arginine–nitric oxide–polyamine metabolism in periodontal disease

Arginine–Nitric Oxide–PolyamineMetabolism in Periodontal DiseaseLeyla Ozer,* Serenay Elgun,* Burcu Ozdemir,† Beste Pervane,† and Nurdan Ozmeric†

Background: Arginine is converted to nitric oxide (NO) viaNO synthase and to ornithine via arginase. Ornithine decar-boxylase (ODC) catalyzes the conversion of ornithine to poly-amines. Arginase can inhibit NO production, and NO caninhibit ODC activity as part of an early inflammatory response.This study examines the arginine-NO-polyamine pathway al-teration in saliva and gingival biopsy samples of patients withgingivitis or periodontitis and healthy controls and evaluatesthe response to periodontal treatment.

Methods: This study includes nine gingivitis patients, 15chronic periodontitis patients, and 11 healthy age-matchedcontrols. Periodontal clinical measurements, gingival biop-sies, and saliva samples were obtained before treatment(BT) and 1 month after periodontal treatment (AT). Arginaseand ODC activities and NO levels were determined spectro-photometrically.

Results: The BT salivary and gingival NO levels were foundto be highest in the gingivitis group, followed by the healthyand the periodontitis groups, respectively. Salivary NO levelssignificantly increased in the periodontitis group and de-creased in the gingivitis group AT (P <0.05). Gingival NOlevels decreased significantly in the periodontitis and thegingivitis groups AT (P <0.05). Arginase levels were detectedhighest in the gingivitis group and lowest in the periodontitisgroup, both in saliva and gingiva. Only gingival arginase levelssignificantly increased AT (P <0.05). ODC activity was highestin saliva, and lowest in the gingiva of the periodontitis patientsBT. It was found to be significantly higher in the periodontitisgroup AT (P <0.05).

Conclusions: In this study, regarding arginine-NO-poly-amine metabolism, gingival tissue seems to be more informa-tive about periodontal pathogenesis than saliva. At earlyphase of periodontal inflammation, NO arginase and ODClevels were measured as higher than at an established lesionof periodontitis. J Periodontol 2011;82:320-328.

KEY WORDS

Arginase; nitric oxide; ornithine decarboxylase; periodontaldiseases.

It has long been known that peri-odontal diseases have multifacetedpathogenesis. Destruction of the peri-

odontal tissues caused by periodontitishas been reported to be associated witheither host or microbial-derived en-zymes, including arginase, nitric oxidesynthase (NOS), and collagenase.1-5

Arginase is the fifth enzyme of the ureacycle, which hydrolyzes arginine to orni-thine and urea. Arginine is a semies-sential amino acid involved in variousphysiologic processes, such as proteinand urea synthesis. It is also the precur-sor of other compounds, such as creati-nine, nitric oxide (NO), polyamines, andproline. Because arginase and NOScompete for the common substrate, argi-nine, arginase can downregulate NOproduction by decreasing intracellulararginine concentrations. Arginase isfound mainly in the human liver, but itcan also be found in other non-hepatictissues, such as salivary glands. Recentstudies have considered the role of sali-vary arginase activity in periodontal dis-ease pathogenesis, showing a highersalivary arginase activity in patientswith chronic periodontitis compared tohealthy periodontal control patients; how-ever, patients with gingivitis were not in-cluded in the study designs.2,6

NO plays an essential role in many im-portant physiologic and pathologic pro-cesses. NO is synthesized from arginineby a family of isoenzymes called ‘‘nitricoxide synthase.’’ Three isoforms of NOShave been reported in mammalian tis-sues: 1) neural, 2) endothelial, and 3)

* Department of Medical Biochemistry, School of Medicine, Ankara University, Ankara,Turkey.

† Department of Periodontology, Faculty of Dentistry, Gazi University, Ankara, Turkey.

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inducible. The endothelial NOS and neuronal NOSare constitutive enzymes, which produce low NOconcentrations for a short period after receptor stim-ulation. Expression of inducible NOS (iNOS) is acti-vated by inflammatory stimuli, such as bacteriallipopolysaccharides and proinflammatory cytokines,in a variety of cell types including macrophages andneutrophils.7 Once expressed, iNOS can generatelarge amounts of NO for extended periods, and isbelieved to be involved in cytotoxic effects after in-flammation.

The increase in iNOS activity in periodontal tissueshas been reported in inflammatory periodontal dis-ease, which suggests the production and participationof NO in the disease process.4,8 It is suggested thatNO is produced in response to periodontal pathogensand local inflammatory alterations.3,9-11 When NO islocally produced in high amounts by iNOS, it can actas a cytotoxic molecule against microbial pathogensand surrounding cells possibly leading to tissue de-struction.7 Thus, NO has antimicrobial activity andis considered to be an important molecule againstinfectious diseases, such as periodontitis. Becausearginine is used as a substrate by both arginase andNOS, an increase in arginase activity may lead to areduction in the production of NO leading to an in-creased susceptibility to bacterial infections.

L-Ornithine, which is producedby arginase, is apre-cursor for the synthesis of polyamines by the ornithinedecarboxylase (ODC) pathway and for the synthesisof L-proline by enzyme ornithine aminotransferase.Polyamines play a role in cell growth and differentia-tion, whereas synthesis of proline may result in colla-gen production.12,13 Both polyamines and collagenare essential for wound healing in many disease pro-cesses. Polyamines are also involved in the regulationof inflammatory reactions.12,13 Spermine, one of thepolyamines, released by damaged or killed cells issaid to favor cell migration and growth at the site oflocal inflammation.13 Polyamines modulate a time-related inverse relationship between the expressionof NOS and polyamine synthesis at sites of inflamma-tion.14 However, it is known that ODC is the initial andrate-limiting enzyme in the polyamine biosyntheticpathway, which maintains an optimal concentrationof polyamines in cells. It is reported that NO has anantiproliferative effect because it can inhibit bothODC and arginase.15 Thus, there seems to be a com-plex relationship between the arginine-NO pathwayand ODC activity. A limited number of studies havebeen reported about arginine-NO-polyamine meta-bolism in periodontal inflammation and its role inthe development of gingivitis and periodontitis is notyet clear.3,8,10 However, depending on their role ininflammatory processes, polyamine metabolismchanges might be expected in periodontal diseases.

Periodontal disease pathogenesis has not yet beenfully understood. We hypothesized that gingival tissueor salivary arginase, NO, and ODC levels may havea role in different forms of periodontal diseases andperiodontal therapy may affect the levels of arginine-NO metabolism in periodontium.2,3

The purpose of this study is to investigate NO, ar-ginase, and ODC levels and iNOS expression withingingival biopsies and saliva of patients with gingivitisor periodontitis and age-matched healthy controls,and to evaluate the effects of scaling and root planingon these inflammatory mediators.

MATERIALS AND METHODS

Patient SelectionEleven periodontally healthy volunteers (one maleand 10 females, age range: 23 to 45 years; meanage: 28.64 – 9.47 years), nine patients with gingivitis(onemaleand eight females,age range:23 to41 years;mean age: 28.20 – 7.24 years), and 15 patients withchronic periodontitis (five males and 10 females, agerange: 35 to 53 years; mean age: 44.30 – 6.90 years)were randomly selected from the group of patientswho were attended to at Gazi University between2006 and 2008.All subjects were systemically healthy,never-smokers, and had not taken anti-inflammatoryagents, antibiotics, or immunosuppressants duringthe 6 months before the study. Individuals who werepregnant or who required premedication with a sys-temic antibiotic were excluded from the study. Thestudy was performed in accordance with the HelsinkiDeclaration of 1975, as revised in 2000. The studyprotocol has been reviewed and approved by the In-stitutional Review Board at Gazi University, Facultyof Dentistry (28.11.2006-17). All subjects were askedto give an informed written consent to participate, aftera thorough explanation of the procedures and objec-tives of the study. The classification of study groupswas made according to criteria proposed by the1999 International World Workshop for a Classifica-tion of Periodontal Disease and Conditions.16

Clinical ExaminationBefore any treatment, baseline clinical indices wererecorded, and gingival biopsies were obtained frominterdental gingival papilla. The clinical examinationincluded plaque index (PI),17 gingival index (GI),18

probing depth (PD), clinical attachment level (CAL),and bleeding on probing (BOP). The measurementswere made at six sites per tooth (mesio-vestibular,mid-vestibular, disto-vestibular, mesio-lingual, mid-lingual, and disto-lingual) using a periodontalprobe‡ by the same researcher (BP). After recordingclinical indices, saliva, and gingiva at baseline, allsubjects received oral hygiene instructions. Gingivitis

‡ Williams periodontal probe, Nordent Manufacturing, Elk Grove Village, IL.

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and periodontitis patients received phase I periodontaltreatment consisting of scaling and root planing withultrasonic and hand instruments, which was com-pleted within 2 weeks from the beginning of the study.Clinical indices, saliva, and gingival biopsy samplingwere repeated for gingivitis and periodontitis patientsat the fourth week after periodontal phase 1 therapy.

Saliva SamplingThe participants were asked not to eat food and drinkliquids for 2 hours before each separate collection.They were instructed to rinse their mouth for 30 sec-onds with 10 ml of water and to rest for 2 minutes be-fore unstimulated saliva was collected by spitting.19

Approximately 1 ml of unstimulated whole salivawas collected into Eppendorf tubes. Samples werecentrifuged for 10 minutes at 15,000 · g at 4�C to re-move any particulate matter. The supernatants werestored at -20�C to be analyzed later.

Biopsy TechniqueBiopsies were obtained before periodontal therapy,under local anesthesia, from the interdental gingivalpapillae described previously.3 The clinically healthytissue samples were collected from patients undergo-ing crown lengthening procedures. Briefly, sites withGI £1 and PD £4 mm were selected for tissue samplingin healthy individuals, whereas sites with GI >1 and PD<4 mm were selected in patients with gingivitis. Fi-nally, sites with GI >1 and PD >4 mm were chosenfor patients with periodontitis. An inverse bevel inci-sion was used to obtain 1-mm tissue samples fromthe underside of the papillae. We obtained sulcus epi-thelium and some connective tissue at the biopsiesof healthy subjects, whereas pocket epithelium andconnective and granulation tissues were obtainedfrom biopsies of patients with gingivitis and peri-odontitis. In healthy volunteers caution was paid toobtain tissue samples of similar dimensions. Carewas taken to achieve uncomplicated wound healingwithout any esthetical consequences. After washingin sterile 0.15 M saline solution, samples were placedin saline and immediately sent to a laboratory. Biopsieswere repeated for patients with gingivitis and peri-odontitis at 4 weeks after scaling and root planing.20,21

Biochemical ProceduresNitric oxide. NOassay wasperformedby using acom-mercial kit.§ In aqueous solution, NO rapidly degradesto nitrate and nitrite. Spectrophotometric quantitationof nitrite using Griess Reagent is straight forward, butdoes not measure nitrate. This kit uses the reducedform of nicotinamide adenine dinucleotide–dependentenzyme nitrate reductase for conversion of nitrate tonitrite before quantitation of nitrate using Griess re-agent, thus providing for determination of total NOproduction.

Arginase. Arginase activity was measured spectro-photometrically according to Chinard’s method.22

The enzyme activity was determined by measuringthe amount of ornithine produced from the hydrolysisof arginine by arginase and was expressed as interna-tional unit per milliliter. The specific enzyme activitywas referred to the protein amount and calculatedby dividing the mean of enzyme activity by the meanof the protein amount. The specific enzyme activitywas expressed as international unit per milligram ofprotein. Protein contents of the samples were mea-sured by the method of Lowry et al.23

Ornithine decarboxylase. A rapid and sensitivespectrophotometric assay for ODC described byNgo et al.24 was used. It is based on the observationthat putrescine, the product of ODC, reacts with2,4,6-trinitrobenzene sulfonic acid to give a coloredproduct soluble in 1-pentanol, unlike ornithine. Theamount of putrescine produced by the enzyme isdetermined by measuring the absorbance of the1-pentanol extract of the reaction mixture at 420 nm.

Immunohistochemical Staining for iNOSFour-micrometer thick sections of formalin-fixed andparaffin-embedded biopsy samples were processedby the avidin-biotin-peroxidase complex (ABC)method for iNOS, as described previously.3 Deparaf-finization of the sections was followed by the blockingof endogenous peroxidase activity by incubatingthe sections in 3% H2O2 for 10 minutes. After rinsingwith phosphate-buffered saline, the sections weretreated in a microwave by antigen retrieval solutioni

for 15 minutes and then the slides were left to coolat room temperature for 30 minutes. Non-specificbinding was reduced with protein blocking serum¶

for 20 minutes. Sections were incubated with iNOSprimary polyclonal antibody# at room temperature(25�C) for 120 minutes. After rinsing thoroughlywith phosphate-buffered saline, the slides were incu-bated with biotinylated secondary antibody (murine[mouse])** for 30 minutes. The sections were washedwith phosphate-buffered saline followed by treatmentwith ABC†† for 30 minutes. The diaminobenzidine(DAB) tetrachloride‡‡ was used as a chromogen forvisualization of the antibody binding. Finally, the sec-tions were counterstained with hematoxylin, cleared,and mounted. iNOS-positive cells were counted andgraded25 on randomly selected three high-power fieldsfor each slide.

§ Colorimetric Assay for Nitric Oxide Product No. NB 98, OxfordBiomedical Research, Oxford, MI.

i HK087-5K, Biogenex, San Ramon, CA.¶ Rabbit anti-iNOS polyclonal, Zymed, San Francisco, CA.# HK112-9K, Biogenex.** Signet, Dedham, MA.†† HK330-9K, Biogenex.‡‡ Lipshaw Immunon, Pittsburgh, PA.

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Statistical AnalysesThe average of the clinical parameters (PI, GI, BOP,PD, and CAL) and levels of NO, arginase, and ODCwere calculated for all subjects. Our sample sizewas based on those of Lappin et al.4 and Reheret al.26 All calculations were undertaken using a statis-tical software package.§§

The full-mouth clinical parameters were assessedby Wilcoxon signed-rank test and Kruskal-Wallistest was used for comparative evaluations amonggroups. The statistical analysis of sampled sites of PDand CAL was performed by repeated measurementvariance analysis and the significant differences wereexamined using the Duncan test. Kruskal-Wallis testfor comparison of three groups before treatmentand two groups after treatment separately, andWilcoxon test for evaluation of the differences beforeand after the treatment were used for statistical anal-ysis of PI, GI, and BOP of sampled sites.

Wilcoxon signed-rank test for examining the signif-icant differences among groups and Mann-WhitneyU test for evaluation of the differences before andafter the treatment were used for statistical analysisof NO, arginase, and ODC levels. We used Kruskal-Wallis test to analyze the differences among theperiodontitis, gingivitis, and healthy groups. The cor-relations between NO, arginase, and ODC levels andclinical parameters were analyzed with Pearson cor-relation coefficient.

RESULTS

The descriptive measurements of full-mouth clinicalindices including mean and standard deviation valuesare summarized in Table 1. The clinical indices of thesampled sites before and after treatment are shownin Table 2.

The mean salivary NO, arginase, and ODC levels ofthe groups, with their standard errors, are shown inTable 3. Before treatment, salivary NO levels in thegingivitis group were the highest among the groups.Regarding the salivary NO levels, the difference wassignificant only between the periodontitis and gingi-vitis groups (P <0.05). It is the same for salivary argi-nase levels, but the differences were statisticallysignificant between the periodontitis and gingivitisgroups and periodontitis and healthy controls(P <0.05). Salivary ODC activity was higher in thechronic periodontitis group compared to controls(P <0.05). After treatment, NO and ODC levels didnot show a significant difference between the peri-odontitis and gingivitis groups; however, there wasa significant increase in arginase activity.

The mean – SE values of NO, arginase, and ODClevels in gingival biopsy samples are summarized inTable 4. Before treatment the NO and ODC levelswere significantly different in the periodontitis, gingi-

vitis, and healthy groups (P <0.05). Arginase activitydiffered significantly only between the periodontitisand gingivitis groups (P <0.05). After treatment, NOlevels decreased significantly in the periodontitisand gingivitis groups (P <0.05). Arginase levels wereincreased in both groups, but the difference wassignificant only in the periodontitis group. ODC activ-ity was the highest in the gingivitis group before treat-ment. It was found to be significantly higher in theperiodontitis group and, although non-significantly,lower in the gingivitis group after treatment.

Before treatment, in the gingivitis group significantcorrelations were found between gingival arginaseand NO levels (0.684; P = 0.029) and between gingi-val and salivary ODC levels (0.774; P = 0.009). Theperiodontitis group showed significant correlationbetween gingival NO–salivary arginase (0.705; P =0.034) and gingival NO–CAL (0.685; P = 0.042) atbaseline. Significant correlation existed for the peri-odontitis group between PD and CAL, before(0.873; P = 0.002) and after the treatment (0.906;P = 0.001).

iNOS expression of gingival tissues was evaluatedby immunohistochemical staining. The present datademonstrated that inflammatory cells, such as mac-rophages, lymphocytes, and neutrophiles, were iNOSpositive. Immunohistochemical staining results fromthe three study groups are presented in Figure 1and Table 5. iNOS positivity decreased prominentlyafter periodontal treatment.

DISCUSSION

To our knowledge, this study is the first one to evalu-ate involvement of the arginine-NO pathway togetherwith ODC activity in both saliva and gingiva of pa-tients with chronic periodontitis and gingivitis andhealthy individuals.

The periodontal clinical measurements suggestedthat after non-surgical periodontal treatment in boththe periodontitis and gingivitis groups, healing wasuneventful in all subjects resulting in a significant re-duction of PD. There was an improvement in CAL inboth groups after the treatment but the level of statis-tical significance was reached only for the peri-odontitis group. Furthermore, in both patient groupsscaling and root planing resulted in PI, GI, and BOPreductions.

The highest salivary and gingival NO levels weredetected in the gingivitis group followed by thehealthy and periodontitis groups, respectively. Afterperiodontal treatment, salivary NO levels increasedin the periodontitis group significantly and decreasedin the gingivitis group. Similar to NO levels in bothsaliva and gingiva, arginase levels were highest in

§§ SPSS v.10.0, SPSS Chicago, IL.


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the patients with gingivitis and lowest in the patientswith periodontitis. After treatment, only gingival argi-nase levels significantly increased in both patientgroups. ODC levels were highest in saliva of the pa-tients with periodontitis, whereas the lowest levelswere detected in the gingiva of the same patients. Asignificant increase in gingival ODC levels was seenafter treatment.

Previously, Reher et al.26 reported that elevatedsalivary NO levels were correlated with periodontal

disease severity. On the contrary, salivary NO levelsof our chronic periodontitis group were insignificantlylower compared to the clinically healthy group. Sim-ilar to our findings, Aurer et al.27 reported that patientswith periodontitis had lower salivary NO levels thanhealthy individuals. Decrease in salivary NO levelsin patients with periodontitis compared to that in pa-tients with gingivitis and in healthy individuals maylead to a decrease in antibacterial properties of salivaincreasing the susceptibility of periodontal tissues to

Table 1.

Full Mouth Clinical Indices Before and After Treatment (SD and SE)

Groups Time PI (scores from 0 to 3) GI (scores from 0 to 3) BOP (%) PD (mm) CAL (mm)

Periodontitis (n = 15) Before 0.55 – 0.13 0.50 – 0.13 36 – 7 3.27 – 0.15 3.40 – 0.16After 0.29 – 0.07 0.16 – 0.07 32 – 6 2.75 – 0.11 3.02 – 0.15

Before-after P = 0.015 P = 0.003 P = 0.783 P = 0.002 P = 0.012

Gingivitis (n = 9) Before 0.42 – 0.10 0.59 – 0.15 14 – 5 1.94 – 0.19 1.94 – 0.19After 0.35 – 0.05 0.23 – 0.13 10 – 4 1.81 – 0.16 1.81 – 0.16

Before-after P = 0.575 P = 0.114 P = 0.858 P = 0.037 P = 0.037

Healthy (n = 11) Before 0.26 – 0.08 0.21 – 0.10 5 – 3 1.68 – 0.10 1.68 – 0.10

P-G* Before P = 0.480 P = 0.898 P = 0.092 P = 0.000 P = 0.000

P-H† Before P = 0.213 P = 0.287 P = 0.009 P = 0.000 P = 0.000

G-H‡ Before P = 0.904 P = 0.224 P = 0.719 P = 0.524 P = 0.547

P-G§ After P = 0.339 P = 0.594 P = 0.013 P = 0.000 P = 0.000

* Before treatment statistical analysis between the periodontitis and gingivitis groups.† Before treatment statistical analysis between the periodontitis and healthy groups.‡ Before treatment statistical analysis between the gingivitis and healthy groups.§ After treatment statistical analysis between the periodontitis and gingivitis groups.

Table 2.

Clinical Indices of the Sampled Sites Before and After Treatment (SD and SE)

Groups Time PI (scores from 0 to 3) GI (scores from 0 to 3) BOP (%) PD (mm) CAL (mm)

Periodontitis (n = 15) Before 0.45 – 0.11 0.68 – 0.17 41 – 11 6.86 – 0.24 7.36 – 0.31After 0.18 – 0.08 0.27 – 0.12 32 – 10 5.36 – 0.33 5.77 – 0.44

Before-after P = 764 P = 0.395 P = 0.524 P = 0.000 P = 0.029

Gingivitis (n = 9) Before 0.50 – 0.22 1.30 – 0.21 40 – 16 2.90 – 0.57 2.20 – 0.25After 0.30 – 0.15 0.70 – 0.26 20 – 13 2.00 – 0.33 2.00 – 0.33

Before-after P = 492 P = 0.031 P = 0.024 P = 0.000 P = 0.249

Healthy (n = 11) Before 0.18 – 0.06 0.55 – 0.15 18 – 3 2.00 – 0.16 2.00 – 0.16

P-G* Before P = 0.185 P = 0.019 P = 1.000 P = 0.000 P = 0.000

P-H† Before P = 0.432 P = 0.525 P = 0.183 P = 0.000 P = 0.000

G-H‡ Before P = 0.467 P = 0.024 P = 0.361 P = 0.254 P = 0.171

P-G§ After P = 0.663 P = 0.531 P = 0.170 P = 0.000 P = 0.000

* Before treatment statistical analysis between the periodontitis and gingivitis groups.† Before treatment statistical analysis between the periodontitis and healthy groups.‡ Before treatment statistical analysis between the gingivitis and healthy groups.§ After treatment statistical analysis between the periodontitis and gingivitis groups.


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periodontal pathogens.28 It was postulated that somesalivary factors may suppress NO synthesis or de-stroy already produced NO.27 Furthermore, salivaof patients with periodontitis was demonstrated notto stimulate but rather to depress the NO synthesis inpolymorphonuclear leukocytes (PMNL), whereas sa-liva of the healthy individuals stimulated NO synthe-sis.29 Although the exact mechanisms are not yetfully understood, salivary NO reduction was reportedto be more evident in subjects with severely progressedperiodontal destruction.27 Fukada et al.30 evaluatedthe role of NO in bone loss in infection-induced api-cal periodontitis by using an iNOS-deficient micemodel. In their experiment NO deficiency was associ-ated with severe infection-stimulated bone loss.30

iNOS-positive cells in the periodontal tissues were

shown to be lymphocytes, PMNLs, and especiallymacrophages.4 NO is considered responsible for thebactericidal action of macrophages because they ac-tivate their killing potential when producing NO.31

Taking into account that gingivitis is a lesion charac-terized by neutrophils and macrophages, we assumethat the increase in NO levels might be stimulatedby these cells in our gingivitis group. In addition, a sig-nificant decrease of salivary NO observed in subjectswith gingivitis after periodontal treatment fits ourtheory. In contrast to the gingivitis group, significantincrease in salivary NO resulted after treatment inthe periodontitis group, accompanied by clinical im-provements, such as significant PD reduction. Wespeculate that this increase in salivary NO levelmay be related to an effective reduction of the local

Table 3.

Salivary NO, Arginase, and ODC Levels Before and After Treatment (mean – SE of mean)

NO (mmol /g) Arginase (IU/mg) ODC (IU/mg)

Groups Before After Before-After Before After Before-After Before After Before-After

Periodontitis 16 – 5.1 46.2 – 14.1 P = 0.027 53.8 – 20.6 97.1 – 53.3 P = 0.609 107.7 – 34.1189.5 – 40 P = 0.084

Gingivitis 94.2 – 18.1 43.6 – 8 P = 0.028 967.5 – 204.7 844.3 – 169 P = 0.441 50 – 13.4 74.7 – 11.3 P = 0.139

Healthy 61.8 – 18.3 741.8 – 340.8 29.5 – 9.2

P-G* P = 0.000 P = 0.421 P = 0.010 P = 0.000 P = 0.522 P = 0.179

P-H† P = 0.421 P = 0.040 P = 0.121

G-H‡ P = 0.184 P = 0.221 P = 0.211

* Statistical analysis between the periodontitis and gingivitis groups.† Statistical analysis between the periodontitis and healthy groups.‡ Statistical analysis between the gingivitis and healthy groups.

Table 4.

NO, Arginase, and ODC Levels in Gingival Biopsy Samples Before and After Treatment(mean – SE of mean)

NO (mmol /g) Arginase (IU/mg) ODC (IU/mg)

Groups Before After Before-After Before After Before-After Before After Before-After

Periodontitis 23.9 – 6.3 12.2 – 4.1 P = 0.078 4.5 – 0.9 18.1 – 5.3 P = 0.028 8.6 – 6 31.7 – 17.7 P = 0.028

Gingivitis 147.5 – 19.6 96.4 – 9.4 P = 0.038 7.8 – 1.5 13.7 – 5.7 P = 0.678 153.7 – 21.8 145.4 – 29.8 P = 0.767

Healthy 58.5 – 13.1 6.7 – 1.8 65.1 – 17.2

P-G* P = 0.000 P = 0.000 P = 0.000 P = 0.050 P = 0.000 P = 0.000

P-H† P = 0.000 P = 0.121 P = 0.010

G-H‡ P = 0.020 P = 0.800 P = 0.020

* Statistical analysis between the periodontitis and gingivitis groups.† Statistical analysis between the periodontitis and healthy groups.‡ Statistical analysis between the gingivitis and healthy groups.


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inflammatory process associated with tissue destruc-tion also increasing antibacterial potential of saliva.

Salivary arginase levels were found to be lower be-fore treatment in the periodontitis group compared topatients with gingivitis or healthy subjects. It may beconsidered that arginase prevents periodontal tissuesfrom toxic effects of NO. In accordance, arginase wasfound to be highest in the healthy subjects. Arginaseincrease in gingivitis and healthy groups might bea compensatory mechanism in health againstexcessive NO production in these groups. Increasedsalivary arginase activity in turn contributes to a de-

crease in NO production. In addition to this, we ob-served that periodontal treatment contributed alsoto a decline in salivary NO levels in patients with gin-givitis. After treatment, both patient groups reachedsimilar levels of NO in saliva.

Even though the differences were not statisticallysignificant, salivary ODC levels in both the gingivitisand periodontitis groups were increased after treat-ment. Increase in ODC may cause an increase in poly-amines, which are essential for cellular proliferationand collagen production in wound healing.32 Thus, in-creased salivary arginase and ODC levels in the pa-tients with periodontitis in our study may indicatethe healing process initiated by periodontal treatment.

Gheren et al.6 demonstrated that non-surgical peri-odontal therapy significantly reduced salivary argi-nase activity in patients with periodontitis. On thecontrary, Gullu et al.3 reported that both surgicaland non-surgical periodontal treatments significantlyincreased salivary arginase activity in patients withchronic periodontitis and decreased iNOS expression.Our results are particularly in agreement with Gulluet al.3 We also found increased salivary arginaseand increased salivary NO and salivary ODC levelsin patients with periodontitis. When locally producedin high concentrations, NO may act as both a cytotoxic

Figure 1.iNOS(+) inflammatory cells shown in gingival tissue samples (arrows). ABC, DAB chromogen, and Mayer’s hematoxylin counterstain, A) originalmagnification ·400. B) Original magnification ·100. C) Original magnification ·400. e = epithelium.

Table 5.

Ratios of iNOS Positive and NegativeTissue Samples Before and AfterTreatment

Periodontitis

(n = 15)

Gingivitis

(n = 9)Healthy*(n = 6)Groups Before After Before After

iNOS (-) 59% 63% 66% 85% 85%

iNOS (+) 41% 37% 34% 15% 15%

* Not all samples evaluated immunohistochemically.


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molecule against invading pathogens and againstadjacent tissue cells, thus being related to both bene-ficial and harmful effects.10 We conclude that lowerand higher concentrations of NO can initiate des-truction in periodontal tissues, yet a threshold NOlevel might be required in remodeling of periodontaltissues.

Similar to the enzyme levels in saliva, gingival NO,arginase, and ODC levels were higher in the gingivitisgroup compared to the periodontitis and healthygroups at baseline. Increased gingival arginase levelscan downregulate NO production by decreasingintracellular arginine concentrations and can up-regulate ornithine concentration, which may causeincreased ODC activity and production of poly-amines, such as putrescine. Previously, significantcorrelation was demonstrated between ODC activ-ity and intracellular putrescine levels.33 Lamsteret al.34 suggested that putrescine might have animportant homeostatic role in the periodontium.

Five forms of arginase were characterized in humantissues and one of them is from the salivary glands.32

However, although not yet characterized, there area few studies reporting the existence of arginase ingingiva.3,35 In the present study, tissues are alwayswashed with saline to remove blood, saliva, or any-thing else to avoid contamination.

Previously, Gullu et al.3 reported that gingival tissueiNOS expression in patients with chronic periodontitisdecreased after periodontal therapy, whereas arginaseactivity increased. In accordance with their results, wefound that gingival NO levels and iNOS expressiondecreased significantly after scaling and root planingin both groups. In the periodontitis group, gingivalarginase and ODC levels increased significantly afterscaling and root planing.

The immunohistochemical results of our study re-veal higher iNOS expression in tissues obtained fromthe patients with chronic periodontitis than in biopsiesfrom the patients with gingivitis and healthy individ-uals. A previous immunohistochemical study showedthat the number of iNOS cells in the gingiva of patientswith periodontitis disregarding their disease progres-sion was significantly elevated compared to clinicallyhealthy gingival tissues.10 This study demonstratesthat iNOS expression in the gingiva of patients withchronic periodontitis was higher than in the gingivitisand healthy groups. However, tissue NO levels in theperiodontitis group were found to be lower. This differ-ence may be explained by the hypothesis that varioussalivary factors may degrade already produced NO.27

However, NO may react with other free radicals, suchas superoxide anions, producing reactive oxidants,such as peroxynitrite.36 Additionally, periodontaltreatment for both patients with chronic periodontitisand gingivitis resulted in decreased iNOS positive

numbers. Our results were concordant with a previousstudy.3

In the present study, the inconsistent pattern of NOand enzyme levels in saliva might be associated withseveral factors. Compared to saliva, gingiva is at closeproximity to periodontal tissues where the onset ofperiodontal disease happens. In addition, the exactassignment of sources of the enzymes or proteins insaliva is much more difficult compared to tissue sam-ples. The complete mechanism of the arginine-NO-polyamine pathway in saliva or gingival tissues, andan understanding of the factors influencing thismechanism, still remains unclear. The compositionof whole saliva is very complex, and blood, gingivalcrevicular fluid, and extrinsic substances, such asfood debris, bacteria, and bacterial products, are onlysome of its components.37 Therefore, NO, arginase,or ODC levels within saliva might be affected byvarious intrinsic or extrinsic factors.

CONCLUSIONS

Regarding the arginine-NO-polyamine pathway, datacollected from gingival tissue samples are more infor-mative about the pathogenesis of the periodontal dis-eases than saliva samples. At an early or establishedphase of periodontal inflammation, measured NO, ar-ginase, and ODC levels were higher than in advancedperiodontitis lesions.

ACKNOWLEDGMENTS

This study was supported by a research grant fromThe Scientific and Technological Research Councilof Turkey (106S353 SBAG-HD-200). The authorsreport no conflict of interest related to this study.

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Correspondence: Professor Nurdan Ozmeric, Departmentof Periodontology, Faculty of Dentistry, Gazi University,Bishkek Caddesi 84. Sokak 06510 Emek, Ankara, Turkey.E-mail: [email protected].

Submitted April 7, 2010; accepted for publication July 30,2010.


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arginine–nitric oxide–polyamine metabolism in periodontal disease

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