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Clinical Chemistry 43: 2318-2322, 1997;
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(Clinical Chemistry. 1997;43:2318-2322.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Secretory carbonic anhydrase isoenzyme (CA VI) in human serum

Jyrki Kivelä1,2,a, Seppo Parkkila1,3, Abdul Waheed3, Anna-Kaisa Parkkila1,3, William S. Sly3 and Hannu Rajaniemi1

1 Department of Anatomy, University of Oulu, Oulu, Finland.

2 Parolannummi Garrison Hospital, Finnish Defence Forces, Hattula, Finland.

3 Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO.
a Address correspondence to this author at: Parolannummi Garrison Hospital, P.O. Box 5, FIN-13701 Parolannummi, Finland. Fax 358–3-1814–4612; e-mail jyrki.kivela{at}pp.inet.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Carbonic anhydrase VI (CA VI) is a secretory isoenzyme that, by analogy to {alpha}-amylase, is produced in the salivary 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 corresponding saliva samples. The presence of CA VI in serum was confirmed by Western blotting, which under reducing conditions identified a 42-kDa polypeptide band corresponding 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Carbonic anhydrase VI (CA VI) is a secretory enzyme that was initially described in the ovine parotid gland and saliva (1).1 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 molecular 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 physiological 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 periodicity, 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) interferes 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 salivary 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 differential 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 confirmed the presence of CA VI in serum by using a sensitive Western blotting method with chemiluminescent substrate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 0010 to 0900, during which time the subjects were awakened only for sample collection. Saliva was collected at 3-h intervals and the sampling was staggered ~45 min from one participant to the next. The subjects chewed paraffin wax for 7 min to stimulate saliva secretion, saliva from the first 2 min of chewing being swallowed and the rest collected by spitting into 10-mL tubes containing 200 µL of 0.2 mol/L benzamidine in deionized (d)H2O to prevent proteolysis. The samples were frozen and stored at -20 °C until assayed. After thawing, a 1-mL aliquot from each sample was centrifuged (15 000g) at 4 °C for 10 min and the supernatant was used for the immunofluorometric analysis of CA VI. After obtaining the saliva samples, 1-mL blood samples were obtained through an intravenous catheter from the cephalic vein in the cubital fossa. After the blood samples were centrifuged at 2000g for 20 min, serum was frozen and stored at -20 °C until assayed.

The samples were collected after informed consent, and all procedures were in accordance with the Helsinki Declaration of 1975 (as revised in 1983).

antiserum
CA VI was purified from human saliva by inhibitor affinity chromatography as described in detail previously (3)(5). The production and characterization of the antiserum to CA VI have similarly been described earlier (11). Briefly, affinity-purified CA VI was subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE) in tube gels. The polypeptide band, which was slightly stained with Coomassie brilliant blue R-250, was cut and the gel pieces used to immunize a rabbit. The specificity of the antiserum was tested by dot blot and Western blot analyses, which showed no cross-reactivity with human CA I, CA II, {alpha}-amylase, or IgA.

antigen labeling and fluoroimmunoassay procedure
The labeling of CA VI has been described previously (12). For the present study, the enzyme was labeled with 0.12 mg of Eu labeling reagent according to the manufacturer's instructions (Wallac). The enzyme was first pretreated by buffer exchange with a gel filtration procedure. CA VI (200 µg in 900 µL of Tris-SO4 buffer, pH 7.0, containing 0.4 mol/L NaN3, 1 mmol/L benzamidine, and 200 mL/L glycerol) was applied to a PD-10 column (Pharmacia) preequilibrated with 25 mL of labeling buffer (9 g/L NaCl, 50 mmol/L NaHCO3, pH 8.5), and a 1-mL enzyme fraction was collected after the void volume. The actual labeling consisted of a single step in which the pretreated enzyme was mixed with the 0.12 mg of Eu labeling reagent and incubated for 15 h at room temperature. Free Eu3+ was removed by gel filtration on a PD-10 column equilibrated with 25 mL of Tris buffer, pH 7.75, containing 9 g/L NaCl, 50 mmol/L Tris, and 0.5 g/L NaN3. Fractions of 1 mL were collected and measured for fluorescence with a 1234 Delfia Research Fluorometer (Wallac). For long-term storage, highly purified, heavy metal-free bovine serum albumin was added to the fraction containing the enzyme peak to a final concentration of 1.0 g/L. The labeled CA VI was stored at 4 °C.

The principle of the time-resolved immunofluorometric assay for CA VI has also been described and characterized earlier in detail (12). The steps were as follows: Sheep anti-rabbit IgG-coated microtitration strips (Wallac) were washed with 200 µL of the Delfia wash solution (Wallac). Anti-CA VI diluted 1:50 000 in Neo hTSH (thyrotropin) assay buffer (Wallac) was added to the microtitration wells (200 µL/well). After incubation at room temperature for 4 h with continuous gentle shaking, the wells were washed six times with the wash solution. Eu3+-labeled CA VI (92 µL, diluted appropriately in assay buffer), calibrators, saliva (50 µL, 1:50 dilution), or serum (10 µL, undiluted) samples were added to the wells, and the incubation was brought to 200 µL/well (all dilutions with Neo hTSH assay buffer). The mixture was incubated at room temperature with shaking for 20 h, followed by washing of the wells six times with the wash solution and addition of 200 µL of enhancement solution (Wallac) to each well. After intense shaking for 5 min, the fluorescences were measured with the research fluorometer (Wallac). The mean intraassay CV of the present series was 4.6% and the interassay CV determined in three assays was 10.1%.

sodium dodecyl sulfate (sds)-page and western blot
Aliquots of 5 µL of saliva, 0.5 µL of serum, and 0.5 µg of purified human IgG (Sigma) were subjected to SDS-PAGE under nonreducing or reducing conditions. The reagents for SDS-PAGE were from Bio-Rad Labs. and Sigma, and the electrophoreses were performed in a Mini-Protean unit (Bio-Rad Labs.) at a constant current of 50 mA/gel for 40 min, according to Laemmli (13), with a 90 g/L acrylamide separating gel and a 40 g/L acrylamide stacking gel.

The proteins were electrophoretically transferred from the gel onto a polyvinylidene fluoride (PVDF) membrane (Millipore) with a constant voltage of 30 V for 1.5 h in a Xcell II Mini-Cell apparatus (Novex). After the transblotting the PVDF membranes were stained with Coomassie brilliant blue R-250, and the lanes containing the molecular mass calibrators were cut. The sample lanes were destained in methanol, rinsed in Tris-buffered saline with Tween 20 (TBST) buffer (containing 10 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, and 0.5 mL/L Tween 20), and incubated for 30 min with 1:10 diluted cow colostrum in TBST buffer. Then the sheets were incubated for 1 h with anti-CA VI serum or normal rabbit serum diluted 1:5000 in TBST buffer, washed five times for 5 min with TBST buffer, and incubated for 1 h with peroxidase-conjugated goat anti-rabbit IgG (Sigma) diluted 1:1000 in TBST buffer. After washing four times for 5 min in TBST buffer, the polypeptide bands were visualized by chemiluminescence reaction, performed as described in detail by Leong and Fox (14).

isolation of immunoglobulin–ca vi complex from serum
A human blood sample (1 mL) was collected from the cephalic vein and centrifuged at 4000g for 10 min at 4 °C. A serum sample (5 µL) was added to 45 µL of PBS and mixed for 2 h at 4 °C with 50 µL of protein A immobilized on Sepharose (Sigma). After centrifugation at 15 000g for 2 min, the supernatant was saved as an unbound fraction, and the protein A–Sepharose conjugate was washed two times with 400 µL of PBS. Finally, the bound proteins were eluted with 400 µL of 0.1 mol/L glycine-HCl solution, pH 2.5, and the eluted material was mixed appropriately with 1 mol/L Tris base to neutralize the pH of the solution. The eluted proteins were concentrated on Centricon P-10 tubes (Amicon) to a 100 µL volume, and analyzed by SDS-PAGE followed by Western blotting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To elucidate the presence of CA VI in serum, we collected blood samples from four subjects at 3-h intervals throughout a 24-h period, measured serum concentrations of CA VI by a time-resolved immunofluorometric assay, and compared the results with those measured from the corresponding saliva samples. The results indicated that serum contains detectable amounts of CA VI (Fig. 1 A), although the enzyme concentrations were much lower (~1/22) than those of saliva samples, the mean ± SEM values after breakfast being 0.20 ± 0.02 mg/L in serum and 4.29 ± 0.57 mg/L in saliva. Fig. 1B shows that salivary CA VI concentrations varied greatly among the subjects. The enzyme concentrations were very low during the sleeping period in all subjects and increased rapidly in the morning after awakening and eating breakfast. The serum enzyme concentrations also showed much intraindividual variation, but the circadian rhythm was not as evident as in saliva.



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  		Figure 1. Serum (A) and salivary (B) CA VI concentration patterns in four subjects during a 24-h period.

The salivary enzyme concentrations are low during sleep but rise markedly after waking up and eating breakfast at 0900. The serum enzyme concentrations also show much variation in each individual, but the circadian periodicity is not as evident as in saliva samples. The bars indicate sleeping time.

The presence of CA VI in serum was also confirmed by Western blotting. Under nonreducing conditions, the antibody detected a high-molecular-mass band >130 kDa in all serum samples studied (Fig. 2 B), suggesting that CA VI associates with some other serum protein(s). After reduction, two major polypeptide bands of 51 kDa and 42 kDa were identified in serum samples (Fig. 2C ), the latter band being antibody specific and corresponding to the molecular mass of the monomeric CA VI (3)(4). The 51-kDa band was found to be a nonspecific reaction for IgG heavy chain, since normal rabbit serum identified a polypeptide of the same molecular mass in a Western blot of the purified human IgG (Fig. 2C ). By contrast, the anti-CA VI antibody identified a strong 42-kDa polypeptide in Western blots of the saliva samples under nonreducing conditions (Fig. 2A ). In addition, minor 58-, 36-, and 33-kDa polypeptide bands were occasionally seen in saliva blots as in earlier studies (3)(5).



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  		Figure 2. Western blots of saliva and serum samples electrophoresed under nonreducing (A, B) or reducing (C) conditions.

A1–D1 and A2–D2 samples were collected between 0600 and 0900 and 1200 and 1500, respectively. The anti-CA VI serum identified a major 42-kDa polypeptide in saliva, the strongest signal seen in the samples collected in the afternoon. In the nonreduced serum samples, anti-CA VI serum identified a high-molecular-mass band >130 kDa, which was reduced into two polypeptides of 42 and 51 kDa in reducing conditions. The normal rabbit serum (NRS) also showed an identical 51-kDa polypeptide band in Western blots of serum and purified human IgG.

The above results from the Western blots suggested that CA VI may associate with IgG in serum. To confirm the presence of this association, we isolated IgG-CA VI complexes from serum with a protein A–Sepharose conjugate. After the electrophoretic transfer, a Coomassie brilliant blue R-250 staining of the PVDF membrane showed a strong 51-kDa polypeptide corresponding to the IgG heavy chain (Fig. 3 ). In the same bound fraction, anti-CA VI serum identified 42- and 36-kDa polypeptide bands corresponding to the monomeric and deglycosylated forms of CA VI, respectively (3). These results indicate that CA VI is associated with IgG in serum, whereas it mainly occurs as a monomeric enzyme in saliva.



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  		Figure 3. Association of CA VI with IgG in serum.

The serum sample was mixed with protein A–Sepharose, and unbound (lane 1) and bound (lane 2) fractions were isolated. Aliquots of 5 µL from both fractions were subjected to SDS-PAGE under reducing conditions, followed by Western blotting. A strong 51-kDa polypeptide band of IgG heavy chain is present in the bound fraction stained with Coomassie brilliant blue R-250 (protein). When anti-CA VI or normal rabbit sera (NRS) were used, the second antibody identified the 51-kDa band of IgG heavy chain. The anti-CA VI serum specifically identified the major 42- (arrows) and minor 36-kDa polypeptide bands of CA VI in the bound fraction.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CA VI is an abundant secretory protein expressed in the serous acinar and demilune cells of the salivary glands and some unidentified epithelial cells of the rat lacrimal gland (15), but not in any tissues of the lower gastrointestinal tract (16). The tissue-specific expression of CA VI suggests that it could be a good marker protein to determine the exocrine function of the serous salivary glands. Since the development of the sensitive and robust time-resolved immunofluorometric assay for human CA VI, quantifying enzyme concentrations in complex biological fluids such as saliva has become possible (12).

According to our original hypothesis, at least small amounts of CA VI leak from the salivary glands or are absorbed from the alimentary canal into the bloodstream because of its high expression and secretion in the salivary glands. Our present results showed that 10-µL aliquots of the morning serum samples contained on average seven times more CA VI (2.0 ng/10 µL of serum) than the reported detection limit of the present fluoroimmunoassay (0.3 ng/200 µL well) (12), indicating that the method is sensitive enough for serum analyses. By analogy to the salivary CA VI concentrations, the serum concentrations also showed a marked intraindividual variation during the 24-h period. The changes of serum CA VI concentrations may be linked both to the periodicity in the expression and to the rapid clearance from the bloodstream, the latter being supported by the recent findings that CA VI bears unique Asn-linked oligosaccharides terminating with GalNAc-4-SO4, known to enhance the clearance of lutropin from the circulation via a specific receptor in liver (17). The marked variation in enzyme concentrations indicates that all CA VI analyses should be planned carefully and taken at the same time in the morning.

The presence of CA VI in serum could be confirmed by using a sensitive Western blotting method. In nonreducing conditions, the anti-CA VI antibody identified only a single high-molecular-mass band >130 kDa. After reduction, the high-molecular-mass polypeptide was reduced into two polypeptides of 42 and 51 kDa, corresponding to monomeric CA VI and immunoreactive IgG heavy chain, respectively. Therefore, the Western blotting results suggested that the protein associated with CA VI could be IgG. This observation was further confirmed by isolating IgG-CA VI complex from the serum with protein A affinity resin. Western blotting of the unbound and bound fractions revealed that CA VI was indeed associated with IgG, which may protect the enzyme from proteolytic degradation or target it to cells that do not express CA VI.

Laboratory testing for serum {alpha}-amylase is very commonly used in the diagnosis of diseases of the pancreas and in the investigation of pancreatic function. Most amylase assay methods are based on determination of the enzyme activity and cannot differentiate P-type and S-type amylase isoenzymes; therefore, more specific methods involving electrophoresis, wheat germ inhibitor of S-type amylase, or monoclonal antibodies have been developed (18). Because both CA VI and S-type amylase are produced in the serous elements of the salivary glands and probably share the same secretory pathways, studying whether CA VI measurements can help to assess the contribution of the salivary glands to the increased amylase concentrations will be challenging. The present results indeed provide a basis for further studies to determine the serum CA VI concentrations in patients with different salivary gland disorders.


   Acknowledgments
 
We thank Eva Kontula for help with the sample collection. We also acknowledge the skillful technical assistance of Lissu Hukkanen. This work was supported by grants from the Finnish Dental Society and the Finnish Defence Forces to J.K., from the Sigrid Juselius Foundation to S.P., from the Academy of Finland and the Maud Kuistila Foundation to A.-K.P., and from the US Public Health Service DK40163 and GM34182 to W.S.S.


   Footnotes
 
1 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.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Fernley RT, Wright RD, Coghlan JP. A novel carbonic anhydrase from the ovine parotid gland. FEBS Lett 1979;105:299-302. [Web of Science][Medline] [Order article via Infotrieve]
  2. Feldstein JB, Silverman DN. Purification and characterization of carbonic anhydrase from the saliva of the rat. J Biol Chem 1984;259:5447-5453. [Abstract/Free Full Text]
  3. Murakami H, Sly WS. Purification and characterization of human salivary carbonic anhydrase. J Biol Chem 1987;262:1382-1388. [Abstract/Free Full Text]
  4. Aldred P, Fu P, Barrett G, Penschow JD, Wright RD, Coghlan JP, Fernley RT. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry. Biochemistry 1991;30:569-575. [Medline] [Order article via Infotrieve]
  5. Parkkila S, Kaunisto K, Rajaniemi L, Kumpulainen T, Jokinen K, Rajaniemi H. Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human parotid and submandibular glands. J Histochem Cytochem 1990;38:941-947. [Abstract]
  6. Parkkila S, Parkkila A-K. Carbonic anhydrase in the alimentary tract. Roles of the different isozymes and salivary factors in the maintenance of optimal conditions in the gastrointestinal canal. Scand J Gastroenterol 1996;31:305-317. [Web of Science][Medline] [Order article via Infotrieve]
  7. Parkkila S, Parkkila A-K, Lehtola J, Reinilä A, Södervik H-J, Rannisto M, Rajaniemi H. Salivary carbonic anhydrase protects gastroesophageal mucosa from acid injury. Dig Dis Sci 1997;42:1013-1019. [Web of Science][Medline] [Order article via Infotrieve]
  8. Parkkila S, Parkkila A-K, Rajaniemi H. Circadian periodicity in salivary carbonic anhydrase VI concentration. Acta Physiol Scand 1995;154:205-211. [Web of Science][Medline] [Order article via Infotrieve]
  9. Lott JA, Ellison EC, Applegate D. The importance of objective data in the diagnosis of pancreatitis. Clin Chim Acta 1989;183:33-40. [Web of Science][Medline] [Order article via Infotrieve]
  10. Salt WB, Schenker S. Amylase—its clinical significance: a review of the literature. Medicine 1976;155:269-289.
  11. Parkkila S, Kaunisto K, Rajaniemi H. Location of the carbonic anhydrase isoenzymes VI and II in human salivary glands by immunohistochemistry. Botré F Gros G Storey BT eds. Carbonic anhydrase. From biochemistry and genetics to physiology and clinical medicine 1991:254-257 VCH Verlagsgesellschaft Weinheim. .
  12. Parkkila S, Parkkila A-K, Vierjoki T, Ståhlberg T, Rajaniemi H. Competitive time-resolved immunofluorometric assay for quantifying carbonic anhydrase VI in saliva. Clin Chem 1993;39:2154-2157. [Abstract]
  13. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685. [Medline] [Order article via Infotrieve]
  14. Leong ML, Fox GR. Enhancement of luminol-based immunoblot and western blotting assays by iodophenol. Anal Biochem 1988;172:145-150. [Web of Science][Medline] [Order article via Infotrieve]
  15. Ogawa Y, Toyosawa S, Inagaki T, Hong S-S, Ijuhin N. Carbonic anhydrase isozyme VI in rat lacrimal gland. Histochem Cell Biol 1995;103:387-394. [Web of Science][Medline] [Order article via Infotrieve]
  16. Parkkila S, Parkkila A-K, Juvonen T, Rajaniemi H. Distribution of the carbonic anhydrase isoenzymes I, II, and VI in the human alimentary tract. Gut 1994;35:646-650. [Abstract/Free Full Text]
  17. Hooper LV, Hindsgaul O, Baenziger JU. Purification and characterization of the GalNAc-4-sulfotransferase responsible for sulfation of GalNAc 1,4GlcNAc-bearing oligosaccharides. J Biol Chem 1995;270:16327-16332. [Abstract/Free Full Text]
  18. Rauscher E, Gerber M. Pancreatic {alpha}-amylase assay employing the synergism of two monoclonal antibodies. Clin Chim Acta 1989;183:41-44. [Web of Science][Medline] [Order article via Infotrieve]



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