as a diagnostic fluid , saliva has several advantages over blood [ 1 , 2 ] . saliva is inexpensive and easier to collect , and sufficient volume can be obtained to allow performance of a variety of analyses . for patients , particularly children , the noninvasive sample collection reduces anxiety and discomfort and simplifies procurement of repeated samples for time - series analyses . saliva contains a wide array of proteins and peptides that are responsive to pathological conditions . advanced instrumentation and refined analytical techniques have been successfully applied for discovering oncological , hormonal , immunological , and cardiovascular [ 7 , 8 ] biomarkers that can be informative for early detection and assessment of progression of oral and systemic diseases . of particular interest , saliva is known to contain detectable levels of endothelins , an important risk marker for cardiovascular disease . endothelins are a family of potent vasoconstrictor peptides consisting of three distinct isoforms , endothelin-1 ( et-1 ) , endothelin-2 ( et-2 ) , and endothelin-3 ( et-3 ) coded by distinct genes . the mature endothelins are produced through cleavage of the big endothelin ( bet ) precursors by endothelin - converting enzymes [ 11 , 12 ] . endothelin-1 , the most studied isoform , has been implicated in several diseases , particularly in the progression of cardiovascular diseases [ 13 , 14 ] . it has been known for two decades that et-1 , et-2 , and et-3 are present in saliva [ 15 , 16 ] . however , only recently have levels of saliva et-1 been related to conditions such as chronic heart failure , upper gastrointestinal diseases , vibration - induced white finger , and oral cancer [ 20 , 21 ] . the relationship between saliva and plasma et-2 and et-3 isoforms is comparatively less well understood . because of the emerging significance of all three isoforms in health and disease , notably the role of et-2 in the cardiovascular system , in ovulation , immunology , and cancer , we sought to extend the data on the relationships between the three endothelin isoforms in concurrent saliva and plasma samples . ethylenediaminetetraacetic acid ( edta ) , trifluoroacetic acid ( tfa ) , phenylmethyl sulfonyl fluoride ( pmsf ) , 3,4-dichloroisocoumarin , molecular weight cut - off filters ( 30  kda ) , endothelin-1 ( et-1 , human ) , endothelin-2 ( et-2 , human ) , and endothelin-3 ( et-3 , human ) were obtained from sigma aldrich ( oakville , ontario ) . big endothelin-1 ( bet-1 , human ) was obtained from bachem bioscience ( american inc . , acetonitrile , acetone , methanol , and hydrochloric acid were purchased from sigma aldrich ( oakville , ontario ) . amber glass vials and screwcaps with septa were purchased from chromatographic specialities ( brockville , ontario ) . deionized water was obtained from a super - q plus high purity water system ( millipore corporation , bedford , ma ) . compressed gaseous nitrogen was of uhp grade quality and was supplied by matheson gas products ( whitby , ontario ) . anonymous , paired human plasma and saliva samples ( from males ) certified free of hiv , hep - a , and hep - b were purchased commercially ( innovative research inc . both plasma and saliva samples were treated on site with pmsf ( final , 1.7  mg / ml ) and edta ( final , 10  mg / ml ) to stabilize endothelins , shipped to our laboratory in dry ice , and stored at 80c until further use . thirty ( 30 ) sample pairs of good quality and in sufficient amounts for analysis were retained for this study . briefly , plasma ( 250  l ) and saliva ( 1  ml ) samples were treated with ice - cold 3,4-dichloroisocumarin solution in isopropanol to prevent conversion of bet-1 to et-1 during the sample processing . the samples were deproteinized with ice - cold acid - acetone mix ( acetone  :  1  n hcl  :  water , 40  :  1  :  5 ) and centrifuged at 9000  g for 10  min , and the supernatants obtained were concentrated by evaporation under nitrogen flow . deproteinization and concentration stages were repeated once more to ensure the removal of abundant proteins . samples were then loaded onto 30  kda molecular weight cut - off filters ( prewashed with deionised water ) and centrifuged at 5000  g for 30  min . these filters were then washed with 50% methanol and centrifuged at 5000  g for 30  min . filtrates were dried under nitrogen and reconstituted in 75  l of 30% acetonitrile in 0.2% tfa / h2o for hplc - fluorescence analysis . the hplc unit consisted of a gilson solvent delivery system ( mandel scientific , guelph , on ) , a gilson autosampler ( model 231 xl ; middleton , wi ) , a supelcosil lc-318 reverse - phase column ( 25  cm length , 4.6  mm i d , 5  m particle size , and 300   pore dimension ; supelco , oakville , on ) , and a rf 551 model fluorescence detector ( shimadzu , japan ) . endothelins ( 20  l injection volume ) were separated using a gradient elution with acetonitrile / water mobile phase at a flow rate of 1  ml / min and detected at ex 280  nm and em 340  nm . blanks were analyzed after each set of four samples in order to assess the extent of analyte carryover . student 's t - test or mann - whitney rank sum test was carried out as appropriate using sigmastat v 11.0 ( spss inc . , correlation between different endothelin isoforms in plasma and saliva was tested using pearson 's product moment correlation revealing p and r values . we used hplc - fluorescence to measure simultaneously the isoforms bet-1 , et-1 , et-2 , and et-3 ( figures 1(a)-1(b ) ) in time - matched plasma and saliva sample pairs ( n = 30 ) obtained from anonymous individuals ( table 1 ) . our results confirm and extend previous reports of the presence of endothelins in saliva [ 15 , 16 ] and of a relationship between saliva and circulating endothelins [ 1719 ] , and the correlated measurements provide additional insight into the relationship of et-2 to the other two isoforms . the recoveries , analytical precision , and accuracy of the hplc - fluorescence procedure   are comparable to values reported by walczak et al . for hplc with electrospray tandem mass spectrometry detection : recoveries of endothelins from spiked plasma are between 60% ( et-2 ) and 97% ( et-1 ) , depending on the endothelin isoforms ; analytical precision is on the order of 4% for replicate peptide standards ; analytical accuracy is 20% for replicate measurements of plasma samples ; limit of detection is 0.20.5  pmol ; and linearity is 1100  pmol on the column ( 20  l injection volume ) . both methods detect the separated peptides directly ( i.e. , on the basis of autofluorescence of aromatic amino acids by hplc [ 23 , 25 ] or from mass ion fingerprints in ms / ms ) , which is different from elisa detection of immunoreactive endothelin in total plasma [ 26 , 27 ] . for this reason , we have verified identities of analytes by pulling down of et-1 and et-3 with monoclonal antibodies during sample processing , confirming the correspondence between immunoreactive endothelins in plasma and the peptides measured by direct autofluorescence detection after separation on column . it should be noted that the aim of our study was not to explore the physiological significance of variations of saliva and plasma endothelin levels , within and between individuals , and for this reason we have not requested any information on the health status , cardiovascular or buccal , of the donors . salivary secretions and saliva volume will be affected by water and food uptake and by medication . these factors , together with the health status of subjects and sample quality , can contribute to the variance in observed saliva endothelin concentrations and relationship to plasma levels . furthermore , sample collection procedures , sample preparation methods , matrix effects , and the analytical approach used are likely to impact the absolute values of estimates . finally , because all samples analyzed for endothelin content in our small study were from male donors , our data do not address potential gender differences . notwithstanding those limitations , our observations reveal that levels of all endothelin isoforms were significantly correlated between the saliva and plasma matrices in the subjects studied : bet-1 ( r = 0.38 , p = 0.041 ) , et-1 ( r = 0.53 , p = 0.003 ) , et-2 ( r = 0.51 , p = 0.004 ) , and et-3 ( r = 0.75 , p < 0.001 ) ( table 1 , figure 2 ) . our results indicate a slight increase in the ratios of endothelin levels in plasma versus saliva with age of the subjects ( et-1 , plasma / saliva ratio in 1840 years versus 4163 years , 6.11  2.16 versus 8.67  2.51 , p = 0.006 ) , which is potentially attributable to saliva osmolality changes with age [ 28 , 29 ] . endothelin levels varied on average by fivefold between individuals , and this applied to all four isoforms measured , in plasma as well as saliva ( 90th/10th percentiles ) . in general , high bet-1 and et-1 levels were predictive of high et-3 levels within an individual ( table 2 ) . within plasma , a significant positive correlation was seen for bet-1 versus et-1 ( r = 0.39 , p = 0.033 ) , bet-1 versus et-3 ( r = 0.46 , p = 0.011 ) , and et-1 versus et-3 ( r = 0.57 , p = 0.001 ) . within saliva , a significant positive correlation was seen for bet-1 versus et-1 ( r = 0.75 , p < 0.001 ) . correlations between bet-1 versus et-3 ( r = 0.31 , p = 0.091 ) and et-1 versus et-3 ( r = 0.27 , p = 0.144 ) in saliva were not significant . there were no significant correlations between et-2 and other isoforms in either plasma or saliva . whole saliva contains secretions from the major parotid , submandibular and sublingual glands , the palate , buccal and labial mucosa , and so on . expression of preproet-1 and preproet-3 as well as the eta and etb receptors has been detected by rt - pcr analyses of submandibular glands of rats . endothelin-1 has also been detected in striated duct cells of human salivary glands by immunohistochemical analysis . however , serum molecules that are not part of the normal salivary secretory constituents , such as proteins , drugs , and hormones , can also reach saliva by passive diffusion [ 1 , 2 , 3436 ] . we have measured six- to eightfold higher concentrations of endothelins in plasma by comparison to saliva , and hence the concentration gradient may result in diffusion of the peptides from the capillaries of the mucosa into the salivary fluid . if the intercept of the linear regressions of saliva versus plasma endothelin concentrations is taken as an estimate of the contribution by salivary glands , then at least half ( bet-1 , et-1 , and et-2 ) to two - thirds ( et-3 ) of the salivary endothelins may originate from plasma ( figure 2 ) . correlation among the different endothelin isoforms within individuals and the large variance of endothelin levels between individuals may be due to a number of factors , including common regulatory mechanisms , genetic polymorphisms , and physiological status . correlation between bet-1 and et-1 should be expected because of the direct precursor - product relationship . furthermore , endothelin converting enzyme-1 , which cleaves bet-1 to et-1 , shares some regulatory elements with the preproet-1 gene , such that when preproet-1 is transcriptionally activated , ece-1 tends to be activated as well . in healthy individuals , et-1 plays an important role in the modulation of vasomotor tone , in conjunction with nitric oxide , but also in regulating cellular proliferation and differentiation in tissues during growth , development , and repair [ 39 , 40 ] . there is good evidence for coordinated regulation of et-1 and et-3 . in individuals with high basal et-1 production , activation of preproet-3 expression through a feedback control mechanism may compensate for the pressor effects of the high circulating levels of et-1 . endothelin-3 stimulates nitric oxide synthase expression and no production through binding to the etb receptor , and increase of et-1 with a decrease of et-3 , due to endothelial dysfunction , has been reported in patients with pulmonary arterial hypertension of various etiologies [ 42 , 43 ] . it is also possible that high or low transcriptional activity of both preproet-1 and preproet-3 genes within an individual is determined by genetic polymorphism or epigenetic conditioning of transacting elements with common cis - acting regulatory sequences between the two genes , but this remains to be investigated . in our analyses , the strongest associations were seen between saliva et-3 and plasma et-3 ( r = 0.75 , p < 0.001 ) and between saliva bet-1 and saliva et-1 ( r = 0.75 , p < 0.001 ) . an overall relationship between et-3 and bet-1 and its mature product et-1 was observed in plasma and saliva but was stronger in plasma ( figure 3 ) . the similar ratio of bet-1 to et-1 in plasma ( 3.35 versus 3.45  pmol / ml ) and saliva ( 0.55 versus 0.52  pmol / ml ) suggests a relative stability of the peptides during sampling and sample processing . however , although saliva et-2 correlated with plasma et-2 ( r = 0.51 , p = 0.004 ) , the poor correlation between et-2 and the other endothelin isoforms , within plasma ( positive , p > 0.05 ) and within saliva ( negative , p > 0.05 ) , is in line with the concept of et-2 regulatory pathways distinct from those of et-1 and et-3 . in conclusion , our data confirm an overall positive correlation between plasmatic and salivary bet-1 , et-1 , et-2 , and et-3 levels and between the bet-1 , et-1 , and et-3 within each of the two compartments . the isoform et-2 correlates poorly with et-1 and et-3 , within saliva and within plasma . salivary et isoform profiles portray the plasmatic profiles and support the view of coordinated regulation of et-1 and et-3 , but distinct regulatory pathways for et-2 .
although saliva endothelins are emerging as valuable noninvasive cardiovascular biomarkers , reports on the relationship between isoforms in saliva and plasma remain scarce . 
 we measured endothelins in concurrent saliva and plasma samples ( n = 30 males ; age 1863 ) by hplc - fluorescence . results revealed statistically significant positive correlations among all isoforms between saliva and plasma : big endothelin-1 ( bet-1 , 0.55  0.27 versus 3.35  1.28  pmol / ml ; r = 0.38 , p = 0.041 ) , endothelin-1 ( et-1 , 0.52  0.21 versus 3.45  1.28  pmol / ml ; r = 0.53 , p = 0.003 ) , endothelin-2 ( et-2 , 0.21  0.07 versus 1.63  0.66  pmol / ml ; r = 0.51 , p = 0.004 ) , and endothelin-3 ( et-3 , 0.39  0.19 versus 2.32  1.44 pmol / ml ; r = 0.75 , p < 0.001 ) 
 . correlations of bet-1 , et-1 , and et-3 within each compartment were positive in both plasma ( p < 0.05 ) and saliva ( p  0.1 ) , whereas et-2 was not significantly correlated with other isoforms in either plasma or saliva . for all isoforms , concentrations varied on average fivefold between individuals ( 90th/10th percentiles ) ; individuals with high plasma endothelin levels generally had high saliva endothelin levels . 
 our results reveal that salivary et isoform profiles portray the plasmatic profiles and support the view of coordinated regulation of et-1 and et-3 , but distinct regulatory pathways for et-2 .