Secretory carbonic anhydrase isoenzyme (CA VI) in human serum Jyrki Kivela ¨, 1,2 * Seppo Parkkila, 1,3 Abdul Waheed, 3 Anna-Kaisa Parkkila, 1,3 William S. Sly, 3 and Hannu Rajaniemi 1 Carbonic anhydrase VI (CA VI) is a secretory isoenzyme that, by analogy to -amylase, is produced in the sali- vary glands and delivered into saliva. To determine whether CA VI is transferred into the circulation and is detectable in human serum, we collected blood samples from four healthy subjects at 3-h intervals throughout a 24-h period and measured concentrations of CA VI by a specific time-resolved immunofluorometric assay. All serum samples contained CA VI, the concentrations being 22 times lower in serum than in the correspond- ing saliva samples. The presence of CA VI in serum was confirmed by Western blotting, which under reducing conditions identified a 42-kDa polypeptide band corre- sponding to the monomeric CA VI. The described time- resolved immunofluorometric assay for CA VI might be useful to identify or exclude diseases of the salivary glands in the differential diagnosis of patients whose serum amylase concentrations are increased. Carbonic anhydrase VI (CA VI) is a secretory enzyme that was initially described in the ovine parotid gland and saliva [1]. 4 It was first purified by Feldstein and Silverman [2] from rat saliva and by Murakami and Sly [3] from human saliva. It is a glycoprotein with apparent molecu- lar mass of 42 kDa [3, 4]. CA VI has been localized in the serous acinar and demilune cells of the human parotid and submandibular glands [5], from where it is secreted into saliva. Recent studies have shed light on the physio- logical role of CA VI, suggesting that it may protect against excess acidity in the mouth and upper alimentary canal [6, 7]. Our previous studies with a time-resolved immunofluorometric assay for CA VI have indicated that salivary enzyme concentrations follow a circadian period- icity, declining to a very low concentration during sleep [8]. The variations of the salivary amylase activity parallel the CA VI concentrations and show an identical decline during sleep, suggesting that both enzymes are secreted via similar mechanisms and are possibly present in the same secretory granules. Assays of amylase activity in serum are largely used in the diagnosis of diseases of the pancreas, where the majority of the pancreatic amylase (P-type) is produced. However, the salivary amylase isoenzyme (S-type) inter- feres in the analyses and results in poor specificity for tests of total amylase activity [9]. Neoplasias, ruptured ectopic pregnancy, and salivary gland lesions caused by infection, irradiation, obstruction, surgery, and tumor have all been reported to produce a significant S-type hyperamylasemia [10]. Because the concentrations of sal- ivary amylase and CA VI show high correlation and follow the similar circadian periodicity, analysis of serum CA VI concentration could be a useful tool in the differ- ential diagnosis of patients with increased serum amylase concentrations. In this report, we studied whether CA VI can be detected in normal human serum. First we measured the serum and salivary CA VI concentrations from four subjects at 3-h intervals during a 24-h period with a time-resolved immunofluorometric assay, and then con- firmed the presence of CA VI in serum by using a sensitive Western blotting method with chemilumines- cent substrate. Materials and Methods collection of saliva and serum samples To study the concentrations of CA VI, serum and saliva samples were collected from four healthy male volunteers throughout the 24-h period. The participants stayed at home during the collection procedure. The study protocol involved meals at 0900, 1230, and 1830, and sleep from 1 Department of Anatomy, University of Oulu, Oulu, Finland. 2 Parolannummi Garrison Hospital, Finnish Defence Forces, Hattula, Fin- land. 3 Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO. *Address correspondence to this author at: Parolannummi Garrison Hos- pital, P.O. Box 5, FIN-13701 Parolannummi, Finland. Fax 358 –3-1814 – 4612; e-mail jyrki.kivela@pp.inet.fi. 4 Nonstandard abbreviations: CA VI, carbonic anhydrase isoenzyme; SDS- PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; and TBST, Tris-buffered saline with Tween-20. Received March 11, 1997; revision accepted June 10, 1997. Clinical Chemistry 43:12 2318 –2322 (1997) Enzymes and Protein Markers 2318 Downloaded from https://academic.oup.com/clinchem/article-abstract/43/12/2318/5640744 by guest on 24 July 2020