A comparison of 4 methods of data presentation for lysosomal enzyme activity in gingival crevicular fluid

In previous studies, we have emphasized the importance of considering the methods used for analysis of gingival crevicular fluid (GCF). This study evaluated 4 different approaches for data presentation of lysosomal enzyme activity in GCF. GCF was collected from patients displaying at least 2 mm of clinical attachment loss at a minimum of 3 sites in the mouth (DA), and patients who did not display clinical attachment loss of 2 mm or more at any site in the mouth (DI), during a 3-month interval following entry into a longitudinal trial. GCF was collected by the timed intrasulcular placement of precut filter paper strips. 16 to 28 individual GCF samples were collected from each patient. The lysosomal enzymes studied were B-glucuronidase (BG) and arylsulfatase. The mean values for the DA and DI groups at baseline and 3 months are reported. The results indicate that when the data is expressed as total enzyme activity (unit activity) per 30-s collection (UA) or UA x GCF volume (microliter) per mm of probing depth, the DA group demonstrated significantly greater mean values than the DI group at baseline and 3 months. In contrast, when the data was expressed as concentration (UA/microliter), or UA per mm of probing depth, differences between the DA and DI groups were observed only at the 3-month evaluation. The difficulty in using concentration when reporting GCF lysosomal enzyme activity is emphsized by comparison of the data from the DA group and the high and low enzyme activity subsets of the DI group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracytoplasmic enzymes in gingival crevicular fluid of patients with aggressive periodontitis

Castro CE, Koss MA, López ME. Intracytoplasmic enzymes in gingival crevicular fluid of patients with aggressive periodontitis. J Periodont Res 2011; 46: 522–527. © 2011 John Wiley & Sons A/S Background and Objective: Biochemical parameters of crevicular fluid could provide evidence of periodontal tissue disease. The aim of this study was to analyze enzymes in crevicular fluid in aggressive localized and generalized periodontitis. Material and Methods: One hundred and twenty‐four subjects were classified as having localized (n = 36) or generalized aggressive periodontitis (n = 38) and subclassified into moderate and severe groups. Controls were 50 periodontitis‐free subjects. Activities of the enzymes lactate dehydrogenase, neutrophil elastase, alkaline phosphatase and aspartate aminotransferase were determined. Data were analyzed using one‐way ANOVA and Tukey’s test. Results: Among the subjects with localized aggressive periodontitis, values of lactate dehydrogenase and alkaline phosphatase increased notably in moderate and severe periodontitis compared with control subjects. Values for aspartate aminotransferase increased with the severity of the disease, and neutrophil elastase was increased in the moderate and severe states. In generalized aggressive periodontitis, lactate dehydrogenase showed higher values than in control subjects in both periodontal subgroups. Alkaline phosphatase and neutrophil elastase showed higher significant differences between moderate and severe periodontitis compared with the control group. Aspartate aminotransferase showed differences between the severe and moderate periodontitis groups compared with the control group. Of all the enzymes analyzed, only lactate dehydrogenase showed higher values in localized than in generalized aggressive periodontitis. Conclusion: Lactate dehydrogenase may distinguish localized and generalized aggressive periodontitis. Alkaline phosphatase increases from moderate to severe states in both types of periodontitis. Aspartate aminotransferase and neutrophil elastase only increase with strong evidence of periodontal destruction.     

龈下菌斑中肽酶活性和龈沟液中碱性磷酸酶水平相关性的研究

目的 :探讨成人牙周炎龈下菌斑中三种牙周致病菌产生的肽酶高低与龈沟液中碱性磷酸酶水平的相关性及其在牙周炎活动性诊断中的意义。方法 :利用Periocheck椅旁快速检测 6 0例成人牙周炎患者共 72个位点、15例健康对照 15个位点龈下菌斑中肽酶活性 ,同时测量龈沟液中ALP水平。结果 :成人牙周炎患者肽酶活性、ALP水平显著高于健康对照组 ,并与探诊深度、附着丧失、牙龈指数呈正相关关系 ,同时二者之间具有一定的相关性。结论 :龈下菌斑肽酶活性和龈沟液中ALP水平相关 ,二者均反映牙周炎的严重程度 ,可用于牙周炎活动性的监测     

Granulocyte elastase activity in static and flow gingival crevicular fluid

OBJECTIVES: This study aimed to evaluate the volume of gingival crevicular fluid (GCF) and granulocyte elastase activity in static GCF (sGCF) and flow GCF (fGCF) from subjects with various periodontal conditions. METHODS: Eleven periodontally healthy, 10 gingivitis and 12 periodontitis subjects were recruited and the sites investigated consisted of healthy sites from healthy subjects (HH); healthy (HG) and gingivitis sites (GG) from gingivitis subjects; and healthy (HP), gingivitis (GP) and periodontitis sites (PP) from periodontitis subjects. fGCF samples were collected either 1 min or 5 min following sGCF collection by paper strip technique. GCF volume was determined by Periotron 6000 and granulocyte elastase activity was assayed with a specific substrate [l-pyroglutamyl-l-prolyl-l-valine-p-nitroanilide(pGluProVal-pNA)]. RESULTS: At baseline, no significant differences existed in clinical and GCF parameters between the two matched sites for subsequent collection of fGCF samples either 1 min or 5 min after sGCF sampling in all subjects. The flow exudate in HG and HP sites quickly replenished to sGCF levels, while a delayed replenishment was found in HH sites, despite the similar sGCF volumes of these sites. The GCF volume and elastase levels in the fGCF at 1 min were higher in GP sites than in GG sites (P < 0.05). Overall, depletion of elastase levels in the fGCF at 1 min was observed in all subjects, whereas elastase levels in the fGCF at 5 min had replenished to sGCF levels in HP, GP, PP sites and GG sites, but had remained at a lower level in HH and HG sites. An overall positive correlation was found between sGCF and fGCF for GCF volume and elastase activity (P < 0.001); however, this correlation varied with GCF parameters and with site conditions of the subjects concerned. CONCLUSIONS: This study shows that patterns of dynamic changes in GCF flow and elastase activity varied under different periodontal conditions. Assessment of both sGCF and fGCF may allow better insight into the dynamic change of the target components in GCF.     

The effect of sequential sampling on crevicular fluid volume and enzyme activity

Gingival crevicular fluid (GCF) volume and constituents in static samples were compared to volume and constituents in subsequent GCF samples collected during a 60-min interval. Using deep intracrevicular placement of precut filter paper strips, GCF was collected from interproximal and facial sites from patients with gingivitis (N = 14; 28 interproximal sites, 28 facial sites) and chronic adult periodontitis (N = 11; 26 interproximal sites, 18 facial sites). The strips were inserted for 30 s at 0, 4, 8, 30 and 60 min. The amount of fluid on each strip was determined and microspectrophotometric techniques were used to assess cytoplasmic and lysosomal enzyme activity. Within each group of sites, mean GCF volume showed minimal fluctuation with repeated sampling. In contrast, the static GCF sample contained the greatest amount of total enzyme activity, and differences were detected between groups. The interproximal sites and the gingivitis-facial sites displayed a similar pattern of change in total enzyme activity during the test period. The highest total enzyme activity was observed in the first sample and decreased at 4 and 8 minutes. At 30 and 60 min, the amount of enzyme either remained at the level detected at 8 min, or displayed a mild tendency to recover towards baseline. A different pattern of total enzyme activity was observed for the periodontitis-facial sites, where a significant decrease was first observed at 30 min. Enzyme concentration was higher in the facial sites than the interproximal sites, and enzyme concentration was generally highest in the static samples. The concentration data, however, is difficult to interpret since a number of sites demonstrated a converted GCF volume of 0 microliter. Our data suggests that total enzyme activity and enzyme concentration are generally greater in the static GCF samples compared to subsequent samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Granulocyte elastase in gingival crevicular fluid

The granulocyte elastase activity and the immuno-reactive (antigenic) granulocyte elastase of gingival crevicular fluid (GCF) were studied in 16 periodontitis patients and in 10 gingivitis patients. The elastase activity was measured with a low molecular weight substrate specific for granulocyte elastase. The antigenic elastase was determined with specific antibodies against granulocyte elastase. Intracrevicular sampling of GCF with paper strips for 30 s seemed to provide representative values of elastase. The elastase activity correlated with probing depth and attachment loss and appeared to be a measure of the degree of tissue destruction. Antigenic elastase represents the number of granulocytes in GCF and should thus be related to the degree of inflammation. The periodontitis patients and the gingivitis patients both had a similar degree of inflammation as measured by antigenic elastase per microliter GCF and gingival index. The elastase activity per microliter GCF, however, was higher in the periodontitis group. Elevated granulocyte elastase activity in GCF seems to be independent of inflammation and could thus be an indicator of patients at risk for periodontitis.     

Phylloquinone in gingival crevicular fluid in adult periodontitis

Abstract. Phylloquinone is a lipid soluble vitamin which is an absolute growth requirement for black-pigmented anaerobes, many of which are implicated in the aetiology of periodontal diseases. This cross-sectional study aimed to detect the levels of phylloquinone in GCF from healthy and diseased sites in subjects with adult periodontitis, in order to investigate further its potential role in the disease process. The sample consisted of eighteen patients with adult periodontitis. Periodontal probing depths, attachment levels and gingival indices were recorded from one healthy and one diseased site in each subject. GCF was sampled and the amount of phylloquinone in each sample was determined using reverse-phase high performance liquid chromatography coupled to electrochemical detection. The mean amount of phylloquinone in accumulated GCF from diseased sites was 406 pg/site and 80 pg/site from healthy sites ( p =0.013). When the amounts of phylloquinone in GCF were expressed as concentrations the values were 228 ng/ml and 3350 ng/ml for diseased and healthy sites respectively ( p =0.084). These findings suggest the levels of phylloquinone in GCF differs in periodontal health and disease in subjects with adult periodontitis. The total phylloquinone at diseased sites may provide the nutritional requirements favouring the growth of black-pigmented anaerobes.     

Relationship of changes in interleukin-8 levels and granulocyte elastase activity in gingival crevicular fluid to subgingival periodontopathogens following non-surgical periodontal therapy in subjects with chronic periodontitis

OBJECTIVES: To determine the effect of scaling and root planing (SRP) on the interrelations of subgingival periodontopathogens and both interleukin-8 (IL-8) and granulocyte elastase activity in gingival crevicular fluid (GCF), and to assess their relations to the short-term treatment response in management of chronic periodontitis. MATERIAL AND METHODS: GCF and subgingival plaque were collected from 16 subjects with untreated chronic periodontitis at baseline and 4 weeks after SRP. IL-8 levels were determined by ELISA. Granulocyte elastase activity was analyzed with a specific substrate, pGluProVal-pNA, and the maximal rate of elastase activity (MR-EA) was calculated. 5 DNA-probes were used to detect the presence of A. actinomycetemcomitans (A. a.), B. forsythus (B.f.), P. gingivalis (P.g.), P. intermedia (P.i.), and T. denticola (T.d.), with a sensitivity = 103 cells/paper point. RESULTS: IL-8 and MR-EA levels in GCF decreased significantly after SRP (p < 0.001) with a corresponding reduction of total count of the species. Of the sites with probing depth (PD) >/= 5.0 mm and co-infection by B.f., P.g., P.i. & T.d. at baseline, the sites without persistent co-infection of these species after SRP exhibited a significant reduction of IL-8 levels (p < 0.02), MR-EA levels (p < 0.02) and PD (p < 0.01). No such change was found in the sites where such a co-infection persisted. Moreover, reduction of IL-8 levels in those pocket sites was accompanied by a concomitant reduction of MR-EA (p < 0.02) and PD (p < 0.01), while no significant change in MR-EA levels and PD was noted in those pocket sites that exhibited an increase of IL-8 levels after SRP. At baseline, the former group of sites showed significantly higher IL-8 levels than the latter group of sites (p < 0.02). CONCLUSIONS: IL-8-related granulocyte elastase activity was related to the change in infection patterns of the target periodontopathogens following scaling and root planing. Varying initial IL-8 levels in GCF and a corresponding shifting change of granulocyte elastase activity in GCF may characterize the different short-term treatment responses.     

Relationship of collagenase and cathepsin G activity in gingival crevicular fluid

Human neutrophil cathepsin G has been identified as a potent proteolytic activator of latent human neutrophil collagenase in vitro. In order to examine the role of cathepsin G in the activation mechanism of latent human neutrophil collagenase in vivo, gingival crevicular fluid was collected from periodontal pockets of patients with adult periodontitis and the relationship of cathepsin G to the proportion of endogenously active collagenase and total collagenase activity was determined. The changes in these parameters were monitored before and after periodontal therapy and compared to control values obtained for periodontal sites without clinical signs of inflammation or increased pocket depth. A significant decrease in cathepsin G and collagenase activity in gingival crevicular fluid collected from initially deep periodontal pockets was observed in response to scaling and root planing ( P < 0.025, Wilcoxon signed rank test). Also the proportion of endogenously active collagenase decreased ( P < 0.05). There was a significant correlation of cathepsin G and total collagenase activity. However, no correlation of cathepsin G activity and endogenously active collagenase was observed. The results indicate the existence of several distinct activation pathways for latent human neutrophil collagenase in vivo and suggest that, apart from cathepsin G, other proteolytic activation cascades and/or non-proteolytic activaton pathways participate in the activation of latent human neutrophil collagenase in vivo.     

Alkaline phosphatase activity in gingival crevicular fluid during canine retraction

OBJECTIVES: The aim of the study was to investigate alkaline phosphatase activity in the gingival crevicular fluid (GCF) during orthodontic tooth movement in humans. SETTING AND SAMPLE POPULATION: Postgraduate orthodontic clinic. Ten female patients requiring all first premolar extractions were selected and treated with standard edgewise mechanotherapy. EXPERIMENTAL VARIABLE: Canine retraction was done using 100 g sentalloy springs. Maxillary canine on one side acted as experimental site while the contralateral canine acted as control. OUTCOME MEASURE: Gingival crevicular fluid was collected from mesial and distal of canines before initiation of canine retraction (baseline), immediately after initiation of retraction, and on 1st, 7th, 14th and 21st day and the alkaline phosphatase activity was estimated. RESULTS: The results show significant (p < 0.05) changes in alkaline phosphatase activity on the 7th, 14th and 21st day on both mesial and distal aspects of the compared experimental and control sides. The peak in enzyme activity occurred on the 14th day of initiation of retraction followed by a significant fall in activity especially on the mesial aspect. CONCLUSIONS: The study showed that alkaline phosphatase activity could be successfully estimated in the GCF using calorimetric estimation assay kits. The enzyme activity showed variation according to the amount of tooth movement.     

吸烟对牙周炎患者龈沟液中弹性蛋白酶水平的影响

目的比较吸烟与非吸烟牙周炎患者和健康人龈沟液中细胞外弹性蛋白酶EA-s和细胞内弹性蛋白酶(EA-p)水平的变化。方法选择慢性牙周炎患者41例,共146个探诊出血(BOP)、牙周袋探诊深度(PD)≥4mm、附着丧失(AL)≥2mm的牙周炎位点,将其分为吸烟组79个,非吸烟组67个。同时选择牙周健康者31人作为对照,共85个探诊不出血,牙龈指数(GI)≤1,PD≤3mm,AL≤1mm的位点,同样分为2组,吸烟组45个,非吸烟组40个。观察牙周治疗前、后牙周临床指标菌斑指数(PLI),GI,PD,AL,BOP和龈沟液中EA-s、EA-p水平的变化。结果牙周炎患者中,吸烟组的GI,AL和EA-s水平低于非吸烟组(P<0.05),其余指标差异无显著性(P>0.05),健康者的各项指标差异均无显著性(P>0.05)。无论是吸烟组还是不吸烟组,牙周炎患者的EA-s,EA-p水平均高于健康者(P<0.05)。结论吸烟会降低牙周炎患者龈沟液中EA-s水平,但对EA-p水平影响不大。吸烟对健康人EA水平无显著影响。     

Leptin levels in gingival crevicular fluid in periodontal health and disease

BACKGROUND AND OBJECTIVE: A high concentration of leptin is associated with healthy gingival tissue, and the concentration of leptin decreases as periodontal disease progresses. However, to date, the leptin concentration in gingival crevicular fluid has not been documented. Hence, the present study was carried out to explore the presence of leptin in gingival crevicular fluid in periodontal health and disease, and to probe further into its possible role in periodontal disease progression. MATERIAL AND METHODS: A total of 45 adult patients were selected, based on their body mass index, for the study. They were categorized into three groups of 15 patients each, based on their periodontal tissue status, as follows: group I (clinically healthy gingiva with no loss of attachment); group II (chronic gingivitis with no loss of attachment); and group III (chronic periodontitis). Gingival crevicular fluid samples of 1 microL were collected extracrevicularly using white color-coded 1-5 microL calibrated volumetric microcapillary pipettes from one site in each person, and samples were analyzed for leptin using a commercially available enzyme-linked immunosorbent assay kit. RESULTS: The concentration of leptin in gingival crevicular fluid of patients in group I (2292.69 pg/mL) was statistically higher (p < 0.05) than in those of groups II (1409.95 pg/mL) and III (1071.89 pg/mL). This suggests a negative correlation of gingival crevicular fluid leptin concentration with clinical attachment loss (p < 0.05). CONCLUSION: As periodontal tissue destruction increased, there was a substantial decrease in gingival crevicular fluid leptin concentration. This observation extends our knowledge of the protective role of leptin in periodontal health.     

Presence of cortisol in gingival crevicular fluid A pilot study

Abstract. Cortisol is one of the primary mediators of the stress response, in the main having immunosuppressive effects. An important component of the host response in periodontal inflammation is gingival crevicular fluid (GCF), with constituents mainly derived from serum, Cortisol like many other steroids, is present in saliva but its occurrence in GCF does not seem to be documented, Unstimulated whole saliva was collected and GCF was sampled on filter disks. The samples were analysed by a modified RIA method for serum in such a way that small volumes and low concentrations could be measured. Our findings suggest that the total concentration of cortisol in GCF might be estimated to levels below 1/10 of that in serum. However, what appears as a distinctive feature is the considerable variation of the cortisol concentrations for individual teeth. To our knowledge, this is the first time cortisol has been measured in gingival crevicular fluid, and this opens Ihe prospects for further in vivo research.     

Association of early onset periodontitis microbiota with aspartate aminotransferase activity in gingival crevicular fluid

OBJECTIVES: The objective of this study was to determine the relationship between the activity of the enzyme aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) using the colorimetric PerioGard (PTM) test and the subgingival microflora in early onset periodontitis lesions. MATERIAL AND METHODS: The study population consisted of 25 otherwise healthy individuals exhibiting early onset periodontitis (EOP). In each patient four experimental sites were identified comprising one deep periodontal pocket (PD >5 mm) randomly chosen in each quadrant. Bacterial samples were obtained from the experimental sites, consecutively cultured anaerobically and in 10% CO(2) using selective and nonselective media. Isolates were characterized to species level by conventional biochemical tests and various identification kits. Clinical measurements as well as AST activity, assessed either as positive or negative using the PTM, were recorded at the same sites. RESULTS: Sixty-two sites exhibited AST positive and 38 AST negative activity. Analysis of bacterial counts using the ANOVA (Mann Whitney U-test) showed that Streptococcus intermedius, Peptostreptococcus micros, Campylobacter concisus, Bacteroides forsythus, Camplobacter gracilis, Campylobacter rectus and Selenomonas sputigena were significantly higher in sites with AST-positive activity. The odds ratio of having high prevalence of S. intermedius, P. micros, C. concisus, B. forsythus, C. gracilis, C. rectus and S. sputigena in the presence of a positive AST site was very high (range: 3.5-17.0). Streptococcus sanguis, Actinomyces naeslundii, Gemella morbillorum, Capnocytophaga gingivalis, Veillonella parvula, Fusobacterium varium, Eubacterium lentum and Prevotella oralis were detected in significantly higher proportions in sites with AST negative activity and manifested a negative odds ratio in the presence of AST positive sites. The logistic regression analysis revealed that smoking and bleeding upon probing showed a significant association with AST activity, while plaque and suppuration were not found to be significant predictors of AST activity. The co-infection of Porphyromonas gingivalis, B. forsythus and P. micros, or P. gingivalis, B. forsythus and C. rectus were found to be significantly associated with the AST activity (p<0.001). AST positive sites revealed significantly higher occurrence of co-infections by P. gingivalis, B. forsythus, S. sputigena or by P. gingivalis, B. forsythus, S. intermedius than AST negative sites (p<0.001). P. gingivalis, B. forsythus, A. naeslundii co-infection was found significantly higher in the AST negative sites (p<0.001). CONCLUSIONS: The present study found a high level of agreement between the presence of putative periodontal pathogens and positive AST scores at periodontal sites that clinically were considered to be potentially disease active. Prospective studies should be performed to confirm the findings.     

Chemokine RANTES in gingival crevicular fluid of adult patients with periodontitis

Background, aims: This study presents the first evidence on the presence of the chemokine RANTES in the gingival fluid crevicular (GCF) of patients with periodontitis. RANTES is a chemokine that selectively attracts and activates macrophages and lymphocytes. Leucocytes play a critical rôle in the host response to the subgingival microflora. Method: In this study, the presence de RANTES in GCF was determined in samples obtained from adult patients with periodontitis and from control subjects with clinically healthy gingiva. GCF was collected from different probing depths (<3 mm, 4–6 mm, >6 mm) (n=72); and active (n=12) and inactive sites (n=12). An active site was defined as attachment loss >2 mm, as determined by sequential probing and the tolerance method. GFC was collected for 30 s using Periopaper^® strips, and RANTES was quantified by ELISA. Results: The presence of RANTES was detected exclusively in the group of patients with periodontitis, presenting a total amount of 40.43±16 pg and a concentration 67.80±41 pg/μl. RANTES concentration was significantly higher in probing depth <3 mm than in probing depth >6 mm (87.24 versus 51.87, p=0.014). Total amount and concentration in the GCF samples from active sites were higher that in inactive sites (p>0.05). Conclusions: The finding that RANTES is found only in patients with periodontitis, may represent a general feature of chronic inflammatory in periodontal diseases. Finally, RANTES may be implicated in the biological mechanisms underlying the pathogenesis and progression of periodontal disease.     

Characterization of bone resorbing activity in gingival crevicular fluid from patients with periodontitis

BACKGROUND: In attempts to elucidate factors stimulating bone resorption in patients with different inflammatory diseases in the vicinity of the skeleton, e.g., peridontal disease and rheumatoid arthritis, we are investigating the presence of bone-resorbing activity in a variety of inflammatory exudates. The aim of the present study was to characterize the bone-resorbing activity present in patients with periodontitis. METHODS: Bone-resorbing activity was assessed in gingival crevicular fluids (GCFs) collected from patients with periodontitis and from patients with no signs of gingivitis. Bone-resorbing activity was evaluated by analyzing the capacity of GCFs to stimulate the release of minerals and the breakdown of bone matrix proteins in cultured neonatal mouse calvariae. The concentrations of IL-1alpha, IL-1beta and PGE2 were determined with ELISA and RIA techniques, respectively. RESULTS: GCF eluates from 24 different healthy sites caused a 1.23+/-0.05 fold stimulation of 45Ca release, whereas GCF eluates from 45 different diseased (periodontitis) sites caused a 2.46+/-0.10 fold stimulation. The effect on 45Ca release was time- and concentration-dependent, inhibited by 3 different osteoclast inhibitors and associated with enhanced release of 3H from [3H]-proline-labelled bones. The activity in GCF causing enhanced 45Ca release was unaffected, or in some samples partially reduced, by ultrafiltration using a filter with a molecular weight cut-off of 3000 Daltons. The bone-resorbing activity was temperature sensitive (+90degrees C, 10 min). The concentrations of prostaglandin E2 (PGE2) in the diluted GCF eluates, used in the bone resorption bioassay, were too low to be responsible for the release of 45Ca. Antisera specifically neutralizing human IL-1a inhibited the stimulatory effect of GCF pooled from several diseased sites. The specific, recombinant human IL-1 receptor antagonist completely inhibited the effect of pooled GCFs. GCF eluates from diseased sites contained human IL-1alpha and IL-1beta at concentrations of 1838+/-294 pg/ml and 512+/-91 pg/ml, respectively. CONCLUSIONS: These data show that GCF contains activity(ies) stimulating osteoclastic bone resorption in vitro. The factor primarily responsible for this activity seems to be IL-1alpha, but IL-1alpha is not the sole activator of bone resorption in GCF.     

Periodontitis‐specific molecular signatures in gingival crevicular fluid

Xiang XM, Liu KZ, Man A, Ghiabi E, Cholakis A, Scott DA. Periodontitis‐specific molecular signatures in gingival crevicular fluid. J Periodont Res 2010; 45: 345–352. © 2010 John Wiley & Sons A/S Background and Objective: Periodontitis is currently diagnosed almost entirely on gross clinical manifestations that have been in situ for more than 50 years without significant improvement. The general objective of this study was, therefore, to evaluate whether mid‐infrared spectroscopy can be used to identify disease‐specific molecular alterations to the overall biochemical profile of tissues and body fluids. Material and Methods: A total of 190 gingival crevicular fluid samples were obtained from periodontitis (n = 64), gingivitis (n = 61) and normal sites (n = 65). Corresponding infrared absorption spectra of gingival crevicular fluid samples were acquired and processed, and the relative contributions of key functional groups in the infrared spectra were analysed. The qualitative assessment of clinical relevance of these gingival crevicular fluid spectra was interpreted with the multivariate statistical analysis‐linear discriminant analysis. Results: Using infrared spectroscopy, we have been able to identify four molecular signatures (representing vibrations in amide I, amide II/tyrosine rings and symmetric and asymmetric stretching vibrations of phosphodiester groups in DNA) in the gingival crevicular fluid of subjects with periodontitis or gingivitis and healthy control subjects that clearly demarcate healthy and diseased periodontal tissues. Furthermore, the diagnostic accuracy for distinction between periodontally healthy and periodontitis sites revealed by multivariate classification of gingival crevicular fluid spectra was 98.4% for a training set of samples and 93.1% for a validation set. Conclusion: We have established that mid‐infrared spectroscopy can be used to identify periodontitis‐specific molecular signatures in gingival crevicular fluid and to confirm clinical diagnoses. Future longitudinal studies will assess whether mid‐infrared spectroscopy represents a potential prognostic tool, recognized as key to advancement of periodontics.     

Collagenase activity in gingival crevicular fluid of patients with juvenile periodontitis

The nature and origin of collagenases in gingival crevicular fluid of juvenile periodontitis patients was investigated. Gingival crevicular fluid collected from deep untreated periodontal pockets of juvenile periodontitis patients was found to contain only vertebrate collagenase (EC 3.4.24.7) activity that cleaved soluble type-I and -III collagens into 3/4 and 1/4 length fragments, as analyzed by SDS-polyacrylamide gel electrophoresis. Type II collagen was degraded at a markedly slower rate. This substrate specificity is indicative of collagenases produced by fibroblasts, epithelial cells and macrophages. We have previously found that collagenase in gingival crevicular fluid of adult periodontics patients appears to be mainly derived from polymorphonuclear leukocytes (PMN). The reasons for the apparent difference in collagenase source between the groups were investigated. We examined whether the pathogen characteristic for juvenile periodontitis, Actinobacillus actinomycetemcomitans, can release collagenase from normal human PMNs. All 10 A. actinomycetemcomitans strains tested, freshly isolated from the subgingival plaque of juvenile periodontitis patients, caused release of collagenase from PMNs in vitro. These results suggest that the lack of normally functioning PMNs in the periodontium of juvenile periodontitis patients may result in a colonization of bacteria that activate the resident periodontal cells to produce increased amounts of collagenase.