unextracted plasma, the use of EDTA during incubation at 4 ° may not be adequate to prevent conversion of both tracer and endogenous peptide and standards to angiotensin fragments with variable reactivities in the radioimmunoassay 3. Blood collection directly into ethanol or into syringes containing 2,3-dimercaptopropanol-EDTA 2 before immediate addition to ethanol rapidly ensures irreversible inactivation of enzymes concerned with angiotensin metabolism. Analysis of 6 human and 9 sheep arterial blood samples collected in this manner showed that the predominant immunoreactive peptide is angiotensin II, being 85% of the total activity in man and 95% in the sheep. In contrast, 6 human venous blood samples showed

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Fig. 2. Chromatography of human whole blood, reconstituted plasma and plasma incubated with [l~I]angiotensin II and EDTA (pH 4.5) for I5 min at 4 ° before extraction, and showing the location (cm) of the 1~I radioactivity (counts/min). Fig. 3. Chromatography of human venous blood and showing the localisation (cm) of peptide in terms of angiotensin II (ng).

Biochim. Biophys. Acta, 194 (I969) 322-324

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that angiotensin II comprised only 28% of the total immunoreactive material. The major immunoreactive peak had the mobility of the 3-8 hexapeptide, while further reactive material was observed near the solvent front (Fig. 3). The results agree with biological observations that angiotensin I becomes converted to angiotensin II in the pulmonary circulation and extraction across tissue vascular beds removes about two-thirds of the circulating angiotensin II1', 1~. The substantial proportion of immunoreactive fragments in venous blood has obvious implications in the interpretation of radioimmunoassay values based on venous blood samples. A further situation in which angiotensin fragments may be relevant is their presence or generation during the serum isolation procedure for the protective cofactor recently reported by HAAS et al. ~4. Such fragments may protect angiotensin II from degradation during its passage through the vascular bed, since in vitro competitive inhibition of certain angiotensinases by angiotensin fragments is known15. The possible occurrence and generation of angiotensin II fragments with varying immunological and biological activities will need to be carefully examined in all situations concerned with measurement and investigation of angiotensin II.

Department of Medicine, Monash University, Prince Henry's Hospital, Melbourne (Australia)

M. D. CAIN

Howard Florey Laboratories of Experimental Physiology, University of Melbourne, Victoria (Australia)

J. P. COGHLAN

K . J . CATT

I K. J. CATT, M. D. CAIN AND J. P. COGHLAN, Lancet, 2 (1967) lOO5. 2 G. W. BOYD, J. LANDON AND W. S. PEART, Lancet, 2 (1967) lOO2. 3 D. J. GOCKE, L. M. SHERWOOD, I. OPPENHOFF, J. GERTEN AND J. H. LARAGH, J. Clin. Endocrinol., 28 (1968) 1675. 4 K. J. CATT, M. D. CAIN, P. Z. ZIMMET AND E. CRAN, Brit. Med. J., I (1969) 819. 5 T. L. GOODFRIEND, D. L. BALL AND D. B. FARLEY, J. Lab. Clin. Med., 72 (1968) 648. 6 L. B. PAGE, E. HABER, A. Y. KIMURA AND A. PURNODE, J. Clin. Endocrinol., 29 (1969) 200. 7 F. M. DIETRICH, Immunochemistry, 4 (1967) 65. 8 K. J. CATT AND J. P. COGHLAN, Australian J. Exptl. Biol. Med., 45 (1967) 269. 9 K. J. CATT, J. P. COGHLAN AND B. ROMBERG, Nature, 215 (1967) 395. IO R. VANDONGEN AND R. D. GORDON, Circulation Res., 23 (1968) 397. I I M. B. VALLOTTON, L. B. PAGE AND E. HABER, Nature, 2i 5 (1967) 714 . 12 K. K. F. NG AND J. R. VANE, Nature, 218 (1968) 144. 13 P. BIRON AND C. G. HUGGINS, Life Sci., 7 (1968) 965. 14 E. HAAS, H. GOLDBLATT, L. LEWIS AND E. C. GIPSON, Am. J. Physiol., 215 (1968) 142o. 15 A. B. KURTZ AND E. D. WACHSMUTH, Nature, 221 (1969) 92.

Received July 7th, 1969 Biochim. Biophys. Acta, 194 (1969) 322-324