OULU 1999
HUMAN SALIVARY CARBONIC
ANHYDRASE ISOENZYME VI
Physiology and association with the experience of dental caries
JYRKI
KIVELÄ
Department of Anatomy and Cell Biology
OULUN YLIOPISTO, OULU 1999
HUMAN SALIVARY CARBONIC
ANHYDRASE ISOENZYME VI
Physiology and association with the experience of dental
caries
JYRKI KIVELÄ
Academic Dissertation to be presented with the assent
of the Faculty of Medicine, University of Oulu, for public
discussion in the Auditorium of the Department of
Anatomy and Cell Biology, on February 26th, 1999, at
12 noon.
Copyright © 1999
Oulu University Library, 1999
OULU UNIVERSITY LIBRARY
OULU 1999
ALSO AVAILABLE IN PRINTED FORMAT
Manuscript received 18.1.1999
Accepted 20.1.1999
Communicated by
Doctor Silvia Pastoreková
Professor Jorma Tenovuo
ISBN 951-42-5140-7
(URL: http://herkules.oulu.fi/isbn9514251407/)
ISBN 951-42-5133-4
ISSN 0355-3221
(URL: http://herkules.oulu.fi/issn03553221/)
Dedicated to my family
Kivelä, Jyrki, Human salivary carbonic anhydrase isoenzyme VI. Physiology and
association with the experience of dental caries.
Department of Anatomy and Cell Biology, University of Oulu, FIN-90220 Oulu, Finland
1999
Oulu, Finland
(Manuscript received 18 January 1999)
Abstract
The carbonic anhydrases (CAs) participate in the maintenance of pH homeostasis in various
tissues of the human body by catalyzing the reversible reaction CO2 + H2O <=> HCO3
−
+ H+.
Carbonic anhydrase isoenzyme VI (CA VI) is secreted into the human saliva by the serous acinar
cells of the parotid and submandibular glands. The present work was undertaken in order to gain
an understanding of the physiological role of CA VI in the oral cavity.
CA VI concentrations were compared with other salivary characteristics and with the clinical
dental status of the subjects. Saliva samples were collected under strictly controlled conditions
from 209 young, healthy men and their CA VI concentrations determined by means of a specific
time-resolved immunofluorometric assay. Salivary secretion rate, pH, buffering capacity, α–
amylase activity level and counts of lactobacilli and mutans streptococci were also determined.
Salivary CA VI concentrations showed positive correlations with salivary secretion rate (r = 0.20,
p = 0.003) and amylase activity level (r = 0.46, p < 0.001), but not with pH, buffering capacity, or
counts of mutans streptococci or lactobacilli. Salivary CA VI concentration, pH and buffering
capacity correlated negatively with the number of decayed, missing or filled teeth (DMFT index).
The correlation between salivary CA VI concentration and DMFT index was closest in the
subjects with poor oral hygiene. No correlation was found between salivary secretion rate or
amylase activity and the DMFT index.
The location of CA VI in the enamel pellicle, a thin layer of proteins on dental surfaces
providing a protective interface between the tooth surface and the external environment, was
demonstrated in samples of extracted teeth using immunostaining with anti-CA VI antibody.
Immunostaining for salivary α-amylase, which was used as a positive control, produced virtually
the same staining patterns. The presence of CA VI in the natural enamel pellicle was confirmed by
Western blotting of pellicle proteins. Histochemical staining of enamel pellicle formed in vitro
showed that the bound enzyme retains its CA activity.
To determine whether CA VI is transferred into the circulation, blood and saliva samples were
collected from four healthy male volunteers at 3-h intervals throughout a 24-h period and assayed
for CA VI concentration. CA VI was present in all the serum samples, although its concentration
was about 22 times lower than in the saliva. The presence of CA VI in serum was confirmed using
a sensitive Western blotting method. Western blotting also showed that serum CA VI is associated
with IgG, which may protect the enzyme from proteolytic degradation or target it to sites that do
not contain CA VI.
The present results suggest that salivary CA VI is not involved in regulation of the actual pH
or buffering capacity of the saliva, but it does seem to have a specific role in the oral cavity. High
salivary concentrations of CA VI appear to be associated with low caries experience. Since active
CA VI is located in the enamel pellicle, it may function locally in the microenvironment of the
dental surfaces and accelerate the neutralization of the acid metabolic products of bacterial plaque.
Keywords: buffering capacity, pH, secretion, serum
Acknowledgements
This work was carried out at the Department of Anatomy and Cell Biology, University
of Oulu, at Parolannummi Garrison Hospital, Hattula, and at the Research Institute of
Military Medicine, Finnish Defence Forces, during the years 1995-1998.
I wish to express my sincere gratitude to my supervisor, Professor Hannu Rajaniemi,
M.D., Ph.D., Head of the Department of Anatomy and Cell Biology, whose expert
scientific knowledge and generous support have been invaluable for the success of my
research. The whole work was based on his long-term vision of the importance of
salivary carbonic anhydrase in the oral cavity.
I am much indebted to my other supervisor, Professor Seppo Parkkila, M.D., Ph.D.,
for introducing me to the interesting field of carbonic anhydrase research and for his
intense commitment to this work. He has devoted long hours of his time to numerous
discussions on the topic, and we have become good friends during these years.
I am very grateful to Professor Jorma Tenovuo, D.D.S., Ph.D., and Dr. Silvia
Pastoreková, Ph.D., for their valuable comments and constructive criticism during the
completion of this thesis.
I wish to thank my co-authors: Dr. Anna-Kaisa Parkkila, M.D., Ph.D., Professor
Abdul Waheed, Ph.D., Professor William S. Sly, M.D., and the undergraduate
researchers Jukka Leinonen and Arto Toivanen. I owe my thanks to Captain Jussi
Metteri, M.Sc., for performing the statistical analyses and for introducing me to the
world of statistics.
I am grateful to the Surgeon General of the Finnish Defence Forces, Brigadier
General M.C. Timo Sahi, M.D., Ph.D., who has encouraged and supported medical
research by maintaining research facilities and by granting financial assistance. I also
wish to thank the preceding Surgeon General of the Finnish Defence Forces, Lieutenant
General M.C. Kimmo Koskenvuo, M.D., Ph.D., for authorizing me to begin the research.
The Surgeon General for Dental Services, Finnish Defence Forces, Lieutenant
Colonel M.C. Tuomo Nuuja, D.D.S., Ph.D., has granted me his full support from the
very beginning of the study, which I gratefully acknowledge. I also wish to thank
Lieutenant Senior Grade M.C. Vesa Jormanainen, M.D. for helpful discussions
concerning military medical research.
I wish to thank Lieutenant Colonel M.C. Jyrki Tikkinen, M.D., previously Senior
Medical Officer of the Armoured Brigade, Colonel Esko Janatuinen, previously
Commanding Officer of the Armoured Brigade, and Colonel M.C. Seppo Rehunen,
M.D., Ph.D., Medical Officer-in-Chief, Western Command, for their constant support
and encouragement during these years.
I also thank all the staff at the Department of Anatomy and Cell Biology at the
University of Oulu, at Parolannummi Garrison Hospital, and at the Central Laboratory of
Oulu University Hospital. Especially, I wish to acknowledge the skilful technical
assistance of Ms Lissu Hukkanen and Ms Airi Elomaa. The language of this manuscript
has been carefully revised by Mr. Malcolm Hicks, M.A., whose assistance I
acknowledge.
Grants from the Finnish Dental Society, the Häme Fund of the Finnish Cultural
Foundation and the Finnish Defence Forces are gratefully acknowledged. Permission to
reproduce the original articles was kindly granted by the Scandinavian Physiological
Society, the American Association for Clinical Chemistry, Inc., and S. Karger AG.
Finally, I extend my deepest thanks to my dear wife Lilli and to my children Antti,
Eeva and Sigrid. Lilli’s comprehensive support and the love and patience of the whole
family have made this work possible.
Pähkinämäki, January 1999
Jyrki Kivelä
Abbreviations
BSA
bovine serum albumin
CA
carbonic anhydrase
CA2
carbonic anhydrase II gene
CA6
carbonic anhydrase VI gene
cAMP
cyclic adenosine 3′,5′ monophosphate
CAs
carbonic anhydrases
cDNA
complementary deoxyribonucleic acid
cfu
colony forming unit
CPI
community periodontal index
CV
coefficient of variation
Cys
cysteine
DAB
3,3’diaminobenzidine tetrahydrochloride
dd.
dentes
DELFIA
dissociation enhancement lanthanide fluorescence immunoassay
dH2O
deionized water
DMFT
decayed, missing or filled teeth
ECL
enhanced chemiluminescence
GPI
glycosyl phosphatidylinositol
HCA
human carbonic anhydrase
HCA6
human carbonic anhydrase VI gene
IgA
immunoglobulin A
IgG
immunoglobulin G
IgM
immunoglobulin M
kDa
kiloDaltons
LB
lactobacillus, lactobacilli
mRNA
messenger ribonucleic acid
NRS
normal rabbit serum
PAGE
polyacrylamide gel electrophoresis
PBS
phosphate-buffered saline
PBST
phosphate-buffered saline with Tween-20
P-type
pancreatic type
PVDF
polyvinylidene fluoride
SD
standard deviation
SDS
sodium dodecyl sulphate
sEGF
salivary epidermal growth factor
SEM
standard error of mean
sIgA
secretory immunoglobulin A
SM
mutans streptococci
S-type
salivary type
TBST
Tris-buffered saline with Tween-20
TR-IFMA
time-resolved immunofluorometric assay
List of original publications
This thesis is based on the following original articles, which are referred to in the text by
Roman numerals I-IV:
I
Kivelä J, Parkkila S, Metteri J, Parkkila A-K, Toivanen A & Rajaniemi H (1997)
Salivary carbonic anhydrase VI concentration and its relation to basic
characteristics of saliva in young men. Acta Physiol Scand 161:221-225.
II
Kivelä J, Parkkila S, Waheed A, Parkkila A-K, Sly WS & Rajaniemi H (1997)
Secretory carbonic anhydrase isoenzyme (CA VI) in human serum. Clin Chem
43:2318-2322.
III
Kivelä J, Parkkila S, Parkkila A-K & Rajaniemi H (1999) A low concentration of
carbonic anhydrase isoenzyme VI in whole saliva is associated with caries
prevalence. Caries Res, in press.
IV
Leinonen J, Kivelä J, Parkkila S, Parkkila A-K & Rajaniemi H (1999) Salivary
carbonic anhydrase isoenzyme VI is located in the human enamel pellicle. Caries
Res, in press.
Contents
Abstract
Acknowledgements
Abbreviations
List of original publications
1. Introduction
15
2. Review of the literature
17
2.1. Historical aspects
17
2.2. Carbonic anhydrases
18
2.2.1. General aspects
18
2.2.2. Carbonic anhydrase isoenzyme II
19
2.2.3. Carbonic anhydrase isoenzyme VI
20
2.2.4. Other carbonic anhydrase isoenzymes
21
2.3. Salivary homeostasis
23
2.3.1. Composition and secretion of whole saliva
23
2.3.2. Salivary buffering capacity and pH
25
2.3.3. Role of saliva in protecting the epithelial surfaces of the upper
alimentary canal
26
2.3.4. Role of saliva in protecting the dental hard tissues
27
2.4. The enamel pellicle
27
3. Aims of the research
29
4. Materials and methods
30
4.1. Subjects (I, III)
30
4.2. Clinical examination (III)
30
4.3. Collection of saliva samples (I, III)
31
4.4. Microbial tests (III)
31
4.5. Tests for salivary pH, buffering capacity and secretion rate (I, III)
31
4.6. Tissue samples and collection of the enamel pellicle (IV)
31
4.7. Collection of serum (II)
32
4.8. Purification of CA VI. Antisera and immunoreagents (I-IV)
32
4.9. SDS-PAGE and Western blots (II, IV)
33
4.10. Isolation of the immunoglobulin-CA VI complex from serum (II)
33
4.11. Immunohistochemistry (IV)
34
4.12. Demonstration of CA activity on the enamel surface (IV)
34
4.13. Antigen labelling and fluoroimmunoassay procedure for CA VI (I-III)
35
4.14. Determination of amylase activity (I, III)
35
4.15. Statistical methods (I, III)
36
5. Results
37
5.1. Correlation of salivary CA VI concentration with the basic characteristics of
human whole saliva (I)
37
5.2. Effect of smoking on salivary characteristics (I)
38
5.3. CA VI in human serum (II)
38
5.4. Association between salivary CA VI concentration and dental caries (III) 39
5.4.1. Clinical and microbial findings
39
5.4.2. Correlations between clinical findings and analytical salivary
characteristics
39
5.5. CA VI in the enamel pellicle (IV)
42
5.5.1. Presence of CA VI in the enamel pellicle formed in vivo
42
5.5.2. Binding of CA VI to enamel in vitro
42
5.5.3. CA activity on the enamel surface
43
6. Discussion
44
7. Conclusions
49
References
1. Introduction
The carbonic anhydrases (CA; EC 4.2.1.1) are evolutionarily old enzymes, expressed in
most organs of the human body (Maren 1967, Hewett-Emmet & Tashian 1991, Sly & Hu
1995a). They participate in a variety of physiological processes involving pH regulation,
CO2 and HCO3
−
transport, ion transport, and water and electrolyte balance (Tashian
1992, Sly & Hu 1995a). In the mammalian body, CAs maintain pH homeostasis in
various tissues and biological fluids by catalyzing the reversible hydration of carbon
dioxide, CO2 + H2O <=> HCO3
−
+ H+ (Maren 1967, Tashian 1989, Brown et al. 1990,
Swenson 1991, Tashian 1992, Sly and Hu 1995b, Kaunisto et al. 1995, Parkkila S and
Parkkila A-K 1996, Lai et al. 1998). Eight isoenzymes with CA activity have been
identified in mammals to date, and all of them are expressed in the alimentary tract
(Lönnerholm et al. 1985, Parkkila S et al. 1994, Fleming et al. 1995, Parkkila S &
Parkkila A-K 1996, Pastoreková et al. 1997, Türeci et al. 1998). Two isoenzymes are
known to be expressed in the salivary glands, cytoplasmic CA II, a high-activity
isoenzyme that is not secreted into the saliva but may catalyze the production of salivary
bicarbonate (Case et al. 1982, Parkkila S et al. 1990), and secretory CA VI, which is
produced by the serous acinar cells of the parotid and submandibular glands (Fernley et
al. 1979, Feldstein & Silverman 1984, Murakami & Sly 1987, Parkkila S et al. 1990,
Ogawa et al. 1992, 1993, Parkkila S et al. 1994). The presence of CA activity in human
saliva was reported almost 60 years ago (Becks & Wainwright 1939, Rapp 1946), but its
physiological role has remained undefined. It has been proposed that CA VI may serve
to regulate the pH in saliva by utilizing the bicarbonate provided by CA II in the major
salivary glands (Feldstein & Silverman 1984, Kadoya et al. 1987, Sly & Hu 1995a).
Salivary CA VI may also have a protective effect against excess acidity on the mucosa
of the oesophagus and stomach (Rees & Turnberg 1982, Parkkila S et al. 1994, 1997).
Saliva is responsible for the maintenance of homeostasis on oral surfaces (Ericson &
Mäkinen 1986, Mandel 1989, van Houte 1994), and its importance for dental health is
demonstrated by the rampant caries seen in patients with grave salivary hypofunction
(Dreizen & Brown 1976, Mandel 1989). Saliva contains inorganic compounds and
multiple proteins that affect conditions in the oral cavity and on the tooth surfaces
(Tenovuo 1989). One essential factor in maintaining oral homeostasis is salivary
buffering capacity (Kleinberg & Jenkins 1964, Jensen 1986, Mandel 1987, van Houte
1994). Three major buffer systems contribute to the total buffering capacity of saliva: a
16
carbonic acid/bicarbonate system, a phosphate system, and a system based on proteins
(Leung 1951, Lilienthal 1955, Leung 1961, Izutsu & Madden 1978, Helm et al. 1982,
Ericson & Mäkinen 1986, Birkhed & Heintze 1989). The carbonic acid/bicarbonate
system, based on the equilibrium CO2 + H2O <=> HCO3
−
+ H+, is physiologically the
most important buffer system in the oral cavity (Leung 1951, Lilienthal 1955, Leung
1961, Izutsu 1981, Helm et al. 1982, Birkhed & Heintze 1989).
The human oral cavity, as the entrance to the alimentary canal, is exposed to widely
varying conditions due to the differing physical and chemical properties of the ingested
food and drinks. The metabolism of the abundant microbial flora also increases acidity
on dental surfaces, mainly in the form of lactic, acetic, formic and propionic acids
(Muntz 1943, Stephan 1944, Kleinberg 1970, Sandham & Kleinberg 1970, Geddes 1975,
1981, Abelson & Mandel 1981). Increased acidity on dental surfaces will lead to
demineralization if the removal of excess acid is not rapid enough. Taken together, the
mechanisms responsible for oral homeostasis, particularly pH homeostasis, are of vital
importance to the protection of the oral cavity from pathological conditions. Knowing
that CA isoenzymes serve to maintain pH homeostasis in various biological fluids and
that CA VI is abundantly secreted into the saliva, we undertook this study to explore the
possible physiological role of CA VI in the human oral cavity.
2. Review of the literature
2.1. Historical aspects
Saliva has been a disregarded substance throughout history, even though human life
without saliva is a misery. Until the end of the 17th century, the salivary glands were
regarded as emunctories, sieving excrementous substances from the blood, particularly
the evil spirits of the brain (Garrett 1975), and up to the early 19th century physicians
who practiced according to the premise that all diseases were due to disorders of the four
“principal humours” (phlegm, blood, yellow bile and black bile) would prescribe doses
of calomel, mercurous chloride, to cause massive salivation, probably to cleanse the
system from noxious substances. Only the strongest survived both illness and cure, and
preventive medicine meant keeping out of the hands of physicians. (Mandel 1987.) In
the course of the present century salivary research has attracted an ever-increasing
interest, and the importance of saliva for the physiology of the mouth and upper
alimentary canal is now well established.
The first notion of the existence of carbonic anhydrases arose in the late 1920’s,
when a red cell substance catalyzing the reversible hydration of carbon dioxide, CO2 +
H2O <=> HCO3
−
+ H+, was recognized in studies on the rate of escape of carbon dioxide
from haemolyzed blood (Henriques 1928). A few years later the discharge of CO2 from
the lung capillaries and its uptake in tissues was found to proceed much more rapidly
than was calculated from the rates of the non-catalyzed reactions, and the substance
responsible for this was shown to be an enzyme and named carbonic anhydrase
(Meldrum & Roughton 1932, Edsall 1968, Carter 1972). The enzyme was soon isolated
and partially purified (Meldrum & Roughton 1933), and in 1939 it was found to have a
molecular weight of about 30 kDa and to contain one zinc ion per molecule (Keilin &
Mann 1939).
Although CA activity was likewise observed in human saliva for the first time almost
60 years ago (Becks & Wainwright 1939, Rapp 1946), only few studies have been
carried out on the physiological role of salivary CA. In 1974, Szabó reported higher
mean salivary CA activity levels in caries-free children than in children with active
caries. Great advances were achieved in the late 1970’s when Fernley et al. described a
novel CA expressed in the ovine parotid gland (Fernley et al. 1979). Over the following
18
10 years, the enzyme was first purified from rat saliva by Feldstein and Silverman
(1984) and later from human saliva by Murakami and Sly (1987) and Kadoya et al.
(1987), and designated CA VI. In 1991, Aldred et al. cloned and characterized the cDNA
encoding for HCA VI. The next major step in research into salivary CA was the
development of specific immunofluorometric and radioimmunoassays for human
salivary CA VI (Parkkila S et al. 1993b, Fernley et al. 1995), which allowed accurate
quantification of CA VI in difficult matrices such as saliva and serum.
2.2. Carbonic anhydrases
2.2.1. General aspects
Eight isoenzymes with CA activity have been identified and characterized in mammals
(CAs I-VI, IX, and XII). The cDNAs encoding for the known human isoenzymes have
been cloned and characterized (Henderson et al. 1973, Lin & Deutsch 1973, Butterworth
et al. 1991, Edwards 1991, Aldred et al. 1991, Okuyama et al. 1992, Nagao et al. 1993,
Pastoreková et al. 1997, Türeci et al. 1998), and the crystallographic structures of
isoenzymes CA I-V have been determined (Eriksson & Liljas 1991, Stams et al. 1996,
Boriack-Sjödin et al. 1995). CAs are formed of a single polypeptide, and in the native
form the molecule contains one tightly bound Zn2+, which is essential for catalytic
activity (Lindskog 1982). The refined structures of the cytoplasmic isoenzymes (CA I, II
and III), as determined crystallographically, appear to be quite homologous (Eriksson &
Liljas 1991), having a 10-stranded β sheet that bisects the molecule. The zinc ion is
located near the centre of the molecule, at the bottom of a cavity 15 Å wide and 15 Å
deep which forms the active site of the enzyme. Three histidine residues ligate the zinc
ion to the β sheet structure, the fourth and fifth ligand sites of the zinc ion probably
being occupied by a water molecule and a hydroxyl ion (Kannan et al. 1977).
The carbonic anhydrases catalyze the reversible hydration of carbon dioxide, CO2 +
H2O <=> HCO3
−
+ H+, and participate in various biological processes involving the
maintenance of pH homeostasis, CO2 transfer and ion exchange (Tashian 1989, 1992).
CAs can also act upon a large variety of other substances which undergo hydration of
aldehydes (Pocker & Meany 1965, 1967) or hydrolysis of aromatic esters (Schneider &
Liefländer 1963). The physiological significance of functions other than those associated
with the interconversion of CO2 and HCO3
−
has nevertheless remained undefined. CAs
are found in almost all organisms, from algae and bacteria to mammals. In addition to
this wide expression among different species, they are distinctive because of their great
diversity in tissue distribution, cellular and subcellular location (Table 1), and in
biological functions (Tashian 1989, 1992).
19
Table 1. Summary of the subcellular location of CA isoenzymes.
Isoenzyme
Subcellular location
CA I
cytoplasmic
CA II
cytoplasmic
CA III
cytoplasmic
CA IV
membrane-associated
CA V
mitochondrial
CA VI
secreted
CA IX
transmembrane protein
CA XII
transmembrane protein
2.2.2. Carbonic anhydrase isoenzyme II
CA II is one of the most efficient enzymes known, having a turnover number of 1.3-1.9
× 106 /s under physiological conditions (Khalifah 1971, Sanyal & Maren 1981, Wistrand
1981). The human CA2 gene is 17 kb long and is located on chromosome 8, like the
genes for CA I and III (Nakai et al. 1987, Tashian 1989). CA II is the most widely
distributed isoenzyme of the CA gene family, being present in virtually every human
tissue or organ (Tashian 1992). It was first found in erythrocytes, where it is abundantly
expressed and is involved in the hydration of CO2 (Meldrum & Roughton 1932, 1933,
Wistrand 1981).
CA II is expressed in many organs of the digestive system (Parkkila S & Parkkila A-
K 1996). It is located in the serous acinar cells of the parotid and submandibular glands
and thought to release bicarbonate ions into the saliva (Parkkila S et al. 1990, 1991b,
Ogawa et al. 1993, Parkkila S et al. 1994). The squamous epithelium of the oesophagus
appears to contain CA II, and it may participate in endogenous bicarbonate production in
the oesophagus (Meyers & Orlando 1992, Parkkila S et al. 1994). In the stomach, it is
expressed in the parietal and surface epithelial cells of the gastric mucosa, where it
regulates the acidity of the gastric juice (Davenport & Fisher 1938, Davenport 1939,
O’Brien et al. 1977, Parkkila S et al. 1994, Parkkila S & Parkkila A-K 1996). The
parietal cells of the gastric glands secrete protons to acidify the gastric juice (Sato et al.
1980, Kumpulainen 1981, Lönnerholm et al. 1985, Swenson 1991, Sasaki et al. 1993),
and the gastroduodenal surface epithelial cells secrete bicarbonate below the mucous gel
layer covering the epithelia to protect the epithelium from being itself digested
(Richardson 1985, Allen & Carrol 1988, Swenson 1991, Parkkila S & Parkkila A-K
1996, Takeda et al. 1997). CA II has been found in the non-goblet epithelial cells of the
mammalian colon (Lönnerholm et al. 1985, Parkkila S et al. 1994), in which it is
implicated in the regulation of NaCl reabsorption (Binder et al. 1987, Goldfarb et al.
1988, Charney & Egnor 1989, Swenson 1991).
In the liver, CA II has been demonstrated in the hepatocytes and the epithelium of the
bile ducts (Dodgson et al. 1984, Carter et al. 1989, Parkkila S et al. 1994), its best-
20
known physiological function being to produce HCO3
−
for alkalization of the bile
(Swenson 1991). It is also expressed in the epithelial cells of the gallbladder, where it is
involved in bile concentration and acidification (Juvonen et al. 1994, Parkkila S &
Parkkila A-K 1996), and in the pancreas, where it is located in the epithelial duct cells.
Its role in the secretion of bicarbonate into the pancreatic juice is well established
(Kumpulainen & Jalovaara 1981, Spicer et al. 1982, Kumpulainen 1984, Swenson
1991).
CA II is well documented in renal tubular cells, where it contributes to urinary
acidification (Wistrand 1980, Wåhlstrand & Wistrand 1980, Kumpulainen 1984, Sly &
Hu 1995a,b, Lai et al. 1998), and it has an essential role in the bone resorption, to the
extent that hereditary CA II deficiency causes osteopetrosis, renal tubular acidosis and
cerebral calcification (Sly et al. 1983, 1985, Sly & Hu 1995a,b). CA II is expressed in
the central nervous system and is involved in control of the production of cerebrospinal
fluid (Maren 1967, Maren & Broder 1970, Parkkila A-K et al. 1994, 1995, 1997, Catala
1997). Recent immunohistochemical studies have shown that it is also expressed in some
endocrine cells of the human pituitary and adrenal glands (Parkkila A-K et al. 1993,
Sasano et al. 1994, Parkkila A-K et al. 1996). In addition to the locations listed above,
CA II has been demonstrated in the type II pneumocytes of the lung (Fleming et al.
1994), various cells in the tissues of the male reproductive tract (Kaunisto et al. 1990,
Parkkila S et al. 1991a, Kaunisto et al. 1995) and in the human placenta and foetal
membranes (Mühlhauser et al. 1994).
2.2.3. Carbonic anhydrase isoenzyme VI
CA VI is the only known secreted isoenzyme of the CA gene family, and has several
properties that distinguish it from the well-characterized cytoplasmic isoenzymes. Its
reported molecular weight is 39-46 kDa (Feldstein & Silverman 1984, Kadoya et al.
1987, Murakami & Sly 1987, Fernley 1991a,b, Parkkila S et al. 1991b, Ogawa et al.
1992). The enzyme molecule has two N-linked oligosaccharide chains, which can be
cleaved by endo-###-N-acetylglucosaminidase F but not by endo-###-N-
acetylglucosaminidase H, indicating that the oligosaccharides are of a complex type
(Murakami & Sly 1987). Neuraminidase has no effect on the endo-###-N-
acetylglucosaminidase F-digested protein, suggesting that HCA VI has no O-linked
oligosaccharide which contains neuraminidase-sensitive sialic acid residues (Murakami
& Sly 1987). The complete amino acid sequence of ovine CA VI was determined by
Fernley et al. (1988), and the complete nucleotide sequence for human CA6 cDNA by
Aldred et al. (1991). The HCA6 gene is located on chromosome 1 (Sutherland et al.
1989, Aldred et al. 1991). The HCA VI protein has a sequence identity of 35 % to HCA
II, while residues involved at the active site of the enzyme are conserved. HCA VI has
three potential N-glycosylation sites and two cysteine residues (Cys25 and Cys207)
(Aldred et al. 1991), the latter presumably forming a disulphide bond, as in the ovine
enzyme (Fernley et al. 1988).
Immunohistochemical studies have demonstrated the expression of CA VI
exclusively in the acinar cells of the mammalian parotid and submandibular glands
21
(Kadoya et al. 1987, Parkkila S et al. 1991b, Ogawa et al. 1992), where it is secreted
into the saliva. Salivary concentrations of ovine and human CA VI have been
investigated using a radioimmunoassay (Fernley et al. 1991, 1995) and a time-resolved
immunofluorometric assay (TR-IFMA) (Parkkila S et al. 1993b, 1995).
Radioimmunoassay of ovine CA VI showed that its mean ± SD concentration in sheep
parotid saliva is 5.61 ± 3.01 mg/l in the normal conscious animal, while feeding
increased the concentration to 33.0 ± 19.0 mg/l (Fernley et al. 1991, Fernley 1991a).
Nerve stimulation and cholinergic drug administration have been used to demonstrate
that both parasympathetic and sympathetic pathways may control the CA VI
concentration in the sheep saliva (Fernley et al. 1991). Radioimmunoassay methods have
shown the mean ± SD concentration of CA VI in human parotid saliva to be 47.0 ± 39.2
mg/l, in which case it represented about 3 % of total protein in the parotid saliva
(Fernley et al. 1995). The mean ± SD concentration of CA VI in human whole saliva,
measured by TR-IFMA, was 6.8 ± 4.3 mg/l (Parkkila S et al. 1993b). In addition,
secretion of CA VI into the saliva was observed to follow a circadian periodicity, its
concentrations being very low during sleep and rising rapidly to the daytime level after
awakening and breakfast (Parkkila S et al. 1995).
The physiological function of CA VI has remained undefined, but it is thought to
have a specialized role in the maintenance of bicarbonate levels in the saliva (Feldstein
& Silverman 1984, Kadoya et al. 1987, Fernley 1988, Sly & Hu 1995a). This suggestion
is in line with an earlier finding that children suffering from caries have lower salivary
CA activity compared to caries-free children (Szabó 1974). A new insight into CA VI
research has been provided by the recent observations that CA VI probably maintains its
activity in the harsh environment of the gastric lumen and that patients with verified
oesophagitis or oesophageal, gastric or duodenal ulcers have reduced salivary CA VI
concentrations relative to patients with a non-acid peptic disease (Parkkila S et al. 1997).
Parkkila S et al. (1990, 1994, 1997) have proposed that CA VI and CA II may form a
mutually complementary system for the regulation of pH homeostasis on the epithelial
surfaces of the upper alimentary canal.
CA VI also appears to be involved in the physiology of taste function. In a recent
study Thatcher et al. (1998) identified gustin, a salivary protein associated with the
function of taste buds, as human CA VI by protein sequencing, activity profiles and
other physical data.
2.2.4. Other carbonic anhydrase isoenzymes
CA I is a well characterized cytoplasmic enzyme with a molecular weight of about 30
kDa (Bundy 1977, Lindskog et al. 1984). It is a low-activity isoenzyme expressed in
mammalian erythrocytes, the epithelium of the colon, the capillary endothelium, the
corneal endothelium, the lens of the eye, the islets of Langerhans, the placenta and foetal
membranes (Lönnerholm et al. 1985, Venta et al. 1987, Sasaki et al. 1993, Parkkila S et
al. 1994, Mühlhauser et al. 1994). A striking feature of this isoenzyme is that although it
is one of the most abundant proteins in mammalian red cells, no haematological
abnormalities have emerged in its absence as the result of a mutation, and it is not
22
expressed in the red cells of certain species, e.g., ruminants and felids, making the
assignment of a physiological role in erythrocytes problematic (Tashian et al. 1971,
Kendall & Tashian 1977, Tashian et al. 1980, Tashian 1992).
CA III is a cytoplasmic, very low-activity isoenzyme expressed primarily in the type
I fibres of the skeletal muscle (Tipler et al. 1978, Holmes 1976), where its exact
physiological function has remained undefined. Smaller amounts of CA III have been
detected in the human uterus, urine bladder, lung, cardiac muscle (Jeffery et al. 1980),
human myoepithelial cells (Väänänen & Autio-Harmainen 1987), equine thymus
(Nishita & Matsushita 1989), guinea pig salivary glands and mouse colon (Spicer et al.
1990). CA III is the second isoenzyme to be demonstrated in the rodent liver (Carter &
Jeffery 1985, Spicer et al. 1990), but only traces of this hormonally regulated CA
isoenzyme have been found in the adult human liver (Jeffery et al. 1980, Carter et al.
1984). The presence of CA III in hepatocytes has aroused interest in its specific function.
Cabiscol & Levine (1995) have demonstrated that it functions in an oxidizing
environment and that it is the most oxidatively modified protein in the liver known so
far.
CA IV was the first membrane-associated CA isoenzyme to be described. It is
thought to facilitate the reversible hydration of CO2 at sites where CO2 and HCO3
−
flux
across membranes needs to be very rapid (Sly & Hu 1995a, Parkkila S & Parkkila A-K
1996). HCA IV contains a 27-amino acid COOH-terminal extension which may serve as
a recognition signal for cleavage and transfer to a glycosyl phosphatidylinositol (GPI)
link, through which CA IV molecules are anchored to the plasma membrane (Zhu & Sly
1990, Ghandour et al. 1992, Waheed et al. 1992a, Sly & Hu 1995a, Parkkila S &
Parkkila A-K 1996). CA IV has been purified from bovine lung microsomes (Whitney &
Briggle 1982) and human kidney (Wistrand & Knuuttila 1989), and in a catalytically
active form from human lung and kidney (Zhu & Sly 1990) and rat lung (Waheed et al.
1992a). It has been found to function in the plasma membranes of the proximal
convoluted tubule and thick ascending limb of Henle in the rat kidney (Brown et al.
1990, Rector et al. 1998), and it is present in the endothelial cells of the choriocapillaries
of the human eye (Hageman et al. 1991) and in the endothelial cells of rat brain
capillaries (Ghandour et al. 1992). CA IV is expressed on the apical surface of epithelial
cells in the colon (Fleming et al. 1995), in the luminal plasma membrane of the human
gallbladder epithelium (Parkkila S et al. 1996), and on the plasma face of endothelial
cells of the pulmonary microvasculature (Fleming et al. 1993). Waheed et al. (1992b)
have demonstrated that the membrane-associated CA in skeletal muscle is CA IV. This
same isoenzyme is also expressed on the apical surface of the epididymal duct, where it
is known to play a major role in the acidification of the epididymal fluid (Parkkila S et
al. 1993a). It has been demonstrated that the expression of CA IV in the epididymis is
regulated by androgens and oestrogen (Caflisch 1990, Caflisch & DuBose 1990,
Kaunisto 1998). The gene for CA IV is located on chromosome 17 (Okuyama et al.
1992, 1993).
CA V is the mitochondrial CA isoenzyme, uniquely located in the mitochondrial
matrix. Its presence has been established in mammalian liver and pancreas (Nagao et al.
1993, Sly & Hu 1995a, Parkkila A-K et al. 1998). The cDNA for human mitochondrial
CA V has been cloned from a human liver cDNA library, and its gene has been localized
to chromosome 16 (Nagao et al. 1993). CA V is thought to participate in two metabolic
23
processes in the mitochondria of hepatocytes: ureagenesis and gluconeogenesis,
supplying bicarbonate for the first urea cycle enzyme, carbamyl phosphate synthetase I
in ureagenesis and for pyruvate carboxylase in gluconeogenesis (Dodgson 1991). CA
inhibitors have been observed to retard both of these processes in the livers of guinea
pigs and rats (Dodgson et al. 1983, Metcalfe et al. 1985, Dodgson 1991). The expression
of CA V in pancreatic β-cells and the observation that the CA inhibitor acetazolamide
inhibits glucose-stimulated insulin secretion have led to a proposal that CA V may have
a role in the regulation of insulin secretion (Parkkila A-K et al. 1998).
CA VII is a putative isoenzyme, the gene for which has been isolated from a human
genomic library (Montgomery et al. 1991, Tashian 1992, Sly & Hu 1995a). The gene is
about 10 kb long and located on chromosome 16, and the predicted amino acid
frequency shows 50, 56, 49, and 37 % homology with human CA I, II, III, and VI,
respectively.
CA IX is an integral transmembrane isoenzyme expressed in the human gastric
mucosa, the cryptal enterocytes of the duodenum and jejunum, the gallbladder mucosa
and the bile ducts (Pastorek et al. 1994, Pastoreková et al. 1997, Saarnio et al. 1998b).
CA IX has also been found in human tumours derived from cervix uteri, kidney, colon,
oesophagus and other organs (Závada et al. 1993, Liao et al. 1994, 1997, McKiernan et
al. 1997, Turner et al. 1997, Saarnio et al. 1998a). Its subcellular location in the
basolateral membranes of cells and its intense expression in proliferative cells have led
to the suggestion that it may be involved in intercellular communication and/or cell
proliferation (Pastoreková et al. 1997, Saarnio et al. 1998b). The active site domain of
the CAs is completely conserved in CA IX, suggesting that it could also participate in
carbon dioxide/bicarbonate homeostasis (Opavský et al. 1996).
CA XII was the second transmembrane CA isoenzyme to be described, having
recently been identified in a human renal cell carcinoma. Northern blot analysis of
normal tissues has demonstrated CA XII mRNA only in the kidney and intestine. Türeci
et al. (1998) have shown that in 10 % of patients with renal cell carcinoma the CA XII
transcript was expressed at higher levels in the tumour than in the surrounding normal
kidney tissue, suggesting that it is the second catalytically active membrane-associated
CA isoenzyme that is overexpressed in certain cancers. The cDNA encoding for CA XII
has been cloned and characterized, and its gene has been mapped to chromosome 15.
(Türeci et al. 1998.)
2.3. Salivary homeostasis
2.3.1. Composition and secretion of whole saliva
Whole saliva is a mixture of the secretions of the parotid, submandibular, sublingual and
minor salivary glands and gingival crevicular fluid. An outline of the secretions of the
individual salivary glands is shown in Table 2. Saliva contains inorganic compounds and
multiple proteins that affect conditions in the oral cavity and locally on the tooth
surfaces. It is involved in the clearance of food debris and provides inorganic ions for the
24
neutralization of the acid and alkaline metabolic products of oral bacteria and for the
remineralization of the enamel. It also brings various defence mechanisms, including
leukocytes, secretory IgA (sIgA), agglutinating proteins and a number of enzymes, to the
actual sites of microbial growth on the tooth and mucosal surfaces (Tenovuo 1989,
Lamkin and Oppenheim 1993, Johnsson et al. 1993, Lagerlöf and Oliveby 1994, Edgar
et al. 1994, Wolinsky 1994).
Table 2. Types of secretion produced by the salivary glands (Ross et al. 1989).
Secretion type
Salivary glands
serous
mucous
Parotid
+++
−
Submandibular
++
+
Sublingual
+
++
Minor
−(+)
+++
In addition to the secretions from the salivary glands, several other factors, including
oral bacteria, desquamated epithelial cells, crevicular fluid and leukocytes, contribute to
the composition of whole saliva (Söderling 1989). The majority of the salivary IgG is
passively diffused from the serum, mainly through the gingival crevices (Challacombe et
al. 1978, Grönblad & Lindholm 1987), while many innate proteins, e.g. lysozyme,
lactoferrin and myeloperoxidase, are partly derived from degenerating leukocytes
migrating into the oral cavity again predominantly via the gingival crevices (Schiött &
Löe 1970, Raeste 1972, 1976, Bennett & Kokocinski 1978, Friedman et al. 1983,
Kowolik & Grant 1983). Furthermore, monocytes and macrophages actively secrete
lysozyme, which may be blended with saliva via the crevicular fluid (Nord et al. 1971,
Gordon et al. 1974, Bennett & Kokocinski 1978).
Salivary secretion can be assessed under resting and stimulated conditions, and a
significant correlation has been reported between the flow rates of chewing-stimulated
and unstimulated saliva (White 1977, Heintze et al. 1983). Normal values for the
salivary secretion rate stimulated by chewing paraffin wax have been estimated to be 1-3
ml/min, and values below 0.7 ml/min have been considered to indicate hyposalivation.
For resting salivary flow, these values are 0.25-0.35 ml/min and < 0.1 ml/min,
respectively. (Ericsson & Hardwick 1978, Lagerlöf & Tenovuo 1994.) Stimulated
salivary flow rate is slightly higher in men than in women, probably due to the larger
size of the salivary glands in men (Parvinen & Larmas 1982). Ageing does not seem to
affect the flow rate of stimulated whole saliva in healthy, unmedicated men, whereas a
postmenopausal decline can often be observed in women (Kullander & Sonesson 1965,
Baum 1981, Parvinen & Larmas 1982, Heintze et al. 1983). (Tenovuo 1992.) The
concentrations of most salivary components depend on the secretion rate. At increased
salivary secretion rates the concentrations of sodium, calcium, chloride and bicarbonate
increase while potassium and fluoride concentrations remain unchanged and phosphate
and iodide concentrations decrease (Dawes 1969, Shannon 1973, Dawes 1974, Ferguson
25
1989). The total protein concentration in saliva also increases with increased flow rate
(Dawes 1969, Shannon 1973). Salivary immunoglobulins show deviating responses to
stimulation (Söderling 1989), IgA correlating negatively with salivary secretion rate
whereas IgG has been reported to be fairly independent of stimulation (Grönblad 1982,
Brandtzaeg 1989).
Salivation is initiated by the salivary centres in the medulla oblongata, which receive
afferent signals from the sensory terminals of the oral and nasal cavities and from the
higher centres in the brain (Garrett 1987). The secretion of saliva is regulated by the
autonomic nervous system (Asking & Gjörstrup 1980, Helm et al. 1982, Garrett 1987,
Olsen et al. 1988, Calvert et al. 1998), and its composition follows circadian rhythms
(Dawes 1972, 1975, Ferguson & Botchway 1979, Parkkila S et al. 1995). Water and
electrolyte secretion are mainly controlled by parasympathetic activity, whereas protein
synthesis and exocytosis are mainly controlled by sympathetic activity (Garrett 1987,
Jensen et al. 1991, Nederfors & Dahlöf 1992, Nederfors et al. 1994, Nederfors & Dahlöf
1996). Two basic stimulus-response coupling pathways are thought to be involved in the
secretion of saliva (Putney 1986). The Ca2+ pathway regulates ion and water flux, and to
some extent protein secretion, while the other pathway, involving cyclic adenosine 3′,5′
monophosphate (cAMP), controls primarily enzyme secretion (Putney 1986). β–
adrenoceptor activation has been shown to stimulate the exocytotic discharge of α–
amylase in the rat parotid gland by a mechanism in which cAMP functions as a
messenger (Rasmussen & Tenenhouse 1968, Schramm & Selinger 1975).
2.3.2. Salivary buffering capacity and pH
Salivary buffering capacity is a factor of primary importance in maintaining oral
homeostasis (Kleinberg & Jenkins 1964, Jensen 1986, Mandel 1987, Birkhed & Heintze
1989). The main buffer systems known to contribute to the total buffering capacity of
saliva are the bicarbonate and phosphate systems and those based on proteins (Leung
1951, Lilienthal 1955, Leung 1961, Izutsu & Madden 1978, Helm et al. 1982, Ericson &
Mäkinen 1986, Mandel 1987). These systems have different pH ranges of maximal
buffering capacity, the phosphate and bicarbonate systems having pK values of 6.8-7.0
and 6.1-6.3, respectively, whereas the proteins contribute to the salivary buffering
capacity at very low pH values only (Ericson & Mäkinen 1986). Most of the salivary
buffering capacity operative during food intake and mastication is due to the bicarbonate
system, which is based on the equilibrium CO2 + H2O <=> HCO3
−
+ H+ (Leung 1951,
Ericsson 1959, Lilienthal 1955, Leung 1961, Izutsu 1981, Helm et al. 1982, Birkhed &
Heintze 1989, Lagerlöf & Oliveby 1994). The concentration of bicarbonate in the saliva
is greatly increased at increased flow rates (Dawes 1969, Shannon 1973, Dawes 1974,
Abelson & Mandel 1981, Ferguson 1989, Söderling 1989). Another essential feature of
this buffer system under the conditions prevailing in the oral cavity is the phase
conversion of carbon dioxide from a dissolved state into a volatile gas. When acid is
added, this phase conversion considerably increases the efficacy of the neutralization
reaction, as there is no accumulation of the end products but complete removal of the
26
acid (Birkhed & Heintze 1989), a phenomenon referred to as “phase buffering” (Ericson
& Mäkinen 1986, Birkhed & Heintze 1989).
Phosphate makes a minor contribution to the total salivary buffering capacity
relative to bicarbonate. Its system is in principle analogous to that of bicarbonate but
without the important phase buffering effect. Within the pH range of the oral cavity, the
phosphate buffer is based on the reversible reaction H2PO4
−
<=> HPO4
2−
+ H+ (Ericson
& Mäkinen 1986). The concentration of HPO4
2−
in saliva is relatively independent of the
salivary secretion rate, and thus the capacity of the phosphate buffering system does not
increase during food intake or mastication.
Evaluation of the salivary buffering effect based on proteins has produced
controversial results, but in general the effect has been regarded as insignificant, or at
least of minor importance (Lilienthal 1955, Ericson & Mäkinen 1986, Birkhed &
Heintze 1989), although data suggesting a deviating conclusion have also been presented
(Leung 1961, Izutsu & Madden 1978).
2.3.3. Role of saliva in protecting the epithelial surfaces of the upper
alimentary canal
The epithelia of the human oral cavity and oesophagus are exposed to widely varying
conditions due to the differing physical and chemical properties of the ingested food, in
addition to which the epithelium of the oesophagus is challenged by acid reflux from the
stomach. The saliva is responsible for the luminal defence of the epithelia of the upper
alimentary canal. A number of salivary proteins are known to bind to the epithelial
surfaces of the oral cavity, including salivary mucins, amylase, salivary cystatins and
acidic proline-rich proteins (Bradway et al. 1989, 1992). This epithelial pellicle provides
a lubricatory film and an effective barrier against desiccation and environmental factors,
and it is also thought to protect the epithelial cells from proteases emanating from
bacteria attached to the mucosal surfaces and from degenerating polymorphonuclear
leukocytes (Mandel 1987, 1989, Vaahtoniemi et al. 1992).
The role of immunoglobulins in saliva is far from clear. Evidence has been presented
for binding of salivary immunoglobulins IgA and IgM to the mucosal epithelial cells
(Boackle & Suddick 1980). sIgA is the primary immunoglobulin in saliva, and it may
promote microbial aggregation (Liljemark et al. 1979, Cohen & Levine 1989), while
specific sIgA antibodies may inhibit microbial enzymes involved in colonization or
pathogenic processes (Tomasi 1983, Gregory et al. 1990, Rudney 1995).
It has been suggested that salivary epidermal growth factor (sEGF) and prostaglandin
E2 may play essential roles in maintaining the integrity of the mucosa of the upper
alimentary canal (Li et al. 1993, Rourk et al. 1994, Wu-Wang et al. 1995, Yang et al.
1996, Sarosiek et al. 1996, Namiot et al. 1997), and even that the influence of sEGF may
extend to the mucosa of the ileum (Rao et al. 1997).
Salivary bicarbonate secretion is known to be of vital importance for the maintenance
of oesophageal pH homeostasis (Rees & Turnberg 1982, Helm et al. 1982, 1984,
Sarosiek & McCallum 1995, Sarosiek et al. 1996), and recent findings suggest that CAs
are also involved in this process. It has been proposed that salivary CA VI catalyzes the
neutralization of excess acid in the mucous layer covering the oesophageal and gastric
27
epithelial cells (Parkkila S & Parkkila A-K 1996, Parkkila S et al. 1997). Three cytosolic
isoenzymes (CA I, II, and III) and one membrane bound isoenzyme (CA IV) have been
identified in human oesophageal epithelial cells, where they probably play a significant
part in protecting the oesophageal mucosa from acid injury (Christie et al. 1997).
2.3.4. Role of saliva in protecting the dental hard tissues
Dental enamel is the hardest tissue in the human body, and the main challenge to it
comes from acidic conditions in the oral cavity, which can cause dissolving of the
mineral contained in it, i.e. dental caries or erosion. The metabolism of the microbial
flora on the dental surfaces produces considerable amounts of acid, mainly in the form of
lactic, acetic, formic and propionic acids (Clarke 1924, Muntz 1943, Stephan 1944,
Fitzgerald & Keyes 1960, Kleinberg 1970, Sandham & Kleinberg 1970, Geddes 1975,
Geddes 1981). Moreover, various foods and drinks add to the acid charge on these
surfaces. Saliva can be considered the oral tissue fluid of the enamel, and the
maintenance of homeostasis on the dental surfaces is totally dependent on salivary
factors, including inorganic compounds and multiple proteins (Kleinberg & Jenkins
1964, Abelson & Mandel 1981, Jensen 1986, Ericson & Mäkinen 1986, Mandel 1987,
1989, Mäkinen 1989, Tenovuo 1989, van Houte 1994, Edgar & Higham 1995, Hall et al.
1997). The importance of saliva for dental health is demonstrated by the rampant caries
seen in patients with grave salivary hypofunction (Dreizen & Brown 1976, Birkhed &
Heintze 1989, Mandel 1989, Peeters et al. 1998). Saliva is involved in the clearance of
food debris, detached epithelial cells and microbes. It also provides inorganic ions for
the neutralization of acid microbial metabolic products and for remineralization of the
enamel, and brings various defence mechanisms, including bicarbonate ions, leukocytes,
sIgA, agglutinating proteins and a number of enzymes, to the actual sites of microbial
adherence and growth on the tooth surfaces (Edgar 1976, Boackle & Suddick 1980,
Cohen & Levine 1989, Hay & Moreno 1989, Lamkin & Oppenheim 1993, Johnsson et
al. 1993, Lagerlöf & Oliveby 1994, Edgar et al. 1994, Wolinsky 1994). Despite
numerous clinical studies, no distinct correlations have been found between the
concentrations of any particular salivary proteins, alone or in combination, and the
prevalence of caries (Gråhn et al. 1988, Rudney 1995, Kirstilä et al. 1998, Dodds et al.
1997). (Tenovuo 1989.)
2.4. The enamel pellicle
The enamel pellicle is a thin layer of proteins covering the enamel, the formation of
which is initiated by the adsorption of specific salivary proteins to the hydroxyapatite
surface (Kousvelari et al. 1980, Al-Hashimi & Levine 1989, Cohen & Levine 1989,
Lamkin & Oppenheim 1993, Lamkin et al. 1996). This adsorption is assumed to be
dependent on the chemical characteristics of the surface as well as the properties of the
particular proteins (Moreno et al. 1984, Al- Hashimi & Levine 1989, Rykke et al. 1990,
28
Jensen et al. 1992, Raj et al. 1992, Johnsson et al. 1993, Lamkin & Oppenheim 1993,
Skjorland et al. 1995, Lamkin et al. 1996). The accumulation of additional organic
material on the initially formed basal layer is thought to constitute the subsequent phase
of pellicle development (Lamkin et al. 1996, Hannig 1997).
The enamel pellicle evidently prevents demineralization of the surface
hydroxyapatite, increasing its acid resistance, although the mechanisms responsible for
this effect have remained elusive (Zahradnik et al. 1976, 1977, 1978, Kousvelari et al.
1980, Meurman & Frank 1991, Featherstone et al. 1993, Wolinsky 1994). It is also
thought to prevent calculus formation on the dental surfaces by controlling the
precipitation of calcium phosphate from supersaturated saliva (Hay & Moreno 1989).
3. Aims of the research
The overall aim of this research was to elucidate the physiological role of salivary CA
VI in the oral cavity. The specific goals were:
− to examine the possible correlations between the CA VI concentration and some
basic salivary characteristics,
− to examine the influence of smoking habits on salivary CA VI concentration,
− to investigate the possible correlation between salivary CA VI concentration and
caries experience,
− to determine whether CA VI binds to the enamel pellicle, and
− to find out whether CA VI is transferred into the blood circulation.
4. Materials and methods
4.1. Subjects (I, III)
The voluntary subjects were selected at a health examination for conscripts at
Parolannummi Garrison Hospital in January 1996. All the subjects were in good health
and were not taking any medication. A conscious effort was made to select cases in
which the subjects had adequate oral hygiene with average or poor dental status, or poor
oral hygiene with comparatively good dental status. The final group of subjects consisted
of 209 healthy men ranging from 18 to 24 years of age (mean 19.8 years).
Smoking habits were ascertained on a 5-grade scale: non-smoker, less than 5
cigarettes per day, 5-9 cigarettes per day, 10-20 cigarettes per day, and more than 20
cigarettes per day.
The research was approved by the ethical committee of the Finnish Defence Forces,
and informed consent was obtained from each subject.
4.2. Clinical examination (III)
The clinical examinations were carried out by the author. The cariological status of each
subject was recorded in terms of the DMFT index (number of decayed, missing or filled
teeth: Klein et al. 1938, Burt 1981). All the permanent teeth were examined. Only
cavitated lesions were included in the D component. Teeth filled because of fractures,
unerupted teeth and teeth extracted for orthodontic reasons were excluded from the
DMFT index. The robust DMFT index was considered appropriate for the measurement
of total lifetime caries experience in this cross-sectional study. It is relatively insensitive
to the influence of behavioural differences in seeking dental treatment and to possible
differences in the caries therapy received. Periodontal status was recorded in terms of the
Community Periodontal Index (CPI), in which each dental sextant (dd. 18-14, 13-23, 24-
28, 38-34, 33-43 and 44-48) is examined for gingival bleeding and calculus (Ainamo et
al. 1982). The code CPI = 0 indicates a healthy periodontium, and CPI = 1 and CPI = 2
indicate gingival bleeding and calculus, respectively, in at least one sextant.
31
4.3. Collection of saliva samples (I, III)
The saliva samples were collected from groups of 15-20 volunteers per morning. The
subjects did not eat or smoke during the 8-h period before sampling. The first sample
was collected 30 min after awakening, between 6.00 and 6.30. Saliva secretion was
stimulated by chewing paraffin wax for seven minutes. During the first two minutes the
saliva was swallowed, and the rest was collected by spitting into 10-ml tubes containing
200 µl of 0.2-M benzamidine in dH2O to prevent proteolysis, and subjected immediately
to microbial tests and measurement of the secretion rate, pH and buffering capacity.
After having breakfast, the subjects returned to the Garrison Hospital, and another saliva
sample was collected in a similar fashion as the first, between 7.00 and 7.30. These
samples were subjected to the same tests as the first ones, except for the microbial tests.
4.4. Microbial tests (III)
The Dentocult LB test was used to obtain a salivary lactobacillus count. A high LB count
reflects sugar in the diet and acidic, cariogenic conditions in the mouth (Larmas 1975,
1985, Bratthall & Carlsson 1989). The Dentocult SM Strip mutans test was used to
assess mutans streptococci in the saliva (Jensen & Bratthall 1989, Bratthall & Carlsson
1989), providing an estimate of the intensity of cariogenic infection on the dental
surfaces (Clarke 1924, Fitzgerald & Keyes 1960, van Houte & Green 1974, Schaeken et
al. 1987, Lindquist et al. 1989). The LB and SM tests were performed and the values
expressed according to the manufacturer’s instructions (Orion Diagnostica, Espoo,
Finland).
4.5. Tests for salivary pH, buffering capacity and secretion rate (I,
III)
Immediately after sampling, salivary secretion rate in ml/min was calculated, and pH
was measured using a pH meter (Schott model CG 837). Buffering capacity was
determined by a slight modification of the method of Ericsson (1959). In brief, 1 ml of
fresh saliva was added to a tube containing 3 ml of 5-mM HCl, shaken, and measured for
pH. After these tests, the saliva samples were frozen and stored at -20°C until the
enzyme assays were performed.
4.6. Tissue samples and collection of the enamel pellicle (IV)
To investigate the presence of CA VI in the enamel pellicle, permanent non-carious
human teeth were obtained from the Department of Oral Surgery, University of Oulu,
and Parolannummi Garrison Hospital. These had been extracted on orthodontic
32
indications and stored in sterile saline at 4°C until used for the experiments. The teeth
were cut using the Exakt Standard Cutting Grinding System (Exakt, Norderstedt,
Germany). Prior to extraction, the teeth examined for in vivo-formed enamel pellicle had
been polished with pumice and the pellicle allowed to reform for 2 h, during which time
no ingestion of food or liquids (except water) had been permitted.
Samples of the enamel pellicle were collected from two volunteers into Eppendorf
tubes containing 300 µl of 0.01-M EDTA, pH 7.5, and 3 µl of 0.2-M benzamidine. The
samples were stored at -80°C until used. Paraffin-stimulated saliva samples for
immunoblotting were collected from the same two volunteers, centrifuged at 16,000 × g
for 10 min at room temperature and the supernatants stored at -80°C until used for
electrophoresis.
4.7. Collection of serum (II)
To find out whether CA VI is transferred into the circulation, saliva and serum samples
were collected from four healthy male volunteers at 3-h intervals throughout a 24-h
period. The protocol involved meals at 9.00, 12.30 and 18.30, and sleep from 0.10 to
9.00. During sleep, the subjects were woken up only for collection of the samples, still at
3-h intervals. The saliva samples were collected, frozen and stored as described in
section 4.3. After collecting the saliva samples, 1-ml blood samples were obtained
through an intravenous catheter from the cephalic vein in the cubital fossa. These
samples were centrifuged at 2000 × g for 20 min, after which the 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).
4.8. Purification of CA VI. Antisera and immunoreagents (I-IV)
The affinity chromatography purification of human CA VI and production of a rabbit
anti-CA VI serum are described by Parkkila S et al. (1990, 1991).
Reagents for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-
PAGE) and transblotting were obtained from Bio-Rad Laboratories (Richmond, CA).
For the immunoblots of saliva and pellicle proteins (IV), polyclonal rabbit antibody to
human salivary α-amylase, purified human salivary α-amylase, biotin-conjugated swine
anti-rabbit IgG and peroxidase-conjugated streptavidin were purchased from Sigma
Chemical Company (St. Louis, MO), and peroxidase-conjugated donkey anti-rabbit IgG
from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Purified human
IgG was obtained from the Sigma Chemical Company (St. Louis, MO) (II).
33
4.9. SDS-PAGE and Western blots (II, IV)
To confirm the presence of CA VI in the in vivo-formed enamel pellicle (IV), samples of
saliva and pellicle protein were subjected to SDS-PAGE under non-reducing conditions
using 4 % concentrating and 10 % separating polyacrylamide gels (Laemmli 1970).
Parallel experiments were performed for α-amylase, as a positive control. The separated
proteins were transferred to nitrocellulose sheets as described earlier (Parkkila S et al.
1990), and these were briefly washed with 0.1 % Tween-20 in phosphate-buffered saline
(PBST) and blocked with 20 % goat colostrum in PBST for 16 h at 4°C. After washing
in PBST, the sheets were incubated with the primary antibody for 3 h at room
temperature. Anti-CA VI serum and normal rabbit serum (NRS) were both diluted
1:2500 in 3 % bovine serum albumin (BSA) in PBST, and antibody to salivary α–
amylase 1:15,000. The sheets were then washed extensively with PBST, treated again
with 20 % goat colostrum in PBST for 1 h at room temperature, washed briefly with
PBST, and incubated with peroxidase-conjugated anti-rabbit IgG diluted 1:20,000 for 1
h at room temperature. After extensive washing of the sheets in PBST, the bands were
visualized by the enhanced chemiluminescence (ECL) method (Amersham International
plc, Aylesbury, UK) according to the manufacturer’s instructions.
To confirm the presence of CA VI in serum (II), aliquots of 5 µl of saliva, 0.5 µl of
serum and 0.5 µg of purified human IgG were subjected to SDS-PAGE under non-
reducing or reducing conditions. The electrophoreses were performed according to
Laemmli (1970), using 4 % concentrating and 9 % separating polyacrylamide gels. The
proteins were electrophoretically transferred from the gel onto a polyvinylidene fluoride
(PVDF) membrane (Millipore Corporation, Bedford, MA) at a constant voltage of 30 V
for 1.5 h in a Xcell II Mini-Cell apparatus (Novex, San Diego, CA). After the
transblotting the PVDF membranes were stained with Coomassie brilliant blue R-250
and the lanes containing the molecular weight standards were cut out. The sample lanes
were destained in methanol, rinsed in TBST buffer containing 10-mM Tris-HCl, pH 7.5,
150-mM NaCl and 0.05 % Tween 20 and incubated for 30 min with 1:10 diluted cow
colostrum in TBST buffer. The sheets were then 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 using a chemiluminescent reagent (Leong
& Fox 1988).
4.10. Isolation of the immunoglobulin-CA VI complex from serum
(II)
A 1-ml human blood sample from the cephalic vein was centrifuged at 4000 × g for 10
min at 4°C and 5 µl of the serum was added to 45 µl of phosphate-buffered saline (PBS)
and mixed for 2 h at 4°C with 50 µl of protein A immobilized on Sepharose (Sigma).
After centrifugation at 15,000 × g for 2 min, the supernatant was saved as an unbound
fraction and the protein A-Sepharose conjugate was washed twice with 400 µl of PBS.
34
Finally, the bound proteins were eluted with 400 µl of a 0.1-M glycine-HCl solution, pH
2.5, and the eluted material was mixed appropriately with 1-M Tris base to neutralize the
pH of the solution. The eluted proteins were concentrated in Centricon P-10 tubes
(Amicon, Beverly, MA) to a volume of 100 µl, and analyzed by SDS-PAGE followed by
Western blotting.
4.11. Immunohistochemistry (IV)
To demonstrate CA VI in the in vivo-formed enamel pellicle, the halves of the crowns of
the extracted teeth were immunostained by a biotin-streptavidin complex method as
follows (Sternberger 1970, Guesdon et al. 1979):
1. Rinsing in 0.1-M PBS.
2. Pre-treatment with goat colostrum for 40 min to block the non-specific binding of
the first antibody, and rinsing in PBS.
3. Incubation for 1 h with the primary rabbit antibody or NRS, both diluted 1:70 in 1 %
BSA-PBS.
4. Treatment with goat colostrum for 40 min and rinsing in PBS.
5. Incubation for 1 h with biotin-conjugated swine anti-rabbit IgG diluted 1:300 in 1 %
BSA-PBS.
6. Treatment with goat colostrum for 5 min and rinsing in PBS.
7. Incubation for 30 min with peroxidase-conjugated streptavidin diluted 1:600 in PBS.
8. Incubation for 5 min in 3,3’-diaminobenzidine tetrahydrochloride (DAB) (9 mg
DAB in 15 ml of PBS plus 10 µl of 30 % H2O2).
The halves of the crowns were washed three times for 10 min in PBS after steps 3, 5
and 7. The entire procedure was carried out at room temperature.
The immunostaining was repeated using samples of the in vitro-formed enamel
pellicle. The halves of the crowns were polished with pumice and incubated in paraffin-
stimulated saliva (300 µl / crown half), in purified human CA VI (3 or 9 µg/ml in PBS),
or in PBS in a rotary shaker for 2 h at room temperature. The samples were
immunostained for CA VI as described above.
To compare the results with those for amylase which is known to be present in the
enamel pellicle, parallel samples were immunostained using α-amylase antibody in
conjunction with the biotin-streptavidin complex method as described above.
4.12. Demonstration of CA activity on the enamel surface (IV)
To examine the activity of the enamel-bound CA VI, the halves of the crowns of the
extracted teeth were stained for CA activity by a slight modification of the histochemical
method of Hansson (1967). The samples were polished with pumice and incubated either
in paraffin-stimulated saliva or with purified human CA VI (30 µg/ml in PBS) in a rotary
shaker for 2 h at room temperature. The staining was performed as follows:
35
1. Incubation for 1 min with a washing buffer solution containing 9 ml of 67-mM
KH2PO4 and 1 ml of 67-mM Na2HPO4 in one litre of physiological saline.
2. Rinsing for 8 min with Hansson’s medium, produced by adding a freshly prepared
solution of 0.75 g of NaHCO3 in 40 ml of dH2O to a solution containing 1 ml of
0.2-M CoSO4, 6 ml of 0.5-M H2SO4 and 10 ml of 67-mM KH2PO4. The samples
were repeatedly flushed with the medium to promote air contact, and carbon dioxide
was blown over the surface of the medium for 10 min prior to the addition of the
NaHCO3 solution and during the flushing.
3. Incubation for 1 min with the washing buffer.
4. Incubation for 5 s with a freshly prepared solution of 1 % (NH4)2S in dH2O.
5. Incubation for 1 min with the washing buffer.
Control experiments were performed using samples incubated in PBS in the absence
of CA VI and saliva and stained as above, and using samples incubated in saliva or CA
VI and stained with Hansson’s medium in the presence of the CA inhibitor sodium
acetazolamide (Diamox, Lederle Parenterals, Carolina, Puerto Rico, USA) at a final
concentration of 50 mM.
4.13. Antigen labelling and fluoroimmunoassay procedure for CA VI
(I-III)
After measuring the pH and buffering capacity, the samples were frozen, stored at
-20 °C and thawed just before the measurement of CA VI concentration and amylase
activity. A 1-ml aliquot from each sample was centrifuged at 15,000 × g for 10 min at
4 °C and the supernatant assayed for CA VI concentration and amylase activity. Both
enzyme assays were performed without knowing the clinical data on the subjects.
Purified CA VI (Parkkila S et al. 1990) was labelled with 0.12 mg of Eu labelling
reagent and the fluoroimmunoassay procedure was performed as described earlier
(Parkkila S et al. 1993b). The fluorescence was measured with a 1234 DELFIA research
fluorometer (Wallac, Turku, Finland). In the series discussed in papers I and III, the
mean intra-assay coefficient of variation (CV) was 6.2 %, and the inter-assay CV,
determined in 17 assays, was 10.3 %. In the paper II the mean intra-assay CV was 4.6 %,
and the inter-assay CV between 3 assays, was 10.1 %. This assay based on determining
the CA VI concentration was considered to be superior to the previously used CA
activity assay (Krebs & Roughton 1948, Gloster 1955, Szabó 1974), which does not
distinguish CA VI from CA II derived from contaminating red blood cells.
4.14. Determination of amylase activity (I, III)
The saliva samples were assayed for amylase activity using the α-amylase EPS-test
(Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s
instructions. The measurements were performed using a BM/Hitachi 911 Automatic
36
Analyzer (Naka Works, Hitachi Ltd., Ibaraki-Ken, Japan), and the values were expressed
as U/l. The inter-assay CV determined in 10 assays was 1.4 %.
4.15. Statistical methods (I, III)
The statistical evaluations were performed by linear regression analysis, one-way
analysis of variance and non-parametric analysis of variance. Statistically significant
differences were accepted at the p < 0.05 level. All reported p-values were calculated
using the t-test. The SAS package (Statistical Analysis System, Cary, NC) was used to
perform the statistical analyses.
5. Results
5.1. Correlation of salivary CA VI concentration with the basic
characteristics
of human whole saliva (I)
The mean (± SEM) rate of saliva secretion, determined for 209 young, healthy men, was
1.8 ± 0.0 ml/min (range 0.6-4.4 ml/min), the mean pH of the samples 7.3 ± 0.0 (6.1-7.7)
and the mean buffering capacity 4.1 ± 0.1 (2.7-6.3). The secretion rates correlated
positively with pH (r = 0.38, p < 0.001) and buffering capacity (r = 0.43, p < 0.001), and
a close correlation was found between salivary pH and buffering capacity (r = 0.67, p <
0.001).
CA VI concentrations in the saliva samples collected before and after breakfast were
in the range 0.1-10.0 mg/l and 0.6-16.0 mg/l, respectively, the mean concentrations
being 1.8 ± 0.1 mg/l and 5.0 ± 0.2 mg/l. Since a close positive correlation was found
between the enzyme levels in the two successive samples (r = 0.71, p < 0.001), only the
second samples were subjected to further analyses. A positive correlation was observed
between the CA VI concentrations and salivary secretion rates (r = 0.20, p = 0.003),
whereas no significant correlation was found between CA VI levels and pH or buffering
capacity.
The most abundant enzyme in human whole saliva, α-amylase (Mason & Chisholm
1975), was measured as a control enzyme, and its activity levels in the second saliva
samples were also assayed and correlated with the basic salivary characteristics and CA
VI levels. The mean amylase activity was 225 ± 10 × 1000 U/l (1-818 × 1000 U/l). No
significant correlation was found between amylase activity and salivary secretion rate,
pH, or buffering capacity. On the contrary, amylase activity did correlate positively with
CA VI concentrations (r = 0.46, p < 0.001).
38
5.2. Effect of smoking on salivary characteristics (I)
According to their own declarations, 97 subjects did not smoke, 19 smoked less than 5
cigarettes per day, 29 smoked 5-9 cigarettes per day, 61 smoked 10-20 cigarettes per
day, and 3 smoked more than 20 cigarettes per day. Since no significant differences
emerged among the subgroups of the subjects, only smokers versus non-smokers were
evaluated. The smokers (n = 112) had a slightly lower average salivary pH, 7.2 ± 0.0
(6.1-7.6) versus 7.3 ± 0.0 (6.3-7.7), p = 0.007, and buffering capacity, 3.9 ± 0.1 (2.7-6.1)
versus 4.2 ± 0.1 (2.9-6.3), p = 0.035, than the non-smokers (n = 97), whereas no
significant differences were seen in mean salivary secretion rate, CA VI and amylase
levels.
5.3. CA VI in human serum (II)
The presence of CA VI in serum was demonstrated using blood samples collected from
four subjects at 3-h intervals throughout the 24-h period. The serum concentrations of
CA VI were measured using a time-resolved immunofluorometric assay (Parkkila S et
al. 1993b), and the values were compared with the CA VI concentrations measured in
the corresponding saliva samples. The serum was found to contain detectable amounts of
CA VI, although the enzyme concentrations were only about 1/22 of those of the saliva
samples. After breakfast, the mean (± SEM) concentration of CA VI was 0.20 ± 0.02
mg/l in serum and 4.29 ± 0.57 mg/l in saliva. Although salivary CA VI concentrations
varied greatly among the subjects, they were very low during the sleeping period in all
cases and increased rapidly in the morning after waking up and having breakfast. Much
intra-individual variation was seen in the serum enzyme levels, although the circadian
rhythm was less evident than in the saliva.
The presence of CA VI in the serum was confirmed by Western blotting. Under non-
reducing conditions, anti-CAVI antibody detected a high molecular weight band of
greater than 130 kDa in all the serum samples studied, suggesting that CA VI is
associated with some other serum protein(s). After reduction, two major polypeptide
bands of 51 kDa and 42 kDa were identified in the serum samples, the latter band being
antibody-specific and corresponding to the molecular weight of the monomeric CA VI
(Murakami & Sly 1987, Aldred et al. 1991). The 51-kDa band appeared to be a non-
specific IgG heavy chain reaction, since NRS identified a polypeptide of the same
molecular weight in a Western blot of purified human IgG. By contrast, the anti-CA VI
antibody identified a strong 42-kDa polypeptide band in Western blots of the saliva
samples under non-reducing conditions. In addition, the minor, differentially
deglycosylated forms of CA VI were occasionally seen in the saliva blots, as in earlier
studies (Murakami & Sly 1987, Parkkila S et al. 1990).
The results of the Western blots implied that CA VI may be associated with IgG in
serum. To confirm this association, a protein A-Sepharose conjugate was used to isolate
the IgG-CA VI complex from the serum. After electrophoretic transfer, Coomassie
brilliant blue R-250 staining of the PVDF membrane showed a strong 51-kDa
polypeptide band corresponding to the IgG heavy chain. In the same bound fraction,
39
anti-CA VI serum identified 42-kDa and 36-kDa polypeptide bands, corresponding to
the monomeric and deglycosylated forms of CA VI, respectively (Murakami & Sly
1987). These results indicate that CA VI is associated with IgG in serum, whereas it
mainly occurs as a monomeric enzyme in saliva.
5.4. Association between salivary CA VI concentration and dental
caries (III)
5.4.1. Clinical and microbial findings
The DMFT index was determined as an indication of the caries experience of each
subject. The mean index was 7.3 ± 0.4 (0-28) and the median 7.0. A healthy
periodontium (CPI = 0) was found in 106 subjects, and gingival bleeding (CPI = 1), a
clinical indicator of poor oral hygiene, in 50. Calculus (CPI = 2) was found in 53
subjects.
The counts of salivary lactobacilli, indicating cariogenicity of the diet, and the counts
of mutans streptococci, reflecting the intensity of cariogenic infection, were determined
using commercially available tests. Both the mean LB and SM counts were between 105
and 106 cfu/ml. The distribution of frequencies for LB was < 104 cfu/ml 35.4%,
104 – 105 cfu/ml 23.0%, 105 – 106 cfu/ml 26.3%, and > 106 cfu/ml 15.3%, and that
for SM 24.4%, 11.0%, 20.1% and 44.5%, respectively. No significant correlations were
found between salivary CA VI concentrations and LB or SM counts. As expected, the
subjects with high microbial counts had higher caries experience. The LB counts
displayed a higher correlation in linear regression analysis than the SM counts, the r
values being 0.50 and 0.32, respectively (p < 0.001 in both cases).
5.4.2. Correlations between clinical findings and analytical salivary
characteristics
The correlations between the DMFT index and salivary characteristics are shown in
Table 3. The data obtained from the immunofluorometric assay supported the initial
hypothesis that CA VI may have a protective effect against dental caries, since the
DMFT index and CA VI concentration showed a statistically significant negative
correlation (r = -0.22, p = 0.001), which was increased in the group of subjects with CPI
= 1 (r = -0.43, p = 0.002). In contrast, no statistically significant correlation was found
between the DMFT index and amylase activity.
40
Table 3. Correlations between salivary variables and DMFT indices in linear regression
analysis.
Group of subjects
All, n = 209
CPI = 0, n = 103
CPI = 1, n = 50
Variable
r
p
r
p
r
p
CA VI concentration
-0.22
0.001
-0.11
0.288
-0.43
0.002
Buffering capacity
-0.24 < 0.001
-0.23
0.017
-0.29
0.039
pH
-0.21
0.002
-0.17
0.083
-0.25
0.081
Rate of saliva secretion
-0.09
0.215
-0.01
0.897
-0.21
0.136
Amylase activity
-0.11
0.120
-0.03
0.791
-0.16
0.272
To widen the analysis, the samples collected after breakfast were classified into three
CA VI concentration categories: the 66 subjects with the lowest CA VI levels (< 3.0
mg/l), the 83 subjects with moderate levels (3.0-6.0 mg/l), and the 60 subjects with the
highest levels (> 6.0 mg/l). The mean DMFT values in these categories are shown in Fig.
1a. To compare the results with those for another salivary enzyme, amylase activity was
determined in the same saliva samples, which were classified correspondingly into three
categories: 70 subjects with amylase activities of < 150 × 1000 U/l, 70 with activities of
150-250 × 1000 U/l, and 69 with activities of > 250 × 1000 U/l. The mean DMFT
values in these categories are shown in Fig. 1b.
Fig. 1. DMFT values (mean ± SEM) in categories classified according to (a) CA VI
concentrations, p = 0.002; (b) amylase activity levels, p = 0.5.
0
1
2
3
4
5
6
7
8
9
10
<3
3-6
>6
CA VI concentration (mg/l)
DM
F
T
1a
n=6
6
n=8
3
n=6
0
0
1
2
3
4
5
6
7
8
9
10
<150
150 – 250
>250
Amylase activity (x 1000 U/l)
DM
F
T
b
n=7
0
n=7
0
n=6
9
41
Two groups of samples were taken for further analyses, the 52 with low LB and SM
counts (both < 105 cfu/ml) and the 65 with high counts (both > 105 cfu/ml). Statistically
significant differences in the mean DMFT indices among the subjects grouped according
to their CA VI levels were seen in both groups (low microbial counts, p = 0.018, and
high microbial counts, p = 0.011). Subjects with low microbial counts and low CA VI
concentrations displayed a mean DMFT index value (7.4 ± 1.8) close to that for the
subjects with high microbial counts and high CA VI concentrations (8.5 ± 1.0). Without
any classification according to the CA VI concentration, the mean DMFT index values
in the groups of low and high microbial counts were 4.2 ± 0.7 and 11.4 ± 0.6,
respectively.
To extend the analysis, two groups of subjects were distinguished on the basis of the
CPI values: the 103 subjects with a healthy periodontium (CPI = 0), and the 50
individuals who had gingival bleeding but no calculus (CPI = 1). When the samples in
these groups were classified according to CA VI concentration, as before, significant
differences in the mean DMFT indices were found only in the second group, suggesting
a greater protective action of CA VI in subjects with neglected oral hygiene (Fig. 2). In
this group (CPI = 1) linear regression analysis revealed a close negative correlation
between salivary CA VI concentration and DMFT index, whereas no correlation was
found in the group with CPI = 0 (Table 3).
Fig. 2. DMFT values (mean ± SEM) in categories classified according to CA VI
concentrations. (a) Healthy periodontium (CPI = 0), p = 0.6, n = 103; (b) Gingival
bleeding, no calculus (CPI = 1), p = 0.008, n = 50.
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
<3
3-6
>6
CA VI concentration (mg/l)
DM
F
T
2a
n=
3
4
n=
3
7
n=
3
2
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
<3
3-6
>6
CA VI concentration (mg/l)
DM
F
T
b
n=
1
6
n=
2
0
n=
1
4
42
5.5. CA VI in the enamel pellicle (IV)
5.5.1. Presence of CA VI in the enamel pellicle formed in vivo
To determine whether CA VI is naturally associated with the pellicle, the halves of the
crowns of the extracted teeth having enamel pellicle that had formed in vivo were
immunostained for CA VI. Parallel samples were immunostained for salivary α-amylase
as a positive control, since amylase is known to be present in the pellicle (Al-Hashimi &
Levine 1989). Both enzymes showed virtually identical staining patterns on the dental
surfaces, indicating that CA VI is present in the enamel pellicle in the same manner as α–
amylase. Control staining using NRS showed no positive reaction.
Immunoblotting of the pellicle proteins confirmed the presence of CA VI in the
enamel pellicle formed in vivo. Anti-CA VI antibody recognized the major 39-kDa
polypeptide band of CA VI in the pellicle proteins, salivary proteins and purified CA VI
preparation. The minor bands of CA VI, 37 kDa and 34 kDa, which represent the
differentially glycosylated forms of the enzyme (Murakami & Sly 1987), were seen in
the immunoblot of purified CA VI. Antibody to salivary α-amylase recognized a doublet
57/60-kDa polypeptide band in the pellicle proteins, salivary proteins and purified
salivary α-amylase preparations.
5.5.2. Binding of CA VI to enamel in vitro
To examine the ability of CA VI to bind to the enamel surface, polished crown quarters
were incubated with purified CA VI in vitro and immunostained using anti-CA VI
antibody. A specific CA VI immunoreaction was observed on the dental surface,
suggesting that the enzyme can indeed bind to the enamel. Two concentrations of
purified CA VI were used, 3 mg/l and 9 mg/l, and the intensity of the immunostaining
was seen to be dependent on the CA VI concentration during the incubation. Salivary α–
amylase again served as a positive control protein, giving similar staining results. No
immunoreaction was seen in the samples incubated with CA VI or salivary α-amylase
and immunostained using NRS, nor in the samples incubated in PBS and immunostained
using CA VI or salivary α-amylase antibodies.
To confirm that salivary CA VI can bind to the enamel in vitro, polished crown
halves were incubated in whole saliva, washed to remove all unbound proteins, and
immunostained using anti-CA VI serum. A positive signal for CA VI was detected on
the enamel surface. Anti-α-amylase antibody served as a positive control, as before.
Polished crown halves incubated in whole saliva and immunostained using NRS showed
no positive immunoreaction.
43
5.5.3. CA activity on the enamel surface
To determine whether CA VI bound to enamel retains its enzymatic activity, polished
crown halves were incubated in vitro either in saliva or with purified CA VI. Both
specimens showed positive histochemical staining for CA activity. No staining was
detected in the control samples incubated in PBS. When acetazolamide, a CA inhibitor,
was added to Hansson’s medium, a marked decrease was observed in the intensity of the
staining reaction.
6. Discussion
The carbonic anhydrases form a family of zinc metalloenzymes that participate in
controlling the ion, fluid and acid base balance in various organs. The only known
secreted isoenzyme is CA VI, several milligrams of which are secreted daily and pass
into the gastrointestinal canal (Parkkila S et al. 1993b). Its physiological role has
remained unclear, however. To explore the role of CA VI in the oral cavity, we
determined its concentrations in human whole saliva and correlated them with basic
salivary characteristics (I). A highly sensitive and specific time-resolved
immunofluorometric assay (Parkkila S et al. 1993b) was used to determine the salivary
CA VI concentrations. This method was considered to be superior to the previously used
CA activity assay (Szabó 1974), which does not distinguish CA VI activity from the CA
II activity present in any contaminating red blood cells. Because the CA VI
concentrations in saliva follow a circadian periodicity and are under the control of the
autonomic nervous system (Parkkila S et al. 1995), it is of utmost importance that
quantitative studies should be carefully standardized regarding the sleeping period and
meal times, and that the time of collecting the saliva samples is fixed and properly
related to the meals. The appropriate time for sample collection appears to be in the
morning, immediately after breakfast. Since the investigation reported in papers I and III
was carried out among young male soldiers, the daily rhythm and other factors that
might influence CA VI secretion could be well standardized. The soldiers had spent 4
weeks in the garrison prior to collection of the saliva samples and had thus adapted to the
regular daily rhythm. In women, the phase of the menstrual cycle may cause variation in
the composition of saliva, as the female sex steroids are known to influence salivary pH
and buffering capacity during pregnancy (Laine et al. 1988).
The results showed that CA VI levels increased markedly after the first sampling (I),
suggesting that reflexes related to the meal had enhanced enzyme secretion. Despite the
careful standardization, salivary CA VI concentrations showed a high inter-individual
variation, which probably reflects differences in enzyme expression. This conclusion is
supported by the finding of a significant positive correlation between the enzyme
concentrations in the first and second samples. These differences cannot be explained by
proteolysis of the enzyme, as a protease inhibitor was used and all the samples were
collected and handled identically. In addition, CA VI seems to be a very stable enzyme
45
which can withstand the harsh conditions in the alimentary canal, and non-degraded
enzyme can be detected even in the gastric juice (Parkkila S et al. 1997).
A significant positive correlation was observed between salivary amylase activity
levels and CA VI concentrations (I). Combined with an earlier report that salivary
amylase activity levels and CA VI concentrations follow a parallel circadian periodicity
(Parkkila S et al. 1995), this finding suggests that both enzymes are secreted via similar
mechanisms and may possibly be present in the same secretory granules. Since the role
of the autonomic nervous system in amylase secretion is well established, it is
conceivable that the parasympathetic/sympathetic pathways also control CA VI secretion
(Asking & Gjörstrup 1980, Olsen et al. 1988, Nederfors & Dahlöf 1992, Parkkila S et al.
1995).
To find out whether CA VI is transferred to the circulation, we collected serum and
saliva samples from four healthy male volunteers throughout the 24-h period (II). Small
amounts of CA VI were detected in serum using the previously mentioned
fluoroimmunoassay. In view of its high expression and secretion in the salivary glands,
CA VI may be leaked from the salivary glands or absorbed from the alimentary canal
into the bloodstream. By analogy to the salivary CA VI concentrations, serum levels also
showed a marked intra-individual variation during the 24-h period. These changes in
serum CA VI concentrations may be linked both with the periodicity in expression and
with rapid clearance from the bloodstream, the latter being supported by the recent
findings that CA VI contains unique Asn-linked oligosaccharides terminating in
GalNAc-4-SO4, which is known to enhance the clearance of lutropin from the circulation
via a specific receptor in the liver (Fiete et al. 1991, Hooper et al. 1995).
The presence of CA VI in serum was confirmed using a sensitive Western blotting
method (II). Under non-reducing conditions, the anti-CA VI antibody identified only a
single high molecular weight band of greater than 130 kDa, but after reduction, the high
molecular weight polypeptide was reduced to two polypeptides of 42 and 51 kDa,
corresponding to monomeric CA VI and the immunoreactive IgG heavy chain,
respectively. The Western blotting results therefore suggest that the protein associated
with CA VI could be IgG. This observation was further confirmed by isolating the IgG-
CA VI complex from the serum using 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 which do
not express CA VI.
Laboratory testing for serum α-amylase is commonly used in the diagnosis of
diseases of the pancreas and in the investigation of pancreatic function (Lott et al. 1976,
Salt & Schenker 1976). As most amylase assay methods are based on determination of
the enzyme activity and cannot differentiate P-type and S-type amylase isoenzymes,
more specific methods have been developed (Rauscher & Gerber 1989). Since both CA
VI and S-type amylase are produced in the serous elements of the salivary glands and
probably share the same secretory pathways, it will be a challenging prospect to study
whether CA VI measurements can help to assess the contribution of the salivary glands
to elevated amylase levels. The present results (II) provide a basis for further
investigations to determine serum CA VI concentrations in patients with different
salivary gland disorders.
46
In the study of salivary CA VI concentrations and their correlation with basic
salivary characteristics (I), over half of the subjects examined were smokers (112 out of
209). Our findings were in accordance with earlier reports that there is no significant
difference in salivary secretion rate between non-smokers and smokers, but that smokers
have markedly decreased salivary pH and buffering capacity (Heintze 1984, Parvinen
1984, Wikner & Söder 1995). Previous research has shown that smoking reduces the
secretion of epidermal growth factor into the saliva (Jones et al. 1992). We did not find
that smoking influenced the secretion of either CA VI or amylase. This confirms an
earlier observation that smoking has no effect on amylase activity levels in saliva
(Nagaya & Okuno 1993), although the present data do not justify any conclusions
regarding the long-term effects of smoking on salivary enzyme secretion, due to the
young age of the subjects.
The observation, that no correlation exists between salivary CA VI concentration and
pH or buffering capacity (I), suggests that CA VI is not involved in the regulation of the
actual salivary pH. A weak positive correlation was observed between salivary secretion
rate and CA VI concentration, but this is not specific to CA VI since the overall protein
content of saliva is known to increase with an increased salivary secretion rate (Shannon
1973). The correlation between salivary secretion rates and amylase activity levels did
not reach statistical significance, however, suggesting that CA VI concentration and
salivary secretion are more specifically interrelated.
The large inter-individual variation in salivary CA VI levels is an important finding
from the clinical point of view. In addition, evidence has been presented to show that the
relative levels of many salivary constituents remain unchanged over time (Rudney et al.
1985, Wu et al. 1993). To determine the possible clinical effects of CA VI in the oral
cavity, we compared its concentrations in whole saliva with the dental status of the
subjects (III). The results were in accordance with the well-established notion that
salivary pH and buffering capacity are negatively correlated with the subjects’ caries
experience as measured using the DMFT index and counts of salivary lactobacilli and
mutans streptococci positively correlated with caries experience. Our data were also in
line with previous observations of no correlation between the salivary secretion rate and
the DMFT index (Birkhed & Heintze 1989, Russell et al. 1990, 1991). A novel and
interesting finding was that the salivary CA VI concentration exhibits a negative
correlation with the DMFT index. This is of special interest as previous research has
revealed no distinct correlation between the concentration of any particular salivary
protein and the prevalence or incidence of caries (Rudney 1995, Dodds et al. 1997,
Kirstilä et al. 1998). In the present research we found no correlation here between
salivary amylase activity and the DMFT index, suggesting that CA VI plays a specific
role in the natural defence systems against dental caries. Interestingly, this protective
effect emerged mainly in subjects with gingival bleeding without dental calculus (CPI =
1), the group that can be considered to have neglected their oral hygiene. Figure 2a
demonstrated that adequate oral hygiene can compensate for a low salivary CA VI
concentration, and conversely, poor oral hygiene does not inevitably lead to high caries
experience provided that the salivary CA VI concentration is high (Fig. 2b). Poor oral
hygiene combined with a low salivary CA VI concentration can result in extensive caries
experience (Fig. 2b).
47
The rate of dissolution of dental hard tissues as a consequence of caries is dependent
on the extent and duration of the decrease in pH on the dental surfaces. Salivary pH and
buffering capacity are known to be central factors protecting the teeth from caries
(Birkhed & Heintze 1989, Russell et al. 1990, 1991, Wolinsky 1994), and our results
suggest that, salivary CA VI concentration is an equally important factor in this respect
(III). However, our results did not confirm the earlier proposal (Feldstein & Silverman
1984) that CA VI may control salivary pH or buffering capacity (I). An interesting
alternative is that it may attach to the enamel pellicle, like several other salivary proteins
(Kousvelari et al. 1980, Al-Hashimi & Levine 1989, Cohen & Levine 1989, Lamkin &
Oppenheim 1993, Lamkin et al. 1996) and function as a local pH regulator. In the
microenvironment of dental surfaces, it could accelerate the neutralization of excess
acidity locally by catalyzing the reaction H+ + HCO3
−
=> H2O + CO2, which constitutes
the main buffering system in the saliva (Lilienthal 1955, Birkhed & Heintze 1989,
Wolinsky 1994) (Fig. 3). To test this hypothesis, we explored the location and activity of
CA VI in the enamel pellicle, the thin layer of salivary proteins associated with the
hydroxyapatite of the enamel. Our results demonstrated that CA VI is indeed a natural
component of the pellicle (IV). Its binding to and activity on the enamel surface was
directly confirmed by incubating pieces of enamel in saliva or in solutions of purified
CA VI in vitro. In the enamel pellicle CA VI is located at the optimal site to catalyze the
conversion of salivary bicarbonate and microbe-delivered hydrogen ions to carbon
dioxide and water. The present results indicate that active CA VI is located in the enamel
pellicle, suggesting that it may accelerate the removal of acid from the local
microenvironment of the tooth surface.
Fig. 3. Hypothetical model for the function of CA VI on dental surfaces.
48
CA VI possesses several properties that make it compatible for functioning in the
microenvironment of dental surfaces. First, it is a very stable enzyme, retaining its
activity in an acidic environment (Parkkila et al. 1997). Its well-established stability may
be attributable to its structural properties. Sheep CA VI contains an intramolecular
disulphide bond linking Cys 25 and Cys 207 (Fernley et al. 1988), and these two
cysteines are conserved in the human enzyme, in which they presumably also form a
disulphide bond (Aldred et al. 1991). Such intramolecular disulphide bonds are a
common feature of secreted proteins, in which they stabilize the molecular
conformation. Second, CA VI is a glycoprotein, possessing two sites of N-linked
glycosylation (Murakami & Sly 1987, Aldred et al. 1991). The exact function of these
oligosaccharide side chains is not known, but it has also been suggested that they may
participate in stabilizing the functional conformation of the enzyme (Fernley 1991a). The
oligosaccharide side chains of CA VI may also be involved in protein-protein and/or
protein-hydroxyapatite interaction, linking the enzyme to the enamel pellicle. The third
unique property of CA VI is that its secretion follows a circadian periodicity (Parkkila et
al. 1995). Its salivary levels are very low during sleep and rise rapidly after awakening.
The increase in salivary CA VI concentration during the daytime obviously increases the
amount of pellicle-bound CA VI, as our results suggest (IV), ensuring that the excess
acidity arising from increased bacterial metabolism during the daytime can rapidly be
neutralized on the dental surfaces.
Several factors, including diet, oral hygiene and genetic properties of the saliva and
teeth, play a role in the aetiology of dental caries (Birkhed & Heintze 1989, Holbrook
1993, van Palenstein Helderman et al. 1996, Watson et al. 1997). The importance of
salivary factors in the host defence against caries is salient among patients suffering
from severe salivary gland dysfunction. Previous investigations have shown that the
saliva brings various defence mechanisms to the actual site of microbial adherence and
growth on the dental surfaces (Tenovuo 1989, Lamkin and Oppenheim 1993, Johnsson
et al. 1993, Lagerlöf and Oliveby 1994, Edgar et al. 1994, Wolinsky 1994). Taken
together, our results indicate that salivary CA VI also plays an essential role in the oral
cavity. Its role does not appear to be antibacterial, but rather protective, contributing to
conservation of the dental hard tissues under the unfavourable influence of microbial
metabolism.
7. Conclusions
− The mean (± SEM) salivary concentration of CA VI in young men, measured after
breakfast, 1.5 h after awakening, is 5.0 ± 0.2 mg/l.
− Salivary CA VI concentration is positively correlated with salivary α-amylase
activity. A weak positive correlation also exists with salivary secretion rate.
− CA VI is not directly involved in the regulation of actual salivary pH or buffering
capacity, nor does it have any association with counts of mutans streptococci or
lactobacilli in the saliva.
− Smoking habits have no influence on salivary CA VI levels in young men.
− High salivary CA VI concentrations are associated with lower caries experience,
particularly in subjects with neglected oral hygiene.
− Enzymatically active CA VI is present in the enamel pellicle, suggesting that, in the
local microenvironment of the dental surface, it may reduce demineralization of the
dental hard tissues by catalyzing the neutralization of excess acidity through the
reaction H+ + HCO3
−
=> H2O + CO2.
− Low levels of CA VI are present in human serum, where the enzyme is associated
with IgG.
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