Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry Mass Spectrometric Identification and Quantification of Hemorphins Extracted from Human Adrenal and Pheochromocytoma Tissue *Anne Cerpa-Poijak, lJeIle Lahnstein, ~Kenyn E. Mason, ~George A. Smythe, and *~Mark W. Duncan * Cooperative Research Centre for Biopharmaceutical Research, Darlinghurst; ~ Biomedical Mass Spectrometrv Unit, University of New South Wales, Sydney, New South Wales; and tNucleic Acid and Protein Chemistry Unit, Department of Plant Science, University of Adelaide, Glen Osmond, South Australia, Australia Abstract: The hemorphins are a family of recently identi- fied opioid receptor binding peptides derived from the proteolytic processing of the ~3, y, 5, and chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocy- toma tumors. The hemorphins were quantified and struc- turally characterized using mass spectrometry. High con- centrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue. Key Words: Hemorphins—Ad- renal tissue—Angiotensin converting enzyme inhibi- tors—Opioid receptor binding peptides. J. Neurochem. 68, 1712—1719 (1997). The adrenal gland is involved in the regulation of blood pressure through the production of epinephrine and norepinephrine. In addition to these two catechol- amines, the adrenal medulla synthesizes several pep- tides, notably the opioid peptides, i.e., enkephalins and /9-endorphins. Furthermore, the secretory granules of the chromaffin cells of the adrenal medulla contain chromogranin proteins, notably chromogranin A, a precursor of several biologically active peptides in- cluding vasostatin, pancreastatin, and chromatostatin (Winkler et al., 1986). Chromogranin A is reportedly a reliable marker in the diagnosis of the adrenal tumor, pheochromocytoma, because elevated circulating lev- els are found in tumor patients (Grandal et al., 1991). The biological roles of these adrenal peptides are poorly understood, but it is likely that they play some role in the regulation of blood pressure (McCubbin, 1993). In the work presented here we have used mass spec- trometry to characterize peptides extracted from pheo- chromocytoma tissue and normal adrenal tissue. The most abundant peptides extracted from adrenal tissue (both pheochromocytoma and normal) were the he- morphins. Hemorphins are a family of endogenous opi- oid peptides derived from the proteolytic processing of the /9, y, & or c chains of hemoglobin (Branti et al., 1986). Hemorphins-4 and -5 were first identified in the in vitro digests of bovine blood using gastric enzymes, and their opioid activity was demonstrated by guinea pig ileum assay (Branti et al., 1986). Fur- thermore, Piot et al. (1992) demonstrated that hemor- phin fragments can be generated through proteolysis of bovine hemoglobin by pepsin. In subsequent studies, hemorphins LVV-hemorphin- 6 and -7 were identified in vivo in human pituitary extracts (Gldmsta et al., 1991) and in the CSF of pa- tients with cerebral bleeding (Glämsta et al., 1992). The biological activity of these extracted hemorphins was demonstrated in the guinea pig ileum assay as well as by receptor binding studies. The hemorphins have been demonstrated to have a low affinity for the classical opioid ~t and ó receptors and a somewhat higher affinity for the K receptors (Liebmann et al.. 1989). Furthermore, the affinity of hemorphins for the Received September 24, 1996; revised manuscript received De- cember 2, 1996; accepted December 3, 996. Address correspondence and reprint requests to Dr. M. W. Duncan at Biomedical Mass Spectrometry Unit, University of New South Wales, Sydney, New South Wales 2052, Australia. Abbreviations used: ACE, angiotensin converting enzyme: ESI. electrospray mass spectrometry; 4HCCA, a-cyano-4-hydroxycin- namic acid; LC/MS, liquid chromatography in-line to electrosprav mass spectrometry; LC/MS/MS, liquid chromatography in-line to tandem mass spectrometry; MALDI, matrix-assisted laser desorption ionization; MS/MS, tandem mass spectrometry; PMSF, phenylmelh- ylsulfonyl fluoride; TFA, trifluoroacetic acid: TLCK, L-l-chlOro-3- (4-tosylamido)-7-amino-2-heptanone hydrochloride: TOF. time of flight. 1712