Saliva nitric oxide levels in relation to caries experience and oral hygiene

The aim of the present study was to determine the relationship between nitric oxide (NO) concentration/rate in the unstimulated whole saliva (UWS) and stimulated whole saliva (SWS) with the decay-missing-filled teeth (DMFT) and simplified oral hygiene (OHI-s) scores. Forty adults were included in the study. Half of the participants (n = 20) had high DMFT-OHI-s compared to the other half. UWS and SWS flow rates, initial and final pHs were also measured. NO concentrations in the UWS and SWS of high and low DMFT-OHI-s groups were determined using modified Griess reaction and NO rates were calculated. The two groups revealed no significant differences in their salivary flow rates and their initial pH. NO concentrations/rates in the UWS and SWS of high and low DMFT-OHI-s groups were not statistically different (p > 0.05). There was no significant correlation between NO concentration or NO rate and other tested variables (DMFT-OHI-s, initial pH and final pH). However, a significant correlation was found between UWS NO rate and UWS flow rate (r = 0.921, p = 0.0001) and SWS NO rate and between SWS flow rate (r = 0.921, p = 0.0001). It could be concluded that neither NO concentration nor NO rate correlates with the dental status. As the exposure to any salivary component (including NO) depends not only on its concentration but also on the rate of production of such concentration, it would be of value when determining individuals’ salivary components to consider their rate values rather than their absolute concentrations.

Journal of Advanced Research (2011) 2, 357–362 Cairo University Journal of Advanced Research SHORT COMMUNICATION Saliva nitric oxide levels in relation to caries experience and oral hygiene Enas H Mobarak a b a,* , Dalaal M Abdallah b Restorative Dentistry Department, Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt Pharmacology and Toxicology Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt Received 21 January 2011; revised April 2011; accepted May 2011 Available online 21 June 2011 KEYWORDS Caries experience; Nitrate; Nitric oxide; Nitrite; Salivary flow rate (SFR) Abstract The aim of the present study was to determine the relationship between nitric oxide (NO) concentration/rate in the unstimulated whole saliva (UWS) and stimulated whole saliva (SWS) with the decay-missing-filled teeth (DMFT) and simplified oral hygiene (OHI-s) scores Forty adults were included in the study Half of the participants (n = 20) had high DMFT-OHI-s compared to the other half UWS and SWS flow rates, initial and final pHs were also measured NO concentrations in the UWS and SWS of high and low DMFT-OHI-s groups were determined using modified Griess reaction and NO rates were calculated The two groups revealed no significant differences in their salivary flow rates and their initial pH NO concentrations/rates in the UWS and SWS of high and low DMFT-OHI-s groups were not statistically different (p > 0.05) There was no significant correlation between NO concentration or NO rate and other tested variables (DMFT-OHI-s, initial pH and final pH) However, a significant correlation was found between UWS NO rate and UWS flow rate (r = 0.921, p = 0.0001) and SWS NO rate and between SWS flow rate (r = 0.921, p = 0.0001) It could be concluded that neither NO concentration nor NO rate correlates with the dental status As the exposure to any salivary component (including NO) depends not only on its concentration but also on the rate of production of such concentration, it would be of value when determining individuals’ salivary components to consider their rate values rather than their absolute concentrations ª 2011 Cairo University Production and hosting by Elsevier B.V All rights reserved * Corresponding author Tel.: +20 37600889/20101641166; fax: +20 233385775 E-mail address: enasmobarak@hotmail.com (E.H Mobarak) 2090-1232 ª 2011 Cairo University Production and hosting by Elsevier B.V All rights reserved Peer review under responsibility of Cairo University doi:10.1016/j.jare.2011.05.005 Production and hosting by Elsevier Introduction Saliva is one of the primary needs for lifelong conservation of the dentition against dental caries Multiple anticariogenic functions of the saliva are related to its fluid characteristics that is mainly includes dilution and washing effects Also, they are related to its specific components such as neutralization of acids, maintaining supersaturated calcium/phosphate concentrations and antibacterial defense [1] Normally, the daily production of saliva ranges between 0.5 and 1.0 l It is composed of more 358 than 99% water and less than 1% solids, mostly proteins and electrolytes The final composition of the whole saliva in the mouth is strongly dependant on the salivary flow rate The concentrations of sodium, chloride and bicarbonate ions have been reported to be increased in stimulated whole saliva (SWS) [1] On the other hand, the unstimulated whole saliva (UWS) composition was reported to be more important for the control of carious lesions development than that of the SWS [2] Recent studies showed that nitrate and nitrite in saliva play a role in the maintenance of certain oral protective functions, in particular, the production of nitric oxide (NO) [3,4] NO represents a free radical gas and a noxious chemical in the atmosphere, but exists in small well-controlled concentrations in the body [5] Actually, it is also one of the most powerful antibacterial compounds [6] acting either through inhibition of bacterial growth or through enhancement of macrophage-mediated cytotoxicity NO easily penetrates the cell membrane and hence induces its microbial damage through several mechanisms, such as inhibition of various iron containing DNA synthases [7], combination with iron sulfur centers of mitochondrial enzymes essential for their respiration [8] and combination with superoxide to form peroxy nitrous acid and the highly reactive hydroxyl radical [9] NO formation requires nitrite, a potential substrate that is found in saliva as a product of nitrate reduction Nitrate in saliva is thus derived from both metabolic and dietary sources so that after its absorption in the gut it is actively transported with the blood to the salivary glands and secreted into saliva [12] Nitric oxide can be measured using various direct and indirect methods (e.g., gas and liquid chromatography, electron paramagnetic resonance, mass spectrometry, spectrophotometry, electrochemistry) The short half-life and low concentrations of NO in-vivo reduce the practicality of these methods for evaluation of biological samples Additionally, these procedures are generally unsuitable for the clinical laboratory due to instrumentation requirements and inexpedience in processing large number of samples The difficulties inherent to quantification of NO can be eliminated by measuring its stable metabolites, in particular, nitrite and nitrate Numerous techniques for detection of these anions have been reported, including spectrophotometric, fluorescent, chemiluminescent, and chromatographic assays The simplest and most frequently applied method employs colorimetric detection with (Griess reagent) However, the conventional Griess reaction has a limitation due to its inability to detect nitrate Moreover, the usage of a reducing metal such as cadmium is time consuming as it requires an extra step Cadmium as a toxic metal needs cautious handling and proper hazardous waste disposal Currently, reduction can be achieved using vanadium to overcome all the mentioned demerits [10] Lately, it has been reported that salivary NO might be an important intra oral defense mechanism against caries pathogens Additionally; its oral production rate was thought to be dependent on the salivary flow rate [11] Nevertheless, only two studies concerning the relation between NO concentration (lM/L) and the past caries experience could be traced; though that, their results were contradicting [12,13] In addition, none has tested the salivary NO rate (lM/min) with regards to the flow rate of either the UWS or SWS The present study was conducted to determine if there is a relationship between NO concentrations/NO rate and subjects’ oral hygiene and past caries experience This relation could be E.H Mobarak and D.M Abdallah of value for caries prediction and diagnosis especially that there is a general belief that past caries experience is a good predictor for future caries [14] Subjects and methods Screening and selection of subjects All patients attending the Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt, over one month were screened for participation in the present study Participants were eligible if they had either poor oral hygiene and high DMFT or good oral hygiene and low DMFT This research has been approved by the local research ethics committee and informed consents have been taken Participants had to have inclusive criteria of being 20–30 years age, apparently in good health, non smokers and not taking any local or systemic medication in the previous two months that might affect their saliva composition Those who had the inclusion criteria and accepted to sign informed consent (n = 57) were stratified according to their gender and whether they had high OH and DMFT scores or not Ten participants were randomly selected from each of these four strata Clinical examination Following the European criteria [15,16], the level of dental caries status for each individual was determined by the same person using the DMFT score In addition, the simplified oral hygiene index (OHI-S) (debris index and calculus index) was used to determine the oral hygiene status [17] A patient was considered with low DMFT-OHI-s when his DMFT score was 62 and his OHI-S score was 61 While the participant was considered with high DMFT-OHI-s, when the DMFT score was P8 and OHI-s score was P4 For those with high DMFT, the percentage of D component had to be more than 75% of the DMFT scores while those of low DMFT score had not to have any D component in their DMFT scores On the day of collecting the samples, participants (n = 40) were asked not to brush their teeth in the morning and to be fasting for at least six hours before the sampling time Measuring unstimulated and stimulated SFR The time of sampling was from to 10 am Two samples were taken from each participant The participant sat in an upright position and was asked to relax with no movement or talking for few minutes to eliminate the effect of the sympathetic tone [18] The UWS and SWS flow rates were obtained following the procedures proposed by Navazesh [19] and Navazesh and Kumar [18] The UWS and SWS flow rates were determined immediately after collection Measuring the initial and calculation of final pH The stimulated saliva sample was poured in a small beaker, after calculating its volume, to measure its pHs (pHep, Hanna instrument, Italy) The reading was recorded as the initial salivary pH value One millimeter of stimulated saliva was then mixed with ml of HCl (0.005 M), after 30s, the pH was measured again and recoded as the final pH [20] Nitric Oxide levels and dental status 359 Determination of nitric oxide concentration Statistical analysis Saliva samples were coded before measuring the NO and decoded thereafter Nitric oxide was determined in saliva according to the method described by Miranda et al [10] Data were described in terms of range, medium and mean ± standard deviation (SD) Comparison of quantitative variables between different groups in the present study was done using Mann Whitney U test Correlation between NO levels and other variables were done using Spearman rank correlation (R) A probability value (p-value) less than 0.05 was considered statistically significant All statistical calculations were done using the computer programs: Microsoft Excel version (Microsoft Corporation, NY, USA) and the SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 15 for Microsoft Windows Principle Nitric oxide is relatively unstable in the presence of molecular oxygen, with an apparent half life of approximately 3–5 s and is rapidly oxidized to nitrate and nitrite totally designated as NOx A high correlation between endogenous nitric oxide production and nitrite/nitrate (NOx) levels has been established Measurements of these levels provide a reliable and quantitative estimate of nitric oxide output in vivo The assay determines the total nitrite/nitrate level based on the reduction of any nitrate to nitrite by vanadium followed by the detection of total nitrite (intrinsic + nitrite obtained from reduction of nitrate) by Griess reagent The Griess reaction entails formation of a chromophore from the diazotization of sulfanilamide by acidic nitrite followed by coupling with bicyclic amines such as N-(1-naphthyl) ethylenediamine The chromophoric azo derivative can be measured colorimetrically at 540 nm Results Data of both tested groups Table shows the characteristic data of both tested groups Statistical analysis revealed no significant difference between both groups regarding the UWS and SWS flow rates and the initial pH (p > 0.5) Regarding the final pH, the difference was statistically significant (p = 0.04) Procedure In the Eppendorf tube, 0.75 ml cold absolute ethanol was added to 0.75 ml saliva then was left for 48 h in the refrigerator to attain complete protein precipitation The mix was then centrifugated at 4000 rpm at 12 °C for 30 using cooling centrifuge (Heraeus, Germany) Only 250 ll of the obtained supernatant was used to which 250 ll vanadiumyrichloride (Aldrich, USA) was added followed by rapid addition of 125 ll sulfanilamide (2% (w/v) in 5% HCl, Sigma, USA) and 125 ll of N-(1-Naphthyl) ethylenediamine dihydrochloride (0.1% (w/v) in distilled water, Fluka, USA) The mixture was left at room temperature for 30 then the absorbance of the pink colored chromophore was measured at 540 nm using a double beam spectrophotometer (UV-150-02, Shimadzu, Japan) against a blank treated in the same manner to the test but using 250 ll distilled water instead of the sample The standard was treated exactly as the supernatant and measured against a blank reagent containing 250 ll distilled water NO levels of both groups are presented in Table For the NO concentration regardless the salivary flow rate, the mean value was higher in the group of high DMFT-OHI-s (79.4 ± 21.6 for UWS and 68.9 ± 13.8 for the SWS) than in the group of low DMFT-OHI-s (77.7 ± 16.1 for the UWS and 66.6 ± 9.8 SWS) However, these differences were not statistically significant (p = 0.86 for UWS and p = 0.50 for SWS) On the other hand, when the NO rate was calculated with regards to the salivary flow rate (lM/L min-1), the results were reversed; where the NO rate mean value was higher in the low DMFT-OHI-s group (25.1 ± 15.8 for the UWS and 153.9 ± 91.1 for the SWS) than the other group (22.8 ± 11.2 for the UWS and 133.2 ± 95.9 for the SWS) Yet, these differences were not also statistically significant (p = 0.68 for UWS and p = 0.74) for (SWS) Calculation of NO concentration (lM/L) Correlation between the NO and other variables The level of total nitrite/nitrate (NOx) in the saliva was expressed as lM and was calculated using the following formula: NOx(lM) = AT/As · n · DF where AT is the absorbance of the test sample; As is absorbance of the standard sample; n is concentration of the standard (lM) and DF is the dilution factor = 1.5/0.75 = Table shows the correlation coefficient and significance between NO levels and tested variables There were no statistically significant correlations between the salivary NO concentrations in the UWS or SWS and any of the variables namely; DMFT, OHI-s, initial and final pHs, and UWS and SWS flow rates There was also no statistically significant correlation between NO rate and DMFT, OHI-s, Initial and final pH A significant correlation between the UWS NO rate and the UWS flow rate was found SWS NO rate had a statistically significant correlation with the SWS flow rate Moreover, a statistically significant correlation between the UWS NO rate and SWS NO rate was found Calculation of the NO rate (lM/min) The exposure of the dental tissue to the NO does not only depend on its concentration in the saliva either unstimulated or stimulated but also on the rate of such exposure Accordingly, the actual NO secreted in the saliva per individual with regards to his UWS and SWS flow rate (NO rate) was calculated This was done according to the following equations: NO rate in UWS (lM/min) = NOx lM/ml · UWS flow rate (ml/min) NO rate in SWS = NOx lM/ml · SWS flow rate (ml/min) NO concentration and rate of both groups Discussion Dental caries is the most common disease in the oral cavity The need for its control is mandatory especially in developing countries where an overall increase in the frequency of dental 360 E.H Mobarak and D.M Abdallah Table Basic characteristics of the study subjects DMFT OHI-s UWS flow rate (ml/min) SWS flow rate (ml/min) Initial salivary pH Final salivary pH First group (n = 20) (High DMFT and OHI-s) mean ± SD Second group (n = 20) (Low DMFT and OHI-s) mean ± SD p-Value 11.5 ± 5.2 2.9 ± 1.3 0.3 ± 0.1a 2.0 ± 1.4b 8.0 ± 0.1c 7.5 ± 0.2 1.5 ± 1.4 0.9 ± 0.9 0.3 ± 0.2a 2.3 ± 1.1b 8.0 ± 0.2c 7.7 ± 0.2 0.00 0.00 0.42 0.36 0.23 0.04 Same letters within rows mean no statistical significance DMFT = decayed missed filled per tooth score; OHI-s = simplified oral hygiene score; UWS = unstimulated whole saliva; SWS = stimulated whole saliva Table Nitric oxide levels of both groups Subjects-groups Mean ± SD (Range, median) NO concentration in UWS (lM/ml) NO concentration in SWS (lM/ml) NO rate in UWS (lM/min) NO rate in SWS (lM/min) High-DMFT/OHI-s 79.4 ± 21.6 (54.2–159.1, 76.5) 77.7 ± 16.1 (53.3–131.9, 76.8) 0.86 68.9 ± 13.8 (48.9–96.0, 76.5) 66.6 ± 9.8 (37.8–79.9, 65.8) 0.50 22.8 ± 11.2 (7.3–54.0, 19.8) 25.1 ± 15.8 (6.2–73.8, 22.0) 0.68 133.2 ± 95.9 (49.5–400.9, 103.5) 153.9 ± 91.1 (53.6–375.4, 120.9) 0.74 Low-DMFT/OHI-s p-Value DMFT = decayed missed filled per tooth score; OHI-s = simplified oral hygiene score; UWS = unstimulated whole saliva; SWS = stimulated whole saliva caries has been reported [21] Knowing that any infectious disease can only occur when the pathogenic organisms are sufficient in number to surmount the intra-oral defense mechanisms, and that tooth decay is an infectious disease caused by acid attacks resulting from bacterial sugars fermentation, would clarify the essentiality of the role of the innate-host defense mechanism against dental caries NO might be one of the important intra oral defense mechanisms against caries pathogens [4] Though cross-sectional analytical studies might suffer from the lack of blinding [22], this was not the case in the present study as the dentist had coded the samples before measuring NO concentrations and the decoding was done thereafter DMFT score was used in present study for caries status determination to make the results comparable with Bayindir’s et al earlier study [12] However, some differences were encountered including doubling the sample size, and measuring the salivary flow rates and initial and final pHs Having cut offs for DMFT scores, P8 for high DMFT group and 62 for those with low DMFT, were used to have a clear distinction between both groups For the same reason, it was specified that those of high DMFT score should have the main component of D and those with low DMFT score not having any D component The other major difference between the two studies was the usage of modified Griess reaction rather than the conventional one used by the other study These differences may explain why the outcomes of the present study contradict with Bayindir’s et al [12] The age group of this study was chosen for two reasons, first to be comparable with the Bayindir et al [12] study Secondly, since DMFT values are very much age dependant, It would be expected to be more reliable to group the study subjects according to their DMFT at this age group rather than at younger age In the current study, smokers were excluded as prior investigation showed inhibition of NO production by acute or chronic cigarette smoking [23] Moreover, subjects were also chosen not to be taking any local (e.g., mouthwashes) or systematic medications (such as antibiotics) that might affect their salivary components for at least two months, because it was reported by Dougall et al [24] that the salivary production of nitrite was reduced following the use of broad spectrum antibiotics Additionally it was found that NO was absent in Germ-free rates [25] The patients were also selected from those who did not take vegetables in the last meal before fasting, for not less than h, as it was reported by Olin et al [26] that salivary NO concentration showed significant increase for up to three hours after ingestion of Nitrate rich food Meanwhile, others were not able to prove that relation [27,28] For many years it was accepted to have samples from the SWS to be used in caries research including analysis of its composition, bacteriological investigation (such as counting Streptococcus mutans and lactobacilli spp.), salivary initial and final pH and its buffering capacity This was because of its expected functional role during food intake However, recently, Bardow et al [29] brought the importance of the UWS composition in caries process to light Accordingly, in the present study, the NO concentration was measured in the UWS and SWS as each of them has a role in caries control and their flow rates are quite different Moreover, on reviewing literatures, no study has been published that measured the NO concentration in each of them for the same individual Measurement of the UWS and SWS flow rates, initial and final pH for the participants, which were found to be within the nor- Nitric Oxide levels and dental status 361 Table Correlation coefficient and significance between NO levels and tested variables NO levels Test variables Correlation coefficient UWS NO concentration DMFT OHI-s Initial PH Final PH UWS SWS 0.050 0.420 0.274 0.121 0.395 0.040 SWS NO Concentration DMFT OHI-s Initial PH Final PH UWS SWS 0.097 0.035 0.035 0.081 0.106 0.089 UWS NO rate DMFT OHI-s Initial PH Final PH UWS SWS 0.009 0.112 0.001 0.055 0.921** 0.404* DMFT OHI-s Initial PH Final PH UWS SWS 0.245 0.132 0.288 0.447* 0.471* 0.921** SWS NO rate UWS = unstimulated whole saliva, SWS = stimulated whole saliva * p < 0.05 ** p < 0.0001 mal ranges [30,31], allowed us to focus on NO concentration difference per se between both groups The higher salivary NO concentration in UWS, independently from the salivary flow rate, in the group of high DMFT/OHI-s in the current should be interpreted with caution as the difference was not statistically significant The other researcher [12,13] had contradicting results This diversity of the results may be attributed to the difference in the methodology For example, in Doel et al [13] study the salivary samples were obtained by using a swab This method of sampling was reported to be the least accurate one for salivary testing [18] Additionally, no prior investigations had been done for the NO concentration in the SWS to compare our finding with The reported significant correlation between the NO rate values in UWS and the UWS flow rate itself and the NO rate in the SWS and the SWS flow rate means that it could be speculated that the measurement or calculation of each is enough as they are very correlated with each other However, further investigations are required to support this speculation In the present study, there was a considerable overlap between the two subject groups regarding both NO concentration and rate values This makes it difficult to consider NO a host defense mechanism when caries increases or oral hygiene deteriorates as mentioned earlier by Bayindir et al [12] Despite the previous findings, it is worth mentioning that the increase in intake of nitrate rich food, which is mainly in green vegetables especially leafy ones such as lettuce and spinach, may contribute to overall protective effect against cariogenic pathogens affecting hard tissue [32] Further longitudinal clinical investigation to verify such findings is still required Conclusion Under the conditions of this study it could be concluded that Neither NO concentration nor NO rate correlates 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was determined by the same person using the DMFT score In addition, the simplified oral hygiene index (OHI-S) (debris index and calculus index) was used to