Effect of cigarette smoking on oral elastase activity in adult periodontitis patients

9012_IPC_AAP_553060 2/15/00 11:02 AM Page 58 Effect of Cigarette Smoking on Oral
Elastase Activity in Adult Periodontitis
Nathalie C. Pauletto,* Kirsti Liede,† Anja Nieminen,† Hannu Larjava,* and Veli-Jukka Uitto*
Background: We have previously reported that elastase activ-
ity in oral fluids is significantly increased in most adult peri-odontitis patients. In some patients, however, elastase levelsremain low despite the presence of deep periodontal pockets.
In this study we explored whether or not smoking is related tothe unexpected low elastase values in these patients.
Methods: We determined what proportion of the periodonti-
tis patients that showed low oral elastase values were smokers.
Paraffin-stimulated saliva or oral rinse samples (3 ml of water, 30 second rinse) were assayed for elastase activity by incubat- ing with 1 mM succinyl-alanyl-alanyl-valine-p-nitroanilide for epithelial cells, following their activation.1 20 hours at 37°C, and the color formation read with a spec- trophotometer. Neutrophil numbers were analyzed by staining the cells in the oral rinse smear samples.
Results: In 2 patient groups, one in Helsinki, Finland (n =
studied for their possible use as indica- 46) and the other in Vancouver, British Columbia (n = 25), 63% and 83%, respectively, of the adult periodontitis patients who had one or more pockets ≥6 mm and had low oral elastase val- ues (increase of optical density <0.5) were smokers. Non-smok- ing periodontitis patients had elevated neutrophil numbers com- pared to healthy subjects, while the smoking patients showed ies indicate that neutrophil elastase lev- no significant change. Next we analyzed elastase levels in stim- els in gingival crevicular fluid, saliva, or ulated whole saliva in a group of smokers (n = 300) and those who had quit smoking (n = 102). Smokers had significantly lower oral elastase levels than former smokers in both advanced and moderate periodontitis groups. In this subject group, 56% normal levels following healing.13-16 Our of all smokers with periodontitis (at least one pocket ≥6 mm) had oral elastase values less than 0.5 U while only 31% of those periodontitis patients have elevated oral patients who had quit smoking had low values.
Conclusions: Cigarette smoking leads to lowered elastase
and neutrophil levels in the oral cavity. The oral neutrophil elas- tase assay, therefore, cannot be used to measure the periodon- surement was tested for detection of peri- tal status of smokers. J Periodontol 2000;71:58-62. KEY WORDS
point of 0.5 U, about 28% of the patientswith one or more pockets ≥6 mm tested Periodontal pockets/diagnosis; periodontitis/diagnosis;
neutrophils; elastase; smoking/adverse effects;
tobacco/adverse effects; saliva/enzymology.
tory factor for that finding. Smoking hasmany tissue effects that appear to play * Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of † Department of Periodontology, Institute of Dentistry, University of Helsinki, Helsinki, demonstrates that oral elastase valuesand neutrophil counts are lower in smok-ing periodontitis patients than in non-smokers with the same degree of dis- 9012_IPC_AAP_553060 2/15/00 11:02 AM Page 59 Pauletto, Liede, Nieminen, Larjava, Uitto
ease. It also shows that when the smoking is stopped, ifest.16 Change in optical density of 0.5 (0.5 U) was the oral elastase values return to the level of non- taken as the cut-off value for the positive test result.
Both free elastase in the oral fluid samples and elas-tase released from neutrophils during the procedure MATERIALS AND METHODS
Study Population
Neutrophil Count
A total of 493 adults, aged 31 to 80, were included in Following the collection of the oral fluid samples, 10 the study. Initially 2 groups consisting of 46 and 25 µl of the samples were smeared on a glass slide and patients with moderate to severe periodontitis were fixed with cytospray.§ The cells were stained using a analyzed for oral rinse elastase activity at the Univer- modification of the Wright-Giemsa technique. Two sity of Helsinki and the University of British Columbia, drops of 0.15% Wright-Giemsa stain‡ were placed on respectively. The number of gingival pockets >5 mm the fixed smears for 1 minute followed by a 30-sec- in depth varied from 5 to 28. Twenty subjects with ond rinse under tap water. The number of neutrophils healthy periodontium who were non-smokers served (PMN) and epithelial cells in the samples was mea- as controls. Subsequently, a group of adults who were sured by counting 40 randomly selected squares of either smokers (n = 300) or former smokers (n = 102) 0.5 cm2 under light microscopy.࿣ Accuracy and repro- were randomly selected from a large pool of subjects ducibility of the measurements were assessed by participating in a cancer prevention study of the repeated counting of 10 randomly selected slides.
National Public Health Institute of Finland.19 The exclu-sion criteria were cancer, severe coronary disease, Statistical Analysis
chronic renal hypofunction, hepatic cirrosis, antico- Significance of the differences in the test groups was agulant medication, A or E vitamin supplement intake, analyzed by one-way analysis of variance test and alcohol abuse. All subjects had at least 15 natural (ANOVA). Spearman rank correlation test was used teeth. They were analyzed for their periodontal condi- to analyze the relationship of elastase activity and num- tion and salivary elastase activity. In this group, 95% of the smokers and 88% of the former smokers had probing depths ≥5 mm or more. None of the patients We have repeatedly observed that most of the patients were undergoing periodontal therapy at the time of the with moderate to advanced periodontitis have high oral study. The patients were examined and tested either elastase values. In this study, 80% of the patients, both by periodontists (KL ar A.N.) or a periodontal gradu- smokers and non-smokers, who had gingival pockets >5 mm had oral elastase activity higher than 0.5 U, a Oral Elastase Assay
cut-off value for the test selected based on our previ- In the first study elastase was measured either in stim- ous studies.16,17 When exploring reasons for the low ulated saliva (Helsinki group) or in oral rinse samples elastase activity in the remaining patients, we observed (Vancouver group). Stimulated saliva was collected by that in 2 periodontitis patient groups, one examined in allowing the subjects to chew paraffin for 30 seconds Helsinki and the other in Vancouver, 83% and 63% of and then collecting the whole saliva. For oral rinse the patients, respectively, who had low oral elastase specimens, subjects rinsed their mouths with 3 ml of values were smokers (Table 1). Data analysis showed tap water for 30 seconds. In the study on oral elastase that periodontitis patients who smoked had a signifi- of smokers, stimulated whole saliva samples were col- cantly lower salivary elastase than non-smoking pa- lected as above. Our previous study has shown that tients with similar disease severity (Fig. 1A). Because both whole saliva and oral rinse samples measure oral elastase is primarily derived from neutrophils, we equally well the activity of neutrophil elastase that is investigated whether the number of these cells was transferred into the oral cavity along with crevicular altered in oral rinse samples of the smokers compared fluid.16 The collected samples were stored at −20°C to non-smokers with a similar degree of periodontitis.
until assayed. After thawing, the samples were cen- Indeed, while the number of neutrophils in periodon- trifuged at 2,700 × g for 10 minutes and 100 µl of the titis patients who did not smoke was about twice that supernate was incubated for 20 hours at 37°C in 0.01 of the healthy group, the neutrophil count was only M tris-HCl-0.2 M NaCl-buffer, pH 7.5, containing 1 mM slightly elevated in the smoking periodontitis group succinyl-alanyl-alanyl-valine-p-nitroanilide‡ as the sub- (Table 2). In this population there was a significant strate for neutrophil elastase.20 The intensity of the correlation between the elastase activity and the num- color formed was read spectrophotometrically. Based ber of neutrophils in the rinse specimens (r = 0.358, on our earlier studies, the 20-hour incubation time wasselected because some oral samples with a low elas- ‡ Sigma Chemical Co., St. Louis, MO.
§ Fisher Scientific Ltd., Nepean, ON.
tase activity required a longer incubation time to man- ࿣ Zeiss, Axioskop, Oberkochen, Germany.
9012_IPC_AAP_553060 2/15/00 11:02 AM Page 60 Oral Elastase in Smokers
P <0.01; Spearman rank correlation test). By com-parison, the number of epithelial cells in the rinse spec- Percentage of Smokers in Periodontitis
imens was increased in both non-smoking and smok- Patients With Low Oral Elastase Levels
ing periodontitis patients (Table 2).
In order to determine the relation of oral elastase values to smoking frequency, 402 subjects weregrouped as heavy smokers (>10 cigarettes per day); moderate smokers (<10 cigarettes per day); and for- mer smokers. In both smoking groups, patients withat least one pocket ≥6 mm had markedly lower elas- tase values than similar patients who were formersmokers (Fig. 1B). The same trend was observed in patients with 4 to 5 mm pockets. Nosignificant differences were observedin oral elastase levels of smokers andnon-smokers without periodontalpockets. The overall effect of smok-ing on oral elastase levels of peri-odontitis patients was highly signifi-cant (P <0.001; ANOVA). It is evidentthat the salivary elastase levels ofpatients who had quit smoking arenot significantly different than thosesubjects who had never smoked, inboth periodontitis (0.85 ± 0.79 ver-sus 1.10 ± 0.59) and healthy groups(0.34 ± 0.58 versus 0.20 ± 0.41).
with one or more pockets ≥6 mm andlow elastase values were calculated,we observed that 56% of all smokershad elastase values less than 0.5 Uwhile only 31% of patients who hadquit smoking had low elastase val-ues. When smokers were excludedfrom the initial study populations thatconsisted of moderate to severe peri- Figure 1.
Oral elastase activity in cigarette smokers with adult periodontitis. A. Elastase activity was
measured in stimulated whole saliva of 18 non-smoking and 27 smoking periodontitis patients with at least one 6 mm gingival pocket. B. Oral elastase activity in 300 smokers and 102 former
smokers.The results are mean ± S.E.M. patients with lower than predicted oralelastase activity.
Neutrophil and Epithelial Cell Counts in Smoking and
Oral elastase activity has the potentialto serve as an important adjunctive Non-Smoking Periodontitis Patients
method in screening for periodontitisand monitoring treatment outcomes.
water rinse samples is a non-invasive,easy to perform, and relatively spe- * Significant difference compared to healthy group (P <0.01; ANOVA test).
9012_IPC_AAP_553060 2/15/00 11:02 AM Page 61 Pauletto, Liede, Nieminen, Larjava, Uitto
released from neutrophils during the experimental pro- alpha 1-protease inhibitor have been found to be cedure provides an easy way to approximate the num- lower in the gingival crevicular fluid of smokers than ber of oral leukocytes which is known to increase with non-smokers.41 Thus, all the information on the effects periodontitis.21 The drawback is that a certain pro- of smoking on neutrophils and tissue elastase levels portion of patients do not test positive with this method, obtained from previous studies and the present appear even though they have deep periodontal pockets. Fol- to point to the conclusion that in periodontitis patients lowing our first studies we assumed that the unex- who smoke, neutrophils and possibly macrophages, pected negative results may be partially explained by rather than migrating via the gingival crevice to the limited sensitivity of the test that reflects the sum of oral cavity, are accumulated in periodontal tissue where active elastase derived from all periodontal sites dur- they release their constituents leading to increased ing the sample collection period, and partially by indi- degradation of connective tissue components. This vidual fluctuation of neutrophil migration rate into the hypothesis needs to be explored in detail. While the pri- oral cavity. In certain periodontitis patients, however, mary goal of periodontal therapy in individuals who the low elastase values were thought to reflect the smoke should be directed to cessation of cigarette actual disease process that deviates from the normal smoking, other approaches aiming at minimizing the inflammatory response. In this study we were able to harmful effects of leukocyte products on the peri- demonstrate that tobacco smoking decreases signifi- odontium should also be considered. In this context, cantly the oral elastase levels. We observed that heavy drugs that stabilize neutrophils and inhibit elastase and smokers had clearly lower oral elastase activity than other proteases should also be tested for their benefi- moderate smokers or non-smokers with similar sever- cial effects in periodontal treatment.
ity of periodontitis. It is well demonstrated that ciga- To conclude, the present study shows that a signif- rette smoking has a significant impact on the risk for icant proportion of periodontitis patients who have low developing periodontal disease. Both the incidence and elastase levels in oral fluids are smokers. When the severity of periodontitis is greater in smokers than in oral elastase test is performed exclusively on adult non-smokers.22-25 The odds ratio for moderate smok- periodontitis patients who do not smoke tobacco, very ers (15 to 30 packs per year) is reported to be 2.8 and good sensitivity values can be obtained. The low oral for heavy smokers (≥30 packs per year), 4.8.24,25 elastase values of smokers should not be interpreted Tobacco users do not respond to periodontal treat- to indicate lack of periodontitis activity. In contrast, it ment as well as non-smokers, reflecting the general may indicate an abnormal leukocyte function and impairment of the tissue repair process.26-28 The effect of smoking seems reversible, because the risk of devel-oping periodontitis is clearly reduced upon cessation ACKNOWLEDGMENTS
of smoking.29 Accordingly, in our study the elastase This study was supported by grants from the Medical values for former smokers were comparable with those Research Council of Canada and the Finnish Dental There are several ways that tobacco substances can act on oral tissues. Tobacco causes constriction and REFERENCES
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