periodontal disease is a major oral health - related problem that affects a large number of patients all over the world . in this context , advanced periodontitis , with clinical attachment loss ( cal ) of 5 mm or more , affects approximately 1015% of the adult population worldwide . based on some observations , the most important risk factors for periodontal disease include poor oral hygiene , tobacco use , excessive alcohol consumption , stress and diabetes . tissue - destroying active cytokines have been reported to be responsible for the loss of connective tissue attachment and bone loss . different kinds of cytokines are synthesized in inflamed periodontal tissues by lymphocytes , monocytes and non - immune cells such as fibroblasts and epithelial and endothelial cells . cytokines are soluble glycoproteins that function as local signaling molecules to control and coordinate cell behavior and function . a hormone - like protein , leptin , which initially attracted attention due to its important roles in regulating weight , metabolism and function of reproductive organs , might play a role in some inflammatory conditions through its direct effect on innate and adaptive immune cells . leptin is an adipose - derived hormone secreted in proportion to the size and number of adipocytes . evidence shows that plasma leptin levels increase in obese subjects and decrease after weight loss . although leptin is predominantly synthesized by the adipose tissue , other tissues also produce this adipokine at low levels . leptin inhibits appetite , stimulates energy expenditure , and it can modulate lipid and bone metabolism , coagulation , hematopoiesis , function of pancreatic beta cells , insulin sensitivity , etc . . moreover , leptin regulates the immune system and inflammatory response in various organs [ 711 ] . leptin is predominantly expressed in the adipose tissue and is considered an important signaling molecule for the regulation of food intake and consumption of energy . in addition , leptin is produced in placenta , granulocytes , stomach and salivary glands . leptin is a 16d non - glycosylated peptide hormone classified as a cytokine , with structural similarities to cytokine families with long and intertwined chains , such as interleukin-6 . therefore , it has been suggested that leptin might modulate the host response to infectious agents and play a role in mounting inflammatory responses in the body through induction of synthesis of other proinflammatory cytokines and stimulation of phagocytosis by macrophages . the biologic functions of leptin include its antilipemic effects , its effects on reproduction , hematopoiesis , angiogenesis , blood pressure , osteogenesis and homeostasis of the lymphoid organs and t lymphocyte system . johnson and serio   in 2001 showed the presence of leptin in healthy gingival tissues and in the gingiva with marginal inflammation , which decreased with the progression of inflammation and an increase in pocket depth . in their study , apart from the adipose cells , the gingiva was also reported to be a source of synthesis of leptin . karthikeyan and pradeep   in 2006 reported a significant decrease in leptin levels in the gcf with an increase in the destruction of periodontal tissues . in addition , the same researchers in another study showed that contrary to the gcf , the serum leptin levels had a direct relationship with chronic periodontitis . in another study by sattari et al ,   in 2011 leptin was not reported to have an inflammatory role in periodontal diseases . overall , it appears that the expression of leptin gene can have an important role in the modulation of inflammatory processes during infection , similar to the response of other cytokines to tissue injuries and infection . the present study was carried out to compare the salivary leptin levels in healthy individuals and patients with advanced periodontitis . a total of 16 advanced periodontitis patients with a mean age of 32.063.70 years ( range 2637 years ) were selected among the patients referred to the department of periodontology at tehran university of medical sciences ( tums ) , school of dentistry . healthy control group consisted of 16 individuals with a mean age of 32.003.44 years ( range 2839 years ) . periodontal parameters examined in this study were plaque index ( pi ) , probing pocket depth ( ppd ) , cal and bleeding on probing ( bop ) . the subjects underwent clinical examination by a periodontist using a williams probe ( hu - friedy , chicago , il , usa ) . patients with advanced periodontitis , intraoral radiographic evidence of bone loss and cal more than 5 mm ( according to the american academy of periodontology international workshop for classification of periodontal diseases , 1999 ) and healthy individuals were evaluated . the exclusion criteria were systemic diseases , recurrent infections , allergic reactions , cancer , use of medications affecting periodontal status during the previous three months , use of antibiotics during the previous two months , history of acute psychological stress during the previous year , history of transplantation , drug abuse , history of radiotherapy , smoking , pregnancy , breastfeeding , menopause , acute malnutrition and history of periodontal therapy in the previous six months . then , the study protocol was explained to the subjects and written informed consent was obtained from those who agreed to participate in the study . 1 ) . whole saliva collection the patients were asked to abstain from eating and drinking ( except for water ) for one hour before collecting unstimulated whole saliva samples . to obtain more accurate results , the subjects were asked to abstain from smoking , chewing gums and any activity that stimulates saliva secretion for one hour before collection of samples . before collecting the samples , the patients rinsed their mouth with drinking water and evacuated their mouth without excessive movements of their head and remained seated for five minutes . then , after a deep swallowing action , they evacuated the saliva from their mouth by bending positioned to collect saliva samples , leaving their mouth slightly open so that saliva slowly flowed into the test tube . the saliva samples were stored in a freezer at 40c until all samples were collected . the salivary leptin levels were determined based on the instructions providedin the kit ( r&d systems , minneapolis , mn , usa ) . spearman s correlation coefficient was used to evaluate the correlation between the salivary leptin level and various clinical parameters . spearman s correlation coefficient was used to evaluate the correlation between the salivary leptin level and various clinical parameters . the age , sex and bmi of patients and healthy controls are depicted in table 1 . there was no significant difference ( p>0.05 ) in the mean age between the study groups . regarding the mean bmi , the current results found no significant difference ( p>0.05 ) in the mean bmi between the study groups ; the mean bmi was 22.291.42 kg / m in the periodontitis group , and this value was 21.941.36 kg / m in the healthy control group . the differences in clinical periodontal parameters in patients and healthy controls are summarized in table 2 . the mean values of pi , ppd , cal and bop were significantly higher ( p<0.001 ) in the periodontitis group compared to the healthy control group . the mean  sd salivary levels of leptin in the healthy individuals and patients with advanced periodontitis were 33.586.64 ( range 21.042.5 ) and 18.086.01 pg / ml ( range of 8.027.0 ) , respectively . in addition , the mean  sd salivary levels of leptin in the healthy females and males with advanced periodontitis were 18.85.87 pg / ml ( range 26.012.5 ) and 17.726.26 pg / ml ( range 8.027.0 ) , respectively ( table 3 ) . the results of two - way anova showed that the effect of sex on salivary levels of leptin was not significant ( p=0.91 ) ; however , the effect of advanced periodontitis on salivary levels of leptin was significant compared to healthy individuals ( p<0.0001 ) . the interaction effect of sex and advanced periodontitis on salivary leptin levels was not significant ( p=0.98 ) . age , sex and bmi of subjects in the study groups not significantly different ( p>0.05 ) . sd : standard deviation clinical periodontal parameters in all parameters p<0.001 descriptive statistics of salivary leptin levels in healthy subjects and patients with advanced periodontitis correlation between leptin levels and clinical periodontal parameters leptin is a hormone - like protein , which initially attracted attention due to its significant role in regulating weight and metabolism of the human body . however , subsequent research showed that leptin affects the body s defense mechanisms , including t cells , macrophages and endothelial cells . in addition , some studies have called it an acute - phase inflammatory protein . on the other hand , the salivary glands of mammals secrete leptin and high levels of epidermal growth factor and other growth factors daily into the oral cavity . since cells in the area , which have been traumatized ( or affected by periodontal disease ) can also synthesize growth factors with a role in the disease progress or wound healing , evaluation of mechanisms related to their protective effects on the course of periodontitis or other oral complications is of great importance . based on the available evidence , the salivary cytokines play a direct role in immune mechanisms in the oral cavity . for example , leptin , which is a member of il-6 family prevents a decrease in the synthesis of mucin in salivary glands after the activation of lipopolysaccharides . therefore , salivary leptin plays a role in the effect of mucin on preventing the growth of bacteria . such preventive effects of leptin on bacterial growth have been reported in the respiratory mucosa of mice , indicating the mucosal secretion of cytokines and their important role in preserving health . the salivary hormones not only modulate the activity of immune cells , but also they play a role in the activity of the oral immune system against prokaryotes . recently , use of saliva instead of blood for different diagnostic purposes has attracted attention   because the process of saliva sample collection is non - invasive and is preferred by many individuals . in this context , in the present study the salivary leptin levels were determined in healthy subjects and patients with advanced periodontitis . based on the results of the current study , there was a decrease in leptin salivary levels by 50% in periodontitis patients compared to healthy controls . the majority of previous studies have evaluated the serum levels of leptin in healthy individuals and during periodontal disease , using gcf . to the best of our knowledge , only a few studies have compared the salivary levels of leptin in healthy subjects and patients with periodontitis . purwar et al ,   in 2015 evaluated leptin levels in saliva and serum of patients with chronic periodontitis and healthy controls . they concluded that the level of salivary leptin in chronic periodontitis patients was significantly lower than that in healthy controls , consistent with the results of the current study . they also noticed that the level of serum leptin increased in patients with periodontitis compared to healthy volunteers . in another study in 2015 , purwar et al ,   found that appropriate non - surgical therapy can increase the leptin levels in the saliva of chronic periodontitis patients . selvarajan et al ,   in 2015 examined and compared the gcf concentration of leptin in patients with periodontal disease and healthy subjects . they observed a substantial decrease in leptin levels in gcf as the periodontal disease progressed . however , with the progression of periodontal disease , the protective effects of leptin on gingival tissues decrease due to a decrease in its concentration . karthikeyon and pradeep   in 2007 reported a significant decrease in the leptin concentrations of gcf with an increase in periodontal tissue destruction , with the highest and lowest leptin concentrations in the gcf in healthy subjects ( 2658 pg / ml ) and patients with chronic periodontitis ( 1312 pg / ml ) , respectively . in their study , gcf samples were evaluated , which are derived from the gingival blood vessels ; however , in the current study , salivary samples from healthy subjects and patients with advanced periodontitis were evaluated . on the other hand , johnson and serio   in 2001 evaluated leptin concentrations in healthy gingival tissues with a gingival sulcus index of 3 mm and in inflamed gingiva with periodontal pockets > 3 mm , with the use of elisa technique . the results showed the highest leptin concentrations in the gingiva adjacent to the sulcus with a maximum depth of 3 mm . based on the results , leptin was present in the healthy and inflamed human gingival tissues and its concentration decreased with an increase in pocket depth and aggravation of periodontal disease , consistent with the results of the present study , in which the salivary leptin levels were higher in healthy subjects compared to patients with advanced periodontitis . bozkurt et al ,   in 2006 evaluated leptin levels in the gcf and the effect of smoking on these levels . it appears that smoking disrupts the mechanism regulating leptin levels . in the current study , smokers were excluded from the study . therefore , the effect of smoking , as a confounding factor , on the results was eliminated . in another study , the concentration of leptin was determined in the gcf in healthy periodontium , in periodontium with chronic gingivitis and in chronic periodontitis . based on the results , there was an inverse correlation between leptin concentration in the gcf and the progression of periodontal disease , with leptin levels decreasing in the gcf with the progression of periodontal disease . aydin et al ,   in 2005 evaluated salivary and plasma leptin levels in healthy young subjects and reported lower salivary levels compared to plasma levels , with slightly lower salivary and plasma leptin levels in males compared to females . based on the results , there were no significant differences in salivary leptin levels between males and females in our study , consistent with the results of previous studies . another study showed a decrease in gcf leptin levels at one and 24 hours after orthodontic tooth movement , with an increase after 168 hours , which was attributed to changes in the periodontium after orthodontic tooth movement . the worsening of gingival tissue status during the early stages of orthodontic treatment is manifested by a decrease in gcf leptin levels . gangadhor et al ,   in 2011 reported a significant decrease in gingival leptin concentrations and a significant increase in plasma leptin levels concomitant with the progression of gingival disease . in their study , a decrease in gingival leptin levels in patients with periodontitis was attributed to its release into the blood serum . since adipocytes are considered a source for the synthesis of leptin , the presence of leptin in the gingival tissue , as reported by the researchers , was an interesting finding because no evidence is available at present in relation to the synthesis of leptin by cells such as fibroblasts or epithelial cells in the gingiva . in addition , since it is not clear which cells produce leptin in the gingiva , its presence in the gingiva might be attributed to its entrapment in the gingival tissue subsequent to its release into the bloodstream . in their study , a significant decrease in salivary leptin levels in patients with advanced periodontitis was observed in comparison to healthy subjects , consistent with the current study ; however , saliva samples were evaluated in the current study while samples were taken from gingival tissues in their study . in the current study , samples were not taken from the plasma of healthy subjects and patients with severe periodontitis . however , lower salivary leptin levels have been reported compared to plasma levels [ 33,36 , 37 ] . therefore , it is not possible to make a correct estimate of leptin plasma levels through determination of salivary leptin levels , which might be explained by the saturated leptin transfer mechanism in the salivary glands . higher salivary leptin levels indicate its protective role in gingival tissues . however , the exact mechanism of its protective role has not been explained and this finding might be accidental , without any cause - and - effect relationship . in addition , the mechanism of its decrease in the saliva of periodontal patients has not been elucidated . it appears that leptin is used as a substrate during the inflammatory process ; thus , its concentration decreases . in the current study , salivary samples were immediately evaluated after being catabolized by the enzyme using the elisa technique ; thus , it is possible that the leptin stored in the cells was released by the enzymatic effect and included in calculations in addition to free leptin . however , further studies are recommended to elucidate the mechanisms involved in changes in salivary leptin levels in periodontal diseases . in general , saliva contains a combination of materials secreted into the gingival sulcus , i.e. gingivo - crevicular transudate , saliva from the salivary glands , debris , bacteria , epithelial cells , hormones and mucus . the gcf is filtered by plasma and enters into the oral cavity by seepage from the very fine blood vessels in the gingival sulcus . it contains components similar to immunoglobulins found in human plasma ; however , their concentration is 8001000 times less than those in the plasma . in addition , gcf contains high levels of igg and igm and small amounts of iga , which have enriched glandular saliva to some extent . this fluid has been used in many studies to estimate leptin concentrations in healthy gingival tissues and in gingival tissues affected by periodontitis . the effect of saliva on wound healing has been shown in animals [ 4042 ] and salivary cytokines such as leptin might have a role in the antimicrobial capacity of the saliva , in association with epidermal growth factor . leptin might affect some biological mechanisms as well , including immune and inflammatory reactions , hematopoiesis , angiogenesis , osteogenesis , and wound healing . high concentrations of leptin in the gcf in healthy periodontal status , too , might have a protective role in this respect because leptin affects the immune system . nokhbehsaim et al ,   in 2014 evaluated the serum levels of leptin in patients with periodontitis with and without diabetic mellitus type ii . the authors believe that the discrepancies observed between various studies could be attributed to the differences in the sample size of studies , differences in types of samples used for each study , differences in sample collection methods and the inflammatory state of patients . thus , determination of salivary levels of leptin can be done as a simple and non - invasive technique to determine the susceptibility to advanced periodontitis .
objectives : leptin is a hormone - like protein produced by the adipose tissue . 
 it plays an important role in protection of host against inflammation and infection . 
 some studies have reported changes in leptin levels in the gingival crevicular fluid ( gcf ) , saliva and blood serum of patients with periodontal disease compared to healthy individuals . 
 the aim of the present study was to compare the salivary leptin levels in patients with advanced periodontitis and healthy individuals.materials and methods : in this case - control study , the salivary samples of healthy individuals and patients with advanced periodontitis with clinical attachment loss > 5 mm were obtained using a standardized method and the leptin levels were measured in the salivary samples by means of elisa . 
 the effects of the periodontal status and sex on the salivary leptin levels of both groups were statistically analyzed by two - way anova.results:the means  standard deviation ( sd ) of salivary leptin levels in healthy subjects and patients with advanced periodontitis were 34.276.88 and 17.875.89 pg / ml , respectively . 
 statistical analysis showed that the effect of sex on the salivary leptin levels was not significant ( p=0.91 ) , while the effect of advanced periodontitis on the salivary leptin levels was significant compared to healthy individuals ( p<0.0001).conclusions : in patients with advanced periodontitis , the salivary leptin levels were significantly lower compared to healthy individuals . 
 thus , assessment of salivary leptin can be done as a non - invasive and simple method to determine the susceptibility of patients to advanced periodontitis .