THE EFFECT OF ORAL CONTRACEPTIVES ON THE CONCENTRATION OF SOME SALIVARY SUBSTANCES TN WOMEN IMVAR
MAGNUSWN.
THWILI)
Dept. Cot-Cw~ology and Laboratory
ERICSON
and A.
HIJGOSON*
for Clinical Chcmistq. Faculty University of Giiteborg. Giiteborl. Sweden
of Odontolog)
Summar! Samples of stimulated whole saliva. as well as individual parotid and subtnandibulal gland secretions, were collected repeatedly from twelve 23~ 26 years old women during one 01 two menstrual cycles before administration of oral contraceptives (Follinyl@. Recip, Sweden; norgestrel 0.5 mg and ethinyloestradiole 0.05 mg) and for about 2 months after the start of the hormone administration. Although large individual variations in concentration of various substances were found in rcsponse to the hormone administration, some of the parameters showed systematic and significant changes. In stimulated whole saliva. protein. sialic acid. hexosamine. fucose. hydrogen ion concentration and total electrolyte concentration decreased. The secretion rates for both parotid and submandibular secretions increased. The sodium and hydrogen ion concentrations increased in parotid secretion and sodium in submandibular secretion. The diffcrcnces in concentration could not be explained by differences in secretion rate. Variations in concentrations were for some parameters smaller during the hormone period than during the control period. indicating a stabilizing cficct of the hormones.
1963: Hugoson. 1970) and during the ovulatory phase of the menstrual cycle subclinical signs of giniival inflammation arc more pronounced (Lindhe and Attstriim. 1967). It has also been reported that administration of oestradiol benzoate or oestriol (Liu, 1967)daily for 6 weeksand oestradiol benzoate daily for 13 weeks (Liu. Lin and Zullo. 1969) induced ;I significant increase of caries in female rats. These observations have not been fully explained in relation to the aetiology of the diseases. Clinically, wide variations exist between persons in the rdtc ofplayuc formation as well as in the frequency of different types of streptococci in early dental plaque. Such variations could bc explained partly bv variations in eating habits and partly by inter-individual variations in the micro-environment. such as variations in the composition of the saliva induced b! c.g. sex hormones. The aim of the present investigation was to study the effect of oral contraceptives 011 the composition of human saliva.
The physiology of the salivary glands is complex. A number of different factors have a regulatory effect of the function of the salivary glands (see Emmelin and Zotterman. 1972). The elTect of female sex hormones has been studied to some extent and both morphological and functional changes have been reported in animals (Lacassagne. 1940; Buillard and Delsuc, 1941; Shackleford and Klapper. 1961: Travill. 1966) and in man. Sex hormones have ;I regulatory effect on the concentration of electrolytes in human saliva. Hugoson (1972) found that the potassium and calcium concentrations of both resting and stimulated parotid saliva increased during pregnancy in women and clccreased after delivery. In contrast. the sodium conccntration decreased during pregnancy. Marder, Wotman and Mandcl (1972) found no change in calcium concentration in parotid secretion, whereas in the submandibular secretion lower values were recorded for calcium during pregnancy than /XJ,Q I)LII‘UIII.In both parotid and submandibular secretions. sodium cotlcentration dccreascd and potassium concentration increased during pregnancy. Stutlics on electrol!tcs in saliva during the menstrual qcle hnvc been made by Puskulian (1972). In submandibular saliva significantly lower values were found for calcium and sodium at ovulation whereas potasxium values were significantly higher. In parotid ul~va. no significant changes occurred for any of these clcctrol! tes. Ovulation was estimated to occur 2 days after midcqclc. The relation between female sex hormones and gingivitis and caries has been subjected to several investigatlons. During human pregnancy there is an aggravation of preexisting gingivitis (L&z and Silness. .--__ for Education. * In\titute Postgraduate Dental .liinkiipinp.
VI IlICRI.\l. 91D
!VlW’HODS
;I Twelve women. aged 23 26 years. consultrng gynaecologist to receive oral contraceptives. volunteered for the investigation. The hormone preparation (Follinvl@. Recip, Sweden; norgestrel 0.5 mg. ethinyl oestradiole 0.05 mg) was administered during 3 week periods separated by a hormone-free week. This rhythm in the administration was followed throughout the entire test period. At the start of the experiment each test person was given one tablet daily from the fifth da! after the first day ofmenstruation. Prior to the hormone treatment saliEiry samples were collected from cdch individual on 4 I2 occasions evenly distributed over one to three menstrual cycles. During the
Sweden. II’)
Ingvar Magnusson. Thorild Ericson
120
and A.
Hugoson
first 2 months of hormone treatment-514 saliva samples were collected. As a reference, 4-10 samples of stimulated whole saliva were collected from each of 5 males, aged 263 I years. during 2 months.
Five ml volumes of paraffin-stimulated whole saliva and ofcitric acid-stimulated secretions from each of the parotid and submandibular glands were collected on each occasion. For collection of the separate parotid secretion, a plastic cup was placed over the orifice of the excretory ducts and held in place by a peripheral suction chamber. The secretions were collected through a Teflon tube from the central chamber of the cups. The submandibular secretion was collected by means of an individually made plastic tray which was passively placed over the orifices of the secretory ducts from the submandibular and sublingual glands. It was held in position by the investigator. All saliva samples were collected in ice-chilled glass tubes, which were sealed with Parafilm to prevent CO,-escape. All samples were collected before lunch between 12 noon and I pm. The pH values were determined within 30 min after collection. After dilution (2: 3) with distilled water, the samples were centrifuged at 20.000 g at 4 C. From the supernatant liquid, sampleswere taken for the chemical analyses.
The following analyses were made on each sample: Protein was analysed by the method of Lowry et al. (1951) with tyrosin as a standard. Siulic clcid was analysed with the thiobarbiturate acid method (Warren, 1959) using n-acetylneuraminic acid as a standard. Methyl-pentosr concentration was analysed by the method of Gibbons (1955) using fucose as a standard. Hesosaminr was determined by a modified Elson-Morgan technique. After hydrolysis in 3 M HCl for 3 hr the acid was removed by lyophilization prior to acetylation and condensation with Ehrlich’s reagent. E~KWO/JTC drtermimtions. The concentrations (mEq/l) of sodium. potassium and total calcium were determined in an Eppendorf flame photometer. The chloride concentration (mEq/l) was measured with a selective chloride electrode (Orion ionalyser). The total electrolyte concentration was measured as the conductivity with a conductivity meter (Radiometer. Copenhagen).
loo DECREASE
50
iii
100
50
%
150
INCREASE.
%
Fig. 1. A longitudinal study of the change in the concentrations of some components in stimulated whole saliK1 caused by administration of female sex hormones to I? women. The mean values of 4-12 measurments made on each individual during normal menstrual cycles. a,,, (c.1’. Table I) are compared to the mean values of 5 14 measurements made during the hormone period. The effect on concentrations is expressed in per cent of the menstruation value. Each marking represents one individual.
for each individual. The difference in mean value of concentration between control and test period is expressed in per cent of the mean values during the
RESCLTS
The mean valuesand standard deviations for the parameters were calculated for stimulated whole, parotid and submandibular saliva. on samples collected several times during a control period of one or two menstrual cycles prior to the administration of hormones. (The individual values may be obtained from us on request.) Generally iarge variations were found in the secretion rates and the concentrations of substances analysed both between individuals and in the same individual. Figure 1 shows the effect of the hormone treatment on the measured parameters in stimulated whole saliva
CONDUC TIVITY
wttl
CH’I SECRETION RATE
II
1I II II
-
I, II
,I II
%
II II
150
lco5oA5olw~ DECREASE
I I
INCREASE
a150
%
Fig. 2. A longitudinal study of the change in concentrations of some components in stimulated parotid saliva caused I~! administration of female sex hormones. Details as in Fig. I.
Effect of oral contraceptives
HEXOSAMINE
CONDUC TIVITY
121
substances
HEXOSAMINE
-
ffli-li
II
t
1 IrnI I 111 I
k”] SECRETION RATE
on salivary
-
I I I
I I
I 1
I I . ..
loo
50
DECREASE,
i;i
50
%
100 INCREASE.
150
>150
%
Fig. 1. A longitudinal study of the change in concentrations of some components in stimulated submandibular saliva cauwi by administration offemalesex hormones. Details as
111Fig. 1. control period. Figures 2 and 3 give the same information for the stimulated parotid and submandibular secretions respectively. In Table I is given the 95 per cent confidence limits for the differences between the mean values obtained for all three secretions prior to and during the administration of hormones. When the conlidcnce interval does not contain zero. either an increase or a dccrcase was induced by the hormone treatment. The significance of the differences between the two sets of data is expressed as t- and p-values. Stimulated whole saliva seems to reflect most clearly the diffcrence between the control and the hormone treatment period. All four organic components. the secre-
100 DECREASE
50
50
150
a30
%
tion rate and total salt concentration. expressed as ion conconductivity. as well as the hydrogen centration show significant changes. Figure 4 shows the separate values for fucose concentration in stimulated parotid saliva measured during the control and hormone periods for tw!o individuals. In one. the standard deviation was reduced from 55.8 to 21.4 whereas in the other case an increase frotn 14.9 to 28.3 was found. In Fig. 5 is shown the difrerences between standard deviations of the measured IO0
P
200
7 5 3 i 8 ?
100 INCREASE
Fig. 5. The efkct of admimstration of female sex hormones on the standard dckiations of the concentrations of bomc components in stimulated whole secretions. The htandard deviations for the values obtained during normal mcnstruatlon cqclcs and the hormone periods arc compared. The effect is expressed as per cent of the standard deviation for the normal menstruation qcle.
300
200
Fi
%
100
ID0
0
0
Fig. 4. Concentration of fucose, pg;ml. in stimulated tid secretions collected on several occasions from two viduals, M.M. and B.M.. during a normal menstrual and during Follinyl@ administration. Mean values and dard deviations are given.
paroindicycle stan-
W P S
W P S W P S W P S
Fucose
Heaosamine
Na+
Total Ca
Sialic acid
W P S W P S
Protein
4.18 040 1.22 1.57 1.36 0.08 1.27 2.87 2.92
5.70 0.33 1.78
1.55 4.34 D44 0.12
1.28
6.66
t
0.5 < 0.4 < 0.2 0.5 < 0.4
MAGNUSWN.
THWILI)
Dept. Cot-Cw~ology and Laboratory
ERICSON
and A.
HIJGOSON*
for Clinical Chcmistq. Faculty University of Giiteborg. Giiteborl. Sweden
of Odontolog)
Summar! Samples of stimulated whole saliva. as well as individual parotid and subtnandibulal gland secretions, were collected repeatedly from twelve 23~ 26 years old women during one 01 two menstrual cycles before administration of oral contraceptives (Follinyl@. Recip, Sweden; norgestrel 0.5 mg and ethinyloestradiole 0.05 mg) and for about 2 months after the start of the hormone administration. Although large individual variations in concentration of various substances were found in rcsponse to the hormone administration, some of the parameters showed systematic and significant changes. In stimulated whole saliva. protein. sialic acid. hexosamine. fucose. hydrogen ion concentration and total electrolyte concentration decreased. The secretion rates for both parotid and submandibular secretions increased. The sodium and hydrogen ion concentrations increased in parotid secretion and sodium in submandibular secretion. The diffcrcnces in concentration could not be explained by differences in secretion rate. Variations in concentrations were for some parameters smaller during the hormone period than during the control period. indicating a stabilizing cficct of the hormones.
1963: Hugoson. 1970) and during the ovulatory phase of the menstrual cycle subclinical signs of giniival inflammation arc more pronounced (Lindhe and Attstriim. 1967). It has also been reported that administration of oestradiol benzoate or oestriol (Liu, 1967)daily for 6 weeksand oestradiol benzoate daily for 13 weeks (Liu. Lin and Zullo. 1969) induced ;I significant increase of caries in female rats. These observations have not been fully explained in relation to the aetiology of the diseases. Clinically, wide variations exist between persons in the rdtc ofplayuc formation as well as in the frequency of different types of streptococci in early dental plaque. Such variations could bc explained partly bv variations in eating habits and partly by inter-individual variations in the micro-environment. such as variations in the composition of the saliva induced b! c.g. sex hormones. The aim of the present investigation was to study the effect of oral contraceptives 011 the composition of human saliva.
The physiology of the salivary glands is complex. A number of different factors have a regulatory effect of the function of the salivary glands (see Emmelin and Zotterman. 1972). The elTect of female sex hormones has been studied to some extent and both morphological and functional changes have been reported in animals (Lacassagne. 1940; Buillard and Delsuc, 1941; Shackleford and Klapper. 1961: Travill. 1966) and in man. Sex hormones have ;I regulatory effect on the concentration of electrolytes in human saliva. Hugoson (1972) found that the potassium and calcium concentrations of both resting and stimulated parotid saliva increased during pregnancy in women and clccreased after delivery. In contrast. the sodium conccntration decreased during pregnancy. Marder, Wotman and Mandcl (1972) found no change in calcium concentration in parotid secretion, whereas in the submandibular secretion lower values were recorded for calcium during pregnancy than /XJ,Q I)LII‘UIII.In both parotid and submandibular secretions. sodium cotlcentration dccreascd and potassium concentration increased during pregnancy. Stutlics on electrol!tcs in saliva during the menstrual qcle hnvc been made by Puskulian (1972). In submandibular saliva significantly lower values were found for calcium and sodium at ovulation whereas potasxium values were significantly higher. In parotid ul~va. no significant changes occurred for any of these clcctrol! tes. Ovulation was estimated to occur 2 days after midcqclc. The relation between female sex hormones and gingivitis and caries has been subjected to several investigatlons. During human pregnancy there is an aggravation of preexisting gingivitis (L&z and Silness. .--__ for Education. * In\titute Postgraduate Dental .liinkiipinp.
VI IlICRI.\l. 91D
!VlW’HODS
;I Twelve women. aged 23 26 years. consultrng gynaecologist to receive oral contraceptives. volunteered for the investigation. The hormone preparation (Follinvl@. Recip, Sweden; norgestrel 0.5 mg. ethinyl oestradiole 0.05 mg) was administered during 3 week periods separated by a hormone-free week. This rhythm in the administration was followed throughout the entire test period. At the start of the experiment each test person was given one tablet daily from the fifth da! after the first day ofmenstruation. Prior to the hormone treatment saliEiry samples were collected from cdch individual on 4 I2 occasions evenly distributed over one to three menstrual cycles. During the
Sweden. II’)
Ingvar Magnusson. Thorild Ericson
120
and A.
Hugoson
first 2 months of hormone treatment-514 saliva samples were collected. As a reference, 4-10 samples of stimulated whole saliva were collected from each of 5 males, aged 263 I years. during 2 months.
Five ml volumes of paraffin-stimulated whole saliva and ofcitric acid-stimulated secretions from each of the parotid and submandibular glands were collected on each occasion. For collection of the separate parotid secretion, a plastic cup was placed over the orifice of the excretory ducts and held in place by a peripheral suction chamber. The secretions were collected through a Teflon tube from the central chamber of the cups. The submandibular secretion was collected by means of an individually made plastic tray which was passively placed over the orifices of the secretory ducts from the submandibular and sublingual glands. It was held in position by the investigator. All saliva samples were collected in ice-chilled glass tubes, which were sealed with Parafilm to prevent CO,-escape. All samples were collected before lunch between 12 noon and I pm. The pH values were determined within 30 min after collection. After dilution (2: 3) with distilled water, the samples were centrifuged at 20.000 g at 4 C. From the supernatant liquid, sampleswere taken for the chemical analyses.
The following analyses were made on each sample: Protein was analysed by the method of Lowry et al. (1951) with tyrosin as a standard. Siulic clcid was analysed with the thiobarbiturate acid method (Warren, 1959) using n-acetylneuraminic acid as a standard. Methyl-pentosr concentration was analysed by the method of Gibbons (1955) using fucose as a standard. Hesosaminr was determined by a modified Elson-Morgan technique. After hydrolysis in 3 M HCl for 3 hr the acid was removed by lyophilization prior to acetylation and condensation with Ehrlich’s reagent. E~KWO/JTC drtermimtions. The concentrations (mEq/l) of sodium. potassium and total calcium were determined in an Eppendorf flame photometer. The chloride concentration (mEq/l) was measured with a selective chloride electrode (Orion ionalyser). The total electrolyte concentration was measured as the conductivity with a conductivity meter (Radiometer. Copenhagen).
loo DECREASE
50
iii
100
50
%
150
INCREASE.
%
Fig. 1. A longitudinal study of the change in the concentrations of some components in stimulated whole saliK1 caused by administration of female sex hormones to I? women. The mean values of 4-12 measurments made on each individual during normal menstrual cycles. a,,, (c.1’. Table I) are compared to the mean values of 5 14 measurements made during the hormone period. The effect on concentrations is expressed in per cent of the menstruation value. Each marking represents one individual.
for each individual. The difference in mean value of concentration between control and test period is expressed in per cent of the mean values during the
RESCLTS
The mean valuesand standard deviations for the parameters were calculated for stimulated whole, parotid and submandibular saliva. on samples collected several times during a control period of one or two menstrual cycles prior to the administration of hormones. (The individual values may be obtained from us on request.) Generally iarge variations were found in the secretion rates and the concentrations of substances analysed both between individuals and in the same individual. Figure 1 shows the effect of the hormone treatment on the measured parameters in stimulated whole saliva
CONDUC TIVITY
wttl
CH’I SECRETION RATE
II
1I II II
-
I, II
,I II
%
II II
150
lco5oA5olw~ DECREASE
I I
INCREASE
a150
%
Fig. 2. A longitudinal study of the change in concentrations of some components in stimulated parotid saliva caused I~! administration of female sex hormones. Details as in Fig. I.
Effect of oral contraceptives
HEXOSAMINE
CONDUC TIVITY
121
substances
HEXOSAMINE
-
ffli-li
II
t
1 IrnI I 111 I
k”] SECRETION RATE
on salivary
-
I I I
I I
I 1
I I . ..
loo
50
DECREASE,
i;i
50
%
100 INCREASE.
150
>150
%
Fig. 1. A longitudinal study of the change in concentrations of some components in stimulated submandibular saliva cauwi by administration offemalesex hormones. Details as
111Fig. 1. control period. Figures 2 and 3 give the same information for the stimulated parotid and submandibular secretions respectively. In Table I is given the 95 per cent confidence limits for the differences between the mean values obtained for all three secretions prior to and during the administration of hormones. When the conlidcnce interval does not contain zero. either an increase or a dccrcase was induced by the hormone treatment. The significance of the differences between the two sets of data is expressed as t- and p-values. Stimulated whole saliva seems to reflect most clearly the diffcrence between the control and the hormone treatment period. All four organic components. the secre-
100 DECREASE
50
50
150
a30
%
tion rate and total salt concentration. expressed as ion conconductivity. as well as the hydrogen centration show significant changes. Figure 4 shows the separate values for fucose concentration in stimulated parotid saliva measured during the control and hormone periods for tw!o individuals. In one. the standard deviation was reduced from 55.8 to 21.4 whereas in the other case an increase frotn 14.9 to 28.3 was found. In Fig. 5 is shown the difrerences between standard deviations of the measured IO0
P
200
7 5 3 i 8 ?
100 INCREASE
Fig. 5. The efkct of admimstration of female sex hormones on the standard dckiations of the concentrations of bomc components in stimulated whole secretions. The htandard deviations for the values obtained during normal mcnstruatlon cqclcs and the hormone periods arc compared. The effect is expressed as per cent of the standard deviation for the normal menstruation qcle.
300
200
Fi
%
100
ID0
0
0
Fig. 4. Concentration of fucose, pg;ml. in stimulated tid secretions collected on several occasions from two viduals, M.M. and B.M.. during a normal menstrual and during Follinyl@ administration. Mean values and dard deviations are given.
paroindicycle stan-
W P S
W P S W P S W P S
Fucose
Heaosamine
Na+
Total Ca
Sialic acid
W P S W P S
Protein
4.18 040 1.22 1.57 1.36 0.08 1.27 2.87 2.92
5.70 0.33 1.78
1.55 4.34 D44 0.12
1.28
6.66
t
0.5 < 0.4 < 0.2 0.5 < 0.4
