1Department of Public Health Dentistry, Kothiwal Dental College & Research Centre, Moradabad, Uttar Pradesh, India; Departments of 2Biochemistry and 3Periodontology, Aligarh Muslim University, Aligarh, Uttar Pradesh, India *Corresponding Author’s e-mail: aliamaan29@gmail.com نازعة هيدروجني الالكتات وبيتا غلوكورونيداز كوامسات لعاب حيوية كيميائية يف التهاب دواعم السن عند املدخنني وغري املدخنني �شيد اأمان علي، رايف لينجي�شا تيلجي، اأميت تريث، عرفان قادر تنرتي، عبدالعليم abstract: Objectives: This study aimed to establish lactate dehydrogenase (LDH) and β-glucuronidase as salivary biomarkers of periodontitis among smokers and non-smokers. Methods: This cross-sectional case-control study was conducted at the Aligarh Muslim University, Aligarh, India, between January and June 2017. A total of 200 participants were divided into four groups based on their periodontal and smoking statuses. Unstimulated mixed saliva samples were collected to estimate LDH and β-glucuronidase levels. In addition, total protein was estimated using Lowry’s method. Results: There was a significant increase in enzyme activity in the periodontitis groups compared to the non- periodontitis groups (P <0.001). However, significantly lower enzyme activity was observed among smokers, irrespective of periodontal status (P <0.001). Nevertheless, a receiver operating characteristic curve analysis indicated the diagnostic potential of both enzymes to be fair-to-excellent. Conclusion: Although smoking was found to significantly alter enzyme activity, LDH and β-glucuronidase were reliable salivary biomarkers of periodontitis among both smokers and non- smokers. Keywords: Periodontitis; Biomarkers; Saliva; Lactate Dehydrogenase; beta-Glucuronidase; Smoking; Tobacco Use; India. كيميائية حيوية لعاب كوا�شمات غلوكورونيداز وبيتا الالكتات هيدروجني نازعة من التثبت اإىل الدرا�شة هذه تهدف الهدف: امللخ�ص: األيجاره جامعة يف املقطعية ال�شواهد حلالت الدرا�شة هذه اأجريت الطريقة: املدخنني. وغري املدخنني عند ال�شن دواعم التهاب يف الإ�شالمية، األيجاره، الهند، يف الفرتة ما بني يناير ويونيو 2017. مت تق�شيم ما جمموعه 200 م�شارك اإىل اأربع جمموعات على اأ�شا�ص حالة اللثة والتدخني. مت جمع عينات اللعاب املختلط وغري املحفز لتقدير م�شتويات نازعة هيدروجني الالكتات وبيتا غلوكورونيداز. بالإ�شافة اإىل ذلك، مت تقدير الربوتني الكلي با�شتخدام طريقة لأوري. النتائج: كان هناك زيادة معنوية يف ن�شاط الإنزمي يف جمموعات التهاب اللثة باملقارنة مبجموعات بدون التهاب اللثة )P >0.001(. ومع ذلك، لوحظ وجود ن�شاط اأنزمي اأقل ب�شكل ملحوظ بني املدخنني، بغ�ص النظر عن حالة اللثة )P >0.001(. ومع ذلك، اأ�شار حتليل منحنى خ�شائ�ص الت�شغيل املتلقي اإىل اإمكانات الت�شخي�ص لكل الإنزميات لتكون عادلة اإىل ممتازة. اخلال�صة: على الرغم من اأن التدخني يغري ن�شاط الإنزمي ب�شكل كبري، كانت نازعة هيدروجني الالكتات وبيتا غلوكورونيداز موؤ�رسات حيوية لعابية موثوقة للتهاب اللثة بني املدخنني وغري املدخنني على حد �شواء. ا�شتخدام تدخني؛ غلوكورونيداز؛ بيتا الالكتات؛ هيدروجني نازعة لعاب؛ بيولوجية؛ وا�شمات ال�شن؛ دواعم التهاب املفتاحية: الكلمات التبغ؛ الهند. Lactate Dehydrogenase and β-Glucuronidase as Salivary Biochemical Markers of Periodontitis Among Smokers and Non-Smokers *Syed A. Ali,1 Ravi L. Telgi,1 Amit Tirth,1 Irfan Q. Tantry,2 Abdul Aleem3 clinical & basic research Sultan Qaboos University Med J, August 2018, Vol. 18, Iss. 3, pp. e318–323, Epub. 19 Dec 18 Submitted 8 Feb 18 Revision Req. 28 Feb 18; Revision Recd. 27 Mar 18 Accepted 12 Apr 18 Advances in Knowledge - This study rules out the effect of tobacco smoke on the diagnostic ability of two salivary enzymes—lactate dehydrogenase (LDH) and β-glucuronidase—in risk profiling for periodontal disease. Application to Patient Care - The current findings support the use of these salivary enzymes as biomarkers for periodontal disease. doi: 10.18295/squmj.2018.18.03.009 Periodontal disease is a common inflamm-atory disease caused by the interaction between certain Gram-negative bacterial species and components of the host immune response.1 Chronic periodontal infections trigger the release of a myriad of metabolic byproducts, destructive cellular enzymes and other mediators of tissue destruction at the inter- face between the tooth and the periodontal pocket.2 As a result, the normal histological architecture of the periodontium is disturbed, with persistent inflammation associated with the irreversible loss of mineralised and non-mineralised tissues.1 Syed A. Ali, Ravi L. Telgi, Amit Tirth, Irfan Q. Tantry and Abdul Aleem Clinical and Basic Research | e319 Chronic periodontitis may advance without causing severe oral discomfort; as such, subjects often seek professional care only after the periodontal tissue is considerably damaged.3 Thus, there is an urgent need to diagnose this disease in its initial stages so as to initiate early intervention. Ideally, a diagnostic marker should be highly specific and sensitive and should indicate the presence of a disease process prior to the occurrence of extensive clinical damage.4 At present, a diagnosis of periodontitis is usually made via imaging and clinical assessment of probing pocket depth, bleeding upon probing and clinical attachment level.5 However, the utility of such measurements is limited because these findings may display evidence of previous rather than current disease activity.6 The saliva contains a wide and unique variety of proteins and enzymes with important oral biological functions. The pathogenesis of periodontitis has been linked to alterations in various salivary enzymes, incl- uding lactate dehydrogenase (LDH), alkaline phosphatase, matrix metalloproteinases 8 and 1, aminotransferases, amylase, β-glucuronidases, arginase, chitinases and dip- eptidyl peptidase.7 Metabolic LDH plays a key role in anaerobic glycolysis and its extracellular presence is always related to cell necrosis and tissue breakdown.8 In contrast, β-glucuronidase is a neutrophil-derived lyso- somal acid hydrolase stored in the azurophilic granules. It is active in the degradation of proteoglycans and the ground substance and is considered a marker for azu- rophilic granule release by polymorphonuclear leuko- cyte lysosomes.9 Under these considerations, salivary LDH and β-glucuronidase may play a potential role as salivary biomarkers of periodontal disease.7 Unfortunately, the diagnostic use of these enzymes in periodontitis cases has been hampered as current understanding of the biomolecules present in saliva and their relevance to disease aetiology is still limited. In addition, various factors may cause enzymatic alterations, such as temperature, pH, and enzyme and substrate concentrations of inhibitors or activators.10 For example, tobacco smoke compounds have been found to impair salivary enzyme activities at the molecular level.11 Therefore, this study aimed to attempt to establish LDH and β-glucuronidase as salivary biochemical markers for periodontitis and to assess their diagnostic potential among both smokers and non-smokers. Methods This cross-sectional case-control study was conducted at the Department of Periodontology of the Aligarh Muslim University, Aligarh, Uttar Pradesh, India, between January and June 2017. The required sample size was calculated using G*Power Software, Version 3.0.10 (Heinrich-Heine- Universität Düsseldorf, Düsseldorf, Germany), based on a calculated effect size of 0.565 as per the results of a pilot study involving 20 subjects, with a 5% level of precision, 95% confidence interval and 80% statistical power. The minimum sample size was calculated to be 200 subjects. Patients who had received antiseptic, professional hygiene or periodontal therapies or treat- ment with anti-inflammatory drugs, immunosuppress- ants or corticosteroids in the preceding six months were excluded from the study. In addition, individuals under- going orthodontic treatment or other dental procedures, subjects with acute oral mucosal lesions and/or suspected oral malignancies, those with adverse behavioural habits such as tobacco/paan chewing or alcohol abuse, subjects suffering from any systemic diseases and pregnant, lactating or post-menopausal women were also excluded. A total of 1,306 patients aged 30–50 years old pres- enting to the Department of Periodontology, Dr. Ziauddin Ahmad Dental College & Hospital, Aligarh, Uttar Pradesh, were screened. A simple random sampling method was used to select 200 of these individuals for inclusion in the study. The participants were divided into four groups of 50 subjects each, including non-smokers with no/mild gingivitis, non-smokers with no/mild gingivitis, smokers with chronic generalised periodontitis and smokers with chronic generalised periodontitis [Figure 1]. Unfortunately, it was logistically difficult to find sufficient cases with no gingival inflammation; as such, cases with no/mild gingivitis were considered to constitute the control groups in the study design. Mild gingivitis was diagnosed based on the Silness-Löe gingival index.12 Chronic generalised periodontitis was defined as a probing pocket depth of ≥5 mm, clinical attachment loss of ≥3 mm and moderate, severe or generalised disease progr- ession involving >30% of the mouth. Patients were categorised as smokers or non-smokers based on the updated definition of the Centers for Disease Control and Prevention.13 All smokers reported currently smoking at least five times per day and had a history of smoking of at least five years. Former smokers were not included in the study. A sample of approximately 5 mL of unstimulated mixed saliva was aseptically collected via aspiration from each participant while seated in an upright position. The collection was performed by a single trained expert between 9 and 11 am, five minutes after the mouth was rinsed with 15 mL of water to wash out any exfoliated cells. Eating, drinking and smoking were restricted for at least two hours prior to collection. The saliva was collected in sterile test tubes capped with sterile tinfoil. The samples were transferred to a refrigerated container at 4 °C and then centrifuged for 10 minutes at 1,500 revolutions per minute. Subsequently, estimations of LDH and β-glucuronidase activity were carried out Lactate Dehydrogenase and β-Glucuronidase as Salivary Biochemical Markers of Periodontitis Among Smokers and Non-Smokers e320 | SQU Medical Journal, August 2018, Volume 18, Issue 3 immediately by a single trained biochemist who was blinded to the study design. The biochemical analysis of LDH was performed following strict protocols for salivary enzyme estimation.14 LDH was assayed following the conversion of reduced nicotinamide adenine dinucleotide (NADH) to oxidised NADH at 340 nm in the presence of sodium pyruvate. Briefly, 100 μL of saliva was added to a 2.8 mL reaction mixture consisting of 0.05 M tris buffer at a pH of 7.4, 3.33 mM of magnesium chloride and 1.6 mM of sodium pyruvate. Then, 100 μL of NADH (at a final concentrate of 0.08 mM) was added just before the change in absorbance observed after 3 minutes using a U-2910 spectrophotometer (Hitachi High- Technologies Corp., Tokyo, Japan).14 The results were expressed in IU/L. For the analysis of salivary β-glucuronidase, 50 μL of 0.9% saline solution (as the control), 50 µL of standard 4-methylumbelliferone (Sigma-Aldrich Corp., St. Louis, Missouri, USA) and 50 µL of the salivary samples were separated in designated tubes. In each tube, 100 μL of methylumbelliferyl β-D-glucuronide containing 0.001% bovine serum albumin (BSA) was added and the contents were mixed and incubated at 37 °C for 15 minutes. The reaction was stopped with 2 mL of 0.2 M glycine buffer at a pH of 11.7 and containing 0.2% sodium dodecyl sulphate. After the solution was mixed for 60 minutes, the fluorescence was measured at an excitation wave- length of 360 nm and an emission wavelength of 459 nm. For the standard, the fluorescence of 0.008 mM and 0.016 mM of 4-methylumbelliferone was measured using an RF-5301 spectrofluorophotometer (Shimadzu Corp., Kyoto, Japan), as the fluorescence of 0.008 mM of 4-methylumbelliferone is equivalent to 1 IU of β-glucur- onidase.15 The total protein in the saliva was estimated by means of Lowry’s method using BSA to derive standard and specific enzyme activity.16 Data were analysed using the Statistical Package for the Social Sciences (SPSS), Version 21.0 (IBM Corp., Armonk, New York, USA). Parametric tests (i.e. an unpaired t-test) and a one-way analysis of variance were used to compare the mean enzyme activities of the four independent groups using an F distribution, followed by a post-hoc Tukey’s analysis. A receiver operating char- acteristic (ROC) curve was used to determine the diagn- ostic ability of the enzymes as their discrimination thresholds varied. Binomial logistic regression was then conducted to estimate the frequency of periodontitis among the patients. A P value of <0.050 was considered statistically significant. This study was reviewed and approved by the institutional ethical committee of the Kothiwal Dental College & Research Centre (#KDCRC/IERB/11/2015/34). Written informed consent from the participants was obtained using a pre-designed proforma. All particip- ation in the study was voluntary in nature. Results The patients ranged in age between 30–35 years old (mean: 32.5 years). There were very few female smokers (20.5%). The standard and specific enzyme activity of LDH and β-glucuronidase in each group is presented in Table 1. There was a significant increase in LDH and β-glucuronidase enzyme activity among participants with periodontitis compared to those without perio- dontitis (P <0.001) [Table 2]. However, significantly lower enzyme activity was noted among the smokers, irrespective of periodontal status (P <0.001) [Table 3]. Overall, there was a 23.8% and 25% reduction in LDH Figure 1: Diagram showing the protocol and design used in the current study. Syed A. Ali, Ravi L. Telgi, Amit Tirth, Irfan Q. Tantry and Abdul Aleem Clinical and Basic Research | e321 enzyme activity among smokers with periodontitis and smokers with no/mild gingivitis, respectively. Similarly, there was a 6.8% and 18.1% reduction in β-glucuron- idase activity, respectively. Nevertheless, logistic modelling highlighted a strong linear association between LDH and β-glucuronidase activity and periodontitis, regardless of smoking status (P = 0.046 and 0.010, respectively) [Table 4]. Furthermore, the sensitivity and specificity of the enzymes were established at the 25th, 50th and 75th quartiles. According to the ROC curve analysis, the diagnostic potential of LDH at the 25th and 75th quartiles indicated that this biomarker had fair-to-good potential in predicting perio- dontitis, with the 50th quartile showing excellent potential. Table 1: Enzyme activity of salivary lactate dehydrogenase and β-glucuronidase according to smoking and periodontal status (N = 200) Group Mean LDH activity ± SD Mean β-glucuronidase activity ± SD Standard activity in IU/L Specific activity in nmol/min/ mg of total protein Standard activity in IU/L Specific activity in nmol/min/mg of total protein Non-smokers with no/mild gingivitis 285.34 ± 35.76 237.78 ± 32.46 30.29 ± 9.43 25.24 ± 6.32 Smokers with no/mild gingivitis 213.92 ± 34.70 178.26 ± 32.34 24.81 ± 7.19 20.67 ± 6.56 Non-smokers with chronic generalised periodontitis 1,075.88 ± 253.76 896.56 ± 264.14 91.76 ± 12.05 76.46 ± 10.43 Smokers with chronic generalised periodontitis 819.10 ± 315.59 682.58 ± 274.12 85.53 ± 13.21 71.27 ± 12.71 LDH = lactate dehydrogenase; SD = standard deviation; min = minute. Table 2: Comparison of specific enzyme activity of lactate dehydrogenase and β-glucuronidase according to periodontal status (N = 200) Enzyme Mean specific activity in nmol/min/mg of total protein ± SD P value* No/mild gingivitis Chronic generalised periodontitis LDH 208.02 ± 22.95 789.57 ± 73.87 <0.001 β-glucuronidase 32.4 ± 6.44 269.13 ± 11.57 <0.001 min = minute; SD = standard deviation; LDH = lactate dehydrogenase. *Using an independent t-test. Table 3: Comparison of specific enzyme activity of lactase dehydrogenase and β-glucuronidase according to smoking and periodontal status (N = 200) Enzyme Mean specific activity in nmol/min/mg of total protein ± SD P value* Post hoc Tukey valueNon-smokers with no/mild gingivitis Smokers with no/mild gingivitis Non-smokers with chronic generalised periodontitis Smokers with chronic generalised periodontitis LDH 237.78 ± 32.46 178.26 ± 32.34 896.56 ± 264.14 682.58 ± 274.12 <0.001 4>3>1>2 β-glucuronidase 25.24 ± 6.23 20.67 ± 6.56 76.46 ± 10.43 71.27 ± 12.71 <0.001 4>3>1>2 min = minute; SD = standard deviation; LDH = lactate dehydrogenase. *Using a one-way analysis of variance. Table 4: Logistic regression model of lactate dehydrogenase and β-glucuronidase as possible predictors of periodontitis among smokers and non-smokers (N = 200) Predictor B SE Wald OR (95% CI) P value LDH 0.007 0.005 2.109 1.007 (0.998– 1.016 0.046 β-glucur- onidase 0.394 0.153 6.617 1.482 (1.098– 2.001) 0.010 Constant −19.35 6.746 8.227 - 0.004 SE = standard error; OR = odds ratio; CI = confidence interval; LDH = lactate dehydrogenase. Lactate Dehydrogenase and β-Glucuronidase as Salivary Biochemical Markers of Periodontitis Among Smokers and Non-Smokers e322 | SQU Medical Journal, August 2018, Volume 18, Issue 3 For β-glucuronidase, the 25th and 50th percentiles indic- ated excellent predictive potential, while the 75th perc- entile indicated fair potential [Table 5]. Discussion The pathogenesis of tissue destruction in periodontitis is due to host-bacteria interactions which cause the host cells (mainly polymorphonuclear leukocyte lysosomes) to release granular enzymes which are capable of invading extracellular matrix components.17 Thus, the extra- cellular presence of certain enzymes seems to play an important role in connective tissue damage. The quant- ification of enzyme activity in saliva, such as LDH and β-glucuronidase, can therefore provide important information and contribute to the timely diagnosis of periodontal disease.18 Combining these two markers into a risk profile for periodontal disease may offer improved accuracy in identifying susceptible patients. In the current study, significantly higher LDH and β-glucuronidase activity was noted in the periodontitis groups compared to the non-periodontitis groups. How- ever, Lamster et al. observed variations in β-glucuron- idase enzyme activity among subjects with periodontitis.19 This accentuates the fact that identical clinical cond- itions may reflect different host responses. Therefore, while increased extracellular enzyme activity may have a positive correlation with disease activity, other factors could be involved in the aetiopathology of periodont- itis.19 Such factors may also influence treatment planning. In the current study, relatively low enzyme activity was noted among smokers, including both those with no/mild gingival inflammation as well as those with chronic generalised periodontitis. Such findings are likely related to salivary changes resulting from exposure to inhaled cigarette smoke. Cigarette smoke contains over 4,000 chemicals as well as oxygen free radicals and volatile aldehydes that can cause damage to biomol- ecules.20 Nagler et al. examined the effect of the in vitro exposure of saliva to the gas phase of cigarette smoke; exposing saliva to cigarette smoke for three hours caused a 41% reduction in LDH activity.21 Furthermore, Avezov et al. reported a 34% reduction in LDH activity when salivary samples were exposed to different levels of cigarette smoke.22 Nevertheless, although tobacco smoke significantly altered enzyme activity in the current study, these changes did not invalidate the predictive value of salivary LDH and β-glucuronidase as diagnostic biomarkers. Therefore, enzyme-based salivary diagnosis can be deemed an effective option in periodontitis cases, as influence of smoking on the biomarker status of LDH and β-glucuron- idase can be ruled out. With further studies, changes in enzyme activity may eventually form the basis of a convenient point-of-care diagnostic tool in routine oral health monitoring. This study was subject to certain limitations. First, the analysis focused on the estimation of LDH and β-glucuronidase in the saliva rather than the gingival crevicular fluid, which would better reflect enzyme activity. Second, the methodology did not analyse the utility of these enzymes as markers of disease severity or response to treatment and, ultimately, disease progn- osis. Further studies are necessary to evaluate the reliab- ility of these parameters in this regard. Third, gender matching was not possible due to the unequal distrib- ution of the sample. However, previous research has confirmed that gender does not significantly affect LDH levels, either among patients with periodontal disease or those with normal periodontia.23 Finally, the current study was designed with strict inclusion crit- eria; thus, the exclusion of potential subjects may have hampered the generalisability of the results. Conclusion The current study found that salivary LDH and β-glucu- ronidase enzyme activity significantly increased among patients with periodontal disease in comparison to those with no/mild gingival inflammation. While smoking significantly influenced enzyme activity, these changes were within acceptable limits and did not rule out the use of these enzymes as diagnostic biomarkers of perio- dontitis. However, further research is needed to identify other variables which may influence LDH and β-glucur- onidase enzyme activity. Table 5: Sensitivity, specificity and area under receiver oper- ating characteristic curve of salivary lactate dehydrogenase and β-glucuronidase alterations among periodontitis patients (N = 200) Quartile Percentile value Sensitivity in % Specificity in % Area under ROC curve* LDH 25th 206.75 63.3 90 0.774 50th 298.50 90 90 0.948 75th 841.50 100 66.7 0.819 β-glucuronidase 25th 21.95 98 98 0.980 50th 43.52 98 98 0.980 75th 72.35 100 66.7 0.750 ROC = receiver operating characteristic; LDH = lactate dehydrogenase. *The diagnostic accuracy of the enzymes was interpreted as follows: 1.0–0.91 = excellent; 0.90–0.81 = good; 0.80–0.71 = fair; 0.70–0.61 = poor; 0.60–0.51 = failure. Syed A. Ali, Ravi L. Telgi, Amit Tirth, Irfan Q. Tantry and Abdul Aleem Clinical and Basic Research | e323 c o n f l i c t o f i n t e r e s t The authors declare no conflicts of interest. f u n d i n g No funding was received for this study. References 1. Guthmiller JM, Novak KF. Periodontal diseases. 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