Salivary Calcium and Albumin as Biomarkers of Oral Dryness in Postmenopausal Women: A Case–Control Analysis

ABSTRACT

Objective:

Menopause-associated hypoestrogenism significantly alters oral homeostasis, often leading to xerostomia. Although salivary calcium and albumin are indicators of mineral turnover and mucosal permeability, their specific correlation with subjective and objective oral dryness in postmenopausal cohorts requires further elucidation. The aim was to evaluate salivary calcium and albumin levels in postmenopausal women with and without oral dryness and to compare these findings with the clinical symptoms.

Methods:

This case–control study included three groups (n = 40 in each group): Group I (premenopausal), Group II (postmenopausal without oral dryness), and Group III (postmenopausal with oral dryness). An unstimulated flow rate of less than 0.1 mL/min was used to measure oral dryness. The estimation of salivary calcium via colorimetric spectrophotometry (612 nm), and albumin was quantified using standard biochemical assays. Statistical analysis was performed using analysis of variance, analysis of covariance, and Pearson’s correlation, with sample size justified by power analysis (80% power).

Results:

Group III (postmenopausal women with oral dryness) had significantly higher salivary calcium and albumin levels than both Group II and Group I (P < 0.05). A moderate positive correlation was found between salivary albumin and calcium level. Multiple regression suggested that these biomarker elevations are associated with menopausal status, although age remains a potential confounding factor.

Conclusion:

Elevated salivary calcium and albumin levels serve as significant biochemical markers for oral dryness in postmenopausal women. These findings suggest that increased mucosal permeability and altered mineral transport are key physiological features of the menopausal oral cavity, necessitating targeted preventive dental care.

INTRODUCTION

The oral cavity is thought to be the “window” or “mirror” of the body because various oral manifestations will accompany many of the systemic diseases and physiological changes associated with aging. If the patient has to receive the proper diagnosis and treatment, these changes must be properly recognized.[1] Oral health means much more than healthy teeth. The relationship between oral health and general health has been appreciated in all aspects of the medical field.[2] Oral health considerations in postmenopausal women require special attention, as it is often neglected by clinicians.

Menopause is a natural physiological process that occurs during the fourth to fifth decade of a woman’s life.[3] Menopause is defined by the World Health Organization as “the permanent termination of menstruation caused by the loss of ovarian follicular activity.” During postmenopause (PM), remarkable changes occur in relation to different parts of the human body, of which the oral symptoms were found to be significantly higher in postmenopausal women (43%) than in premenopausal women (6%).[1,4] The term “perimenopause” refers to the time frame in which a woman’s life moves toward menopause. Menstrual irregularities signal the start of the perimenopause phase, and after 12 months of amenorrhea, menopause is retroactively verified.[2] The indications and symptoms of menopause are strongly determined by lifestyle, rural–urban division, and economic status. Menopausal women in both rural and urban areas frequently complain of urogenital symptoms, exhaustion, and body aches, but urban women are more likely to experience hot flashes, psychological issues, mood swings, and sexual dysfunction. Gingival bleeding, receding gums, loose teeth, and a burning feeling in the oral mucosa are some common oral–dental conditions in menopausal women. Although burning sensation is a common sign of many illnesses and clinical conditions, such as lichen planus, candidiasis, viral infections, xerostomia, etc., burning mouth syndrome (BMS) is characterized by pain without any changes to the oral mucosa. Xerostomia is regarded as a pathological condition characterized by abnormal and inadequate salivary production. Xerostomia is present in around one-fourth of the general female population.[4] Saliva, being the reflector of an arena of systemic diseases, its role has gained importance in various diagnostic and research fields.[5] The reported frequency of BMS in postmenopausal women varies across studies, but a commonly cited range is ≈18%–33%.

A healthy person’s daily saliva production ranges from 1 to 1.5 L. In adults, total stimulated salivary flow (SF) ranges from 1 to 3 mL/min, whereas hyposalivation is said to be characterized by an SF <0.7 mL/min. The normal unstimulated SF ranges from 0.25 to 0.35 mL/min, whereas hyposalivation is characterized by an SF of <0.1 mL/min.[6] The knowledge of normal salivary flow rate (SFR) is extremely important while treating dental patients. Early diagnosis and treatment of xerostomia and hyposalivation will preserve the health of oral structures or tissues and thus lower the incidence of dental caries, fungal infections, and other oral diseases that could result from insufficient SFR.[7]

During menopause, decreased estrogen levels have a significant impact on the oral cavity. SFRs among postmenopausal women are reduced to varying degrees in studies, which can lead to mouth discomfort and mucosal dryness. SF decline has been linked to decreased estrogen stimulation of the salivary glands, as well as probable degenerative alterations in glandular tissues. Changes in salivary composition, such as electrolyte, protein, and enzyme levels, have also been recorded. These biochemical changes could be the result of both systemic hormonal impacts and local glandular modifications.[8]

Calcium, an important ion, is required for enamel remineralization and mineral homeostasis. It is mostly found in saliva as free calcium ions and bound forms. Calcium is best known for its role in the formation of bones and metabolism. Calcium hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) accounts for more than 99% of total body calcium and provides strength to bones and teeth. Calcium in the circulatory system, extracellular fluid, muscle, and other tissues plays an important role in vascular contraction and vasodilation, muscular function, neuronal transmission, intracellular signaling, and hormone secretion. Through the process of bone remodeling, bone tissue serves both as a supply and a storage of calcium for these essential metabolic needs. The parathyroid hormone (PTH)–vitamin D endocrine system, which has multiple homeostatic feedback loops, is primarily responsible for controlling calcium metabolism. Most of the calcium found in foods in the typical diet comes from dairy products, such as milk, yogurt, and cheese, which are high in calcium. The intestinal mucosa absorbs calcium by both passive diffusion (paracellular) and active transport (transcellular). Calcium active transport is facilitated by the interaction between calcitriol and the intestinal vitamin D receptor. This transcellular route, which is mostly responsible for calcium absorption at low and moderate consumption levels, is activated by calcitriol.[9]

Calcium metabolism in the oral cavity is primarily controlled by the dynamic exchange of saliva, dental hard tissues, and oral mucosa, which aids in enamel remineralization, buffering capacity, and mineral homeostasis. Saliva normally contains both ionized calcium and protein-bound calcium (mostly albumin), and its concentration is affected by SF, pH, and systemic calcium balance. During menopause, low estrogen levels affect calcium control by increasing bone resorption and serum PTH, which might result in increased calcium release into saliva. Concurrently, lower SF and increased mucosal permeability enhance salivary calcium levels, increasing the risk of xerostomia, dental calculus formation, and impaired oral homeostasis.[10]

Salivary calcium concentration is affected by pH, flow rate, and systemic calcium metabolism.[11] Salivary calcium levels can fluctuate, indicating alterations in salivary gland function or systemic bone metabolism, both of which may be influenced by estrogen deprivation during menopause. Increased salivary calcium levels have been associated with decreased saliva flow, less buffering capacity, and an increased risk of dental calculus formation.[12]

Saliva is saturated with calcium and phosphate ions, which are required to prevent erosion of the enamel surface of teeth.[13] However, the presence of these ions can also result in the spontaneous formation of calcium phosphate particles when ions exceed homeostatic levels. Calcium phosphate particles are formed and deposited during pathological calcification in many diseases. Calcium phosphate particles are found in saliva and gingival crevicular fluid (GCF) in the oral cavity, and their abundance appears to correlate with oral diseases such as periodontitis; calcium phosphate is suspected to be an etiological factor in ectopic calcification-related diseases that involve dental calculus, dental pulp stones, and saliva gland stones.[14,15]

Albumin, on the contrary, is a plasma-derived protein that is often found in low concentrations in saliva. Its presence in saliva is mostly due to transport via the salivary gland epithelium and GCF. Elevated salivary albumin levels may indicate greater vascular permeability or glandular malfunction, which can be caused by hormonal or inflammatory changes. Thus, salivary albumin is a sensitive indication of oral mucosal and glandular integrity. In postmenopausal women, estrogen deprivation alters salivary gland structure and function, causing salivary albumin levels to rise. Estrogen generally promotes epithelial tight-junction integrity, glandular vascular stability, and regulated salivary secretion. When estrogen levels drop, the salivary gland epithelium gets thinner and becomes more porous, allowing albumin—a plasma-derived protein—to easily permeate into saliva. Furthermore, reduced SF (a frequent menopausal shift) concentrates salivary proteins, raising the detectable albumin level. This method differs from inflammation-driven albumin increase, which involves cytokines like interleukin-1 and tumor necrosis factor-alpha, causing vascular dilation and active exudation of plasma proteins. The increase in salivary albumin during menopause is predominantly hormonal rather than inflammatory and is caused by epithelium atrophy, altered protein transport, and decreased SF, not immune-mediated vascular leakage.[10,16]

Salivary concentrations of PTH and overall calcium turnover may be impacted by decreased estrogen levels in postmenopausal women experiencing oral dryness. Estrogen can precipitate calcium in bones and induce its absorption in the colon. Menopausal women with oral dryness have low estrogen levels, which cause the plasma calcium level to fluctuate downward and raise serum PTH. Because of its impact on bones, PTH raises plasma calcium levels to maintain ideal levels. However, the serum calcium level in these patients cannot rise noticeably since the plasma calcium level is controlled by numerous other factors. Consequently, it is plausible that more calcium is eliminated through urine or saliva.[17] The mechanism of saliva secretion is also closely related to the intracellular calcium concentration and is largely dependent on activation of muscarinic receptors present in salivary acinar cells.

Several studies have investigated the relationship between menopause and salivary changes. For example, research has revealed that SFRs decrease with age and menopause; however, the degree of loss varies by demographic. Similarly, studies on salivary electrolytes have shown that postmenopausal women had higher calcium levels, which could be attributed to systemic bone resorption and altered mineral metabolism caused by estrogen insufficiency.[1,18] Furthermore, although indicators such as SF or specific ions have been addressed, there has been little comprehensive study that compares SF, calcium, and albumin levels in postmenopausal women with and without oral dryness. Understanding the interrelationships between these characteristics can shed light on the mechanisms that drive oral alterations during menopause.

Early detection and treatment of xerostomia in postmenopausal women is clinically significant because oral dryness can exacerbate dental caries, periodontal disease, mucosal pain, and opportunistic infections. Identifying symptoms early allows clinicians to use preventive interventions such as saliva replacements, stimulants, remineralizing agents, and lifestyle changes. This prompt action promotes dental health, nutritional intake, and general comfort. Finally, preventive care helps postmenopausal women enhance their long-term dental health and quality of life.

NEED FOR THE STUDY

Postmenopausal hormonal changes can alter salivary composition, but the differences in calcium and albumin levels between women with and without oral dryness are unknown. This investigation is essential to uncover the variations and improve early detection and therapy of xerostomia in postmenopausal women.

MATERIALS AND METHODS

STUDY DESIGN

This presented case control design study was approved by the Ethical Committee of AB Shetty Memorial Institute of Dental Science. Informed consent was obtained from all the patients before the collection of the sample.

SAMPLE SIZE CALCULATION

where Z_α= 1.96, at 95% confidence interval (CI), Z_β = 1.282 at 90% power, σ = 2.88, pooled standard deviation, µ_d = 2.1, mean difference, and n = 40 in each group.

INCLUSION CRITERIA

Control group: Group I (G1)

1. Healthy female subjects 25–45 years.

2. Subjects who have not attained menopause and are not under any medication.

3. Subjects who do not have any systemic disease.

Study group: Group II (G2)

1. Female subjects who are in the age group of 45–70 years.

2. Subjects who have attained their menopause.

3. No menstrual cycle should have occurred for at least 24 months.

4. Female subjects who have attained their menopause, without symptoms of oral dryness.

Study group: Group III (G3)

1. Female subjects who are in the age group of 45–70 years.

2. Subjects who have attained their menopause.

3. No menstrual cycle should have occurred for at least 24 months.

4. Female subjects who have attained their menopause, with symptoms of oral dryness.

EXCLUSION CRITERIA

1. Subjects with a history of any long-term systemic diseases (such as diabetes and Sjogren’s syndrome) or with acute infections.

2. Obese subjects were excluded from the study (body mass index more than 30 kg/m²).

3. Subjects who are under hormone replacement therapy.

4. Subjects under any kind of medication.

Study Setting

This study was a case–control study conducted on subjects reporting to the Department of Oral Medicine and Radiology, AB Shetty Memorial Institute of Dental Sciences, Mangalore, during 2017–2019. Details were collected from the patients, such as the age of menopause.

The study sample included 120 female subjects and was divided into the following groups:

• Group I (G1): 40 healthy female subjects who have not attained menopause—control.

• Group II (G2): 40 female subjects who have attained menopause without oral dryness.

• Group III (G3): 40 female subjects who have attained menopause with oral dryness.

SAMPLE COLLECTION

Institutional ethical clearance was obtained. Informed consent was obtained, and the oral cavity was thoroughly examined, and a comprehensive case history was documented. To reduce diurnal fluctuations in sampling, saliva was collected between 9 AM and 12 PM. The “spit technique” was used to collect the individuals’ unstimulated saliva. An hour before the sample was taken, the subjects were told not to eat, drink, or smoke. They were instructed to tilt their heads forward while seated in the dental chair. They were instructed not to talk or swallow any saliva. For 5–15 min, the individuals were told to spit into a sterile graded container every 60 s. Whole mouth fluid is represented by the salivary sample that was obtained. The collected saliva sample was stored at −20°C for further analysis.

Sialometry

The collected saliva was then measured. Subjects with unstimulated whole saliva flow rates of >0.1 mL/min were considered as Group II, whereas unstimulated whole SFR < 0.1 mL/min was considered as Group III. The collected sample was centrifuged at 3000 rpm for 10 min, and the supernatant was collected and stored at −20°C.

SAMPLE COLLECTION AND HANDLING

Participants were advised not to eat, drink, smoke, or practice oral hygiene for at least 90 min before collection. Saliva collected in clean, preweighed containers when resting or agitated, with little contamination and constant posture and timing. To avoid evaporation or biochemical changes, samples were capped, tagged with a unique identifier, and stored on ice or in a cooled transport box right away. Samples were then weighed or measured for volume. Samples were stored at −20°C/−80°C for biochemical analysis.

Estimation of salivary calcium

Salivary calcium was estimated by the colorimetric method following the manufacturer’s instructions. All reagents were brought to room temperature. Reagents are mixed as described in the table and incubated for 5 min at room temperature. The absorbance was read against the standard and sample for the reagent blank at 612 nm. The amount of salivary calcium was measured by standard graph and expressed as mg/dL.

	Blank (µL)	Standard (µL)	Sample (µL)
Reagent	1000	1000	1000
Sample (µL)	-	-	10
Standard (µL)	-	10	-
Distilled water (µL)	10	-	-

ESTIMATION OF ALBUMIN

Salivary albumin levels were estimated by the enzyme-linked immunosorbent assay method following the manufacturer’s instructions. All reagents were brought to room temperature before the experiment. The absorbance was measured at 630 nm. The amount of salivary albumin was measured by a standard graph and expressed as mg/mL.

STATISTICAL ANALYSIS

Statistical analysis was performed using Statistical Package for the Social Sciences (IBM SPSS Statistics for Windows, version 20.0, IBM Corp., Armonk, NY, USA). Normality is determined using tests like the Shapiro–Wilk test, the Kolmogorov–Smirnov test, or by inspecting Q–Q plots and histograms. These evaluations demonstrated that the data are normally distributed, so parametric tests are used. A one-way ANOVA test was used for analysis of salivary calcium and albumin levels in all groups. Data expressed as mean ± standard deviation. Post hoc Tukey’s test was used for the comparison of study groups to the control group and the study groups with each other (SF, salivary calcium, and salivary albumin). Pearson’s correlation coefficient was used to correlate the changes in the levels of salivary calcium and salivary albumin between the study groups and the control group, and the study groups with each other. Analysis of covariance (ANCOVA) to show that calcium/albumin is independent of age. P < 0.001 was considered statistically significant.

RESULTS

A case–control study was conducted among 120 female subjects: 40 premenopausal women, 40 postmenopausal women without oral dryness, and 40 postmenopausal women with oral dryness.

• Group I (G1): 40 healthy female subjects who have not attained menopause—control.

• Group II (G2): 40 female subjects who have attained menopause without oral dryness.

• Group III (G3): 40 female subjects who have attained menopause with oral dryness.

DEMOGRAPHIC DATA ANALYSIS OF THE GROUPS

The age range of the subjects who comprised this group was 25–45 years. The mean age of the control group was 34.08 ± 5.87 years. Group II: the age range of the subjects who comprised this group was 45–70 years. The mean age of this group was 52.53 ± 4.79. Group III: the age range of the subjects who comprised this group was 45–70 years. The mean age of this group was 63.35 ± 3.84 years [Table 1].

Table 1.

Demographic summary of the study population

	Groups	N	Mean ± standard deviation
Age (years)	Group I	40	34.08 ± 5.87
	Group II	40	52.53 ± 4.79
	Group III	40	63.35 ± 3.84

Analysis of SF, salivary calcium, and salivary albumin levels in premenopausal women, postmenopausal women without oral dryness, and postmenopausal women with oral dryness. Mean SF in Group I, Group II, and Group III subjects: the mean SF in Group I was 3.42 ± 0.61 mL, whereas the mean SF in Group II and Group III was 1.95 ± 0.27 mL and 0.095 ± 0.22 mL, respectively [Table 2].

Table 2.

Salivary flow rate in the study population

	Groups	N	Mean ± standard deviation
Salivary flow (mL)	Group I	40	3.42 ± 0.61
	Group II	40	1.95 ± 0.27
	Group III	40	0.095 ± 0.22

MEAN SALIVARY CALCIUM AND ALBUMIN LEVELS IN GROUP I, GROUP II, AND GROUP III SUBJECTS

The mean salivary calcium levels in Group I were 5.56 ± 1.75 mg/dL, whereas the mean salivary calcium levels of Group II and Group III were 6.42 ± 0.63 mg/dL and 7.65 ± 0.55 mg/dL, respectively. The mean salivary albumin levels in Group I were 0.20 ± 0.08 mg/mL, whereas the mean salivary albumin levels of Group II and Group III were 0.45 ± 0.07 mg/mL and 0.64 ± 0.10 mg/mL, respectively [Table 3].

Table 3.

Mean salivary calcium and albumin levels in Group I, Group II, and Group III subjects

Parameters	Groups	N	Mean ± standard deviation	P value
Salivary calcium levels (mg/dL)	Group I	40	5.56 ± 1.75	<0.001
	Group II	40	6.42 ± 0.63
	Group III	40	7.65 ± 0.55
Salivary albumin levels (mg/mL)	Group I	40	0.20 ± 0.08	<0.001
	Group II	40	0.45 ± 0.07
	Group III	40	0.64 ± 0.10

On comparison of salivary calcium levels (mg/dL) between the three groups, it was observed that Group III had the highest value of 7.6 and Group I had the lowest value of 5.56. This difference was statistically significant with a test value of 71.305 and a P value of <0.001. Post hoc Tukey’s tests were used for comparing Group I and Group II subjects, which showed a mean difference of −0.86 and was statistically significant with a P value of 0.002. On comparing Group I and Group III subjects, a mean difference of −2.09 was noticed, which was statistically significant with a P value of <0.001. Similarly, on comparison of Group II and Group III subjects, a mean difference of −1.23 was seen and was statistically significant with a P value of <0.001.

While comparing salivary albumin levels (mg/mL) among the three groups, it was observed that Group III had the highest value of 0.64 and Group I had the lowest value of 0.20. This difference was statistically significant with a test value of 222.51 and a P value of <0.001. Post hoc Tukey’s tests were used to compare Group I and Group II subjects, which showed a mean difference of −0.24 and was statistically significant with a P value of <0.001. Furthermore, on comparison of Group I and Group III subjects, a mean difference of −0.43 was noticed, which was statistically significant with a P value of <0.001. While comparing Group II and Group III subjects, a mean difference of −0.19 was observed and was found to be statistically significant with a P value of <0.001 [Table 4].

Table 4.

Comparison of salivary calcium and albumin levels between the three groups

Dependent variable	(I) Group	(J) Group	Mean difference (I–J)	Standard error	P value
Salivary calcium levels (mg/dL)	Group I	Group II	−0.86	0.25	0.002
		Group III	−2.09	0.25	<0.001
	Group II	Group III	−1.23	0.25	<0.001
Salivary albumin levels (mg/mL)	Group I	Group II	−0.24	0.02	<0.001
		Group III	−0.43	0.02	<0.001
	Group II	Group III	−0.19	0.02	<0.001

Pearson’s correlation coefficient was used to correlate the salivary calcium and salivary albumin levels between the groups.

• Group I: A positive correlation was observed between the salivary calcium levels and salivary albumin levels (r = 0.775).

• Group II: A positive correlation was observed between the salivary calcium levels and salivary albumin levels (r = 0.417).

• Group III: A positive correlation was observed between the salivary calcium levels and salivary albumin levels (r = 0.505) [Table 5, Figure 1].

Table 5.

Pearson’s correlation to correlate the salivary calcium and salivary albumin levels between the groups

Group	Salivary albumin levels (mg/mL)
Group I
Salivary calcium levels (mg/dL)
Pearson’s correlation	0.775
Sig. (two-tailed)	<0.001
N	40
Group II
Salivary calcium levels (mg/dL)
Pearson’s correlation	0.417
Sig. (two-tailed)	0.007
N	40
Group III
Salivary calcium levels (mg/dL)
Sig. (two-tailed)	0.001
N	40

Figure 1.

Pearson’s correlation to correlate the salivary calcium and salivary albumin levels between the groups

An ANCOVA was conducted to compare salivary albumin, calcium, and SFR between postmenopausal women with and without oral dryness, adjusting for age as a covariate. Albumin levels remained significantly higher in postmenopausal women with oral dryness compared with those without dryness (F = 335.95, P < 0.001). The regression coefficient for group was β = 0.223 (95% CI: 0.199–0.247), indicating that the difference was independent of age. Age showed a statistically significant negative association (β = −0.00285, P = 0.013) [Table 6]. Similarly, calcium levels were significantly elevated in the dryness group independent of age (β = 1.243, standard error [SE] = 0.011, 95% CI: 1.222–1.264; F = 14099.58, P < 0.001). Age showed a significant effect (P = 0.024). SFR remained significantly reduced in the dryness group after age adjustment (β = −1.004, SE = 0.005, 95% CI: −1.014 to −0.995; F = 42858.11, P < 0.001), whereas age was not a significant covariate (P = 0.312). These findings confirm that the biochemical differences between postmenopausal women with and without oral dryness are independent of the 11-year age gap [Tables 7 and 8].

Table 6.

Multiple linear regression table showing that calcium/albumin is independent of age

Dependent variable	Effect	F value	P value	Partial η2P
Albumin	Group	335.20	<0.001	0.814
	Age	6.52	0.013	0.078
Calcium	Group	14,099.58	<0.001	0.995
	Age	5.32	0.024	0.065
Salivary flow rate	Group	4285.81	<0.001	0.998
	Age	1.04	0.312	0.013*

Analysis of covariance (ANCOVA) was used to compare salivary albumin, calcium, and salivary flow rate between postmenopausal women with and without oral dryness after adjustment for age. The group represents postmenopausal women with and without oral dryness. Age was included as a covariate to control for age-related effects. The F value indicates the strength of the association after age adjustment. Partial eta-squared (η²p) represents effect size. Albumin levels were significantly higher in the postmenopausal group with oral dryness compared with those without oral dryness (F = 335.20, P < 0.001, partial η² = 0.814), even after controlling for age. Age also showed a statistically significant but smaller effect (F = 6.52, P = 0.013). Salivary calcium levels differed significantly between groups after age adjustment (F = 14,099.58, P < 0.001, partial η² = 0.995). Age had a modest but statistically significant effect (F = 5.32, P = 0.024). Salivary flow rate also showed a significant group effect after controlling for age (F = 4285.81, P < 0.001, partial η² = 0.998), whereas age was not a significant covariate (F = 1.04, P = 0.312). Overall, these findings indicate that the observed differences in salivary calcium, albumin, and flow rate between postmenopausal women with and without oral dryness are independent of age.

^*P < 0.05 is considered statistically significant

Table 7.

Age-adjusted mean values (estimated marginal mean ± standard error)

Variable	Group II	Group III
Albumin	0.426 ± 0.0063	0.649 ± 0.0063
Calcium	6.41 ± 0.0055	7.65 ± 0.0055
Salivary flow rate	1.949 ± 0.0025	0.945 ± 0.0025

Table 8.

Analysis of covariance adjusting for age between postmenopausal women with and without oral dryness

Dependent variable	Effect	β	SE	95% CI	F value	P value	Partial η2P
Albumin	Group	0.223	0.012	0.199 to 0.247	335.20	<0.001	0.814
	Age	−0.003	0.001	−0.005 to −0.00067	6.52	0.013	0.078
Calcium	Group	1.243	0.011	1.222 to 1.264	14099.58	<0.001	0.995
	Age	−0.0022	0.00095	−0.00413 to −0.0003	5.32	0.024	0.065
Salivary flow rate	Group	−1.004	0.005	−1.014 to −0.995	42858.11	<0.001	0.998
	Age	0.00045	0.00045	−0.00043 to 0.00134	1.04	0.312	0.013

^*P < 0.05 is considered statistically significant

DISCUSSION

Menopause is the loss of ovarian follicular activity that results in the permanent cessation of menstruation. This definition uses both a symptom that can be identified by a woman, which marks the end of menstruation, and a sign that can be measured by the loss of follicular activities resulting in an alteration in levels of hormones. Effectual changes in circulating estradiol level, including an increase at menarche and a decrease at menopause, occur in a woman’s lifetime. The level of estradiol varies among different races, but irrespective of that, the circulating levels of estradiol reduce drastically during the transitional phase of menopause^.[19] Although over 300 different medications, such as antihistamines and decongestants, can cause “dry mouth,” certain physiologic conditions can also result in the same. “Menopause” is one such condition resulting in dry mouth, which is usually referred to as “Xerostomia.”[20]

A case–control study was conducted among 120 female subjects: 40 premenopausal women, 40 postmenopausal women without oral dryness (control), and 40 postmenopausal women with oral dryness. In this study, salivary calcium and albumin levels were estimated in premenopausal women, postmenopausal women without oral dryness, and postmenopausal women with oral dryness. In the current study, the mean age of subjects in the control group (G1) was 34.08 ± 5.872 years, whereas in postmenopausal women without oral dryness (G2), the mean age was 52.53 ± 4.793 years and in postmenopausal women with oral dryness, the mean age was 58.35 ± 3.84 years, which is in accordance with the study by Singh et al.,[21] where the mean age for postmenopausal women with oral dryness was 60.4 years and for postmenopausal women without oral dryness was 57.9 years. In this study, in the PM group, the minimum age was 45 years, and the maximum age was 70 years^.[21] In a woman’s life span, during middle age, the circulating level of estrogen alters, which is responsible for various physiological changes occurring in the body. These bodily changes can, in turn, affect the quality of life and the psychological well-being of the individual. In this study, the subjects were assessed for oral dryness and were categorized as postmenopausal women with and without oral dryness based on sialometry. Sialometry can be used as a diagnostic tool according to Kalk et al.[22] mainly in two ways: (1) collection of whole saliva, corresponding to combined secretions of all salivary glands, and (2) collection of glandular saliva, corresponding to gland-specific saliva. During sialometric analysis, unstimulated flow rates of <0.03 mL/min per gland from the parotid gland, submandibular, and sublingual glands were considered low^.[22] In this study, on the estimation of SFRs, it was found that the postmenopausal women with oral dryness had the least SFR, which was in accordance with the previous studies.[23] The decreased SFR in postmenopausal women with oral dryness in this study was also in conformity with the study by Singh et al.[21] and Mahesh et al.,[21,23] where they also found that there was improvement in the flow rate in individuals who were on hormone replacement therapy, supporting the role of estrogen in alteration of SFR through the estrogen receptors.[2,24] The generally accepted limit for the stimulated SFR is less than 0.5–0.7 mL/min, and for unstimulated SF, it is <0.1 mL/min measured for 5–15 min.[10] To ensure accuracy and reproducibility in sialometry, all instruments used to measure SF, such as preweighed collection tubes, electronic weighing balances, and timing devices, have to be calibrated regularly in accordance with the manufacturer’s guidelines, with documentation kept throughout the study period. Standardizing the collecting process, training all investigators in similar procedures, and conducting pilot measures to determine observer consistency all help to improve interobserver reliability. Furthermore, proper sample processing is required; saliva is required to be collected under controlled settings (ideally in the morning, with participants fasting or refraining from eating, drinking, or oral hygiene for at least 90 min), immediately labeled, and stored in sterile containers.

In the current study, we observed that the severity of oral dryness was higher in Group III postmenopausal women compared with Group I and Group II subjects. The probable reason could be explained by the fact that xerostomia is a subjective sensation that can occur in individuals with changes in the quality of saliva, but not necessarily affecting the quantity. This could lead to a dry mouth feeling without SF alteration. Previous studies carried out have shown that it generally disappears with suitable hormone replacement treatment.[25]

In this study, on comparison of salivary calcium levels among the three groups, it was observed that postmenopausal women with oral dryness had the highest value (7.65 mg/dL), and the premenopausal group had the lowest value (5.56 mg/dL), and this difference is statistically significant (P < 0.001). On comparison of salivary calcium levels among premenopausal (Group I) and postmenopausal women without oral dryness (Group II), the mean difference was statistically significant (P < 0.001). On comparing premenopausal (Group I) and postmenopausal women with oral dryness (Group III), a statistically significant mean difference (P < 0.001) was observed. Furthermore, on comparison of postmenopausal women with oral dryness (Group III) and without oral dryness (Group II), a statistically significant mean difference was seen (P < 0.001). In this study, the mean salivary calcium levels in Group II and Group III were increased compared with the control group, which is in accordance with previous studies.[26] They also concluded that the severity of oral dryness in postmenopausal women is associated with increased levels of salivary calcium. Thus, increased salivary calcium levels showed a positive correlation with oral dryness in postmenopausal women. The studies of Soundarya et al.[27] found an increase in calcium levels in postmenopausal women, and the studies of Agha-Hosseini et al.[28] are in accordance with our study.

In this study, the mean salivary albumin levels in the premenopausal group were 0.20 mg/mL, whereas postmenopausal women without oral dryness and postmenopausal women with oral dryness gave a value of 0.45 and 0.64 mg/mL, respectively. On comparison of salivary albumin levels (mg/mL) among the three groups, it was found that postmenopausal women with oral dryness (Group III) had the highest value (0.64 mg/mL) and the premenopausal group (Group I) had the lowest value (0.20 mg/mL). This difference was statistically significant (P < 0.001). Furthermore, on comparison of premenopausal (Group I) and postmenopausal women without oral dryness (Group II), there was a statistically significant mean difference (P < 0.001). Comparison of postmenopausal women with and without oral dryness also showed a statistically significant mean difference (P < 0.001). The probable explanation that can be thought of is the leakage of serum albumin into the oral cavity, which is estimated as salivary albumin. In individuals with oral changes like xerostomia and mucositis, the permeability of the oral mucosa increases, leading to increased leakage of serum albumin into the oral cavity^.[29] As salivary albumin level estimation in postmenopausal women was not reported in earlier literature, to the best of our knowledge, this is an important finding of this study, generally reported between 0.01 and 0.15 g/dL (or 0.1–1.5 mg/mL).[30,31]

Although the current study predominantly relates the increase in salivary albumin in postmenopausal women to hormonal changes, local periodontal variables also play an important role that must be acknowledged. Salivary albumin is mostly derived from GCF, which increases dramatically in the presence of gingival inflammation. Clinical signs such as bleeding on probing (BOP), higher community periodontal index scores, and increased probing depths indicate sulcular epithelium rupture and increased vascular permeability, allowing plasma proteins such as albumin to escape into saliva. Recent studies have established a robust relationship between periodontal inflammation and higher salivary albumin levels, independent of systemic factors, emphasizing the necessity to account for gingival health when interpreting salivary biomarkers.[32]

In this study, the correlation of salivary calcium levels and salivary albumin levels in each group was determined using Pearson’s correlation coefficient. It was found that there was a positive correlation between the salivary calcium and salivary albumin in the premenopausal group. In postmenopausal women without oral dryness (Group II) and postmenopausal women with oral dryness (Group III), there was a positive correlation. As salivary albumin is the primary carrier for calcium, it can be rightly hypothesized that the serum albumin, which leaks into the oral cavity, shows a concurrent increase with salivary calcium levels, which are also excreted into the oral cavity as a part of the normal regulatory mechanism. Effective remineralization is hampered in a low-salivary-flow environment by decreased saliva, which restricts the transport and buffering action of calcium to the tooth surface. Despite the presence of calcium, prolonged exposure to acid and inadequate clearance make enamel and dentin more vulnerable to demineralization. In addition, stagnant conditions encourage calcium precipitation, which leads to calculus production and plaque retention. In general, calcium’s protective function is reduced, and the balance is shifted away from repair and toward surface damage and mineral deposition.

However, a larger sample size and a long-term assessment in the form of longitudinal studies are necessary to further authenticate the findings of this study. As the composition of saliva and oral dryness is multifactorial in nature, extensive salivary studies are required to comprehend the interrelationship between estrogen, calcium, albumin, and PTH levels and their relationship with OD.

LIMITATIONS

• The assessment of xerostomia was purely based on sialometry, as no established subjective tools—such as the Xerostomia Inventory or Questionnaire—were used to collect patient reports.

• Various possible confounding factors were not assessed, including dietary calcium intake, hydration status, stress, lifestyle habits, and undiagnosed systemic diseases, which could have altered salivary composition.

• The cross-sectional design makes it difficult to determine the causal relationship between menopausal hormone changes, changed salivary biochemistry, and oral dryness.

• Menopausal status is considered in the context of unavoidable age-related changes, as the intrinsic age inequalities between pre- and postmenopausal individuals limit any claim of age independence.

• Because periodontal inflammation was neither objectively measured nor controlled, it may have served as an uncontrolled confounder, which is one of the limitations of the current study. Due to sulcular epithelium rupture and increased vascular permeability, salivary albumin, which is mostly obtained from GCF, is known to rise in the presence of gingivitis or periodontitis. This study did not record clinical periodontal characteristics, including plaque index, clinical attachment level, probing depth, or BOP. To account for this possible confounding effect and to more clearly define the independent influence of menopause-related hormonal changes on salivary biomarkers, future research should include thorough periodontal examination.

• Because of the significant age disparities across groups, residual confounding cannot be eliminated even after ANCOVA adjustment. The significant age difference may have an independent impact on the biochemical functions of the salivary glands, which could explain the observed variations beyond the menopausal state alone.

CONCLUSION

The current study shows that postmenopausal women, particularly those suffering from oral dryness, have a significantly lower SFR, which is accompanied by a progressive increase in salivary calcium and albumin levels, compared with premenopausal women. These findings indicate that hormonal changes associated with menopause have a detrimental effect on both the amount and biochemical composition of saliva. The positive correlation found between salivary calcium and albumin across all groups suggests that alterations in salivary gland function and mucosal permeability may play an important role in the development of xerostomia throughout the postmenopausal period.

Clinically, the results show the use of salivary calcium and albumin as simple, noninvasive indicators for early detection of oral dryness in postmenopausal women. Early detection of these salivary variations can allow for timely preventive and therapeutic interventions, such as salivary stimulation, remineralizing measures, and personalized oral care strategies, lowering the risk of dental caries, mucosal discomfort, periodontal disease, and a decline in oral health-related quality of life. These findings suggest that increased mucosal permeability and altered mineral transport are key physiological features of the menopausal oral cavity, necessitating targeted preventive dental care.

CONFLICTS OF INTEREST

There are no conflicts of interest.

AUTHORS CONTRIBUTIONS

PST: Conceptualisation, sample collection, review and final editing of the manuscript. AK: Formal analysis of the sample, statistical analysis, drafting the manuscript. PGR: Formal analysis of the sample, statistical analysis, review and final editing of the manuscript. RA: Formal analysis of the sample, statistical analysis, review and final editing of the manuscript.

ETHICAL POLICY AND INSTITUTIONAL REVIEW BOARD STATEMENT

The present study was approved by the Ethical committee of AB Shetty Memorial Institute of Dental Science. Informed consent was taken from all the patients before collection of the sample.

PATIENT DECLARATION OF CONSENT

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

DATA AVAILABILITY STATEMENT

All the data generated is included the main manuscript file.

List of abbreviations

• ANOVA Analysis of Variance

• ANCOVA Analysis of Covariance

• BMS Burning mouth syndrome

• BOP Bleeding on probing

• CPI Community Periodontal Index

• ELISA Enzyme-Linked Immunosorbent Assay

• GCF Gingival crevicular fluid

• IL-1 Interleukin-1

• OD Oral Dryness

• PM Post-menopause

• PTH Parathyroid hormone

• SF Salivary Flow

• SFR Salivary flow rate

• SPSS Statistical Package for Social Sciences

• TNF-α Tumor Necrosis Factor-alpha

• VDR Vitamin D receptor

• WHO World Health Organization

Funding Statement

Nil.