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Salivary carbonic anhydrase VI and its relation to salivary flow rate and buffer capacity in pregnant and non-pregnant women
Archives of Oral Biology (2003) 48, 547—551
Salivary carbonic anhydrase VI and its relation to salivary flow rate and buffer capacity in pregnant and non-pregnant women ¨a,b,*, Merja Lainec, Seppo Parkkilaa,d, Hannu Rajaniemia Jyrki Kivela a
Department of Anatomy and Cell Biology, University of Oulu, FIN-90014 Oulu, Finland Institute of Dentistry, University of Helsinki, Helsinki, Finland c Departments of Cariology and Periodontology, Institute of Dentistry, University of Turku, ¨isenkatu 2, FIN-20520 Turku, Finland Lemminka d Institute of Medical Technology, Tampere University Hospital, University of Tampere, ¨nkatu 6, FIN-33520 Tampere, Finland Lenkkeilija b
Accepted 13 March 2003
KEYWORDS Buffering capacity; Carbonic anhydrase; Hormonal regulation; Human; Postpartum; Pregnancy; Saliva; Secretion
Summary Objective: Previous studies have shown that pregnancy may have unfavourable effects on oral health. The pH and buffer capacity (BC) of paraffin-stimulated saliva, for example, have been found to decrease towards late pregnancy. Salivary carbonic anhydrase VI (CA VI) probably protects the teeth by accelerating the neutralization of hydrogen ions in the enamel pellicle on dental surfaces. Since estrogens and androgens are known to regulate CA expression in some tissues, we studied here whether salivary CA VI concentration shows pregnancy-related changes. Design: Paraffin-stimulated salivary samples were collected from nine pregnant women 1 month before delivery and about 2 months afterwards and assayed for salivary CA VI concentration, BC and flow rate. The enzyme concentration was determined using a specific time-resolved immunofluorometric assay. The control group consisted of 17 healthy non-pregnant women. Results: The results indicated that salivary CA VI levels varied markedly among individuals, but no significant differences in mean concentrations were seen between the samples collected during late pregnancy and postpartum. BC values were lower during pregnancy, however. Conclusions: Our findings suggest that CA VI secretion is not significantly affected by the hormonal alterations associated with pregnancy, and confirm the earlier reports that CA VI is not involved in the regulation of actual salivary BC. ß 2003 Elsevier Science Ltd. All rights reserved.
Introduction Saliva does much to maintain oral health, one important function being to counteract pH changes in the oral cavity. The CO2—carbonic acid—bicarbo
Corresponding author. Present address: Central Military Hospital, P.O. Box 50, FIN-00301 Helsinki, Finland. Tel.: þ358-50-3247761; fax: þ358-9-18125827. E-mail address: [email protected] (J. Kivela ¨).
nate system, phosphate system and proteins form the main buffer systems in saliva, although proteins contribute to buffering only at low salivary pH (5.0), when the other two systems are less effective.1 The CO2—carbonic acid—bicarbonate system is responsible for most of the buffering capacity in whole saliva, the bicarbonate concentration increasing with stimulation of flow rate, e.g. during eating.2 The salivary glands are able to produce bicarbonate from CO2, yielding salivary bicarbonate
0003–9969/03/$ — see front matter ß 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0003-9969(03)00096-7
548
levels that are usually slightly lower than the plasma levels, although they may exceed the latter during stimulation as a result of increased CO2 production during increased metabolism of the glandular cells. The carbonic anhydrases (CAs) catalyze the reversible reaction CO2 þ H2 O , HCO3 þ Hþ . There are several carbonic anhydrase isoenzymes, of which CA II and CA VI are expressed in human salivary glands.3 CA II is a high-activity cytosolic enzyme that probably participates in the supply of bicarbonate ions to the saliva, and CA VI is secreted into the saliva. Recent results indicate that low salivary CA VI concentrations are associated with increased caries prevalence.4 Salivary CA VI has been shown to accumulate in the enamel pellicle maintaining its enzymatic activity.5 Based on these findings it has been suggested that in the enamel pellicle CA VI probably catalyzes neutralization of the acid metabolic products of bacteria, thus locally protecting the dental surfaces from excess acidity and caries. It has also been suggested that CA VI may be involved in protecting the upper alimentary tract from acid injury.6 Salivary pH is closely related to the buffer capacity (BC).7 Although salivary bicarbonate and pH increase with flow rate,2,8 no correlation has been found between salivary CA VI concentrations and either pH or BC.7,9 Women generally have lower BC values than men, which may simply result from their lower flow rates, but hormonal involvement in the modulation of salivary buffering properties cannot be ruled out. The salivary acid—base balance is known to be affected by pregnancy, the salivary pH and BC values decreasing gradually towards late pregnancy and recovering promptly after delivery,10—13 but salivary flow rates do not show such variation. The present study was based on the hypothesis that pregnancy may affect the CA VI concentration in saliva, as several hormones, including estrogens and androgens, are known to regulate the expression of the CA isoenzymes.14—18 Late pregnancy and postpartum salivary CA VI concentrations were compared and correlated with salivary BC and flow rate. A group of non-pregnant healthy women served as controls.
Materials and methods Subjects All the participants, nine pregnant women aged 25— 39 years (mean 32.0 years) and a control group of 17 non-pregnant women aged 24—39 years (mean 29.7 years), were healthy non-smokers who had good
J. Kivela ¨ et al.
oral health with a history of regular dental appointments. The subjects participated the study voluntarily, and informed consent was obtained from each subject.
Collection of samples Saliva collection was carried out twice. In the case of the pregnant women, the first sample was obtained 29 19 days (mean and S.D., range 1— 57 days) before delivery and the second one 53 27 days (range 28—109 days) after delivery, i.e. during the lactation period when menstruation had not yet started. An effort was made to collect salivary samples from the non-pregnant women on days with about the same hormonal status, i.e. within the three first days of menstruation for those with natural cycles (N ¼ 9), and on the same day of the pill cycle for those taking oral contraceptives (N ¼ 8). The two samples were taken in successive menstrual cycles for the women with natural cycles and 4 weeks apart for those on oral contraceptives. Saliva was collected at the same time of the day (between 08:30 and 13:00 h) and by the same dentist (M.L.) in all cases. After the subjects had refrained from eating or drinking for 1 h, saliva secretion was stimulated by chewing a standardized piece of paraffin wax (1 g, Orion Diagnostica, Espoo, Finland) habitually for 6 min, the saliva being swallowed for the first minute and thereafter collected by spitting into test tubes.
Analysis of samples Salivary flow rates were measured to an accuracy of 0.1 ml and calculated in ml/min. Salivary BC was determined electrometrically immediately following the collection as described by Ericsson.19 In the case of five of the pregnant women, BC was measured using the chair-side DentobuffÕ Strip method (Orion Diagnostica, Espoo, Finland), because laboratory equipment for Ericsson’s method was not available at the time of collection, the results being expressed as low, intermediate or high (numbered 1, 2 and 3, respectively). The samples were frozen and stored at 20 8C immediately after the electrometric BC determinations. Thawed whole saliva samples were centrifuged at 15,000 g for 10 min at 4 8C and the supernatant was used for CA VI concentration assays, performed using the antigen labelling and fluoroimmunoassay procedure as described in detail by Parkkila et al.3,9 This assay is highly specific, showing no cross-reactivity with CA I, II and IgA, and only slight intra-assay and interassay variation.3,4,7 Using Western blot, anti-CA VI antibody recognizes only a single polypeptide of CA
Salivary carbonic anhydrase VI and pregnancy
549
Table 1 Individual carbonic anhydrase VI concentrations, and salivary buffer capacity and flow rate values during pregnancy and postpartum in nine pregnant women. Carbonic anhydrase VI concentration (mg/l)
Buffer capacity
3rd trimester
3rd trimester
1.93 3.25 2.17 1.45 0.81 5.89 1.19 1.14 8.19 2.89 0.84c
Postpartum 2.28 3.32 2.07 1.43 0.24 3.12 1.26 2.33 4.49 2.28 0.42c
P ¼ 0.283d
Paraffin-stimulated flow rate (ml/min)
a
2 1 3 3 1 7.88b 3.16 4.51 7.71
Postpartum
3rd trimester
Postpartum
2 2 3 3 3 8.18 7.06 7.01 8.04
1.9 2.3 1.6 1.9 1.4 2.5 2.3 1.9 2.7 2.0 0.1c
1.7 2.0 0.8 2.9 1.1 2.6 2.3 1.5 2.8 1.9 0.2c
P ¼ 0.028e
P ¼ 0.282d
a
DentobuffÕ Strip (1, low; 2, intermediate; 3, high). Ericsson’s method (final pH). c Mean S:E:M. d Paired samples t-test. e Wilcoxon signed ranks test. b
VI in the total protein of the human saliva and milk.20
Statistical methods The Statistical Package for Social Sciences, version 9.0 (SPSS, Chicago, IL, USA) was used for the statistical analyses. Associations between the variables were studied by calculating Pearson’s correlation coefficients. Differences between two measurements were tested using the paired samples t-test and the Wilcoxon signed rank test (BC values in pregnant women). P-values lower than 0.05 were regarded as statistically significant.
Findings No statistically significant difference in mean salivary CA VI concentration was found between the samples collected during late pregnancy and post-
partum, but BC values were significantly lower during pregnancy (P ¼ 0:028). No related changes were found in salivary flow rates. The BC values measured by Ericsson’s method were 5:82 2:35 (mean S:E:M:, N ¼ 4) in late pregnancy and 7:57 0:62 postpartum, while those measured using the DentobuffÕ Strip method were lower (N ¼ 2) during pregnancy than postpartum or remained unchanged (N ¼ 3). CA VI concentrations and BC values showed greater intraindividual and interindividual variation than did flow rates (Table 1). Cross-sectional analyses showed that salivary CA VI concentrations in the pregnant women had a positive correlation with salivary flow rate at the first collection (r ¼ 0:78, P ¼ 0:014), but not at the second (r ¼ 0:56, P ¼ 0:115). Since the hormone users (N ¼ 8) and non-users (N ¼ 9) among the non-pregnant women did not differ from each other in salivary buffer capacity or flow rate, these subgroups were combined for all subsequent analyses. Cross-sectional analysis
Table 2 Carbonic anhydrase VI concentrations, buffer capacity and flow rate in two successive salivary samples from 17 non-pregnant women. Variable
First collection mean S.E.M. (range)
Carbonic anhydrase VI 2.99 0.55 (0.42—7.60) concentration (mg/l) Buffer capacity (final pH) 4.75 0.47 (2.68—7.84) Paraffin-stimulated flow rate (ml/min) 2.0 0.2 (0.9—4.5)
Second collection mean S.E.M. (range)
Paired samples t-test significance (P)
2.39 0.36 (0.38—4.98) 0.082 4.73 0.46 (2.90—7.98) 0.893 2.0 0.2 (0.8—4.1) 0.143
550
showed a significant correlation in the non-pregnant women between salivary flow rate and BC, with correlations of 0.72 (P ¼ 0:001) and 0.67 (P ¼ 0:003) at the first and second collections, respectively. Salivary CA VI concentrations were not correlated with BC, and showed a weak positive correlation with salivary flow at the first collection (r ¼ 0:49, P ¼ 0:045), but not at the second (r ¼ 0:28, P ¼ 0:280). Longitudinal analysis showed that the intraindividual changes in BC and flow rate between the two collections were slight. Intraindividual and interindividual variations in CA VI were greater than those in BC or flow rate (Table 2).
Discussion Although salivary volume and composition are constantly changing, repeated salivary samples collected under strictly standardized conditions seem to be fairly constant in the same individual. This is especially true of paraffin-stimulated flow rates and BC values, which have been found to be reproducible over intervals ranging from 1 week to 5 years.8,21 Thus only slight intraindividual variations in BC and flow rate were found in the present nonpregnant women. Although intraindividual variations in CA VI concentrations were also relatively slight, the results varied considerably between individuals. Secretion of CA VI into the saliva is known to follow a circadian periodicity, its concentrations being very low during sleep and rising rapidly to the daytime level after awakening and breakfast.22,23 In our previous study of 209 healthy young men,7 CA VI concentrations were 1:8 0:1 mg/l (mean S:E:M:) when measured early in the morning, before breakfast, and 5:0 0:2 mg/l after breakfast, with a positive correlation between the successive samples. The present saliva samples were collected several hours after waking and measured using the same method as described in the previous reports. CA VI concentrations in the saliva of the non-pregnant women were 3:0 0:6 and 2:4 0:4 mg/l at the first and second collections, respectively. These results suggest that daytime concentrations of CA VI may be somewhat lower in young women than in young men. The previous study was carried out among soldiers, permitting strict control of the conditions for saliva collection, and all the samples were collected at the same time, minimizing the effect of circadian rhythm on enzyme secretion. Although salivary CA VI concentrations are only slightly influenced by flow rate, a weak correlation was seen between the two at the first collection. This may be non-specific, however,
J. Kivela ¨ et al.
since the total protein content of saliva increases with flow rate.24,25 Neither the salivary CA VI concentration nor the flow rate was affected by pregnancy, the CA VI concentrations showing a slight but statistically significant positive correlation with flow rate during pregnancy but not postpartum. BC values were lower during pregnancy than postpartum in six women and remained unchanged in the other three. These findings are in agreement with our previous findings.12,13 The DentobuffÕ Strip method used here gives a rough approximation to the true pH, whereas more accurate values can be obtained by the Ericsson method. It is possible that slight changes in BC were not detected, because the DentobuffÕ Strip is known to underestimate changes in high BC regions.26—28 Pregnancy is known to affect the plasma acid—base balance, and it is not impossible that falls in plasma bicarbonate might contribute to falls in salivary bicarbonate concentrations. Although salivary BC values were lower during pregnancy, no changes were found in CA VI concentrations. This suggests that CA VI is not responsible for BC changes during pregnancy and confirms earlier findings that CA VI is not directly involved in the regulation of actual salivary pH or buffer capacity.7,9 So far, no explanation can be offered for the decrease in pH and BC during pregnancy. In addition to the possible plasma-originated changes in BC, it is also possible that changes in hormonal status might alter the expression of CA II in the salivary glands and thus modify the secretion of bicarbonate into the saliva.
Acknowledgements This work was supported by grants from the Finnish Cultural Foundation and the Finnish Dental Society (J.K.) and from the Sigrid Juselius Foundation (S.P.).
References 1. Bardow A, Moe D, Nyvad B, Nauntofte B. The buffer capacity and buffer systems of human whole saliva measured without loss of CO2. Arch Oral Biol 2000;45:1—12. 2. Dreizen S, Reed AI, Niedermeier W, Spies TD. Sodium and potassium as constituents of human salivary buffers. J Dent Res 1953;32:497—503. 3. Parkkila S, Kaunisto K, Rajaniemi L, Kumpulainen T, Jokinen K, Rajaniemi H. Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II and I in human parotid and submandibular glands. J Histochem Cytochem 1990;38: 941—7. 4. Kivela ¨ J, Parkkila S, Parkkila A-K, Rajaniemi H. A low concentration of carbonic anhydrase isoenzyme VI in whole
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saliva is associated with caries prevalence. Caries Res 1999; 33:178—84. Leinonen J, Kivela ¨ J, Parkkila S, Parkkila A-K, Rajaniemi H. Salivary carbonic anhydrase isoenzyme VI is located in human enamel pellicle. Caries Res 1999;33:185—90. Christie KN, Thomson C, Xue I, Lucocq JM, Hopwood D. Carbonic anhydrase isoenzymes I, II, and IV are present in human oesophageal epithelium. J Histochem Cytochem 1997;45:35—40. Kivela ¨ J, Parkkila S, Metteri J, Parkkila A-M, Toivanen A, Rajaniemi H. Salivary carbonic anhydrase VI concentrations and its relation to basic characteristics of saliva in young men. Acta Physiol Scand 1997;161:221—5. Heintze U, Birkhed D, Bjo ¨rn H. Secretion rate and buffer effect of resting and stimulated saliva as a function of age and sex. Swed Dent J 1983;7:227—38. Parkkila S, Parkkila AK, Vierjoki T, Sta ˚hlberg T, Rajaniemi H. Competitive time-resolved immunofluorometric assay for quantifying carbonic anhydrase VI in saliva. Clin Chem 1993;39:2154—7. Rosenthal S, Rowen R, Vazakas A. Comparative analysis of saliva in pregnant and non-pregnant women. I. Calcium and pH. J Dent Res 1959;38:883—7. Orosz M, Vasko ´ A, Ga ´bris K, Ba ´no ´czy J. Changes in salivary pH and lactobacilli count in pregnant women. Proc Finn Dent Soc 1980;76:204—7. Laine M, Tenovuo J, Lehtonen O-P, Ojanotko-Harri A, Vilja P, Tuohimaa P. Pregnancy-related changes in human whole saliva. Arch Oral Biol 1988;12:913—7. Laine M, Pieniha ¨kkinen K. Salivary buffer effect in relation to late pregnancy and postpartum. Acta Odontol Scand 2000;58:8—10. Ha ¨rko ¨nen PL, Ma ¨kela ¨ SI, Valve EM, Karhukorpi EK, Va ¨a ¨na ¨nen HK. Differential regulation of carbonic anhydrase II by androgen and estrogen in dorsal and lateral prostate of the rat. Endocrinology 1991;128:3219—27. Kaunisto K, Fleming RE, Kneer J, Sly WS, Rajaniemi H. Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis. Biol Reprod 1999;61:1521—6. Holm L, Berg C, Brunstro ¨m B, Ridderstra ˚le Y, Brandt I. Disrupted carbonic anhydrase distribution in the avian shell
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gland following in ovo exposure to estrogen. Arch Toxicol 2001;75:362—8. Zhou Q, Clarke L, Nie R, et al. Estrogen action and male fertility: roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function. Proc Natl Acad Sci 2001;98:14132—7. Lee KH, Finnigan-Bunick C, Bahr J, Bunick D. Estrogen regulation of ion transporter messenger RNA levels in mouse efferent ductules are mediated differentially through estrogen receptor (ER) alpha and ER beta. Biol Reprod 2001;65:1534—41. Ericsson Y. Clinical investigations of the salivary buffering action. Acta Odontol Scand 1959;17:131—65. Karhumaa P, Leinonen J, Parkkila S, Kaunisto K, Tapanainen J, Rajaniemi H. The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk. Proc Natl Acad Sci USA 2001;98:11604—8. Rask PI, Emilson CG, Krasse B, Sundberg H. Dental caries and salivary and microbial conditions in 50—60-year-old persons. Community Dent Oral Epidemiol 1991;19:93—7. Parkkila S, Parkkila A-K, Rajaniemi H. Circadian periodicity in salivary carbonic anhydrase VI concentration. Acta Physiol Scand 1995;154:205—11. Kivela ¨ J, Parkkila S, Waheed A, Parkkila A-K, Sly WS, Rajaniemi H. Secretory carbonic anhydrase isoenzyme (CA VI) in human serum. Clin Chem 1997;43:2318—22. Dawes C. The effects of flow rate and duration of stimulation on the concentrations of protein and the main electrolytes in human parotid saliva. Arch Oral Biol 1969; 14:277—94. So ¨derling E. Practical aspects of salivary analyses. In: Tenovuo J, editor. Human saliva: clinical chemistry and microbiology, vol I. Boca Raton (FL): CRC Press, 1989. p. 1—24. Frostell GA. Colourimetric screening test for evaluation of the buffer capacity of saliva. Swed Dent J 1980;4:81—6. So ¨derling E, Le Bell Y, Alanen P, Kirveskari P. Salivary buffer systems and the DentobuffÕ test. Proc Finn Dent Soc 1985;81:284—7. Wikner S, Nedlich U. A clinical evaluation of the ability of the DentobuffÕ method to estimate buffer capacity of saliva. Swed Dent J 1985;9:45—7.
Salivary carbonic anhydrase VI and its relation to salivary flow rate and buffer capacity in pregnant and non-pregnant women ¨a,b,*, Merja Lainec, Seppo Parkkilaa,d, Hannu Rajaniemia Jyrki Kivela a
Department of Anatomy and Cell Biology, University of Oulu, FIN-90014 Oulu, Finland Institute of Dentistry, University of Helsinki, Helsinki, Finland c Departments of Cariology and Periodontology, Institute of Dentistry, University of Turku, ¨isenkatu 2, FIN-20520 Turku, Finland Lemminka d Institute of Medical Technology, Tampere University Hospital, University of Tampere, ¨nkatu 6, FIN-33520 Tampere, Finland Lenkkeilija b
Accepted 13 March 2003
KEYWORDS Buffering capacity; Carbonic anhydrase; Hormonal regulation; Human; Postpartum; Pregnancy; Saliva; Secretion
Summary Objective: Previous studies have shown that pregnancy may have unfavourable effects on oral health. The pH and buffer capacity (BC) of paraffin-stimulated saliva, for example, have been found to decrease towards late pregnancy. Salivary carbonic anhydrase VI (CA VI) probably protects the teeth by accelerating the neutralization of hydrogen ions in the enamel pellicle on dental surfaces. Since estrogens and androgens are known to regulate CA expression in some tissues, we studied here whether salivary CA VI concentration shows pregnancy-related changes. Design: Paraffin-stimulated salivary samples were collected from nine pregnant women 1 month before delivery and about 2 months afterwards and assayed for salivary CA VI concentration, BC and flow rate. The enzyme concentration was determined using a specific time-resolved immunofluorometric assay. The control group consisted of 17 healthy non-pregnant women. Results: The results indicated that salivary CA VI levels varied markedly among individuals, but no significant differences in mean concentrations were seen between the samples collected during late pregnancy and postpartum. BC values were lower during pregnancy, however. Conclusions: Our findings suggest that CA VI secretion is not significantly affected by the hormonal alterations associated with pregnancy, and confirm the earlier reports that CA VI is not involved in the regulation of actual salivary BC. ß 2003 Elsevier Science Ltd. All rights reserved.
Introduction Saliva does much to maintain oral health, one important function being to counteract pH changes in the oral cavity. The CO2—carbonic acid—bicarbo
Corresponding author. Present address: Central Military Hospital, P.O. Box 50, FIN-00301 Helsinki, Finland. Tel.: þ358-50-3247761; fax: þ358-9-18125827. E-mail address: [email protected] (J. Kivela ¨).
nate system, phosphate system and proteins form the main buffer systems in saliva, although proteins contribute to buffering only at low salivary pH (5.0), when the other two systems are less effective.1 The CO2—carbonic acid—bicarbonate system is responsible for most of the buffering capacity in whole saliva, the bicarbonate concentration increasing with stimulation of flow rate, e.g. during eating.2 The salivary glands are able to produce bicarbonate from CO2, yielding salivary bicarbonate
0003–9969/03/$ — see front matter ß 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0003-9969(03)00096-7
548
levels that are usually slightly lower than the plasma levels, although they may exceed the latter during stimulation as a result of increased CO2 production during increased metabolism of the glandular cells. The carbonic anhydrases (CAs) catalyze the reversible reaction CO2 þ H2 O , HCO3 þ Hþ . There are several carbonic anhydrase isoenzymes, of which CA II and CA VI are expressed in human salivary glands.3 CA II is a high-activity cytosolic enzyme that probably participates in the supply of bicarbonate ions to the saliva, and CA VI is secreted into the saliva. Recent results indicate that low salivary CA VI concentrations are associated with increased caries prevalence.4 Salivary CA VI has been shown to accumulate in the enamel pellicle maintaining its enzymatic activity.5 Based on these findings it has been suggested that in the enamel pellicle CA VI probably catalyzes neutralization of the acid metabolic products of bacteria, thus locally protecting the dental surfaces from excess acidity and caries. It has also been suggested that CA VI may be involved in protecting the upper alimentary tract from acid injury.6 Salivary pH is closely related to the buffer capacity (BC).7 Although salivary bicarbonate and pH increase with flow rate,2,8 no correlation has been found between salivary CA VI concentrations and either pH or BC.7,9 Women generally have lower BC values than men, which may simply result from their lower flow rates, but hormonal involvement in the modulation of salivary buffering properties cannot be ruled out. The salivary acid—base balance is known to be affected by pregnancy, the salivary pH and BC values decreasing gradually towards late pregnancy and recovering promptly after delivery,10—13 but salivary flow rates do not show such variation. The present study was based on the hypothesis that pregnancy may affect the CA VI concentration in saliva, as several hormones, including estrogens and androgens, are known to regulate the expression of the CA isoenzymes.14—18 Late pregnancy and postpartum salivary CA VI concentrations were compared and correlated with salivary BC and flow rate. A group of non-pregnant healthy women served as controls.
Materials and methods Subjects All the participants, nine pregnant women aged 25— 39 years (mean 32.0 years) and a control group of 17 non-pregnant women aged 24—39 years (mean 29.7 years), were healthy non-smokers who had good
J. Kivela ¨ et al.
oral health with a history of regular dental appointments. The subjects participated the study voluntarily, and informed consent was obtained from each subject.
Collection of samples Saliva collection was carried out twice. In the case of the pregnant women, the first sample was obtained 29 19 days (mean and S.D., range 1— 57 days) before delivery and the second one 53 27 days (range 28—109 days) after delivery, i.e. during the lactation period when menstruation had not yet started. An effort was made to collect salivary samples from the non-pregnant women on days with about the same hormonal status, i.e. within the three first days of menstruation for those with natural cycles (N ¼ 9), and on the same day of the pill cycle for those taking oral contraceptives (N ¼ 8). The two samples were taken in successive menstrual cycles for the women with natural cycles and 4 weeks apart for those on oral contraceptives. Saliva was collected at the same time of the day (between 08:30 and 13:00 h) and by the same dentist (M.L.) in all cases. After the subjects had refrained from eating or drinking for 1 h, saliva secretion was stimulated by chewing a standardized piece of paraffin wax (1 g, Orion Diagnostica, Espoo, Finland) habitually for 6 min, the saliva being swallowed for the first minute and thereafter collected by spitting into test tubes.
Analysis of samples Salivary flow rates were measured to an accuracy of 0.1 ml and calculated in ml/min. Salivary BC was determined electrometrically immediately following the collection as described by Ericsson.19 In the case of five of the pregnant women, BC was measured using the chair-side DentobuffÕ Strip method (Orion Diagnostica, Espoo, Finland), because laboratory equipment for Ericsson’s method was not available at the time of collection, the results being expressed as low, intermediate or high (numbered 1, 2 and 3, respectively). The samples were frozen and stored at 20 8C immediately after the electrometric BC determinations. Thawed whole saliva samples were centrifuged at 15,000 g for 10 min at 4 8C and the supernatant was used for CA VI concentration assays, performed using the antigen labelling and fluoroimmunoassay procedure as described in detail by Parkkila et al.3,9 This assay is highly specific, showing no cross-reactivity with CA I, II and IgA, and only slight intra-assay and interassay variation.3,4,7 Using Western blot, anti-CA VI antibody recognizes only a single polypeptide of CA
Salivary carbonic anhydrase VI and pregnancy
549
Table 1 Individual carbonic anhydrase VI concentrations, and salivary buffer capacity and flow rate values during pregnancy and postpartum in nine pregnant women. Carbonic anhydrase VI concentration (mg/l)
Buffer capacity
3rd trimester
3rd trimester
1.93 3.25 2.17 1.45 0.81 5.89 1.19 1.14 8.19 2.89 0.84c
Postpartum 2.28 3.32 2.07 1.43 0.24 3.12 1.26 2.33 4.49 2.28 0.42c
P ¼ 0.283d
Paraffin-stimulated flow rate (ml/min)
a
2 1 3 3 1 7.88b 3.16 4.51 7.71
Postpartum
3rd trimester
Postpartum
2 2 3 3 3 8.18 7.06 7.01 8.04
1.9 2.3 1.6 1.9 1.4 2.5 2.3 1.9 2.7 2.0 0.1c
1.7 2.0 0.8 2.9 1.1 2.6 2.3 1.5 2.8 1.9 0.2c
P ¼ 0.028e
P ¼ 0.282d
a
DentobuffÕ Strip (1, low; 2, intermediate; 3, high). Ericsson’s method (final pH). c Mean S:E:M. d Paired samples t-test. e Wilcoxon signed ranks test. b
VI in the total protein of the human saliva and milk.20
Statistical methods The Statistical Package for Social Sciences, version 9.0 (SPSS, Chicago, IL, USA) was used for the statistical analyses. Associations between the variables were studied by calculating Pearson’s correlation coefficients. Differences between two measurements were tested using the paired samples t-test and the Wilcoxon signed rank test (BC values in pregnant women). P-values lower than 0.05 were regarded as statistically significant.
Findings No statistically significant difference in mean salivary CA VI concentration was found between the samples collected during late pregnancy and post-
partum, but BC values were significantly lower during pregnancy (P ¼ 0:028). No related changes were found in salivary flow rates. The BC values measured by Ericsson’s method were 5:82 2:35 (mean S:E:M:, N ¼ 4) in late pregnancy and 7:57 0:62 postpartum, while those measured using the DentobuffÕ Strip method were lower (N ¼ 2) during pregnancy than postpartum or remained unchanged (N ¼ 3). CA VI concentrations and BC values showed greater intraindividual and interindividual variation than did flow rates (Table 1). Cross-sectional analyses showed that salivary CA VI concentrations in the pregnant women had a positive correlation with salivary flow rate at the first collection (r ¼ 0:78, P ¼ 0:014), but not at the second (r ¼ 0:56, P ¼ 0:115). Since the hormone users (N ¼ 8) and non-users (N ¼ 9) among the non-pregnant women did not differ from each other in salivary buffer capacity or flow rate, these subgroups were combined for all subsequent analyses. Cross-sectional analysis
Table 2 Carbonic anhydrase VI concentrations, buffer capacity and flow rate in two successive salivary samples from 17 non-pregnant women. Variable
First collection mean S.E.M. (range)
Carbonic anhydrase VI 2.99 0.55 (0.42—7.60) concentration (mg/l) Buffer capacity (final pH) 4.75 0.47 (2.68—7.84) Paraffin-stimulated flow rate (ml/min) 2.0 0.2 (0.9—4.5)
Second collection mean S.E.M. (range)
Paired samples t-test significance (P)
2.39 0.36 (0.38—4.98) 0.082 4.73 0.46 (2.90—7.98) 0.893 2.0 0.2 (0.8—4.1) 0.143
550
showed a significant correlation in the non-pregnant women between salivary flow rate and BC, with correlations of 0.72 (P ¼ 0:001) and 0.67 (P ¼ 0:003) at the first and second collections, respectively. Salivary CA VI concentrations were not correlated with BC, and showed a weak positive correlation with salivary flow at the first collection (r ¼ 0:49, P ¼ 0:045), but not at the second (r ¼ 0:28, P ¼ 0:280). Longitudinal analysis showed that the intraindividual changes in BC and flow rate between the two collections were slight. Intraindividual and interindividual variations in CA VI were greater than those in BC or flow rate (Table 2).
Discussion Although salivary volume and composition are constantly changing, repeated salivary samples collected under strictly standardized conditions seem to be fairly constant in the same individual. This is especially true of paraffin-stimulated flow rates and BC values, which have been found to be reproducible over intervals ranging from 1 week to 5 years.8,21 Thus only slight intraindividual variations in BC and flow rate were found in the present nonpregnant women. Although intraindividual variations in CA VI concentrations were also relatively slight, the results varied considerably between individuals. Secretion of CA VI into the saliva is known to follow a circadian periodicity, its concentrations being very low during sleep and rising rapidly to the daytime level after awakening and breakfast.22,23 In our previous study of 209 healthy young men,7 CA VI concentrations were 1:8 0:1 mg/l (mean S:E:M:) when measured early in the morning, before breakfast, and 5:0 0:2 mg/l after breakfast, with a positive correlation between the successive samples. The present saliva samples were collected several hours after waking and measured using the same method as described in the previous reports. CA VI concentrations in the saliva of the non-pregnant women were 3:0 0:6 and 2:4 0:4 mg/l at the first and second collections, respectively. These results suggest that daytime concentrations of CA VI may be somewhat lower in young women than in young men. The previous study was carried out among soldiers, permitting strict control of the conditions for saliva collection, and all the samples were collected at the same time, minimizing the effect of circadian rhythm on enzyme secretion. Although salivary CA VI concentrations are only slightly influenced by flow rate, a weak correlation was seen between the two at the first collection. This may be non-specific, however,
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since the total protein content of saliva increases with flow rate.24,25 Neither the salivary CA VI concentration nor the flow rate was affected by pregnancy, the CA VI concentrations showing a slight but statistically significant positive correlation with flow rate during pregnancy but not postpartum. BC values were lower during pregnancy than postpartum in six women and remained unchanged in the other three. These findings are in agreement with our previous findings.12,13 The DentobuffÕ Strip method used here gives a rough approximation to the true pH, whereas more accurate values can be obtained by the Ericsson method. It is possible that slight changes in BC were not detected, because the DentobuffÕ Strip is known to underestimate changes in high BC regions.26—28 Pregnancy is known to affect the plasma acid—base balance, and it is not impossible that falls in plasma bicarbonate might contribute to falls in salivary bicarbonate concentrations. Although salivary BC values were lower during pregnancy, no changes were found in CA VI concentrations. This suggests that CA VI is not responsible for BC changes during pregnancy and confirms earlier findings that CA VI is not directly involved in the regulation of actual salivary pH or buffer capacity.7,9 So far, no explanation can be offered for the decrease in pH and BC during pregnancy. In addition to the possible plasma-originated changes in BC, it is also possible that changes in hormonal status might alter the expression of CA II in the salivary glands and thus modify the secretion of bicarbonate into the saliva.
Acknowledgements This work was supported by grants from the Finnish Cultural Foundation and the Finnish Dental Society (J.K.) and from the Sigrid Juselius Foundation (S.P.).
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