Abnormal thyroid function has important public health consequences (1). Thyroid hormones play an important role in human body metabolism (2). Subclinical thyroid disease is a common disorder, particularly in middle-aged and elderly individuals and the clinical significance of subclinical thyroid dysfunction is much debated (3,4).
Mixed saliva consists mainly of water (98%), electrolytes, mucus, antibacterial constituents, and various enzymes (5). The function of the saliva glands is regulated by the sympathetic nervous system which influences saliva circulation (6). It is well known that the composition of saliva depends on a number of factors related to physiological, pathological, environmental factors, and the humoral agents, including thyroid hormones (6,7). The mixed saliva composition may differ on account of varying contribution rate and quality from the individual glands, the presence or absence of a stimulatory source, the type of food ingested, the circadian cycles and the presence of infections (7). It is known that saliva could offer an excellent alternative to serum as a biological fluid analyzed for diagnostic purposes. This would be of great biomedical importance. Because it is very easy to collect, offering a cost-effective approach for screening of large populations, and could represent an alternative for patients whose blood is difficult to obtain or when compliance is a problem (8).
Zinc (Zn) is an essential trace element that is present in small amounts in all tissues and body fluids, including saliva (5,7). Zn, an intracellular signaling molecule and an anti-in?ammatory agent, is instrumental in immune response and serves important functions in the body with its antioxidant properties (9). Thyroid hormones could affect the tissue-specific distribution and availability of some minerals. In some studies, alterations in Zn homeostasis have been reported in subjects with thyroid disorders (10). However, there are inconclusive data on alteration in Zn status in patients with thyroid diseases. In addition, to our knowledge, saliva Zn level which is a good indicator of body Zn status has not been investigated yet in patients with thyroid diseases. Therefore, in the present study, we have investigated saliva Zn levels and correlations between saliva Zn and serum thyroid hormones (TSH, free T3 and free T4) levels in patients with thyroid diseases.
This study was performed on 126 patients with thyroid function disorders (21 male, 105 female) aged 18-70 years (mean: 38.19±12.50) and 38 control subjects (7 male, 31 female) aged 18-70 years (mean: 36.3±10.2). Among the subjects who participated in this study, 31(9 male, 22 female) had hyperthyroidism, 30 (2 male, 28 female) - hypothyroidism, 31 (5 male, 26) - subclinical hyperthyroidism and 34 (5 male, 29 female) had subclinical hypothyroidism. Patients with a free T3 of >4.71 pg/mL and free T4 of >1.9 ng/dL were considered to have hyperthyroidism, while those with a free T3 of <1.57 pg/mL and free T4 of <0.8 ng/dL were diagnosed with hyperthyroidism. The diagnosis of subclinical hypothyroidism was established in subjects with TSH of >4.2 µU/mL and serum free T3 and free T4 levels within the reference ranges. Subjects with TSH of < 4.2 µU/mL and serum free T3 and free T4 levels within the reference ranges were considered to have subclinical hypothyroidism. The normal range for serum TSH, free T3 and free T4 levels are 0.27 to 4.2 (µU/mL), 1.57 to 4.71 (pg/mL) and 0.8 to 1.9 (ng/dL), respectively in our laboratory.
Dietary patterns of the subjects were estimated using a food intake questionnaire. All subjects ate ordinary food. Less than 5% of patients with thyroid diseases and control subjects reported tobacco use. Exclusion criteria for the study included malignant disease, diabetes mellitus, chronic liver disease, chronic kidney disease, infectious disease, hypertension, a history of cardiovascular disease and taking Zn supplements. The study protocol was approved by the Ethics Committee of Meram Medical School, University of Selcuk, Konya, Turkey. All patients were informed of the details of the study and written informed consent was obtained from all subjects.
Body height and weight were measured with participants wearing indoor clothing without shoes. Body mass index (BMI) was computed by dividing the body weight (in kilograms) by height (in meters) squared. Blood sampling was performed in the fasting state at 08.00 h. Serum glucose, TSH, free T3 and free T4 levels were measured immediately.
The study of saliva secretion was performed without any stimulus in the morning (09:00h-11:00h) under standard temperature and humidity conditions. All subjects refrained from eating, drinking and smoking for a minimum of 2 h before saliva collection. Subjects were comfortably seated and after a few minutes of relaxation, they were trained to avoid swallowing saliva; later they were asked to lean forward and spit all the saliva they produced for 10 min into a graduated test tube through a glass funnel. Immediately after the collection, the saliva sample was transferred to clean and minerals free microtubes and than centrifuged at 12 000 rpm for 5 min to remove the precipitate. The supernatant was frozen at -80 oC until assay.
Measurement of Zn concentrations
Zn concentrations were determined using atomic absorption spectrophotometer (Rayleigh WFX-320). For the determination of Zn in saliva, the slit width was 0.4 nm, lamp flow was 3.0 mA, wavelength was 213.9 nm. The linear range of Zn standard curve was 0.0-0.5 µg/ml. Detection limit of Zn was 0.007 µg/ml. The test repeatability was assured by rejecting the results if the percentage of Related Deviation Standard (RDS%) was more than 10%. All of the reagents were of analytical grade and water was double distilled. Just before analysis, the samples were allowed to defrost at room temperature and vigorously shaken in a vortex for 30s to re-homogenize it just before analysis. Aliquot (1mL) of saliva samples was diluted with 2 mL of 1% nitric acid (Merck) immediately before the assay. The levels of saliva Zn were calculated after application of absorbencies on suitable calibration curve for each element made from standard solutions. Concentrations of Zn elements were calculated as µg/L mixed saliva volume.
Measurement of other analyses
Serum glucose was measured by commercially available kits based on routine methods on the Synchron LX System (Beckman Coulter, Fullerton CA). Serum TSH, free T3 and free T4 levels were measured by routine chemiluminesans method on E170 analyzer (Roche Diagnostics).
All data are expressed as mean ± standard deviations (SD). Statistical analyses were done using SPSS version 16.0 for Windows. The differences between two means were analyzed by one-way analysis of variance (ANOVA). Differences were considered significant at a probability level of p<0.05.
Clinical and analytical characteristics and saliva Zn levels in patients with thyroid disorder and control subjects are presented in Table 1. As seen, serum free T3 and free T4 levels were significantly higher in hyperthyroid subjects than in controls. BMI and serum TSH levels were significantly higher and serum free T4 levels were significantly lower in hypothyroid subjects than in controls. On the other hand, serum free T3 and free T4 levels were significantly lower and serum TSH levels were significantly higher in hypothyroid subjects than in hyperthyroid ones.
Furthermore, BMI and serum TSH levels were significantly higher in subclinical hypothyroid subjects than in controls. Saliva Zn levels were significantly lower in hypothyroid and subclinical hypothyroid subjects than in control subjects. On the other hand, saliva Zn levels did not correlate with serum thyroid hormone levels (TSH, free T3 and free T4) in the groups (Table 2).
In this study, we found that hypothyroid and subclinical hypothyroid group had a significantly decreased saliva Zn levels compared with the control group. Besides, to our knowledge, saliva Zn levels in patients with thyroid diseases have not been investigated yet. As serum levels of a variety of electrolytes are correlated to saliva levels of these electrolytes, it is possible that some of the factors that affect serum Zn levels will also affect saliva Zn levels (11).
There are a few works in the literature investigating the level of serum Zn in patients with thyroid diseases. Although there are some conflicting findings, generally, the findings of the other investigators support ours. For example, Al-juboori et al. (2) Zhang et al. (12), and Dolev et al. (13) have found that serum Zn levels were lower in hypothyroidism patients than in control subjects. In addition, Buchinger et al. (14) have found that hypothyroid patients had a most significantly reduced Zn content in serum compared to euthyroid patients. However, Aihara et al. (15) have found that there was no difference in plasma Zn concentrations between patients with thyroid diseases and control subjects.
It has been reported that various factors including diet, stress, infection, age, pregnancy, medications and oral contraceptives affect serum Zn levels (11). Possible explanations for these findings were reported to be severely impaired gastrointestinal absorption of Zn and a change in Zn distribution in subjects with hypothyroidism (2). It is known that, a low serum Zn level due to change in Zn distribution may reflect sequestration of Zn by the liver or other tissues. Besides, this status may be due to significant influence of TSH on the variation of the concentration of iodine, selenium and Zn in normal and altered human thyroid tissue (2). On the other hand, changes in body metabolic rate have been shown to be reflected in altered Zn metabolism. For example, thyroxine administration to rats was reported to significantly enhance the rate of Zn-turn-over and altered its distribution in various organs leading to an almost 50% depletion in soft tissue levels of Zn. Zn turnover and utilization were significantly arrested in rats made hypothyroid by iodine treatment (15). We concluded that decreased saliva Zn levels are the cause of hypothyroid disease. There are some limitations in our study. One of these limitations is the small sample size. Besides, parallel measurement of Zn both in plasma and saliva may provide more informative results.
In conclusion, our results show that saliva Zn levels are affected by hypothyroidism. Also, further studies involving larger number of subjects and parallel measurement of Zn both in plasma and saliva may provide additional information regarding the treatment of thyroid diseases.
1. Canaris GJ, Manowitz NR, Mayor G, Ridgway EC. The Colorado thyroid disease prevalence study. Arch Intern Med 2000;28:526-34.
2. Al-Juboori IA, Al-Rawi R, A-Hakeim HK. Estimation of serum copper, manganese, selenium and zinc in hypothyroidism patients. IUFS J Biol 2009;68:121-6.
3. Canaris GJ, Manowitz NR, Mayor G, Ridgway EC. The Colorado Thyroid Disease Prevalence Study. Arch Intern Med 2000;160:526-34.
4. Biondi B, Cooper DS. The clinical significance of subclinical thyroid dysfunction. Endocr Rev 2008;29:76-131.
5. Wang D, Du X, Zheng W. Alteration of saliva and serum concentrations of manganese, copper, zinc, cadmium and lead among career welders. Toxicol Lett 2008;176:40-7.
6. Koczor-Rozmus A, Zwirska-Korczala K, Sadlak-Nowicka J, et al. Evaluation of salivary gland function in women with autoimmune thyroid diseases. Wiad Lek 2003;56:412-8.
7. Burguera-Pascu M, Rodríguez-Archilla A, Burguera JL, et al. Flow injection on-line dilution for zinc determination in human saliva with electrothermal atomic absorption spectrometry detection. Anal Chim Acta 2007;600:214-20.
8. Mata AD, Marques D, Rocha S, et al. Effects of diabetes mellitus on salivary secretion and its composition in the human. Mol Cell Biochem 2004;261:137-42.
9. Prasad AS. Zinc: role in immunity, oxidative stress and chronic inflammation. Curr Opin Clin Nutr Metab Care 2009;12:646–52.
10. Kucharzewski M, Braziewicz J, Majewska U, Gózdz S. Copper, zinc, and selenium in whole blood and thyroid tissue of people with various thyroid diseases. Biol Trace Elem Res 2003;93:9-18.
11. Greger JL, Sickles VS. Saliva zinc levels: potential indicators of zinc status. Am J Clin Nutr 1979;32:1859-66.
12. Zhang F, Liu N, Wang X, Zhu L, Chai Z. Study of trace elements in blood of thyroid disorder subjects before and after 131I therapy. Biol Trace Elem Res 2004;97:125-34.
13. Dolev E, Deuster PA, Solomon B, et al. Alterations in magnesium and zinc metabolism in thyroid disease. Metabolism 1988;37:61-7.
14. Buchinger W, Leopold B, Lind P, et al. Changes in zinc level in the serum, whole blood and erythrocytes in disorders of thyroid function. Wien Klin Wochenschr 1988;100:619-21.
15. Aihara K, Nishi Y, Hatano S, et al. Zinc, copper, manganese, and selenium metabolism in thyroid disease. Am J Clin Nutr 1984;40:26-35.