a number of investigators have proposed an association between periodontitis and psychosocial stress and the majority of studies in a systematic review   found a positive relationship between stress / psychological factors and periodontal disease . however , the relationship between periodontitis and stress - related hormones in saliva is poorly understood . in general , the stress system consists of brain elements , of which the main components are the corticotropin - releasing hormone ( crh ) and locus ceruleus - norepinephrine / autonomic systems , as well as their peripheral effectors , the hypothalamic - pituitary - adrenal ( hpa ) axis , and sympatho - adrenomedullary system . a well - known stress - related hormone is cortisol , while its salivary level reliably reflects hpa activity and has long been used in human psychological studies as a biological marker of stress .
further , dhea is also known as an hpa - related steroid hormone and has a positive correlation with depression severity [ 4 , 5 ] .
smoking is associated with elevated cortisol and dhea levels , as it increases the levels of adrenocorticotropin hormone ( acth ) .
in addition , smoking is also a major risk for periodontitis   and considered likely to be a significant mediator in the relationship between stress - related disorders and the hpa axis .
however , to date scant attention has been given to smoking regarding the association between those hormones and periodontal status .
we hypothesized that measurement of these hormone levels would be useful for screening for periodontitis in patients who never smoked , since smoking is a stronger risk factor for periodontitis . in the present study , we investigated the associations between those levels and periodontitis in never - smokers and smokers .
we also assessed cortisol and dhea as practical candidate biomarkers for screening of periodontal disease by using receiver operating characteristic ( roc ) analyses .
the subjects in this cross - sectional study were recruited from members of two senior citizen colleges in kitakyushu city , japan .
the study sample consisted of community - dwelling , independently living elderly people aged 60 years old and older who attended lectures once a week .
these colleges are part of the adult educational system supported by the government of kitakyushu city , which enrolls students as volunteers for a period of one year .
the course of study focused not only on health topics but also on other topics of interest to elderly people , such as finance and culture .
the study population voluntarily participated in oral and systemic examinations and initially consisted of 231 subjects ( 116 males , 115 females ) residing in fukuoka prefecture , japan .
all of the subjects were independent in daily activities , with none hospitalized at the time of the study , as described previously .
exclusion criteria were as follows : ( 1 ) individuals who chronically used corticosteroids and/or immune suppressor drugs as well as those with immune suppressor diseases ; ( 2 ) individuals with missing questionnaires or saliva data ; ( 3 ) individuals who used antibiotics within the last 6 months , had symptoms of acute illness , or had any apparent oral infections ; and ( 4 ) individuals with fewer than 3 natural teeth .
as a result , we evaluated a total of 171 subjects ( 85 males , 86 females ; mean age 68.4  4.46 years old ) in the present study .
we also assessed the differences between the 171 subjects who completed the study and the 60 subjects had been excluded earlier and found no significant differences regarding the tested variables .
all subjects understood the nature of the research project and provided written informed consent .  
the study protocol was reviewed and approved by the ethics committee of kyushu dental college ( no .
each subject was asked to respond to a survey conducted by a dental nurse that consisted of questions related to general medical condition , medication usage , lifestyle , oral health behavior , and oral hygiene habits , and each was also questioned verbally to obtain information regarding smoking status ( never , past , or current ) .
the subjects were classified as either never - smokers or smokers ( i.e. , past , current ) on the basis of their answers .
furthermore , a method that used face - scale scores was used to evaluate self - rated health status . from those scores ,
the subjects were divided into three groups based on overall health ( moderate , good , and very good ) .
saliva samples were collected from all subjects between 11:00 am and 1:00 pm to minimize any circadian rhythm effects , after they had refrained from oral intake for at least 2 hours prior to collection .
the subjects were first asked to swallow all saliva in the mouth , then chew paraffin for 3 minutes at a constant pace of 60 times per minute , which was monitored with an electric metronome .
collected samples were placed on ice immediately and the salivary flow rate ( ml / min ) was estimated by measuring the volume of saliva collected in the tube .
thereafter , the saliva samples were frozen at 30c until further analysis . the concentration of cortisol in saliva ( ng / ml ) was measured using a salivary cortisol enzyme immunoassay kit ( salimetrics , state college , pa ) , with a lower sensitivity limit of 0.07  ng / ml , while that of dhea ( pg / ml ) was determined using a salivary dhea enzyme immunoassay kit ( salimetrics , state college , pa ) , with a lower sensitivity limit of 10  pg / ml .
periodontal status was evaluated using probing depth ( pd ) , bleeding on probing ( bop ) , and clinical attachment loss ( cal ) .
periodontal examinations were conducted at two sites ( mesio - buccal , mid - buccal ) of all teeth examined , using a standard periodontal probe ( hu - friedy , chicago , il . )
that was inserted into the periodontal pocket parallel to the long axis of all teeth fully erupted in the mouth , according to a modified method described in the third national health and nutrition examination survey ( nhanes iii ) , with some modifications .
the intraclass correlation coefficients between examiners for assessment of pd and cal were 0.72 ( 95% confidence interval ( ci ) 0.560.82 ) and 0.74 ( 95% ci 0.600.84 ) , respectively , while those for intraexaminer were 0.78 ( 95% ci 0.660.87 ) and 0.84 ( 95% ci 0.740.90 ) , respectively .
severe periodontitis was defined as maximum pd  5  mm or maximum cal  6  mm using the mean value of each cutoff point .
further , in order to evaluate extensive periodontitis , we divided the subjects into three categories ( none , low , and high ) according to the number of teeth with maximum pd  5  mm or cal  6  mm , as described previously .
thus , for those with pd , the none group included subjects with no teeth with pd  5  mm , while the low group included those with less than 3 teeth with pd  5  mm , and the high group those with 3 or more teeth with pd  5  mm involved . as for cal ,
the none group included subjects with no teeth with cal  6  mm , while the low group included those with less than 3 teeth with cal  6  mm , and the high group those with 3 or more teeth with cal  6  mm involved . in order to assess differences among the groups ,
a chi - square test was used for categorized variables and a kruskal - wallis test or anova for continuous variables .
if a normal distribution was not present according to the results of a kolomogorov - smirnov test , the former test was used .
receiver operating characteristics ( rocs ) analyses were performed to determine the optimum cutoff values for the concentrations of cortisol and dhea , which were used to screen for severe periodontal status .
subjects with severe periodontal status were dichotomized into periodontitis - negative ( i.e. , none = 0 ) and periodontitis - positive ( i.e. , low and high = 1 ) , based on the pd and cal levels .
this dichotomy was then used to calculate the sensitivity and specificity for the cutoff values of cortisol and dhea .
roc curves were plotted for cortisol and dhea with pd and cal and the area under the roc curve ( auc ) was calculated for each .
the optimum sensitivity and corresponding specificity were determined from the point on the plot that was closest to the top left - hand corner of the axes .
tables 1 and 2 show subject characteristics of the 3 groups divided according to pd and cal stratified by smoking status , which were used to analyze the relationships of cortisol and dhea levels in saliva with periodontal health status .
for pd in never - smokers , there were significant differences among the groups regarding age , bop , and self - rated health status ( based on face - scale score ) , while there were also significant differences for the number of teeth , frequency of tooth brushing , and self - rated health status in the smokers group ( table 1 ) .
as for cal there were significant differences regarding sex and bop in the never - smokers , while there were significant differences between age and self - rated health status in the smokers ( table 2 ) .
as shown in tables 1 and 2 , most of the smokers were male ( approximately 96% ) . among never - smokers ,
the median and 25th and 75th percentile values for cortisol were 1.87 , 1.42 , and 2.89 ( ng / ml ) , respectively , while those for dhea were 59.79 , 32.69 , and 89.52 ( pg / ml ) , respectively .
in contrast , among smokers , the median and 25th and 75th percentile values for cortisol were 2.18 , 1.41 , and 3.01 ( ng / ml ) , respectively , while those for dhea were 46.07 , 30.07 , and 89.77 ( pg / ml ) , respectively . however , there were no significant differences regarding those hormone levels between smokers and never - smokers .
we compared the levels of cortisol and dhea between the with and without extensive periodontitis groups , which were defined by the number of teeth with pd  5  mm or cal  6  mm .
table 3 shows the results of that comparison in the never - smokers , while table 4 shows the results for smokers . as for the levels of both in the never - smokers ,
significant differences were found among the three categories defined by pd and cal , and higher hormone levels were found in subjects with severe pd or cal .
however , in smokers , no significant differences were found among the 3 categories defined by pd or cal .
in contrast , in comparisons of salivary flow rate , there were no significant differences among the three categories for both pd and cal , irrespective of smoking status ( tables 3 and 4 ) .
next we evaluated the usefulness of measuring cortisol and dhea levels to screen for periodontitis in never - smokers with the above - mentioned associations .
for that purpose , we performed roc analysis to discriminate between false - positive and true - positive diagnoses of severe periodontitis ( i.e. , low and high ) at various cutoff levels .
the area under the curve ( auc ) for pd was 0.68 ( 95% ci 0.580.78 , p = .001 ) and 0.69 ( 95% ci 0.590.79 , p <
.001 ) for cortisol and dhea , respectively ( figure 1 ) . for cal ,
the auc was 0.71 ( 95% ci 0.620.81 , p < .001 ) and 0.68 ( 95% ci 0.580.78 , p = .002 ) for cortisol and dhea , respectively ( figure 2 ) .
as shown in table 5 , the optimal sensitivity and specificity of cortisol for pd were 0.63 and 0.71 , respectively , while they were 0.67 and 0.66 , respectively , for dhea , with cutoff values of 2.06 ( ng / ml )   for cortisol and 60.24 ( pg / ml ) for dhea , which indicated a corresponding level in the 55th and 51st percentile , respectively .
as shown in table 6 , the optimal sensitivity and specificity of cortisol for cal were 0.70 and 0.73 , respectively , while they were 0.63 and 0.64 , respectively , for dhea , with cutoff values of 2.12 ( ng / ml ) for cortisol and 61.78 ( pg / ml ) for dhea , which indicated a corresponding level in the 56th and 54th percentile , respectively .
in the present cross - sectional study , we investigated cortisol and dhea levels and periodontal status in elderly subjects and found that levels of the salivary stress - related hormones cortisol and dhea were useful for screening for periodontitis in subjects who had never smoked .  
the association between periodontitis and stress - related hormones has been largely overlooked , with only two known human studies of the associations between cortisol in saliva and periodontitis reported .
one of those was our own survey , while the other was a report by hilgert et al . .
in the latter , the authors found a positive association between salivary cortisol and periodontitis , while hypercortisolemia was independently associated with the severity of periodontitis , as defined by cal ( mean cal  4  mm versus < 4  mm ) , and the extent of periodontitis , as defined by pd ( 26% versus < 26% of sites with pd  4  mm ) or cal ( 30% of sites with cal  5  mm versus < 30% ) .
however , smoking status was removed from the final model of logistic regression analysis in that report , while we did not treat smoking as a factor based on stratification of smoking status . in a recent study , treatment of smoking as a confounding factor resulted in a greatly underestimated magnitude of association .  
smoking is known to be associated with elevated cortisol and dhea levels . in a recent report that compared cortisol profiles of smokers and nonsmokers over the day , cortisol levels were elevated in everyday life among smokers compared with nonsmokers , and the differences in values were quite substantial , averaging 35% or more on both working and weekend days . in the present study ,
cortisol levels were higher in smokers than never - smokers , though the differences were not significant ( 2.18 versus 1.87  ng / ml ) .
furthermore , salivary levels of cortisol and dhea were significantly elevated according to the severity of periodontitis , in our never - smoked subjects , whereas no significant association was observed in smokers .
one possible explanation for these findings might be that the capability of those hormones to differentiate periodontally affected patients from periodontally healthy individuals is weaker than that of smoking .
conversely , since smoking itself is a stronger risk factor for periodontitis than other known risk factors , evaluation of smoking behavior may be a superior screening method for smokers .
measurement of biomarkers in saliva has many advantages , as the procedure is stress - free and noninvasive , and allows for frequent and rapid sampling , whereas diurnal rhythm , artificial changes due to food or drinking substances , and blood - contamination are some of the disadvantages .
thus , as described above in the methods section , the sampling method must be carefully performed . in order to minimize any circadian rhythm effects
, we selected the period between 11:00 am and 1:00 pm for obtaining saliva samples , which has been reported to be stable in regard to daytime hormones levels in nonsmokers .   a periodontal probe is generally used for periodontitis screening during community - based oral health check - ups , though several problems , including cost , burden on the subject , prevention of infection , and manpower needs , have been pointed out . our analysis using roc curves showed acceptable sensitivity and specificity for periodontitis screening with both of the salivary hormones tested in the present study .
on the other hand , progress is being made in the development of various screening tests for periodontitis using enzymes , cytokines , and antimicrobial proteins   in saliva .
we think that it would be better to combine several tests that reflect the multiple risk factors associated with periodontitis and not depend on results of a single test . on the other hand
, it is important to consider the cost - effectiveness when promoting such screening tests for clinical use .
a commercial test kit for cortisol costs approximately us$ 250 and can be used 50 subjects , that is , us$ 5.00 per test . however
, additional costs must be added when considering the required manpower for the assay and other laboratory charges . in the near future , it is anticipated that screening tests used for a large population will be noninvasive and less burdensome for the subjects , with no requirements of specific devices or instruments , or for individual examinations by an expert as well as reasonable cost - effectiveness performance .
a limitation of the present study is that our subjects were generally in good health and noninstitutionalized .
in addition , the percentage of subjects with severely extensive periodontitis among all subjects analyzed was low .
thus , these findings may indicate that the association exists primarily in systemically and orally healthy elderly individuals .
furthermore , it is possible that the periodontal examination method that utilized two sites per tooth in this study , which was based on nhans iii , may underestimate the severity and extent of periodontitis .
additional investigations are necessary to validate and extend the findings using other ages or outpatients .
further , the usefulness of cortisol and dhea as predictors for periodontitis can not be determined from our findings , and a longitudinal study is necessary to determine the relationships of those hormones to the progression of periodontitis .       in summary , we found a significant association between the salivary steroid hormones cortisol and dhea and periodontitis severity in community - dwelling elderly subjects who had never smoked .
these results indicate that these stress - related hormones are useful indicators of the risk for periodontitis , as they showed moderate levels of sensitivity and specificity for periodontitis . within the study limitations regarding smoking status , the present method of determining cortisol and dhea levels may include a possibility as screening test for periodontal disease in the near future . additional approaches to reveal a new type of screening test system for periodontitis as an alternative to the conventional probing method are required .
 
 background . 
 recent reports have found a positive relationship between periodontitis and the hormones cortisol and dehydroepiandrosterone ( dhea ) . 
 we investigated the associations between those levels and periodontitis in never - smokers and smokers of elderly subjects . 
 subjects and methods . 
 cortisol and dhea levels in saliva were determined in 171 subjects ( 85 males , 86 females ) , with clinical examinations including probing depth ( pd ) and clinical attachment loss ( cal ) also performed . 
 results . 
 smoking had effects on cortisol and dhea levels , and those were significantly associated with severe pd and cal in never - smokers . according to roc analysis , the cutoff values of cortisol and dhea to obtain the optimal sensitivity and 
 specificity for detecting severe periodontitis were 2.06  ng / ml and 60.24  pg / ml , respectively , for pd , and 2.12  ng / ml and 61.78  
 pg / ml , respectively , for cal . conclusions . 
 assessment of hormone levels may be a useful screening method for periodontitis , though limited to never - smokers . 