Arylsulphatase activity in human gingival crevicular fluid

Arylsulphatase activity in fluid collected from non-inflamed (n = 5), gingivitis (n = 5) and periodontitis (n = 5) subjects was assayed. The mean volume activity for each group was: non-in-flamed = 768 ± 165 nm/ml per h; gingivitis = 2431 ± 1118 nm/ml per h; periodontitis = 2860 ± 1839 nm/ml per h. The mean total unit activity for each group was: non-inflamed = 0.326 ± 0.076 nm; gingivitis = 1.394 ± 0.411 nm; and periodontitis = 3.571 ± 1.700 nm. Analysis of fluid from isolated sites in periodontitis suggests that reporting total unit activity (absolute amount of enzyme activity) is more meaningful than reporting volume activity (enzyme concentration) for arylsulphatase activity.     

Protease activity in gingival crevicular fluid

Abstract. Our aim was to study protease activity in GCF from inflamed sites with or without tissue destruction. 19 patients with both periodontitis and gingivitis sites and 12 patients having gingivitis alone participated in the study. GCF samples were collected by an intracrevicular washing method. The protease activity was measured as degradation of FITC-conjugated casein. To obtain a semiquantitative estimate of the harvested GCF volume, we measured the transferrin concentration in the wash-fluid. The protease activity was significantly higher in the deep pockets in periodontitis patients than in shallow pockets in the same patients. This difference was still higher when the ratio of protease activity to the amount of transferrin in the sample was plotted. Although protease activity was lower in samples from gingivitis patients than in the deep pockets in periodontitis patients, the difference was not significant. About 90% of the activity could be inhibited by the addition of an excess amount of α-1-antitrypsin (A1AT). This study shows that protease activity is higher in inflamed sites with tissue destruction than in inflamed sites without. Most of this activity could be inhibited by A1AT, which suggests that the activity is due to an imbalance between protease and antiprotease rather than to proteases insensitive to A1AT.     

In vitro release of elastase from human blood and gingival crevicular neutrophils.

Peripheral PMNs were collected from blood, and crevicular PMNs separated by filtration from gingival washings in 13 patients, aged 22-75 y, who had varying degrees of gingivitis and periodontitis. After pre-incubation with cytochalasin B, the same number of crevicular and peripheral cells were incubated either in PBS (with Ca2+ and Mg2+) (spontaneous release) or in the same buffer containing increasing concentrations of FMLP (stimulated release); elastase activity was measured in the supernatant by a fluorometric technique. There was a higher spontaneous release of enzyme from crevicular than from peripheral neutrophils. The average elastase activity in the supernatant of 1 x 10(4) crevicular cells was more than five times higher than that obtained from the same number of peripheral cells. However, stimulated crevicular PMNs liberated smaller amounts of enzyme than did stimulated peripheral PMNs. These results suggest that crevicular PMNs are already releasing elastase, and are consistent with the possibility that lysosomal enzymes contribute to tissue damage during gingivitis and periodontitis.     

Analysis of human gingival tissue and gingival crevicular fluid beta-glucuronidase activity in specific periodontal diseases

BACKGROUND: Beta-glucuronidase (betaG) is one of the enzymes involved in the destruction of non-collagenous components of the extracellular matrix. It is also considered an indicator or predictor of periodontal disease activity. The present study was conducted to determine the presence and the levels of betaG activity in gingival tissue and gingival crevicular fluid (GCF) in periodontal disease and health status. The validity of 2 expressions of data, total betaG activity versus betaG concentration, and the correlations between clinical periodontal status and betaG profile was also evaluated. METHODS: betaG activities in gingival tissues and GCF samples from 57 individuals, divided into 3 equal groups of adult periodontitis (AP), early-onset periodontitis (EOP), and periodontally healthy subjects were spectrophotometrically examined. RESULTS: Both patient groups had higher betaG levels in both gingiva and GCF than controls. Significant differences were observed among all groups when total GCF betaG activities were examined (P <0.05). However, the difference between AP and controls was not significant when concentration values were compared (P >0.05). The highest GCF betaG activity, with both expressions, was detected in EOP group. No absolute correlations between clinical parameters and betaG activity were observed, except for random correlations in the patient groups with mean total betaG activities. Also GCF/gingiva betaG levels and the 2 expressions did not show absolute correlations. CONCLUSIONS: The findings of the present study confirm the relationship between betaG activity and periodontal diseases. The differences in data concerning GCF total betaG activity and betaG concentration may suggest that they are not matching measures. Data presentation seems to be an important factor in GCF/enzyme profile studies.     

Acid-base properties of human gingival crevicular fluid

The pH of human gingival crevicular fluid (GCF) has been reported by many authors to be very alkaline (pH 7.5 - 8.7). This alkalinity could be explained, at least partially, by the fact that all measurements were performed either at low PCO2 or in the absence of CO2. Therefore, we set up a procedure which allows for measurement of the pH of GCF samples from single inflamed sites at controlled PCO2. At a PCO2 of 4.7 kPa (= 35 mmHg) and at 37 degrees C, the pH was 7.96 +/- 0.10 (SEM, n = 9), a value which differs significantly from the value of 8.38 +/- 0.09 measured in the absence of CO2 in the same samples. The non-bicarbonate buffer value of the sample determined by CO2 titration was 6.0 slykes. It is because this value is low that pH varies so greatly with PCO2. At physiological PCO2, the total buffering power becomes very high above pH 8.0, because of the high bicarbonate concentration.     

Polyamine analysis of human gingival crevicular fluid

Crevicular fluid from inflamed periodontitis sites was analyzed for polyamines by high performance liquid chromatography. Putrescine and cadaverine concentrations were positively correlated with site probing depth (p<0.01). Spermidine concentrations were poorly correlated with probing depth (p<0.30), but were significantly higher at moderately inflamed sites (as assessed by the Gingival Index) than at mildly inflamed sites (p<0.02).     

Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils

Hiroshima Y, Bando M, Inagaki Y, Mihara C, Kataoka M, Murata H, Shinohara Y, Nagata T, Kido J. Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils. J Periodont Res 2012; 47: 554–562. © 2012 John Wiley & Sons A/S Background and Objective: Resistin is an adipocytokine that induces insulin resistance and is predominantly expressed in adipocytes and peripheral blood mononuclear cells. Resistin expression increases in inflammatory diseases as well as diabetes mellitus, and is upregulated by bacterial pathogens and proinflammatory cytokines. The aim of this study was to identify resistin in human gingival crevicular fluid, to compare the resistin levels in gingival crevicular fluid between subjects with and without periodontitis and diabetes mellitus and to investigate the regulation of resistin release from human neutrophils by Porphyromonas gingivalis lipopolysaccharide (P‐LPS). Material and Methods: Gingival crevicular fluid samples were collected from patients with chronic periodontitis (n = 24), patients with diabetes mellitus‐related periodontitis (n = 18) and healthy subjects (n = 21). Resistin in gingival crevicular fluid was determined using western blot analysis and an ELISA kit. The glycated hemoglobin (HbA_1c) value was obtained from patients with diabetes mellitus‐related periodontitis by a medical interview. Human neutrophils were cultured with P‐LPS (0–1000 ng/mL), or incubated with inhibitors of actin or microtubule polymerization in the absence or presence of P‐LPS. The medium and cellular fractions were used for determination of resistin by ELISA. Results: The resistin level in gingival crevicular fluid from patients with periodontitis or diabetes mellitus‐related periodontitis was significantly higher than that of healthy subjects. The resistin level in gingival crevicular fluid was correlated with gingival index score, but not blood HbA_1c value. The P‐LPS increased resistin release from human neutrophils, and its induction was decreased by actin polymerization inhibitors. Conclusion: We show, for the first time, the presence of resistin in gingival crevicular fluid. A high resistin level in gingival crevicular fluid samples from periodontitis patients may to some extent be related to P‐LPS‐induced resistin release from neutrophils.     

Alkaline phosphatase activity in gingival crevicular fluid during human orthodontic tooth movement.

Bone remodeling that occurs during orthodontic tooth movement is a biologic process involving an acute inflammatory response in periodontal tissues. A sequence characterized by periods of activation, resorption, reversal, and formation has been recently described as occurring in both tension and compression tooth sites during orthodontic tooth movement. We used a longitudinal design to investigate alkaline phosphatase (ALP) activity in gingival crevicular fluid (GCF) to assess whether it can serve as a diagnostic aid in orthodontics. Sixteen patients (mean age, 15.5 years) participated in the study. The maxillary first molars under treatment served as the test teeth (TT) in each patient; in particular, 1 first molar was to be retracted and hence was considered the distalized molar (DM), whereas the contralateral molar (CM) was included in the fixed orthodontic appliance but was not subjected to the distal forces. The DM antagonist first molar (AM), free from any orthodontic appliance, was used as the baseline control. The GCF around the experimental teeth was harvested from mesial and distal tooth sites immediately before appliance activation, 1 hour after, and weekly over the following 4 weeks. The clinical gingival condition was evaluated at the baseline and at the end of the experimental term. ALP activity was determined spectrophotometrically at 30 degrees C, and the results were expressed as total ALP activity (mUnits/sample). GCF ALP activity was significantly elevated in the DMs and the CMs as compared with the AMs at 1, 2, 3, and 4 weeks; conversely, in the AMs, GCF ALP activity remained at baseline levels throughout the experiment. Moreover, the enzyme activity in the DMs was significantly greater than in the CMs. In the DMs, a significantly greater ALP activity was observed in sites of tension compared with sites of compression. This difference was not seen with the CMs, in which the enzyme activity increased to the same extent in tension and compression sites. These results suggest that ALP activity in GCF reflects the biologic activity in the periodontium during orthodontic movement and therefore should be further investigated as a diagnostic tool for monitoring orthodontic tooth movement in clinical practice.     

Enzyme activity in human gingival crevicular fluid: considerations in data reporting based on analysis of individual crevicular sites

Using a reproducible approach to collection, processing and analysis of gingival crevicular fluid (GCF), this study examined 284 fluid samples from individual crevicular sites for the presence of the enzymes lactate dehydrogenase (LDH), B-glucuronidase (BG) and arylsulfatase (AS). 88 of the sites were from periodontally healthy individuals (probing depth 1-3 mm), while 98 sites from patients with periodontitis were examined before and 2 weeks after scaling and root planing (probing depths 1-3 mm, 4-6 mm and 7-10 mm). This study demonstrated the sensitivity of the enzyme assays. When GCF was collected with a 30-s insertion of the filter strip, 90% of the sites from the control subjects demonstrated LDH activity, 85% demonstrated BG activity and 73% demonstrated AS activity. For the 1-3 mm sites from the patients with periodontitis, 100% of sites from which fluid was collected demonstrated LDH and BG activity, and 90% of sites had AS activity before therapy. After therapy, 100% of sites demonstrated LDH activity, 90% had BG activity and 83% had AS activity. All sites in the 4-6 mm and 7-10 mm categories demonstrated activity of all 3 enzymes. The data were analyzed in terms of enzyme activity/30-s sample and as concentration of enzyme in a standard volume of GCF. Enzyme activity/30-s sample was a different and possibly more sensitive indicator of periodontal pathology than standard clinical parameters. There was a disassociation between clinical parameters and the data for enzyme analysis when it was reported as concentration.     

The concentration of proteins in human gingival crevicular fluid

The amount and concentration of protein present in fluid obtained from individual healthy and inflamed gingival crevices have been examined. It was found that an artefactual hyperbolic relationship existed between protein concentration and fluid volume in fluid originating from healthy crevices, resulting in over-estimation of protein content. In fluid originating from inflamed crevices, no statistically significant correlation existed between protein concentration and fluid volume, which is to be expected for an inflammatory exudate. The fluid dynamics of the crevicular region is therefore probably similar to that of the rest of the body.     

Radioimmunoassay quantification of adrenomedullin in human gingival crevicular fluid

OBJECTIVE: To investigate whether adrenomedullin (ADM), a multifunctional peptide with key roles in host antimicrobial defence and inflammation, was present and quantifiable in human gingival crevicular fluid (GCF) and to study its relationship with periodontal health and disease. DESIGN: GCF samples (30s) were collected using perio-paper strips from one diseased site in 21 subjects with periodontal disease and one healthy site from 19 control subjects with no evidence of periodontal disease. Samples were analysed by radioimmunoassay using a specific anti-human ADM antibody. RESULTS: Measurable adrenomedullin-like immunoreactivity (ADM-LI) was present in all the GCF samples collected. ADM-LI was significantly higher in periodontitis sites (mean 493.6 pg) than in control healthy sites (mean 248.5 pg), p = 0.0016. CONCLUSION: It is concluded that ADM is present in GCF at levels at which it could have an antibacterial role in the gingival crevice and modulate the pathophysiology of periodontal inflammation.     

Glycosaminoglycans in human gingival crevicular fluid during orthodontic movement

Glycosaminoglycans (GAG) in gingival crevicular fluid (GCF) were investigated by cellulose acetate electrophoresis of simultaneously collected samples from the mesial and distal surfaces of teeth in 3 groups of young persons. In a control group, which had not undergone orthodontic treatment, a major band of hyaluronic acid (HA) and a minor band of chondroitin sulphate (CS) were present. No differences in the mean content of either GAG between the mesial and distal surfaces were detected. From teeth undergoing movement by fixed appliances (active group), a raised mean level of CS was present in GCF from the surface towards which movement was directed. Teeth held passively by an appliance following cessation of active movement (retention group) showed raised levels of CS at mesial and distal surfaces. A heparan sulphate-like GAG was commonly present in this group only. No significant increase in the levels of HA were detected at the mesial and distal surfaces of either the active or the retention groups, despite increased GCF flow rates unassociated with more severe gingival inflammation. The GAG composition of GCF, particularly CS, appears to reflect changes occurring in the deeper periodontal tissues of alveolar bone and periodontal ligament during orthodontic treatment.     

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.     

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.     

Osteopontin in gingival crevicular fluid

Osteopontin (OPN) is a major glycosylated phosphoprotein in bone matrix and is produced by several cells including osteoblasts, osteoclasts and macrophages. OPN levels increase in active sites of bone metabolism. Recently, several bone-related proteins were identified in gingival crevicular fluid (GCF) to seek markers of alveolar bone resorption in periodontal disease. In this study, we investigated the existence of OPN in GCF and the correlation between OPN level in GCF and probing depth (PD) of sampling sites in 98 periodontitis patients and 35 healthy subjects. An immunoblotting analysis using 10% polyacrylamide gel showed that two forms of OPN with molecular masses of 54 and 66 kDa and several degraded fragments were detected in most GCF samples from diseased sites (PD > 4 mm). In GCF samples from healthy sites (PD < or = 3 mm), only one form (54 kDa) was observed, but any degraded fragments were not detected. When OPN amounts in GCF samples were determined by ELISA, a weak. but significant correlation was observed between OPN amount in GCF and PD (r=0.32, p=0.0013). These results demonstrate that OPN exists in GCF and that OPN level in GCF increases with the progression of periodontal disease.     

Peptides of human gingival crevicular fluid determined by HPLC-ESI-MS

The acidic-soluble protein content of human gingival crevicular fluid was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), and the eluent deriving from the chromatography separation was directly introduced into an ion-trap mass spectrometer through electrospray ionization (ESI-IT MS). By this technique the molecular weight of peptides/proteins was determined with a precision of approximately 1/10,000 amu. On the basis of the chromatographic behavior and the knowledge of the molecular mass value, some peptides and proteins soluble in acidic solution were unambiguously recognized. Besides high quantities of human serum albumin, alpha-defensins 1-4 and minor amounts of cystatin A, statherin, basic PB salivary peptide and other unidentified components were detected. The presence of alpha-defensins in gingival crevicular fluid is in agreement with their relevant contribution to protein composition deriving from granulocyte secretions. Other peptides and proteins abundant in human saliva, such as proline-rich proteins (PRPs) and histatins, were not detected in gingival crevicular fluid. Further investigations will be necessary to establish the origin of statherin and PB salivary peptide in gingival crevicular fluid.     

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.