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Articles |
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, -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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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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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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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 -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.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amylase assay employing the synergism of two monoclonal antibodies. Clin Chim Acta 1989;183:41-44.
[ISI][Medline]
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The following articles in journals at HighWire Press have cited this article:
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  P. Karhumaa, J. Leinonen, S. Parkkila, K. Kaunisto, J. Tapanainen, and H. Rajaniemi The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk PNAS, September 5, 2001; (2001) 121172598. [Abstract] [Full Text] [PDF]
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 J. Saarnio, S. Parkkila, A.-K. Parkkila, A. Waheed, T. Karttunen, and W. S. Sly Cell-specific Expression of Mitochondrial Carbonic Anhydrase in the Human and Rat Gastrointestinal Tract J. Histochem. Cytochem., April 1, 1999; 47(4): 517 - 524. [Abstract] [Full Text]
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P. Karhumaa, J. Leinonen, S. Parkkila, K. Kaunisto, J. Tapanainen, and H. Rajaniemi The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk PNAS, September 25, 2001; 98(20): 11604 - 11608. [Abstract] [Full Text] [PDF]
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