Background
Metabolic syndrome (MS) is a disease that poses significant risk of developing diabetes mellitus (DM) and cardiovascular disease (CVD). It is indicated by the presence of three or more of the following components: abdominal obesity, raised fasting glucose, dyslipidemia (raised triglyceride (TG) with lowered high-density lipoprotein cholesterol (HDL-C)), and raised hypertension. Waist measurement for abdominal obesity varies between gender and among different populations [
1]. The number of patients with MS is expanding worldwide. The prevalence in developed and developing countries is comparable, ranging from 15.2% to 43.7% [
1‐
4].
Many of the components of MS are connected through physiological pathways. Adiponectin, an anti-atherogenic and anti-inflammatory adipocytokine involved in glucose and lipid metabolism, improves insulin sensitivity [
5]. Adiponectin levels in plasma are inversely correlated with visceral adiposity. Lower levels of adiponectin were observed in patients with high blood pressure, hyperglycemia, low HDL-C, and hypertriglyceridemia, and in obese patients with MS [
6]. Brooks et al. showed that a low level of circulating adiponectin may be used as a possible biomarker for MS [
7]. Leptin, an anti-obesity adipocytokine, regulates body weight by modifying energy levels and increasing metabolic rate while decreasing food intake. Most overweight and obese patients show resistance to leptin at the receptor level, and therefore have higher leptin levels than non-overweight individuals [
8]. Serum leptin levels in patients with MS are higher than those in healthy controls [
9]. Adiponectin and leptin levels show an inverse correlation with each other [
10].
The relationship between protein levels in saliva and plasma makes saliva an attractive diagnostic tool that could be used as an alternative to blood in tests measuring biomarkers [
11]. Currently, assays are available to analyze various salivary parameters. Measurement of adiponectin and leptin in saliva is simple, non-invasive, and may be an acceptable alternative to plasma sampling. Previous studies have reported successful measurement of adiponectin and leptin in saliva from healthy individuals or DM patients [
12‐
15]. To the best of our knowledge, no previous studies have analyzed the levels of these biomarkers in saliva from patients with MS. Thus, the aim of this study was to determine adiponectin and leptin profiles from saliva and plasma in patients with MS compared with those of healthy patients. We aimed to measure correlations between salivary and plasma adipocytokines in both groups to determine if an association exists between these biomarkers and MS.
Materials and methods
Study participants
Patients being seen at the Golden Jubilee Medical Center (Mahidol University) were randomly assessed for MS. All participants were fully informed before completing their written consent document. The protocol and consent forms were approved by The Ethics Committee of Mahidol University and Tokyo Medical and Dental University, which conforms to the Helsinki Declaration (Reference Number: MU-DT/PY-IRB2011/134.3006, TMDU-IRB 2012/1108.860). Waist circumference was measured, and levels of TG, HDL-C, fasting plasma glucose (FPG), and blood pressure (BP) were evaluated from patient medical chart records. MS was diagnosed when three of the following five factors were present: (1) elevated waist circumference (≥85 cm in Thai men and ≥80 cm in Thai women); (2) elevated TG level (≥150 mg/dL); (3) reduced HDL-C (<40 mg/dL in men, and <50 mg/dL in women); (4) elevated blood pressure (systolic BP (SBP) ≥130 or diastolic BP ≥85 mmHg); and (5) elevated FPG (≥100 mg/dL) [
16].
Eighty-two patients newly diagnosed with MS and never receiving medications were included in this study. Patients with systemic diseases as well as those who had received medications were excluded. Forty-six subjects with healthy status according to case selection procedure were recruited as a control group. Demographic data were collected. The height and body weight of participants were also recorded and body mass index (BMI) was calculated.
Plasma collection
On the same date that peripheral vein blood was drawn to determine blood chemistries, a second blood sample was centrifuged to obtain plasma and a 1-mL aliquot was kept frozen (-80°C) for further adipocytokine analyses.
Saliva collection
Unstimulated whole saliva was collected from all patients. Each individual was requested to abstain from eating, drinking, smoking, and brushing his/her teeth for at least 60 min prior to collection. Saliva samples were collected between 9 a.m. and 12 noon. Unstimulated whole saliva was collected using the drooling technique. Each subject rinsed their mouth with water before saliva collection, then the patient was asked to swallow to remove saliva from the mouth. The patient was seated upright, and leaned their head forward over a test tube with a funnel, allowing their saliva to drain into the tube. Whole saliva (~5 mL) was obtained from each individual. During saliva collection, the test tube was placed on ice. At the end of the collection, any remaining saliva in the patient’s mouth was expelled into the test tube.
Saliva processing
Saliva obtained was centrifuged at 15,000 × g (MPW-65R, MPW.Med Instrument, Warszawa, Poland) for 15 min at 4°C to remove insoluble material. Supernatant was divided into 1-mL aliquots in pre-chilled cryotubes. The specimens were immediately frozen (-80°C) until analysis.
Assay for adiponectin and leptin
Saliva and plasma were thawed at room temperature for adipocytokine measurement by enzyme-linked immunosorbent assay (ELISA). A commercial ELISA kit for human adiponectin and leptin (RayBio®, Norcross, GA) was used and the assay was conducted according to the manufacturer’s instructions with minor modification as previously reported [
15]. Each sample of saliva and plasma from the same patient was run in the same experimental set and analyzed in duplicate. Plasma samples required 20,000-fold dilution for adiponectin and 100-fold dilution for leptin; saliva samples were analyzed without dilution.
Data analysis
SPSS 16.0 software for Windows (SPSS Inc., Chicago, IL) was used for statistical analyses. Descriptive statistics were calculated and scatterplots created for each biomarker. All continuous variables were first assessed for normality using the Kolmogorov-Smirnov test and biomarker data were positively skewed as anticipated. Results are displayed as median with interquartile range. Comparison between saliva and plasma adipocytokine was performed using the Wilcoxon signed-rank test. Comparison of saliva or plasma adipocytokines and other variables between healthy patients and patients with MS was performed using the Mann–Whitney U test. Spearman’s Rho correlation coefficients and coefficients of partial correlation controlling for age, gender, and BMI were calculated to examine relationships between saliva and plasma adipocytokines and variables of MS. Logistic regression analysis controlling for the effect of covariates was further performed to ascertain the independent predictive value and impact of variables significantly correlated with plasma adiponectin levels, plasma leptin levels, and MS. A p value of < .05 was considered to be statistically significant.
Discussion
Adiponectin and leptin were successfully detected in the saliva of patients with MS, although lower levels were observed compared with those in plasma. Salivary adiponectin, as well as the salivary adiponectin/salivary leptin ratio, was significantly correlated with its plasma levels, while salivary leptin levels were not. Between patients with MS and healthy individuals, significant differences were seen in adiponectin and leptin levels from plasma, but not from saliva. The levels of salivary adiponectin and leptin were in identical ranges to those found in previous studies that measured the levels in healthy subjects or in DM patients [
13,
14]. Our finding of a correlation between salivary and plasma adiponectin in this study is in agreement with previously published reports [
13,
17,
18]. Toda et al. found that there was a significant correlation between plasma and salivary adiponectin levels in an older group of healthy patients [
13]. Similar results have been recently reported in the studies of Mamali et al. and Akuailou et al., although the measured values of adiponectin were different, possibly due to saliva sample dilution and the characteristics of the patients [
17,
18].
However, uncorrelated salivary and plasma cytokines have been previously reported in other studies [
19,
20]. As in the present study, Aydin et al. saw that the levels of salivary leptin were almost the same as in plasma; no significant correlation between plasma leptin and salivary leptin was shown in all of the healthy patients in the study [
21]. Nevertheless, others have seen a strong correlation between salivary and plasma leptin in healthy individuals, with this correlation significantly higher in plasma than saliva [
12,
22]. Dissimilar results in the correlation between salivary and plasma leptin levels might be explained by the laboratory method used (RIA vs ELISA), molecular structure of leptin, or the expression and secretion of leptin by the salivary glands themselves [
12,
22,
23]. Furthermore, Randeva et al. reported circadian variation in the levels of salivary leptin. It reached low levels in the morning, the time at which saliva samples were collected in this study [
12]. Thus, concentrations of salivary leptin do not always correlate well with plasma concentrations, as is seen with adiponectin. Autonomous production, structure modification, secretory pattern, or varying patient metabolism could reflect the discrepancies in the level and correlation of salivary and plasma leptin.
Regarding the correlation of salivary adiponectin and leptin with age or BMI, only two previous reports have discussed salivary adiponectin [
13,
17]. Similar to our study, others found salivary adiponectin was not significantly correlated with either age or BMI. Contrary to our study, salivary leptin correlation with BMI was described by a single previous study [
12]. One interesting observation from our work was that the salivary adiponectin/salivary leptin ratio was significantly associated with waist circumference, although this association disappeared after controlling for age, gender, and BMI. No previous studies have documented this aspect, so it would be interesting for further studies to clarify this possible association of the salivary adiponectin/salivary leptin ratio with waist circumference. In addition, no previous reports directly investigated about salivary adiponectin and leptin with alcohol consumption, smoking habit or education level. However from our data analysis (data not shown), no statistically significant difference between healthy subjects and patients with metabolic syndrome according to these confounding factors and no correlation of these confounders with salivary levels of adipocytokines were found.
When we considered saliva and plasma adipocytokines between male and female patients, female patients in our study had higher plasma adiponectin and leptin levels compared with the male patients. Although slightly higher salivary adiponectin and leptin levels in men were observed in this study, no significant difference in salivary leptin and salivary adiponectin levels was observed between genders. Results showing that both saliva and plasma leptin levels are higher in female patients compared with male patients have been documented [
12,
21]. However, in the study by Randeva et al., the ratios of salivary and plasma leptin concentrations were higher in men [
12]. Sex steroid hormones, body fat content or genetic differences might be responsible for this difference observed between men and women [
12]. Despite this potential caveat, our findings extend the data of adiponectin and leptin levels in saliva of patients with MS.
Owing to the correlation of plasma adiponectin and leptin with MS components, this study found that plasma adiponectin and plasma leptin had distinct correlations with TG, HDL-C, DBP, and waist circumference. These results are consistent with many previous reports [
24‐
30]. Others have shown there is a positive correlation of plasma adiponectin and HDL-C, and an inverse relationship between this adipocytokine with TG, waist circumference, and low-density lipoprotein-cholesterol (LDL-C) [
31]. Similar to the data from Kawamoto et al. that showed an inverse association of TG and adiponectin [
32], TG and waist circumference in our study were associated with the risk of having a low level of plasma adiponectin, while HDL-C was inversely associated with a high level of plasma leptin. Identical results were reported in a study by Wannamethee et al. where increased leptin levels were independently associated with a decrease in HDL-C [
28]. Existing data for correlation between plasma adiponectin and age are controversial. Plasma adiponectin levels are reported as lower, unchanged, or higher as age increases [
13,
33,
34], whereas plasma adiponectin levels were negatively correlated with BMI, and were higher in female patients than in male patients [
25]. The present study noted a significant inverse association of plasma adiponectin levels with BMI, being higher in women than in men, while no association was observed with age. These results suggest that age, gender, and BMI could be the driving forces behind the correlations. Similar to previous studies, we reported the complex interaction of age, gender, and BMI with each component of MS [
35‐
37]. The effect of sex hormones might explain this discrepancy, as it has been hypothesized that androgen inhibits adiponectin secretion [
38].
Our results are in agreement with those of Schutte et al., who reported significant correlations of plasma adiponectin and plasma leptin with mean arterial pressure that disappeared after the effects of age and BMI were taken into account [
37]. Moreover, our study showed that FPG was clearly not associated with plasma adiponectin and plasma leptin after adjustments for age, gender and BMI. While previous studies have shown that there are strong associations between low adiponectin levels and impaired FPG or insulin resistance, independent of age, gender, or BMI [
24,
29,
33,
34,
39,
40], meta-analysis revealed that higher adiponectin levels are associated with a lower risk of type 2 DM [
41]. A controversial correlation between plasma leptin and BP and impaired fasting glucose or insulin resistance was published [
27‐
29,
40,
42], however Wannamethee et al. found no association of plasma leptin and blood glucose, BP, and total cholesterol, which was identical to the results of this study and those of Wang et al. [
28,
29]. Controversies about correlations between adiponectin, leptin, and blood glucose might be due to different sample populations, and future studies are necessary to verify these results. Finally, the logistic regression model in this study showed that age, BMI, and plasma adiponectin, not salivary adiponectin, levels were significantly associated with the presence of MS. Similar to previous studies showing plasma adiponectin was a strong predictor of MS in both men and women [
7,
24,
25,
43].
The findings of the present study should be considered as preliminary, and subject to some limitations. First, this study was designed to be cross-sectional, which limits its ability to eliminate causal relationships between salivary and plasma adipocytokines and MS. Longitudinal studies are required to validate the diagnostic usefulness of saliva and plasma adiponectin and leptin for the detection of MS. Second, the sample size was not large; the patients who had four or five components of MS were fewer than those who had three components. This might reflect the insignificant difference of plasma adiponectin or leptin levels among each component group, a finding that is different from previous studies where plasma adiponectin levels decreased as components of MS increased [
24,
25], with leptin showing the opposite trend [
33]. Our results need to be confirmed longitudinally and with a greater number of samples in order to find strong associations. Third, selection bias should be considered. The study group is not strictly a random population sample, being influenced by selection criteria and responses that might bias the results obtained from patients with MS versus healthy individuals. Last, another restriction is the lack of data for the homeostasis model assessment of insulin resistance (HOMA-IR). Owing to a laboratory limitation, adequate analysis for HOMA-IR has not been achieved.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ST: designed and performed the experiments, analyzed the data and wrote the manuscript; HW and ST: participated in the design of the study; YI: conceived and designed the experiments, and helped to draft the manuscript. All authors read and approved the final manuscript.