Marek Cigáň

Anton Gáplovský

Ivica Sigmundová

Pavol Zahradník

Roman Dědic

Magdaléna Hromadová

Roman Dĕdic

Photostability of D-π π π π-A nonlinear optical chromophores containing a benzothiazolium acceptor

Keywords: Photostability, benzothiazolium D-π-A NLO chromophores, Trans-cis photoisomerization, Photooxidation, Singlet oxygen Photostability, benzothiazolium D-π-A NLO chromophores, Trans-cis photoisomerization, Photooxidation, Singlet oxygen

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INTRODUCTION

In the past fifteen years, a considerable effort has been focused on the development of organic molecules with enhanced second order nonlinear optical (NLO) properties due to their potential applications in various areas such as optical modulation, frequency doubling and molecular switching [START_REF] Nalwa | Nonlinear Optics of Organic Molecules and Polymers[END_REF][START_REF] Marder | [END_REF][3][4][5] . The most widely studied second-order (quadratic) NLO effects such as second harmonic generation (SHG) arise from high first molecular hyperpolarizabilities β. The most common design of molecules with large β values comprises strong electron-donors and acceptors connected by a π-conjugated system (donor-π-acceptor or ''push-pull'' chromophores) [START_REF] Nalwa | Nonlinear Optics of Organic Molecules and Polymers[END_REF][START_REF] Marder | [END_REF] . In such donor-π-acceptor (D-π-A) systems, the donor and acceptor moieties provide the necessity of ground-state charge asymmetry, whereas the π-conjugated bridge provides a pathway for the redistribution of electron density under the influence of external electric fields [6] .

For the practical application of second-order NLO materials, not only a high molecular quadratic hyperpolarizability β, but also good thermal, chemical and photochemical stabilities are required [7,[START_REF] Murali | Proceedings Symposium IEEE/LEOS Benelux Chapter[END_REF] . Good thermal and photochemical stabilities of NLO chromophores with preserved high hyperpolarizabilities can be achieved by replacement of an aromatic ring with easily delocalizable heteroaromatics [START_REF] Breitung | [END_REF] . In respect of these findings, benzothiazole-derived dyes with a D-π-A setup were promising candidates for NLO applications [10] . Further improvement in β values was achieved by proper introduction of donor and acceptor substituents onto the benzothiazole core due to its nonsymmetric character and quaternization of the benzothiazole 3 nitrogen [11] . Benzothiazolium styryl dye and its derivatives with different donors and acceptors were widely investigated in last century as good laser [12] and NLO dyes [13] . Over the last few years, our research group synthesized a large number of push-pull 3-alkyl-benzothiazolium salts with different number n of ethenylene units in a π-conjugated bridge and various donor and acceptor substituents [11,[14][15][16][17] . The benzothiazolium salts were found to be much more effective NLO-phores in comparison with the corresponding neutral benzothiazoles. It was shown that the length of the π-conjugated bridge between the electron-donating group and benzothiazolium moiety, as well as the donor and acceptor ability, have a significant influence on β. Recently, Coe et al. [18] reported the results of Hyper-Rayleigh scattering (HRS) measurements for 3-methylbenzothiazolium salts (n=1-4; D=N,N-dimethylaminophenyl) in comparison with the analogous 1-methylpyridinium salts. Experimental measurements revealed that the static molecular quadratic hyperpolarizability β 0 increases with the length of polyene chain, and the benzothiazolium salts exhibit larger NLO responses than their pyridinium analogues.

In most cases, donors and acceptors in D-π-A systems are connected via the conjugated bridge containing C=C bonds. However, this bond readily undergoes trans-cis photoisomerization, which may hamper the material efficiency and lifetime. It was previously reported that the primary causes of photochemical instability of NLO chromophores containing C=C bonds are photooxidation and photoisomerization [19][20][21][22][23][24] . In either case, the nonlinear activity is significantly diminished, the charge-transfer (CT) system is reduced, and new absorption features appear elsewhere in the spectrum, usually at a much shorter wavelength. The identity and the number of specific reactions and their relative rates are influenced in a complicated way by the exact identity of the chromophore, the influence of atmospheric environment, the irradiation wavelength, the temperature, etc. Photostability of organic dyes is consequently often considered the Achilles Heel of organic photonic materials [START_REF] Murali | Proceedings Symposium IEEE/LEOS Benelux Chapter[END_REF]20,25,26] and limits their practical applications. Most of the state-of-the-art chromophores which were designed to have high electro-optic coefficients and hyperpolarizabilities cannot be put to use because they are highly unstable and degrade because of a photochemically generated singlet oxygen [START_REF] Murali | Proceedings Symposium IEEE/LEOS Benelux Chapter[END_REF]27] .

Here we report results of a comprehensive investigation of the photochemical stability, particularly the photooxidative stability of seven benzothiazolium iodides with a different length of conjugated bridge and a different acceptor strength of benzothiazolium core caused by withdrawing substituents. The studied compounds 1-7 are dipolar push-pull chromophores Experiments were carried out to determine the role of photoisomerization and photooxidation in the photodegradation of 1-7 and to evaluate the overall stability and availability of such molecules for practical applications.

EXPERIMENTAL

Synthesis

The compounds under study have been prepared by the condensation reactions as is presented in Fig. S1 (Supplementary Material) and has been published in [11]. The pyridine or piperidine have been used as the base. All compounds have all-trans configuration of double bonds in the conjugated bridge.

Experimental techniques

Electronic

Photodegradation measurements

All photochemical measurements were performed using the apparatus described elsewhere (Fig. 7. without ultrasonic horn H) [28] . The light source was a 100-W xenon lamp (300-850 nm;

∫ ∫ ∫ ∫ ∑ ∫ - =       - = - = ∞ ∞ ∑ - λ λ λ λ λ λ λ λ λ λ λ λ λ d d d d d 10 1 0 T 0 0 0 i i X 0 0 T 0 a i i S S S I I I A A I I I I A , (1)
where ∫S 0 dλ and ∫S T dλ denote the total area under the emission spectrum of the light source after passing through a cuvette without and with X, respectively, I 0 and I T the incident and the transmitted photon flux, respectively, A means absorbance (the value of absorbed photon flux during photodegradation depends on time -for detailed calculation of Φ during photodegradation of studied compounds using time-dependent values of I a see eqns. (7) and (10)). The incident photon flux I 0 in all measurements was determined using the integrated form of the equation for Ф 1 O 2 determination for a solution of a sensitizer with known Ф 1 O 2 and an acceptor with known β (eqn.( 5)).

Determination of singlet oxygen quenching rate constants (k Q )

The rate constant k Q for 1 O 2 quenching (relative reactivity index β Q =k d /k Q ; in this case β Q includes both physical and chemical quenching) by 1-7 necessary for more accurate Ф 1 O 2 determination were calculated based on inhibition of DPIBF oxidation (β=1.1×10 -4 M in MeOH [29,30] ) by competitive 1 O 2 quenching [31,32] using the following equations (in the primary model we replaced the concentrations with the corresponding absorbances):

[ ] [ ] [ ] [ ] [ ] [ ] [ ] ) / ln( Q ) / ln( ) / / ( Q t DPIBF 0 DPIBF 0 t DPIBF Q t DPIBF d 0 t DPIBF DPIBF Q t DPIBF DPIBF r Q A A A A k A A k k + - = ε ε (2) F o r P e e r R e v i e w 6 or [ ] [ ] [ ] [ ]               -               -       - + = 1 d / d d / d Q DPIBF Q DPIBF DPIBF 0 DPIBF DPIBF 0 r d Q t A t A k k k ε ε , (3)
and by monitoring the decrease of the DPIBF absorption band at different quencher concentration using Young´s kinetic technique [33] : [29,34] ).

[ ] [ ] Q 1 1 Q 1 Q d Q 0 β + = + = k k S S . (4)

Determination of the quantum yield of singlet oxygen production (Φ 1 O 2 )

Values of Ф 1 O 2 were determined using an equation that describes the change in concentration c A (mol dm -3 ) of a singlet oxygen acceptor (with known β value) after irradiation for a given time t (s) in the presence of a singlet oxygen generator (sensitizer 1-7) that absorbs a photon flux I a (mol s -1 dm -3 ) (Fig. 3) [34,35] :

[ ] [ ] [ ] [ ] [ ] [ ] DMF DMF X DMF DMF d DMF d d d Q(X) r DMF d r DMF ) X ( O a A 2 1 + = + + Φ = - = - Z k k k k I t t c , (5)
[ ]

r DMF Q(X) X k k Z + = β .
In this approach, we involved quenching of 1 O 2 by a sensitizer using parameter Z.

Parameter Z is the sum of two components and includes overall (physical and chemical) quenching of 1 O 2 by acceptor A (2,5-DMF; k r =3.9.10 8 dm -3 .mol -1 .s -1 [34] ) and sensitizer X, and should be used for every sensitizer with high value of k Q (e.g. omitting the parameter Z for sensitizer 3 with high value of k Q leads to a reduction in quantum yield of about 60%). Parameter β is the reactivity index of the acceptor (the ratio between the rate constants for nonradiative unimolecular decay of 1 O 2 for the appropriate solvent k d and acceptor oxidation k r ) [36] . Its value represents the acceptor concentration at which half the reactive 1 O 2 species will be trapped.

Constant k Q is the overall quenching constant for sensitizer X. Integrating the equation ( 5) and deducing acceptor concentrations from absorbance values gives:

t l I A Zl A A Zl A (X) O DMF a a0 DMF a0 a DMF a 2 1 ln ln Φ - + = + ε ε ε , ( 6
)
where A a0 and A a are the acceptor absorbance (e.g. at the position of maximum absorption) before and at various irradiation times, l is the optical path length (cm) and ε is the molar extinction coefficient of the acceptor. The slope of a plot of this equation against irradiation time gives Ф 1 O 2 when the other parameters are known. mol dm -3 ) [37,38] .

Phosphorescence measurements (determination of the energy of a triplet state E T )

Phosphorescence spectra were detected using a home-built set-up for detection of weak near-IR luminescence described in [39]. Excitation pulses of ~ 10 µJ and 4.5 ns wide were provided by the dye laser Lambda Physik FL1000 pumped by the excimer laser ATL Lasertechnik ATLEX 500i. The excitation wavelengths and laser dyes were chosen to match different absorption spectra of the investigated molecules, namely 524nm (Coumarin 307), 540nm The spectra were obtained as integrals of the counts in each of the kinetics at individual wavelengths in the spectral region from 750 to 1350nm and corrected in respect to the spectral sensitivity of the detection set-up as well as to the equivalent absorbed energy. The kinetics were fitted by double exponential decays to obtain phosphorescence lifetimes.

Determination of oxidation potential (E 0' )

Iodides 1-7 were metathesized to their corresponding hexafluorophosphate salts (due to interfering signal of I -anion) by precipitation from the MeOH/MeCN/aqueous KPF 6 .

Electrochemical measurements were done in a three-electrode arrangement using a home-made fast rise-time potentiostat. The instrument was interfaced to a personal computer via an IEEEinterface card (PC-Lab, AdvanTech Model PCL-848) and a data acquisition card (PCL-818) using 12-bit precision. Positive iR compensation just short of oscillations was used in all the measurements. The reference electrode Ag|AgCl|1M LiCl was separated from the test solution by a salt bridge. The working electrode was a glassy carbon electrode with an area of 3.79×10 -3 cm 2 .

The auxiliary electrode was a cylindrical platinum net. Ferrocene was used as an internal reference and all data are referred to the formal redox potential of the ferrocene/ferrocenium (Fc/Fc + ) couple equal to +0.52 V in this system. Oxygen was removed from the solution by a stream of argon, which blanketed the solution throughout the measurements. E 0' values were obtained from the cyclic voltammetric measurements of 0.5mM solutions of 1-7 in acetonitrile and 0.1M tetrabutylammonium hexafluorophosphate supporting electrolyte. Error in the E 0' determination is ± 0.005 V. Estimated values E 0' for 1-3 agree well with the literature data [18] .

Spectral characteristics

The UV-visible absorption spectra of salts 1-7 in MeOH were measured previously [11] . These spectra are dominated by intense low-energy bands in the visible region (λ A 350-500 nm) attributable to π→π* ICT transitions from the terminal electron-donating N,N-dimethyl groups to the central benzothiazolium skeleton. Less intense bands in the spectra are associated with nondirectional π→π* transitions at higher energy. Basic absorption characteristics of ICT transitions (absorption maxima λ A , extinction coefficient log ε, oscillator strength f, ICT energies

E A )
and calculated values of first hyperpolarizabilities β (at the semiempirical PM3 level by the finite-field method) [11] of benzothiazolium NLO chromophores 1-7 are presented in Table 1. ICT bands move predictably to lower energy with increasing conjugation length and with increasing acceptor strength of the benzothiazolium skeleton. An additional acceptor group bound to the benzothiazolium ring (NO 2 or CN) has a controversial effect on the NLO response of 4-7.

Addition of this group to chromophore 1 with single double bond enhances the value of β and addition to chromophore 2 with two double bonds leads to a modest decrease in NLO response. A rising number of double bonds enhances the NLO response of benzothiazolium chromophores.

The nonplanarity (decreasing conjugation strength) together with possible isomerisation in longer linkers [40] could explain the lowered values of log ε and f in absorption spectra of compound 6.

Fluorescence spectra showed very low intensity in MeOH (quantum yields of fluorescence Ф F never exceeded 0,005). This can be attributed to enhanced ICT interaction, which often results in fluorescence quenching due to enhanced probability of nonluminiscent ICT and TICT (twisted intramolecular charge transfer) state population in polar solvents [41] . The same effect was observed in a few push-pull D-π-A salts and it has also a practical application [START_REF] Rettig | Progress in Photochemistry and Photophysics[END_REF] . The evidence of the final relaxed nonluminiscent dark TICT state in the commercial benzothiazolium push-pull laser dye LDS 751 (which is structurally very close to dye 2) was recently confirmed experimentally using the FS TR SEP FD method (femtosecond timeresolved stimulated emission pumping fluorescence depletion) [START_REF] Guo | [END_REF] . Hydrogen bonding effect of the solvent can also contribute to the non-radiative internal conversion [41] .

Phosphorescence spectra were measured to estimate the energy of triplet states (the position of the main phosphorescence maxima λ P , Table 1) as one of the major factors that 3). All the studied compounds exhibit similar luminiscence spectra with λ P around 852±11 nm and a shoulder around 910 nm as a consequence of the vibrational structure of the emissive state. Bathochromic shift of the main phosphorescence maximum with the length of the conjugated bridge was observed for chromophores 1-3. On the contrary, no such shift was found for compounds 4 and 6 and a hypsochromic shift was obtained for compounds 5 and 7.

Luminescence intensity exhibits very strong dependence on the length of the conjugated bridge and on addition of the acceptor to N-methylbenzothiazolium moiety. It increases significantly with increasing length of the conjugated bridge. This effect was the most pronounced in chromophores 1-3, where compound 1 exhibited only approximately 3% of the total phosphorescence intensity of compound 2, while the phosphorescence intensity of compound 3 was ~ 27 times higher than that of compound 2. This effect was also observed in compounds 4 and 6 as well as in 5 and 7 where the total phosphorescence intensity increased 5 and 7 times, respectively, in the compounds with longer conjugated bridges. Time-resolved luminescence signals were accurately fitted by biexponential functions. We distinguished fast (τ 1 ) and slow (τ 2 ) decay times of approximately 80±10 ns and 1.3±0.1 µs, respectively, with the short component fractional yield of ∼70-75 % from the biexponential fits. The short component of the luminiscence signal is assumed to result from a deactivation of the dark TICT state [START_REF] Guo | [END_REF] or it may be as well a component of phosphorescence. However, the second alternative is quite unusual for most organic triplets. All the respective lifetimes are quite similar, one can only say that longer component is always (for all different excitation wavelengths) slower in sample 3 (see Table 1).

The striking differences in phosphorescence intensities of the different materials are therefore caused by different triplet state quantum yields and not by different quenching of the emitting states. Thus, the extension of the conjugated bridge and an additional acceptor group bonded to the heterocycle have a substantial effect on the triplet state population although they do not significantly influence the energy of the triplet states.

Photostability

As already mentioned, the NLO chromophores photostability is a very important characteristic with regard to their practical usage. There are two dominant processes leading to photodegradation of NLO chromophores containing a carbon-carbon double bond as part of a π-

Values for the photodegradation rate constant (k), the time for 50% degradation (t 0.5 ), the time for 90% degradation (t 0.9 ), the quantum yield for photodecomposition (Φ deg ), the percent of initial isomer in the photostationary state and the contribution of self-sensitized photooxidation by 1 O 2 to overall photodegradation during photoisomerization are summarized in Table 2. (and/or TICT) excited-states population with the nonradiative phantom-state population (from the second one trans-cis isomerization occurs) and moreover the hydrogen bonding effect of MeOH may enhance the non-radiative internal conversion [41] . Addition of the third double bond to the conjugated bridge and an additional acceptor bound to position 6-of N-methylbenzothiazolium decreased the value of Φ deg , although the overall percentage photodegradation slightly increased.

Very rapid cis-trans photoisomerization of the initially formed cis isomer may be responsible for a low contribution of this pathway to the overall photodegradation. Trans-cis photoisomerization thus seems to be a fast (mainly for 1-3) but inefficient (∼1-7 % decrease of initial concentration) photobleaching mechanism for these irradiation wavelengths. Contribution of self-sensitized photooxidation by 1 O 2 (Section 2.4 in RESULTS AND DISCUSSION) as a parallel degradation pathway to the overall photodecomposition during trans-cis photoisomerization of 1-7 is not significant (0-3%).

Photooxidation

There are two possible mechanisms for interaction between an excited dye molecule and dioxygen, which lead to the formation of singlet oxygen (Type II process) and a superoxide anion (Type I process), respectively, both of which can contribute to dye photofading [44,45] . Therefore, we conducted experiments to determine whether 1-7 generate these reactive oxygen species and whether there is evidence of self-sensitized photooxidation.

Production of the superoxide anion radical O 2 .-

To study the generation of superoxide anion radical O 2 .

-after photoexcitation of 1-7, EPR spintrapping was carried out with DMPO as the spin trapper [34,35] . After 20 min of irradiation with a 250-W halogen lamp only 4 showed a very low-intensity EPR signal (Fig. S3 in Supplementary Material) in the region of free radicals (g = 2.004). This signal seemed to represent DMPO-O 2 .

adduct with three coupling constants due to the nitrogen atom and two hydrogen atoms in the β coupling constants agree well with literature data [37,38] . Electron transfer (formation of a superoxide anion radical) and subsequent oxidation reactions (followed by various radical recombinations) or nucleophilic addition of O 2 .

-thus may contribute to the overall degradation of 4.

Production of singlet oxygen 1 O 2

Values for the quantum yield of singlet oxygen production (Φ 1 O 2 ) are summarized in Table 4 and outlined in Fig. 3. Relatively low values of Ф 1 O 2 for all seven derivatives can be attributed to the ICT character of these molecules, which manifests its effect in sensitizer-oxygen contact complexes [46,47] . CT character in the sensitizer can influence the energy, yield, and lifetime of the triplet state, all of which can, likewise, ultimately be reflected in the singlet oxygen yield.

Increasing CT interactions reduce the overall quantum yield of singlet oxygen formation Ф 1 O 2 and the decrease is further enhanced in polar solvents due to the enhancement of CT interactions (stabilization of exciplexes of triplet excited sensitizer and O 2 in moving from non-polar to highly polar solvents). The extent of CT, both within the sensitizer itself (i.e., intramolecular CT) as well as in the sensitizer-oxygen complex (i.e., intermolecular CT), thus have a large adverse effect on the efficiency with which singlet oxygen is generated [48] .

The increasing value of Ф 1 O 2 after addition of the acceptor groups to the Nmethylbenzothiazolium moiety of 1 (simultaneous increase of E 0' ) demonstrates an effect of intermolecular CT (molecules 4 and 5). This behavior does not take effect on going from molecule 2 to structure 6 and 7 and even a slight decrease of Ф 1 O 2 in both cases was observed.

These results suggest a controversial role of a combination of both inter-and intramolecular CT on the 1 O 2 production efficiency, possibly affecting the quantum yield of the triplet state population and/or a triplet state deactivation pattern. As was previously mentioned by Nielsen et al. [48] , one cannot always rely on rule-of-thumb guidelines when attempting to construct either efficient or inefficient 1 O 2 sensitizers and a full investigation of the photophysical properties of the system studied is generally required.

The value of Ф 1 O 2 for 1-3 represents again an unexpected decrease of Ф 1 O 2 of 1-3 with increasing value of E 0 . This increasing tendency of a quantum yield with extension of conjugated conclusions of the emission probability could be done. This behavior is consistent with the fact that the S 0 -S 1 excitation energy changes more with the conjugate chain length than that of S 0 -T 1 [49,50] . Enhancement in rate constants for intersystem crossing for longer chains was observed and attributed to a lowering of the S 1 -T 1 energy gap for longer chains [49,51] . Thus, molecules with longer conjugation lengths seem to have a higher triplet quantum yield. The first excited singlet states of 1-7 should not contribute markedly to overall 1 O 2 production owing to their short lifetimes τ 0 ≤ 10 ns (τ 0 = 1.5/ν 2 max f where ν max is the wavenumber corresponding to the absorption maximum). The 1 O 2 production efficiency of the studied molecules is clearly the greatest for 3 and the least for 1.

Quenching of singlet oxygen

Estimated values of 1 O 2 quenching rate constants k Q (β Q ) (Table 3) exceeded the values expected for an electronic-to-vibrational energy transfer (converting electronic excitation energy of the 1 O 2 molecule into vibration of O 2 and quencher Q) by orders of magnitude. These high values of k Q regarding relatively high triplet energies (E T >130 kJ mol -1 ) and low oxidation potentials (E 0' <0.65 V) can be attributed to a CT-induced quenching of 1 O 2 , where the deactivation of the initially formed singlet encounter complex 1 (Q 1 ∆) EC is enhanced by the formation of a singlet exciplex 1 (Q 1 ∆) CT , which is stabilized by the transfer of electric charge from the quencher to the oxygen molecule [52] . Iodides do not contribute significantly to such high k Q values in protic solvents [53] . The formation of a singlet exciplex 1 (Q 1 ∆) CT is followed by isc to the ground-state CT complex, by chemical reaction, or by separation of free ions. Thus, the overall quenching rate constant k Q can be additively composed of a physical and a chemical component, i.e., k 3. Because of the almost constant value of E T , increasing k Q with extension of the conjugated bridge can be rationalized by the decreasing value of E 0' . This is an established phenomenon [52] . Albeit to the opposite effect of NO 2 (CN) group addition on E 0' , k Q considerably increased after incorporation of these groups to the benzothiazolium moiety. Considering again the relatively constant values of E T , these results indicate a significant role of intramolecular charge-transfer (ICT) in the CT-induced quenching of 1 O 2 . ICT most likely plays an important role not only in the production of 1 O 2 , but also in the quenching of 1 O 2 . The increasing tendency of k Q copies the increasing tendency of ground-state dipole moments µ [11] , although absolute correlation does not exist. Polarizability of the chromophore most likely contributes as another factor to CT-induced quenching of 1 O 2 . An opaque trend of k Q behavior after the exchange of a CN group for an NO 2 group in pairs 4-5 and 6-7 could be explained by different ground state geometry of compound 6 (values of log ε and f in Table 1). Calculated µ of compound 6 probably do not correspond with an objective ground-state dipole moment.

Q = k p + k r . Σ + →  Σ →  ∆ → ← ∆ → ← ∆ + 3 CT 3 3 CT 1 1 EC 1 1 1 Q ) Q ( ) Q ( ) Q ( Q p k Rate constants k Q of 1-

Self-sensitized photooxidation by 1 O 2

In many cases, CT quenching of 1 O 2 additionally competes with chemical reactions, which are often far from negligible [52] . To determine the fraction of chemical reaction with 1 O 2 in the overall quenching of 1 O 2 , we carried out a series of experiments in which the decrease in absorbance of 1-7 due to MB-generated 1 O 2 in the absence of any other quencher was monitored.

According to the mechanism of photooxidation by 1 O 2 , the reaction rate at low concentration

(β r >>[C]) (assuming that [ 1 O 2 ]
is constant) is given by the following equations [33,34] :

[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ][ ] [ ] C O C C C C C C C d C d 2 1 r d r f r f r f r a ) MB ( O a r 2 1 K k k k k k k I I t = = = = + = + Φ = Φ = - β β β . (8)
The photofading rate constant K for the dyes was obtained from the slope of the plot

( ) Kt A A = t 0 / ln , (9)
where The slopes of the plots indicate that the photofading rate constant is the greatest for 4 and the smallest for 7 (Fig. 4B).

K=[C][ 1 O 2 ].
If the rate of 1 O 2 formation k f is known, then we can calculate an objective value of the secondorder rate constant k r (or β r ) for 1 O 2 photooxidative attack. The k f value was obtained by multiplying the known value Ф 1 O 2 for MB in MeOH by the value of I a absorbed by this sensitizer. Results are summarized in Table 3.

Concentrations of chromophores 1-3 did not change after 2 hours on irradiation at the same experimental conditions. This does not necessarily mean that molecules 1-3 do not react with 1 O 2 , but the second-order rate constant k r should not exceed 10 5 dm 3 mol -1 s -1 (β r ≥ 0.1 mol dm -3 ).

To determine the kinetic parameters of self-sensitized photooxidation due to 1 O 2 production and subsequent reaction between 1 O 2 and the sensitizer, we first calculated the timedependent concentration values (c i+1 ) for this photodegradation pathway using the following equations: i=0, 1, 2, …,n ; ∆t = 50000 s (10) and then subtracted the half-life times (t 1/2(1O2) ) of the self-sensitized photooxygenation reaction and the times of degradation of 90% of the compound (t 9/10(1O2) ) at these reaction from the corresponding graphs c i+1 = f(t i ) (Fig. 5) (coefficients α were determined using OO SD 2000 spectrophotometer).

To compare the kinetic parameters of autooxidation by 1 O 2 for molecules with (4-7) and without (1-3) an acceptor group bonded to the heterocycle, the maximum values of k r =10 5 dm 3 mol -1 s -1 (β r ≥ 0.1 mol dm -3 , respectively) for all three chromophores 1-3 was taken. Results are summarized in Table 4. The obtained order of self-sensitized photodegradation for 1-3 coincides with the order of photodegradation after 3 weeks of the exposure of solution of 1-3 (10 -4 mol.dm - 3 ) to daily sunlight. For a more accurate estimation of kinetic parameters describing this The quantum yields (Φ r(1O2) ) of these self-sensitized photodegradations were calculated according to:

[ ] C r O r(102) 2 1 β Φ = Φ . (11)
Values of Φ r(1O2) are expressed as number×[C] to point out their dependence on the concentration of the corresponding chromophore (Table 4). Times t 1/2(1O2) and t 9/10(1O2) are relatively high (except derivate 3 with t 1/2(1O2) ∼ 4 days) and showed strong dependence on addition of an acceptor group to the heterocycle and the length of the conjugated bridge. Both structural changes decrease the overall photostability although the change is not so significant after the addition of an acceptor group to skeleton 2. Self-sensitized photooxidation by 1 O 2 thus makes a slow parallel degradation pathway for irradiation at 300-850 nm and, albeit to the small Ф, contributes with large fraction to the overall photodegradation of 4-7 (and probably also of 1-3).

Exchange of the NO 2 group for CN causes a large increase in the photostability of compound 5, but had only a very small influence on the photostability of 7. This difference could be most likely due to the different ground state geometry of compound 6. The photostabilities of the chromophores can be placed in the order:

1 > 5 > 2 > 7 > 6 > 4 > 3.
It is apparent that a compromise should be made between an increase in NLO response and a decrease in photostability to enable a choice of NLO chromophores for practical applications. (A) and 7 (B) at 300-850 nm (I 0 =2.05±0.05×10 -5 mol s -1 dm -3 ). Absorbed photon flux at t=0 s was I a0 =1.38×10 -5 mol s -1 dm -3 for 2 and I a0 =1.05×10 -5 mol s -1 dm -3 for 7.

  N-methylbenzothiazolium electron-acceptor group, an alkenylene conjugation spacer with 1-3 double bonds and an N,N-dimethylamino group as a donor in the para position of the substituted phenyl group. The acceptor strength of 4-7 is enhanced by introducing an additional withdrawing substituent into the benzothiazole fragment (NO 2 or CN).The chemical structure of the compounds studied is presented in Fig.1.

  photooxidative attack of singlet oxygen ( 1 O 2 ) in this solvent (Sections 2 and 3 in EXPERIMENTAL). All photodegradation measurements were carried out at 25°C in the dark with only the 100-W xenon lamp as the light source. Concentrations of 1-7 never exceeded 2×10 -

5 I 0

 50 =2.05±0.05×10 -5 mol s -1 dm -3 ). The actual concentration of compounds studied in air-saturated solutions during irradiation was measured spectrophotometrically (HP 8452A) at shielding the incident photon flux from the light source. Absorbed photon flux I a (by compound X) was calculated from emission curves for the light source measured on an Ocean Optics SD 2000 spectrophotometer using the following equation:

  Electron paramagnetic resonance (EPR) spectra were recorded on an ERS 230 instrument (ZWG Berlin, Germany), which operates in the X-band (~9.3 GHz) with a modulation amplitude of 0.1 mT and microwave power of 5 mW. A sample containing 2.5×10 -5 mol dm -3 of one of the derivatives studied was continuously irradiated at ~60 W m -2 by a 250-W halogen lamp through a 10-cm water filter for 20 min. The superoxide anion radical generated was indirectly monitored using a spin trap with 5,5-dimethyl-4,5dihydroxypyrrole-N-oxide (DMPO, Sigma; c ~3.5×10-4

8 (

 8 Coumarin 153), and 575nm (Rhodamin 6G). The samples were excited through optically polished bottoms of fluorescence cells. Luminescence was collected by lens assembly through two long-pass filters Schott RG7 and high-luminosity monochromator Jobin Yvon H20IR to the infrared-sensitive photomultiplier Hamamatsu R5509 cooled to -50 °C by liquid nitrogen. The pulses from the photomultiplier were fed through the Becker-Hickl HF AC-26dB preamplifier to the Becker-Hickl MSA 200 photon counter/multiscaler with time resolution of 5 ns per channel.

  and quenching of singlet oxygen 1 O 2 (RESULTS AND DISCUSSION -Sections 2.2 and 2.

1 - 7 , 1 .

 171 photostability of 1-7, MeOH solutions of the compounds were subjected to irradiation with a 100-W Xe lamp.

12 Φ

 12 deg exhibited no concentration dependence, consistent with a first-order photodecomposition process. The values of Φ deg ranged from (0.11-0.14)×10 -4 (2% photodegradation) for 5 to (5.5-5.7)×10 -4 (0.8% photodegradation) for 2, with the percentage photodegradation calculated from the asymptotes of the fitted exponential curves. Small values of Φ deg are most likely connected with the extent of CT character in the molecules studied. Substantial competition of the ICT

  ability of phosphorescence emission by molecules 1-3 although only qualitative

7

 7 showed very strong dependence on the length of the conjugated bridge and an addition of acceptor group to the N-methylbenzothiazolium moiety. Values of k Q increased significantly with both characteristics. Both quenching of 1 O 2 and 1 O 2 production depends on two main parameters: triplet-state energy E T and oxidation potential of the sensitizer.The oxidation process is reversible for all studied compounds and its formal redox potential E 0' is

Fig. 4

 4 Fig. 4 shows the photostability of 4-7 in MeOH against 1 O 2 photooxigenative attack. The linear relationships between ln[C] 0 /[C] t and t indicate that these are first-order kinetics reactions.

17 degradation pathway for 1 - 3 ,

 1713 further experiments with higher irradiation intensities and more effective sensitizer should be carried out.

Finally, the minimal

  photodegradation pathway (0.014×[C] and 0.012×[C]) and the high β values (370×10 -30 esu and 184×10 -30 esu) mean that compounds 2 and 5 are promising for NLO applications. Compound 3, albeit to very large value of β, is not a good candidate for practical NLO applications due to its poor photostability.

18 CONCLUSIONSFig. 1 .

 181 photodegradation of 1-7. Electron transfer (formation of a superoxide anion radical) and subsequent oxidation reactions (followed by various radical recombinations) or nucleophilic addition of O 2 .-may contribute to the overall degradation of compound 4. Both structural modifications increasing NLO response (extension of the conjugated bridge and an additional acceptor group bonded to the heterocycle) led to a decrease in photostability due to manifestation of the main photodegradation pathway (self-sensitized 1 O 2 photooxidative attack). From the values of the kinetic parameters of self-sensitized photooxidation by 1 O 2 , it can be concluded that a compromise should be made between an increasing NLO response and a decreasing photostability to enable a choice of NLO chromophores 1-7 for practical applications. The existence of photostationary states at ~1-2% degradation by photoisomerization and low photochemical quantum yield (Φ = 0.014×[C] and 0.012×[C]) for the main photodegradation pathway, together with high β values mean that 2 and 5 are promising derivatives for NLO applications.

Fig. 2 .

 2 Fig. 2. Changes in the concentrations of 1-3 (A) and 4-7 (B) in MeOH on irradiation with a 100-W Xe lamp (300-850 nm; I 0 =2.05±0.05×10 -5 mol s -1 dm -3 ). Time-dependent concentrations are fitted by single exponential functions [dotted line].

Fig. 3 .

 3 Fig. 3. Kinetic changes in the absorption spectra of 2,5-DMF in MeOH during irradiation of 2

Fig. 4 . 2 (Ф 1 O 2 7 against 1 O 2

 4212712 Fig. 4. (A) Photodegradation of 4 in MeOH due to photooxidation by MB-generated 1 O 2 (Ф 1 O 2 =0.50±0.01, I a =4.5±0.5×10 -6 mol s -1 dm -3 for MB; colour filter with transmittance >550 nm was used to avoid direct photodegradation of 4). (B) Photostability of MeOH solutions of 4-7 against 1 O 2 photooxidative attack (dotted lines -linear regression of experimental data).

Fig. 5 .

 5 Fig. 5. Calculated photodegradation curves of (A) 1-3 and (B) 4-7 in MeOH due to self-sensitized

Φ 1 O 2 - 2 FFig. 1 .

 1221 Fig. 1. Chemical structure of the studied D--A benzothiazolium salts.

Fig. 2 .

 2 Fig. 2. Changes in the concentrations of 1-3 (A) and 4-7 (B) in MeOH on irradiation with a 100-W Xe lamp (300-850 nm; I0=2.05±0.05×10-5 mol s-1 dm-3). Time-dependent concentrations are fitted by single exponential functions [dotted line].

Fig. 3 .

 3 Fig. 3. Kinetic changes in the absorption spectra of 2,5-DMF in MeOH during irradiation of 2 (A) and 7 (B) at 300-850 nm (I0=2.05±0.05×10-5 mol s-1 dm-3). Absorbed photon flux at t=0 s was Ia0=1.38×10-5 mol s-1 dm-3 for 2 and Ia0=1.05×10-5 mol s-1 dm-3 for 7.

Fig. 4 .

 4 Fig. 4. (A) Photodegradation of 4 in MeOH due to photooxidation by MB-generated 1O2 (Ф1O2=0.50±0.01, Ia=4.5±0.5×10-6 mol s-1 dm-3 for MB; colour filter with transmittance >550 nm was used to avoid direct photodegradation of 4). (B) Photostability of MeOH solutions of 4-7 against 1O2 photooxidative attack (dotted lines -linear regression of experimental data).

Fig. 5 .

 5 Fig. 5. Calculated photodegradation curves of (A) 1-3 and (B) 4-7 in MeOH due to self-sensitized 1O2 photooxidative attack during irradiation at 300-850 nm (incident photon flux I0=2.05×10-5 mol s-1 dm-3).

Table 1 .

 1 Photophysical properties of the molecules studied A -the absorption maximum, log εthe extinction coefficient, f -the oscillator strength, E A -the ICT energies, λ Pthe phosphorescence maximum, E T -the energy of triplet state, τ 1 and τ 2 -fast and slow decay time of luminescence signals, β -the first hyperpolarizability

	Dye	λ A (nm)	E A (kJ mol -1 )	log ε	f	λ P (nm)	E T (kJ mol -1 )	τ 1 (ns)	τ 2 (µs)	β (10 -30 esu)
	1	520	230	4.83	1.07	841	142	85±3	1.3±0.4	98.5
	2	562	213	4.78	1.24	851	141	76±4	1.2±0.1	370.1
	3	580	206	4.75	1.33	861	139	79±8	1.4±0.1	744.8
	4	562	213	4.90	0.99	854	140	82±9	1.2±0.1	183.3
	5	551	217	4.91	1.01	863	139	90±8	1.2±0.1	183.7
	6	630	190	4.54	0.53	852	140	83±1	1.2±0.2	317.3
	7	618	194	4.83	1.14	850	142	83±2	1.1±0.1	321.0
	λ

Table 2 .

 2 Photodegradation characteristics of 1-7 on irradiation at 300-850 nm -the photodegradation rate constant, t 0.5 -the time for 50% degradation, t 0.9 -the time for 90% degradation, Φ degthe quantum yield for photodecomposition, % initial -the percent of initial isomer in the photostationary state, % 1 O 2 contribution -the contribution of self-sensitized photooxidation by 1 O 2 to overall photodegradation during photoisomerization

	Dye	k (10 -4 s -1 )	t 0.5 (min)	t 0.9 (min)	Φ deg ×10 -4	% initial	% 1 O 2 contribution
	1	1800	0.07	0.22	4.33	99.6	0.002
	2	1150	0.10	0.33	5.60	99.2	0.060
	3	76.0	1.52	5.05	0.57	97.9	2.2
	4	7.8	15	49	0.15	96.7	3.2
	5	10.0	12	38	0.13	97.9	1.4
	6	9.1	13	42	0.66	92.8	0.6
	7	10.0	12	38	0.18	97.2	1.5
	k

Table 3 .

 3 Parameters for characterization of physical and chemical quenching of 1 O 2 by 1-7 Q -the rate constant for overall 1 O 2 quenching, β Q -the relative reactivity index (includes both physical and chemical quenching) for 1 O 2 quenching, E 0' -the formal redox potential, µ -the ground-state dipole moment, k r -the rate constant for 1 O 2 photooxidative attack, β r -the reactivity index of dye with 1 O 2

	Dye	k Q (10 9 dm 3 mol -1 s -1 )	β Q (10 -5 mol dm -3 )	E 0' vs Fc/Fc + (V)	µ (D)	k r (10 6 dm 3 mol -1 s -1 )	β r (10 -2 mol dm -3 )
	1	0.17	65.0	0.567	2.4	≤ 0.1	≥ 10
	2	1.50	7.3	0.381	4.1	≤ 0.1	≥ 10
	3	5.00	2.2	0.264	7.3	≤ 0.1	≥ 10
	4	1.20	9.2	0.626	12.4	3.2	3.5
	5	0.41	26.8	0.610	7.3	1.5	7.5
	6	3.70	3.0	0.435	12.0	2.2	5.0
	7	7.10	1.6	0.424	6.1	1.5	7.3
	k

Table 4 .

 4 Parameters for characterization of 1 O 2 production by 1-7 and subsequent self-sensitized photooxidation

	Dye	Φ 1 O 2	E 0' vs Fc/Fc + (V)	t 1/2(1O2) (days)	t 9/10(1O2) (days)	Φ r(1O2)
	1	0.0006	0.567	80.5	362	0.0063×[C]
	2	0.0014	0.381	29.5	124	0.014×[C]
	3	0.0098	0.264	4.1	15	0.098×[C]
	4	0.0012	0.626	13.5	60	0.034×[C]
	5	0.0009	0.610	38.8	177	0.012×[C]
	6	0.0011	0.435	25.0	115	0.021×[C]
	7	0.0012	0.424	27.0	116	0.017×[C]

http://mc.manuscriptcentral.com/poc Journal of Physical Organic Chemistry

mol dm-3 , to avoid molecule aggregation and photochemical dimerization processes.

Acknowledgements

This work was supported by the Slovak Research and Development Agency through APVV Grant No. 0259-07, by the Slovak Grant Agency for Science (VEGA No. 1/ 0639/08), UK Grant No. UK/229/2007, Grant Agency of the Czech Republic (GACR 203/08/1157), and by the project MSM 0021620835 from the Ministry of Education, Youth, and Sports of the Czech Republic.


Veli Uitto

-Jukka

Leppilahti Jussi

Ahonen Minna-Maija

Hernández Marcela

Munjal Sarvan

Netuschil Lutz

Uitto Veli-Jukka

Sorsa Timo

Mäntylä Päivi
email: paivi.mantyla@helsinki.fi

Dr Päivi Mäntylä

Oral rinse MMP-8 point-of-care test identifies patients with strong periodontal inflammatory burden

Keywords: oral rinse, biomarker, chair-side test, periodontal diagnostics, MMP- Oral rinse, biomarker, chair-side test, periodontal diagnostics, MMP-8

Objective:

The aim of the study was to determine whether oral rinse MMP-8 levels, measured by three different methods, TIMP-1 levels and elastase activity differentiate subjects with different periodontal condition; and second, to find out if MMP-8 levels were comparable among the methods used.

Methods: MMP-8 levels were analyzed with an immunofluorometric method (IFMA), dentoAnalyzer and commercial ELISA. Also TIMP-1 levels and elastase activity were measured.

For statistical analysis 214 study subjects were categorized into four groups, specified by the presence and number of moderate (4-5 mm) and deep (≥ 6 mm) periodontal pockets, and bleeding on probing percentage.

Results: MMP-8 levels especially measured by dentoAnalyzer and adjusted to the number of teeth per subject differentiated the study group with strong periodontal inflammatory burden from groups with lower levels. This was also verified with receiver operating characteristic (ROC) analysis. Elastase activity associated with higher IFMA and dentoAnalyzer MMP-8 levels. IFMA MMP-8/TIMP and dentoAnalyzer MMP-8/TIMP-1 tended to be higher with the increasing level of periodontal inflammatory burden. TIMP-1 levels decreased with increasing age.

Conclusions: Oral rinse MMP-8 together with TIMP-1 analysis may have potential in complementary periodontal diagnostics. dentoAnalyzer can be applied in quantitative oral rinse chair side biomarker diagnostics.

Introduction

Periodontitis is a very common inflammatory disease in humans. Though bacteria are the initiating factor, periodontitis is not a classical infectious disease. Majority of the causative organisms are also present in the healthy mouth [START_REF] Socransky | Periodontal microbial ecology[END_REF], and the host response modified by environmental and behavioural factors plays a key role in disease development, progression and in the maintenance of the treatment result. Despite the etiology or type of periodontitis, the products of host response and tissue destruction are similar. Markers of inflammatory process from periodontal tissues migrate into the periodontal pockets within a serum originating fluid, gingival crevicular fluid (GCF) (Uitto et al., 2003), and further from gingival crevices/pockets into oral cavity where GCF associates with saliva. The more inflamed the periodontal tissues and the deeper the periodontal pockets are, the more GCF is excreted and the more it contains inflammatory markers (Uitto et al., 2003). This also means that the inflammatory burden of periodontium may be reflected in saliva [START_REF] Uitto | Salivary collagenase. Origin, characteristics and relationship to periodontal health[END_REF][START_REF] Sorsa | The role of gingival crevicular fluid and salivary interstitial collagenases in human periodontal diseases[END_REF].

Unlike in medicine, biochemical testing in dentistry has not yet been taken into everyday clinical practice despite the advantages in supplementation of traditional diagnostics. GCF has been used for site specific diagnostic studies of periodontal disease status, and diagnostic tests for host derived markers have been developed (Uitto et al., 2003;[START_REF] Sorsa | Matrix metalloproteinases (MMPs) in oral diseases[END_REF]. Inflammatory markers from GCF analysed individually or in combination may be valuable in the identification of individual sites or teeth at risk of periodontitis progression. Saliva can also be regarded as a diagnostic fluid but unlike GCF, it mirrors more generally the oral status and is more unspecific.

Whole saliva contains sloughed epithelial cells from oral mucous membranes, nasopharyngeal discharge, food debris, bacteria and their products and, in dentate subjects, its content is also affected by GCF flow from gingival pockets. An oral rinse sample can be regarded as a fluid containing mainly the GCF from all periodontal pockets [START_REF] Sorsa | The role of gingival crevicular fluid and salivary interstitial collagenases in human periodontal diseases[END_REF][START_REF] Uitto | Salivary collagenase. Origin, characteristics and relationship to periodontal health[END_REF]Uitto et al., , 2003)). Thus, analysis of oral rinse may be useful in the diagnosis of general periodontal disease status, early identification of subjects at risk of periodontitis (Uitto et al., 2003), evaluation of periodontal treatment results or the need of hygiene visits during maintenance and in the follow up of the effect of modifying medication over host response (for example low-dose doxycycline, LDD) [START_REF] Reinhardt | Association of gingival crevicular fluid biomarkers during periodontal maintenance with subsequent progressive periodontitis[END_REF]. (MMPs) and their inhibitors and polymorphonuclear leukocyte (PMN) elastase could be candidates as diagnostic markers in oral rinse specimens (Uitto et al., 2003;[START_REF] Sorsa | Matrix metalloproteinases (MMPs) in oral diseases[END_REF]. Among MMPs, especially GCF MMP-8 levels have been found to be increased in periodontitis [START_REF] Mäntylä | Gingival crevicular fluid collagenase-2 (MMP-8) test stick for chair-side monitoring of periodontitis[END_REF][START_REF] Mäntylä | Monitoring periodontal disease status in smokers and nonsmokers using a gingival crevicular fluid matrix metalloproteinase-8-specific chair-side test[END_REF]. The deeper the periodontal pockets are, the higher are the levels of MMP-8 at individual sites, and the levels decrease by periodontal hygiene phase treatment. GCF MMP-8 levels remain low in treated and stable periodontitis while in subjects with tendency to progression of periodontitis or poor treatment response (refractory periodontitis) GCF indicates repeatedly high MMP-8 levels. Though smoking may decrease GCF MMP-8 levels, there may also be smoking subjects who repeatedly have especially high GCF MMP-8 levels and poor treatment response [START_REF] Mäntylä | Monitoring periodontal disease status in smokers and nonsmokers using a gingival crevicular fluid matrix metalloproteinase-8-specific chair-side test[END_REF].

Salivary MMP-8 levels have been suggested serving as a biomarker of periodontitis and applicable as a point-of-care test because MMP-8 levels in saliva have been found to correlate with periodontal disease severity [START_REF] Miller | Salivary biomarkers of existing periodontal disease: a cross-sectional study[END_REF][START_REF] Ramseier | Identification of pathogen and host-response markers correlated with periodontal disease[END_REF]. [START_REF] Herr | Microfluidic immunoassays as rapid saliva-based clinical diagnostics[END_REF] have introduced a microfluidic assay for point-of-care rapid quantitation of biomarkers from human saliva which in validation with MMP-8 identified the periodontitis subjects.

Also salivary elastase-like activity has been concluded to reflect the severity of periodontal destruction because elevated activity levels have been found in untreated periodontitis subjects' saliva, which decrease after periodontitis hygiene phase treatment [START_REF] Nieminen | The effect of treatment on the activity of salivary proteases and glycosidases in adults with advanced periodontitis[END_REF]. The results were similar in the oral water-rinse samples: a significant correlation was noticed between oral rinse elastase activity and the average of community index of periodontal treatment needs (CPITN) [START_REF] Uitto | Oral fluid elastase as an indicator of periodontal health[END_REF].

In our previous study [START_REF] Sorsa | Detection of gingival crevicular fluid MMP-8 levels with different laboratory and chair-side methods[END_REF] we analyzed GCF MMP-8 levels by different methods and found that methods which based on the same monoclonal antibody against MMP-8 [immunofluorometric method (IFMA), MMP-8 specific chair-side dip-stick test and dentoAnalyzer appliance] gave comparable results. In this current paper we have used IFMA, dentoAnalyzer as well as commercially available Amersham ELISA assay to determine MMP-8 levels in oral rinse samples from subjects with different periodontal health status expressed as different levels of periodontal inflammatory burden. Additionally, TIMP-1 levels and detection of elastase-like activity were screened in oral rinse samples by Amersham ELISA and a test utilising a chromogenic substrate [START_REF] Nieminen | The effect of treatment on the activity of salivary proteases and glycosidases in adults with advanced periodontitis[END_REF][START_REF] Uitto | Oral fluid elastase as an indicator of periodontal health[END_REF], respectively. The aim was to find out if these markers analyzed with the applied methods individually or in combination, have diagnostic

Materials and Methods

Patients

In this cross-sectional study 214 adult patients with age range 44-78 years (mean±sd 57.2±7.9) were randomly selected from dental public health clinics in cities of Helsinki and Vantaa.

Patients in need of antibiotic prophylaxis or suffering of a contagious disease were excluded. The protocol was clearly explained to all study subjects and institutional review board-approved informed consents were signed. The study protocol was approved by the ethical committee of the Institute of Dentistry, University of Helsinki and Helsinki University Central Hospital, and the experiments were undertaken with the understanding and written consent of each subject and according to ethical principles, including the World Medical Association Declaration of Helsinki.

All study subjects were examined clinically: bleeding on probing (BOP) was registered dichotomously on four tooth surfaces and pocket probing depths (PPD) by Florida-probe device (Florida Probe Corporation, USA) on six tooth surfaces. PPD was recorded if ≥ 4mm. Examinations were done by two calibrated general dentists. Background characteristics were recorded by using questionnaires. Oral rinse samples were taken from all patients.

Patients were categorized into four groups according to their periodontal inflammatory burden, determined by combining the Periodontal Inflammatory Burden Index (PIBI) described by [START_REF] Lindy | Statin use is associated with fewer periodontal lesions: A retrospective study[END_REF] and BOP%. PIBI is calculated by adding the number of periodontal sites indicating moderate periodontitis (number of periodontal pockets with PPD 4-5 mm = Nmod) to the weighted number of periodontal sites indicating advanced periodontitis (number of periodontal pockets with PPD ≥ 6 mm multiplied by two = Nadv).

Periodontal inflammatory markers are conducted into oral rinse within the GCF. Flow of GCF is increased in inflamed tissues and is also proportional to the area of inflamed epithelial surface of the periodontal sulcus/pocket [START_REF] Armitage | Clinical evaluation of periodontal diseases[END_REF]Weidlich et al., 2001). BOP% indicates inflammation in the periodontal tissues and the PIBI reflects periodontal inflammatory injury [START_REF] Lindy | Statin use is associated with fewer periodontal lesions: A retrospective study[END_REF] ) with the purpose to indicate the GCF flow. We hypothesized that the higher the flow of GCF, the higher is the level of inflammatory markers in oral rinses, and that it reflects the periodontal health status.

According to the categorization methods described above we formed four groups of study subjects: (1) 36 subjects were regarded as periodontally healthy (BOP < 10% of teeth surfaces and no deepened periodontal pockets; (2) 21 subjects with BOP ≥ 10% of teeth surfaces but no deepened periodontal pockets, mild periodontal inflammatory burden, regarded clinically as gingivitis. Patients diagnosed to have deepened periodontal pockets (PPD ≥ 4 mm) were categorized into two groups according the PIBI x BOP %: 104 subjects had PIBI x BOP % 1-100 (group 3; moderate periodontal inflammatory burden) and 53 patients had PIBI x BOP % ≥ 100 (group 4; strong periodontal inflammatory burden). Threshold of 100 was chosen to discriminate moderate inflammatory burden (clinically regarded as minor or early periodontitis) from strong inflammatory burden (clinically more advanced disease).

Oral rinse samples

Oral rinse samples were taken as follows: 1 ml of tap water was placed into the subject's oral cavity with a disposable plastic pipette. Subjects rinsed their mouth with tap water for 1 minute and the rinse was collected into collection tubes [START_REF] Nieminen | The effect of treatment on the activity of salivary proteases and glycosidases in adults with advanced periodontitis[END_REF][START_REF] Uitto | Oral fluid elastase as an indicator of periodontal health[END_REF]. On the same day an aliquot of the specimen was taken for elastase activity testing and the rest of the sample was frozen on the same day for further MMP-8 and TIMP-1 analyses [START_REF] Uitto | Oral fluid elastase as an indicator of periodontal health[END_REF].

MMP-8, TIMP-1 and elastase determinations

All samples were analyzed for MMP-8 levels by three different immunological assays: dentoAnalyzer device [START_REF] Munjal | Immunoassay-based diagnostic point-of-care technology for oral specimen[END_REF][START_REF] Sorsa | Detection of gingival crevicular fluid MMP-8 levels with different laboratory and chair-side methods[END_REF], a time-resolved immunofluorometric assay [START_REF] Hanemaaijer | T. Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells. Regulation by tumor necrosis factor-alpha and doxycycline[END_REF][START_REF] Mäntylä | Gingival crevicular fluid collagenase-2 (MMP-8) test stick for chair-side monitoring of periodontitis[END_REF][START_REF] Mäntylä | Monitoring periodontal disease status in smokers and nonsmokers using a gingival crevicular fluid matrix metalloproteinase-8-specific chair-side test[END_REF] and a commercial ELISA kit by Amersham [START_REF] Munjal | Immunoassay-based diagnostic point-of-care technology for oral specimen[END_REF][START_REF] Sorsa | Detection of gingival crevicular fluid MMP-8 levels with different laboratory and chair-side methods[END_REF]. Briefly, dentoAnalyzer is designed as a portable user-friendly bench-top instrument. The device applies a sandwich based immunoassay technology using ABICAP filters. TIMP-1 levels were measured with Amersham ELISA [START_REF] Tuomainen | Serum matrix metalloproteinase-8 concentrations are associated with cardiovascular outcome in men[END_REF][START_REF] Rautelin | Enhanced systemic matrix metalloproteinase response in Helicobacter pylori gastritis[END_REF]. Amersham ELISA analyses of MMP-8 and TIMP-1 levels were done according to manufacturer's instructions. MMP-8 and TIMP-1 results (ng/ml) were expressed as levels per subject and per total number of teeth/subject. Elastase activity test was done based on the method described by [START_REF] Nieminen | The effect of treatment on the activity of salivary proteases and glycosidases in adults with advanced periodontitis[END_REF] and [START_REF] Uitto | Oral fluid elastase as an indicator of periodontal health[END_REF]. The chromogenic substrate succinyl-alanyl-valine-p-nitroanlilide (Sigma Co, St Louis, MO, USA) was dissolved in a solution with the following final concentrations: 1 mM substrate, 0.1% dimethyl sulfoxide, 0.2 M NaCl and 0.05 M Tris-HCl buffer, pH 7.5. The enzyme activity is expressed as optical density after subtracting the 0 h value. Optical densities were measured at nominal/categorical scale by comparing the colour of test plates to a coloured slide designed for eye comparison of optical densities. Optical densities were analyzed by two observers to minimize chance of misinterpretation. Optical densities were categorized according to previously published results, which proved good correlation between elastase activity and the number of deep periodontal pockets [START_REF] Uitto | Oral fluid elastase as an indicator of periodontal health[END_REF]. 0.2 OD was used as a cut off level for a significant positive elastase activity and levels below the threshold of 0.2 OD were regarded as negative [START_REF] Uitto | Oral fluid elastase as an indicator of periodontal health[END_REF].

Data analysis

Non-parametric Kruskal-Wallis or Mann-Whitney U test for independent samples was used for testing statistical significances of differences between study groups and bivariate non-linear Spearman correlation, for testing of correlations between results obtained by different MMP-8 detection methods. An association of nominal variables in crosstables was tested by Cramér V test.

The diagnostic accuracy of MMP-8 measuring methods used was evaluated using Receiver Operating Charasteristics (ROC) curve analysis. p-values < 0.05 were considered to be statistically significant. Statistical analyses and tests were done by SPSS-software (version 17.0).

Results

Clinical parameters and demographic data

Subject groups, clinical parameters and demographic characteristics are shown in Table 1.

Association between consensus risk factors [START_REF] Genco | Current view of risk factors for periodontal diseases[END_REF] for chronic periodontitis; gender, tobacco smoking and diabetes, and different periodontal indeces were tested (Table 2). In general terms, male gender and tobacco smoking were associated with higher values of all periodontal indeces used in the study: PIBI, BOP%, and PIBI x BOP% (p < 0.001), except for the association between male gender and BOP%, that was insignificant (p = 0.323). However, diabetes associated with 2). Diabetic subjects were not examined as a separate group, and they could belong to any of the study groups 1-4.

Assocation between MMP-8 and periodontal inflammatory burden

MMP-8 levels increased along with periodontal inflammatatory burden (Table 3). Enzyme levels measured by dentoAnalyzer were significantly different among groups (p = 0.018) and differences were more pronounced when MMP-8 levels were adjusted to the number of teeth for both, dentoAnalyzer (Figure 1) and IFMA (p = 0.007 and p =0.032, respectively).

Correlations between MMP-8 detection methods

Correlation between MMP-8 levels measured with IFMA and dentoAnalyzer were tested with Spearman correlation test which gave a correlation coefficient 0.691 (statistically significant at p < 0.01 level). No correlation was found between MMP-8 analysis by dentoAnalyzer or IFMA and Amersham ELISA. MMP-8 levels measured by Amersham ELISA test did not correlate with the values of periodontal parameters.

Statistical outliers

Based on statistical outliers of MMP-8 levels adjusted to the number of teeth (Figure 1), we determined the threshold for peak levels of MMP-8/number of teeth measured by both dentoAnalyzer and IFMA. The thresholds were determined at 15 ng/ml level for dentoAnalyzer results corresponding 80 ng/ml levels for IFMA. Percentages of MMP-8/number of teeth peak levels in each study group are shown in Table 4. The higher the severity of periodontal disease (heavier periodontal inflammatory burden) was, the higher the percentage of peak values of MMP-8/number of teeth, except for group 3 (Table 4). The difference was statistically significant when MMP-8 levels were measured by dentoAnalyzer (p = 0.016; Table 4). The Amersham ELISA MMP-8 levels did not express statistical outliers.

MMP-8/TIMP-1 ratio and TIMP-1 levels

MMP-8/TIMP-1 molar ratios increased together with periodontal inflammatory burden when measured by dentoAnalyzer (p = 0.033, Table 3). only the latter differences were significant (p < 0.001, Figure 2).

Elastase activity

A statistically significant difference (p < 0.001) in MMP-8/number of teeth levels between groups with negative and positive elastase activity test results was detected when MMP-8 was measured by dentoAnalyzer or IFMA but not when measured by Amersham ELISA. The difference of dentoAnalyzer MMP-8/number of teeth median values between elastase activity groups was also tested separately in each patient group and confirmed to be statistically significant (for groups with mild, moderate and strong inflammatory burden, i.e. groups 2, 3, and 4 p < 0.001; for periodontally healthy, i.e. group 1 p = 0.029; Figure 3). dentoAnalyzer MMP-8/number of teeth levels increased in line with study groups (inflammatory burden), except for group 3 with moderate inflammatory burden, as mentioned earlier in the Results and seen in Figure 1. However, the increase was especially pronounced when the study groups were further categorized according to the elastase activity (positive elastase activities: p = 0.002; negative elastase activity p = 0.322 ns; Figure 3).

ROC-curve analysis

A Receiver Operating Charasteristic (ROC) analysis was ran to evaluate the diagnostic sensitivity and specificity of dentoAnalyzer MMP-8/number of teeth. The ability of dentoAnalyzer MMP-8/number of teeth to distinguish between periodontally healthy subjects and subjects with different levels of periodontal inflammatory burden (groups 2, 3 and 4 with mild, moderate and strong inflammatory burden) was tested separately. The distinction was significant between periodontally healthy (group 1) and subjects with strong periodontal inflammatory burden (group 4) (Figure 4). The area under the curve was 0.701 (p = 0.002; 95% confidence interval 0.59-0.81).

Discussion

In this study the subjects were not selected based on a defined periodontal disease status.

Instead, the randomly selected study subjects were categorised based on the level of periodontal inflammatory burden expressed as numbers and depths of deepened periodontal pockets and percentage of bleeding on probing.

MMP-8 levels in oral rinse samples were higher in subjects with the strongest periodontal inflammatory burden than in subjects with less inflammatory changes when oral rinse levels were adjusted to number of teeth, especially when measured with dentoAnalyzer appliance. This finding was confirmed with ROC analysis. Similarly, MMP-8/TIMP-1 ratios increased in subjects with strong periodontal inflammatory burden when MMP-8 was measured by dentoAnalyzer. MMP-8 increments were especially pronounced in subjects showing detectable elastase activity.

The measurement of host originating oral biomarker levels in periodontal diagnostics has faced the problem that it has been extremely difficult to define the "normal" levels of various biomarkers. Biomarkers which are expressed at high levels in periodontal inflammation can be detected at lower levels also in periodontally healthy oral cavity, obviously as an indication of the physiologic turnover of oral tissues [START_REF] Mcculloch | Host enzymes in gingival crevicular fluid as diagnostic indicators of periodontitis[END_REF], Uitto et al., 2003). Besides, the individual expression of biomarkers in healthy state as well as in disease may vary because of genetic variation [START_REF] Mcculloch | Host enzymes in gingival crevicular fluid as diagnostic indicators of periodontitis[END_REF]Uitto et al., 2003).

MMP-8 can be regarded as a central enzyme in periodontitis development and progression [START_REF] Sorsa | Matrix metalloproteinases (MMPs) in oral diseases[END_REF][START_REF] Sorsa | Matrix metalloproteinases: contribution to pathogenesis, diagnosis and treatment of periodontal inflammation[END_REF]. It may be useful in site specific diagnostics to find out if individual sites or teeth are at risk of progression of periodontitis when analysed from GCF. [START_REF] Mäntylä | Monitoring periodontal disease status in smokers and nonsmokers using a gingival crevicular fluid matrix metalloproteinase-8-specific chair-side test[END_REF] found that repeatedly high MMP-8 levels in GCF tested with a MMP-8 specific chair side dip-stick test identified sites/teeth which were at risk of progression of periodontal attachment loss. Site specific MMP-8 testing also identified the subjects with poor response to conventional periodontal treatment, i.e. scaling and root planing. These subjects had multiple sites with an unstable character, and these sites were repeatedly test positive during the 12 month maintenance phase of periodontal treatment. Repeated testing may thus give more reliable diagnostic information than single testing in site specific biomarker diagnostics.

Smoking has been found in several studies to decrease MMP-8 levels in GCF because of the effects on local blood circulation and lowered signs of inflammation [START_REF] Söder | Neutrophil elastase activity, levels of prostaglandin E2, and matrix metalloproteinase-8 in refractory periodontitis in smokers and non-smokers[END_REF][START_REF] Söder | Neutrophil elastase activity, levels of prostaglandin E2, and matrix metalloproteinase-8 in refractory periodontitis in smokers and non-smokers[END_REF] [START_REF] Mäntylä | Monitoring periodontal disease status in smokers and nonsmokers using a gingival crevicular fluid matrix metalloproteinase-8-specific chair-side test[END_REF] detected the highest MMP-8 GCF levels in smoking subjects with poor response to conventional periodontal treatment (scaling and root planing, SRP). Thus it cannot be regarded categorically that all smoking periodontitis patients have lower levels of MMP-8 in GCF and further in oral rinse sample.

Periodontitis is regarded to be cyclic by nature, and part of the dentition may be in active and progressing phase of periodontitis while other teeth may be quiescent or even in the phase of recovery [START_REF] Carranza | The periodontal pocket[END_REF]. Cyclic nature of periodontitis may be even more challenging for oral rinse than for GCF based diagnostics. In an oral rinse sample the GCF is pooled from all periodontal pockets, and the collected oral rinse sample represents GCFs from whole periodontium. For this reason the definition of the cut off level for the biomarker which would be indicative for the deterioration of periodontal health is demanding. For this reason an oral rinse sample biomarker analysis can be used only for a rough assessment of the periodontal inflammatory level. The activity of periodontitis can only be established by monitoring patients longitudinally, and the progression can only be assessed site specifically. In our current study we did not address the question of disease activity or progression. Instead, we wanted to find out, if the surface area for subgingival biofilm accumulation expressed as periodontal pocketing and the simultaneous bleeding on probing, which points out the possibility of an ongoing inflammation, are reflected in an oral rinse sample.

In this current study some subjects with clinically healthy periodontium (no deepened periodontal pockets and bleeding on probing < 10 %) as well as some subjects with mild to moderate inflammatory burden with limited numbers of deepened periodontal pockets indicating gingivitis or mild periodontitis, expressed high levels of oral rinse MMP-8 which was detected by IFMA and dentoAnalyzer. High oral rinse MMP-8 levels in subjects with no clinical periodontal disease and in subjects with mild to moderate periodontal inflammatory burden were statistical outliers or peak values and were expressed only by some individuals in groups of subjects with healthy periodontium or with mild or moderate inflammatory burden. The cut off level for "positive" MMP-8 finding in an oral rinse sample measured by dentoAnalyzer and adjusted to the number of teeth which indicates a person at risk, may be the level above which the statistical outliers or peak values in groups of subjects with mild to moderate periodontal inflammatory burden are situated in box and whiskers plots, i.e. 15 ng/ml. These peak values in periodontally healthy subjects' samples may indicate a proinflammatory condition in subjects which may have an 12 aggressive host response and can be susceptible to develop periodontitis, i.e. subjects with a high "normal" MMP-8 level. In subjects with mild to strong inflammatory burden, oral fluid MMP-8 at least partly originates from inflamed marginal gingival tissues but levels above the risk level (15 ng/ml) may also indicate the subjects who are at risk to progress from gingivitis to periodontitis, or at risk of progression of periodontitis.

MMP-8 levels measured by IFMA and dentoAnalyzer, but not with Amersham ELISA, were comparable. The obvious reason for the high correlation between IFMA and dentoAnalyzer MMP-8 levels is the same monoclonal antibody shared by both methods [START_REF] Hanemaaijer | T. Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells. Regulation by tumor necrosis factor-alpha and doxycycline[END_REF][START_REF] Mäntylä | Gingival crevicular fluid collagenase-2 (MMP-8) test stick for chair-side monitoring of periodontitis[END_REF][START_REF] Mäntylä | Monitoring periodontal disease status in smokers and nonsmokers using a gingival crevicular fluid matrix metalloproteinase-8-specific chair-side test[END_REF][START_REF] Sorsa | Detection of gingival crevicular fluid MMP-8 levels with different laboratory and chair-side methods[END_REF], but based on its association with the severity of periodontal inflammation, this antibody may also be applicable and promising in oral fluid periodontal diagnostics. The antibody used in dentoAnalyzer and IFMA exert high sensitivity to target active forms of MMP-8 [START_REF] Sorsa | Detection of gingival crevicular fluid MMP-8 levels with different laboratory and chair-side methods[END_REF][START_REF] Hanemaaijer | T. Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells. Regulation by tumor necrosis factor-alpha and doxycycline[END_REF], whereas the ELISA might test proform and thus target total MMP-8. IFMA is a method which can be used only in research laboratories and for this reason it is not ideal for rapid chair-side use in a dentist's office (Sorsa et al., 2010). dentoAnalyzer has been designed for chair-side use and it gives numeric quantitative information about the analysis in 15 minutes [START_REF] Munjal | Immunoassay-based diagnostic point-of-care technology for oral specimen[END_REF][START_REF] Sorsa | Detection of gingival crevicular fluid MMP-8 levels with different laboratory and chair-side methods[END_REF].

It has been suggested that the simultaneous measurement of several oral fluid biomarkers could be beneficial in periodontal diagnostics. In this current study oral rinse MMP-8 levels and elastase activity, both enzymes which originate especially from PMN cells, associated with the level of periodontal inflammatory burden. However, it is not necessarily useful to measure many biomarkers with the same cellular origin. In this study we also analysed TIMP-1 levels, which is a tissue inhibitor of MMPs, in oral rinse samples. MMP-8/TIMP-1 ratios had a trend to be higher in the group of subjects with strong inflammatory burden , and the ratio was significantly higher when MMP-8 levels were analysed by dentoAnalyzer.

A novel finding was that oral rinse TIMP-1 levels decrease with increasing age which can be regarded as an indication of age related changes in tissue homeostasis. Age as such has not been regarded as a risk factor for periodontitis. In aging skin, expression of TIMP-1 is decreased with fibroblast senescence [START_REF] Hornebeck | Down-regulation of tissue inhibitor of matrix metalloprotease-1 (TIMP-1) in aged human skin contributes to matrix degradation and impaired cell growth and survival[END_REF]. Aging may have a similar effect in oral mucous membranes and periodontal tissues. Relevant age related changes related to TIMP-1 decrease can also be supported by the findings of [START_REF] Benatti | Influence of aging on biological properties of periodontal ligament cells[END_REF] indicating that aging modulates important biological properties of periodontal ligament cells and favours extracellular matrix remodelling. when the likelihood of a disease is estimated [START_REF] Pauletto | Effect of cigarette smoking on oral elastase activity in adult periodontitis patients[END_REF]Uitto et al., 2003;[START_REF] Sorsa | Matrix metalloproteinases: contribution to pathogenesis, diagnosis and treatment of periodontal inflammation[END_REF]. Very seldom a single biochemical test is diagnostically fully reliable but it may be helpful as an adjunct to clinical examination [START_REF] Mäntylä | Gingival crevicular fluid collagenase-2 (MMP-8) test stick for chair-side monitoring of periodontitis[END_REF][START_REF] Mäntylä | Monitoring periodontal disease status in smokers and nonsmokers using a gingival crevicular fluid matrix metalloproteinase-8-specific chair-side test[END_REF]. No positive test finding in clinic should lead to treatment unless clinical findings are in line, and vice versa, the negative finding shouldn't lead to the neglect of health promoting measures if clinical findings indicate inflammation. Based on our findings, the oral rinse sample analysis of MMP-8 could be clinically useful in rough screening to indentify individuals who are at risk to develop periodontitis or in analysing the individual level of host response. Simultaneous analysis of MMP-8 and TIMP-1 could be beneficial. Oral rinse sample analysis may be useful in defining the optimal period between periodontal maintenance visits after active periodontal treatment. During the active treatment phase it would be possible to monitor the decrease of oral rinse MMP-8 levels, and oral rinse MMP-8 levels at the end of the active treatment phase would show the individual level to keep the host response in control. In this regard, the testing could give valuable information about the control of inflammation which today is based on the clinical findings, i.e. shallower or eliminated periodontal pockets and less bleeding on probing. At best the biomarker level could be monitored by a home test to indicate when the cut off of for possibly unsafe biomarker level is reached. Also the effect of MMP-8 inhibiting low-dose doxycycline (LDD) medication could be monitored by analysing the oral rinse MMP-8 levels to find out when a possible brake in medication would be possible or when the medication should be taken again [START_REF] Golub | Subantimicrobial-dose doxycycline modulates gingival crevicular fluid biomarkers of periodontitis in postmenopausal osteopenic women[END_REF][START_REF] Reinhardt | Association of gingival crevicular fluid biomarkers during periodontal maintenance with subsequent progressive periodontitis[END_REF].

In conclusion, MMP-8 testing of oral rinse samples may be beneficial in periodontal diagnostics, but should be applied at this phase of research with caution. Testing may be adjunctive to other clinical screening methods if it is used correctly. Today it cannot be ruled out that there may be false negative findings because of inadequate knowledge about the safe cut off levels, and the levels may vary between different individuals depending on individual biomarker profiles.

Simultaneous analysis of TIMP-1 may be beneficial.

Figure 1. Boxplots representing the distributions of MMP-8 levels detected by dentoAnalyzer and IFMA adjusted to the number of teeth in study groups (p = 0.007). Threshold line for peak dentoAnalyzer and IFMA MMP-8 levels is drawn at 15ng/ml and 80 ng/ml levels, respectively.

Figure 2. TIMP-1 values grouped according to age of studied subjects (p < 0.001).

Figure 3. Association between the detection of elastase activity and dentoAnalyzer MMP-8/number of teeth levels in study groups. Green represents the category of positive elastase activity and blue negative elastase activity, respectively. dentoAnalyzer MMP-8 levels v/s elastase activity in subject groups 1-4 with healthy peridontium, and with mild, moderate and strong periodontal inflammatory burden, respectively, p < 0.05;. dentoAnalyzer MMP-8 levels v/s all study groups 1-4, in subjects with positive elastase activity, p = 0.002. There were no statistically significant differences between study groups with negative elastase activities.

Figure 4. ROC (receiver operating characteristic) curve analysis for the evaluation of diagnostic sensitivity and specificity of dentoAnalyzer MMP-8/number of teeth levels in different levels of periodontal inflammatory burden (study groups 1-4 with healthy peridontium, mild, moderate and strong periodontal inflammatory burden, respectively). The area under the ROC curve, 95% confidence interval, and p-values are given next to the figure. Association between the detection of elastase activity and dentoAnalyzer MMP-8/number of teeth levels in study groups. Green represents the category of positive elastase activity and blue negative elastase activity, respectively. dentoAnalyzer MMP-8 levels v/s elastase activity in subject groups 1-4 with healthy peridontium, and with mild, moderate and strong periodontal inflammatory burden, respectively, p < 0.05;. dentoAnalyzer MMP-8 levels v/s all study groups 1-4, in subjects with positive elastase activity, p = 0.002. There were no statistically significant differences between study groups having with negative elastase activities.

  p = 0.016) in this study population (Table

  always somewhat unreliable and they should only be supplementary

  Boxplots representing the distributions of MMP-8 levels detected by dentoAnalyzer and IFMA adjusted to the number of teeth in study groups (p = 0.007). Threshold line for peak dentoAnalyzer and IFMA MMP-8 levels is drawn at 15ng/ml and 80 ng/ml levels, respectively.

Figure 4 .

 4 Figure 4. ROC (receiver operating characteristic) curve analysis for the evaluation of diagnostic sensitivity and specificity of dentoAnalyzer MMP-8/number of teeth levels in different levels of periodontal inflammatory burden (study groups 1-4 with healthy peridontium, mild, moderate and strong periodontal inflammatory burden, respectively). The area under the ROC curve, 95% confidence interval, and p-values are given next to the figure. 196x131mm (96 x 96 DPI)

  . Based on this we reasoned to integrate PIBI and BOP% into one index (PIBI x

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  TIMP-1 levels decreased progressively with inflammatory burden and with age group, but

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  al.,

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  Overall CM, McCulloch C (2003). Proteolytic host cell enzymes in gingival crevice fluid. Lopes de Souza MA, Oppermann RV(2001). Evaluation of the dentogingival area during early plaque formation. J Periodontol. 72: 901-910.

	O r a O r a O r a O r a O r a Uitto VJ, Periodontol 2000. 31: 77-104. Weidlich P, Page 18 of 27 Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy 18 Oral Diseases -Manuscript Copy O r a
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Table 1 .

 1 Periodontal indeces and demographic data of patient groups. Medians (IQR) indicated for each parameter.

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						Deleted: x
		Group 1	Group 2	Group 3	Group 4	p-value
		Periodontally	Mild	Moderate	Strong
		healthy	inflammatory burden	inflammatory burden	inflammatory burden
		n = 36	n = 21	n = 104	n = 53
	Teeth (n)	25 (22-27)	26 (22-28)	26 (23-28)	25 (18-27)	0.094
	Age (years)	57 (52-64)	55 (50-57)	55 (52-61)	56 (51-63)	0.507
	BOP (%)	4.2 (1.8-5.9) 13.7 (12.0-16.0)	6.9 (4.8-13.9)	19.7 (14.5-28.2)	<0.001
	PIBI	0	0	2.5 (2-5)	15 (8-26)	<0.001
	PIBI x BOP (%) 0	0	21.0 (10.9-46.1)	263.9 (166.6-487.2)	<0.001

Table 2 .

 2 Association between periodontal indeces and consensus risk factors. Medians (IQR) indicated for each parameter.

Table 3 .

 3 MMP-8 and TIMP-1 levels among patient groups. Medians (IQR) indicated for each parameter. dA= dentoAnalyzer, AE = Amersham ELISA.

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Table 4 .

 4 Percentages of subjects (%) representing peak levels of MMP-8 measured by dentoAnalyzer (dA) and IFMA adjusted to numbers of teeth. Contingency coefficient and Cramer V test was used for the statistical analyses.

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		Peak	Group 1	Group 2	Group 3	Group 4	*p-
		threshold	Periodontally	Mild	Moderate	Strong	value
			healthy	inflammatory	inflammatory	inflammatory
			n = 36	burden	burden	burden
				n = 21	n = 104	n = 53
	IFMA MMP-8/number of teeth >80 ng/ml	8.3%	9.5%	3.8%	15.1%	0.104	Deleted:
			(n = 3)	(n = 2)	(n = 4)	(n = 8)
	dA MMP-8/number of teeth	>15 ng/ml	13.9%	19.0%	13.5%	34.0%	0.016
			(n = 5)	(n = 4)	(n = 14)	(n = 18)

*

Acknowledgements

This study was supported by grants from the Academy of Finland, the Research Foundation of Helsinki University Central Hospital, the Finnish Dental Society Apollonia, and the Fondecyt Foundation of Chile. Timo Sorsa is an inventor of US-patents 5652227, 5736341, 5866432 and 6143476. dentognostics GmbH has been the scientific developer of the dentoAnalyzer. However, dentognostics GmbH does not manufacture, sell or distribute this device.