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Year : 2006  |  Volume : 17  |  Issue : 3  |  Page : 117-20
Evaluation of salivary nitric oxide levels in oral mucosal diseases : A controlled clinical trial.

Department of Oral Medicine and Radiology, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Tamilnadu, India

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Lichen planus is a common dermatologic disease to manifest in the oral cavity. Recurrent aphthous ulcers are the most common ulcers of the oral cavity causing discomfort to the patients. These two diseases have different clinical manifestations which require appropriate treatment after correct diagnosis. Though numerous etiological factors have been proposed for these diseases, their true etio-pathogenesis is not yet established and therefore all therapies are palliative and none is effective universally. In light of this, the role of nitric oxide as a mediator in the etio-pathogenesis of these diseases was considered. The present study was undertaken to note the salivary nitric oxide levels as measured through its product nitrite in oral mucosal diseases like lichen planus and recurrent aphthous ulcers and also to ascertain whether salivary nitric oxide level has a role to play as a pathophysiological mediator in these diseases

Keywords: Lichen planus, recurrent aphthous ulcers, saliva, nitric oxide

How to cite this article:
Sunitha M, Shanmugam S. Evaluation of salivary nitric oxide levels in oral mucosal diseases : A controlled clinical trial. Indian J Dent Res 2006;17:117

How to cite this URL:
Sunitha M, Shanmugam S. Evaluation of salivary nitric oxide levels in oral mucosal diseases : A controlled clinical trial. Indian J Dent Res [serial online] 2006 [cited 2018 Jun 23];17:117. Available from:

   Introduction Top

Oral lichen planus (OLP) is clinically recognized as white striations and patches associated with mucosal atrophy and erosion. Recurrent aphthous ulcers (RAU) are characterized by painful recurring ulcers of the oral mucosa causing extreme discomfort and pain for the patient. A proper diagnosis and appropriate management becomes mandatory to relieve the agony of such patients. Numerous etiological factors like hereditary, dietary endocrine, infectious, allergic, immunological and psychological factors have been proposed. But their true etiopathogenesis is not yet established and therefore all therapies are palliative and most of the time oral physicians have remained helpless due to a lack in proper treatment protocol to control the recurrence of these diseases.

The possible role of nitric oxide as a mediator in the etiopathogenesis ofthese diseases was then considered. Nitric oxide, a free radical gas, is a noxious chemical in the atmosphere but in small controlled concentrations in the body, acts as a physiological and patho­physiological mediator and plays an important role in biological systems. The known biological functions of nitric oxide can be divided into two categories. First, it acts as an endothelial-derived relaxer of vascular smooth muscle, an inhibitor of platelet aggregation and adhesion and a neuronal messenger. Secondly, the nitric oxide synthesized in large amounts by activated macrophage is a cytotoxic molecule influencing the ability of cells to kill bacteria, viruses, protozoa as well as tumour cells. In addition, it is well established that nitric oxide has damaging effects against cellular proteins, DNA and lipids eventually leading to cell death, tissue injury and organ failure.

Bodis S and Haregowin A reported that freshly released human saliva contains measurable and sometimes relatively high levels of nitric oxide [1]. Ohashi et al noted an elevated production of salivary nitric oxide in oral mucosal diseases in their study [2]. The cellular origin, the normal concentration in saliva and the physiological and possible pathological role of nitric oxide in saliva is at present unknown. The aim of the present study was to note the salivary nitric oxide levels as measured through its product nitrite in oral mucosal diseases like lichen planus and recurrent aphthous ulcers in our population and to note whether salivary nitric oxide has a role to playas amediator in these diseases.

   Materials and methods Top

This study was carried out in the Department of Oral Medicine and Radiology, Tamil Nadu Government Dental College, Chennai. The study consisted of three groups; Group-1 formed the control group, Group-2 comprised of subjects with erosive lichen planus and Group-3 constituted subjects with recurrent aphthous ulcers. Twenty healthy adults who attended the clinic without inflammatory oral lesions and systemic diseases were used as controls. Eight males and twelve females in the age group of 20-51years with mean age of 33.4 years formed the control group. Consent was obtained from all patients and volunteers for examination. The clinical confirmation of erosive type of lichen planus was putforth as the criteria of selection of subjects in group 2. Twenty cases of erosive lichen planus consisting of 8 males and 12 females in the age group of 22-51 years with a mean age of 37.7 years were selected. Twenty cases of recurrent aphthous ulcers including 9 males and 11 females in the age group of 20-48 years with the mean age of 29 years were selected. The subjects in the experimental groups were first asked to rinse their mouth with povidone iodine mouth rinse for 2 minutes which ensured a substantial reduction in bacterial count. The patients were then asked to wait for a minute, after which freshly secreted unstimulated saliva, about 1 ml was collected in a sterile container. The sample was then diluted with 10ml of phosphate buffered saline in order to neutralise the pH. After diluting, the mixture was centrifuged for 5minutes at 3 000 rpm and the supernatant fluid was then stored at 20°C until use. A standard curve was obtained using known standard solutions of the substance to be determined reacted with an appropriate reagent so that unknown concentration of the substance to be determined can be obtained from the standard curveby a spectrophotometer. The substance to be determined in our study was nitrite and hence known concentrations of sodium nitrite solutions were prepared for evaluation. Sodium nitrite solutions in varying concentrations from 25p -60 p were prepared.

These solutions were reacted with Griess reagent which was prepared using 1% sulfanilamide, 1% naphthylethylene diamine dihydrochloride and 2.5% phosphoric acid. Griess reagent is very unstable as it reacts with surface atmospheric nitrogen. Hence it was freshly prepared before use. 0.5 ml of the prepared standard solutions of sodium nitrite were reacted with equal volume of Griess reagent in Eppendorf tubes and incubated at room temperature for 10 minutes to ensure that complete reaction takes place. The reacted mixture was then transferred onto plastic cuvettes for measurement in the spectrophotometer which is connected to a computer so that digital readings can be taken. Using these readings taken for the standard solutions, a graph of absorbance versus concentration was plotted, which constituted the standard curve. In a similar manner, samples of the 60 subjects were reacted with Griess reagent and transferred to spectro­photometer and their optical densities (OD) were recorded. The optical densities were then correlated in the standard curve and corresponding concentrations of nitrite were observed. Data was fed into the computer database and analysed using the Social and Preventive Statistical package (SPSS) package. Mean, standard deviation and 95% confidence interval of all groups were calculated. These groups were compared by one-way analysis of variance followed by Duncans R multiple range test andregressiveanalysis.

   Results Top

The correlation of sodium nitrite concentration and optical density for standard solutions was determined [Table - 1]. A correlation co-efficient of 0.998 and a co-efficient determination of 99.8% was obtained. The p value was found to be <0.0001which indicates that there is a highly significant linear relationship between the standard nitrite concentration and the optical density values and that the known value of one of these parameters can lead to a highly significant prediction of the value of the other parameter with an accuracy of 99.8%. The control group had a mean optical density value of 0.1044 t 0.0379, while Group 2 and Group 3 had a mean optical density value of 0.2780 f 0.1503 and 0.1838 f 0.1189 respectively [Table - 2]. Inter group comparison of OD values of all groups were done using one way analysis of variance (ANOVA) [Table - 3], which showed a highly significant difference between the optical densities of all the three groups (p <0.00001) whereas intra group comparison of values revealed that there was no statistically significant difference among them. Inter group comparison by Duncans multiple range test [Table - 4] indicated that the OD values of Group 1 was significantly different from that of Group 3 and Group 2. Further the optical density of Group 3 was significantly lower than Group 2. Summary statistics revealed that Group 1 had a mean nitrite concentration of 4.2000 f 1.8525 while Group 2 recorded a mean value of 12.2750 f 6.9632. Group 3 recorded a mean value of 7.9750 ± 5.4446 [Table - 5].

   Discussion Top

Lichen planus and recurrent aphthous ulcers are two of the most prevalent diseases affecting the oral mucous membrane that have been investigated critically over years. The etiology of both the diseases have immunological factors as a common source. Both T lymphocytes and macrophages implicated in the etiologyoflichenplanus serves to point a possible role of nitric oxide in the mediation and cell injury in these diseases. Inducible nitric oxide synthetase enzyme is capable of producing nitric oxide over a longer period of time by various immunological mediators and a wide variety of cells including macrophages, T cells and natural killer cells. The expression of inducible nitric oxide synthetase and the documented actions of nitric oxide, like disease mediation and cell injury when generated in excess, led to focus the significance of its levels in saliva and the possible role in the etiology and progression of these diseases.

Sessa described three isoforms of nitric oxide synthase (NOS) and that the type 3 or inducible NOS is found on oral mucoua, endothelial cells and salivary gland tissue [3]. The possible cellular source of salivary nitric oxide are from nerve endings, salivary gland endothelial cells or macrophages in response to oral bacterial products. It has been pointed out that there are psychological aspects to the etiology of oral lichen planus and recurrent aphthous ulcers. Levels of salivary endocrine such as cortisone are high in patients with OUP and RAU. Walsh et al have reported the role of stress in exacerbating oral

lichen planus and suggested the involvement of neural and immune interaction in its pathogenesis [4]. Tsuchiya et al have reported that psychological stress causes nitric oxide release in correlation with increase of neural NOS activity [5]. This may be one explanation for the elevated nitric oxide production in patients with OUP and RAU. A careful observation into the histopathological picture of lichen planus reveals the presence of hyperkeratosis, acanthosis, saw tooth rete ridges, degeneration of basal cell layer and a well defined band-like inflammatory cell infiltrate consisting mainly of lymphocytes including CD4 and CD8 cells in the sub epithelial connective tissue.

Imrnunohistochemical studies indicate that activated T cells can lie close to the damaged epithelial basal cells and basement membrane and sometimes in areas adjacentto epithelial erosion as in erosive lichenplanus. Mathews et al have noted that in lichen planus, macrophages are frequently found in contact with lymphocytes displaying signs of activation and that they lie in close proximity to epithelial basal cells where cell damage occurs and play a role in the pathogenesis of this condition [6]. RAU are also characterized histologically with a mixed inflammatory infiltrate with T4 cells predominating in pre-ulcerative and healing phase and T8 cells predominating in the ulcerative phase. Dolby and Allison established the relationship of mast cells to aphthousulcer [7].

Nitric oxide is released after immunological stimulation by an enzyme, the inducible nitric oxide synthase. This enzyme was originally described in macrophages. Kirk et al have demonstrated the induction of nitric oxide synthesis in T lymphocytes [8]. It is also known to be expressed by mast cells. The inducible NOS once induced continues to produce nitric oxide for a much longer period of time than either neural or endothelial NOS. Being readily observed in patients with infectious or inflammatory conditions, the expression of inducible NOS and hence nitric oxide could be increased in cellular infiltrate seen in erosive OUP and RAU when compared with normal mucoua and thus may contribute to elevated levels of nitric oxide in the saliva of these patients. The inducible form of NOS is expressed in response to the release of various cytokines including IL-1, Interferon-7 and tumour necrosis factor (TNFa) and by a wide variety of cells including natural killer cells. Yamanoto et al in their in-vitro studies noted that T lymphocytes from lichen planus tissue produced increased levels of IL-6 and granulocyte macrophage colony stimulating factors (GM-CSF) and can be stimulated to produce more TNFa by IL-1(3, IL-6 and GM-CSF [9]. Invitro studies have shown that keratinocytes from OLP lesions produce IFN-a. IL-6 and TNFa in response to IL-(3. These keratinocytes thus have the potential to produce large amount of cytokines capable of activating infiltrating T cells. These cytokines, interleukins and TNF are capable of activating T cells and thus contribute to the release of nitric oxide. The role of nitric oxide in saliva is stillunclear. Ohashi et al noted the effects of nitric oxide on cultured cells using nitric oxide donating agents and found that it caused severe damage to fibroblasts, keratinocytes and oral epithelial cells in-vitro [2]. Volk et al noted the cytotoxic potential of nitric oxide against various cells [10]. Moncada et al opined that when generated in excess, nitric oxide is a key mediator of cell damage, tissue injury and organ failure [11]. Investigations by Das et al have indicated that free radicals including nitric oxide may play an important role in ulceration induced by several kinds of stress [12]. Therefore nitric oxide is considered to cause erosion and ulceration as a consequence of cell damage. For these reasons, it is proposed that free radicals including nitric oxide represent one route of pathogenesis and that excess of salivary nitric oxide may have pathophysiological implications for erosive and ulcerative lesions in OLP and RAU.

Further research on the role of T lymphocytes in the production of nitric oxide and tong term clinical studies on larger samples and indepth research on nitric oxide need to be undertaken to fully understand its role in oral mucosal diseases.

   References Top

1.BodisS,HaregewoinA:Evidence fortherelease and possible neural regulation of nitric oxide in human saliva, Biochem Biophys Res Common, 194:347­350,1993.  Back to cited text no. 1    
2.Ohashi M, Iwase M, Nagumo: Elevated production of salivary nitric oxide in oral mucosal diseases, J Oral Pathol Med, 28:355-359,1999.  Back to cited text no. 2    
3.Sessa WC, Prichard K, Seyedi N, Wang J: Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression, Circ Res, 74:349-353, 1994.  Back to cited text no. 3    
4.Walsh IT, Savage NW, Ishii T, Seymour GJ: Immuno pathogenesis of oral lichen planus, J Oral Pathol Med, 19:389-396,1990.  Back to cited text no. 4    
5.Tsuchiya T, Kishimoto J, Koyama J, Ozawa T Modulatory effect of L-NAME, a specific nitric oxide synthase (NOS) inhibitor or stress-induced changes in plasma adreno-corticotropic hormone and corticosterone levels in rats, Brain Res, 776: 68-74, 1997.  Back to cited text no. 5    
6.Matthews JB, Basu MK, Potis AJC: Macrophages in oral lichen planus, J Oral Patho1,14: 553-558,1985.   Back to cited text no. 6    
7.Dolby and Allison: Quantitative changes in the mast cell population of Mikulicz's recurrent oral aphthae, J DelitRes, 48[5]:901-903,1969.  Back to cited text no. 7    
8.Kirk ST, Regan MC, Barbul A: Cloned marine T lymphocytes synthesise a molecule with the biological characteristics of nitric oxide, Biochem Biophys Res Commun,173:660-665, 1990.   Back to cited text no. 8    
9.Yamamoto T, Osaki T, Yoned K, Utea E: Cytokine production by keratinocytes and mononuclear infiltration in oral lichen planus, J Oral Pathol Med, 23:309-315,1994.  Back to cited text no. 9    
10.Volk T, Ioamridis I, Hensel M, Degroot H Endothelial damage induced by nitric oxide synergism with reactive oxygen species, Biochem. Biophys Res Common, 213:1196-1203,1995.  Back to cited text no. 10    
11.Mocada S, Palmer RMJ, Higgs EA: Nitric oxide, physiology, pathophysiology and pharmacology Pharmacological Reviews, 43:109-142,1991.  Back to cited text no. 11    
12.Das D, Bandyopadhyay D, Bhattacharjee M, Banerjee RK: Hydroxyl radical is the major causative factor in stress induced gastric ulceration, Free Ruche BiolMed, 23:8-18,1997.  Back to cited text no. 12    

Correspondence Address:
M Sunitha
Department of Oral Medicine and Radiology, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Tamilnadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.29878

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[Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5]

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