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Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (SI00A8), a member of the S100 family of calcium-binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases.

In order to examine the relationship of possible crevicular biochemical parameters to attachment loss (ALOSS), 330 sites from 8 untreated adult patients were monitored longitudinally at 3-month intervals, for up to 1 year. Attachment levels were measured with a force-sensing probe and an acrylic stent in duplicates at each study point. Crevicular samples were collected and used for the determination of the following 11 markers: number of polymorphonuclear leukocytes (PMNs), prostaglandin E2 (PGE2), osteocalcin (OC), alkaline phosphatase (ALP), collagenase (COL), beta-glucuronidase (BG), antigenic and functional elastase (AEL and FEL), alpha-1 antitrypsin (a1AT), alpha-2 macroglobulin (a2M) and aspartate aminotransferase (AST). 10 sites with ALOSS of > or = 1.5 mm per 3 months (active sites) and 43 sites with negligible changes (inactive sites) were identified. Total amounts of ALP, BG and COL were found to be significantly higher in active as compared to inactive sites, prior to significant ALOSS, without any significant differences in crevicular fluid volume and clinical indices. When biochemical parameters were expressed as ratios to the number of PMNs, PGE2/ PMNs was significantly elevated in active sites. The capacity of such individual parameters to distinguish between active and inactive sites was limited. However, linear discriminant analysis using total amounts of PGE2, COL, ALP, a2M, OC and AEL showed more significant diagnostic values (sensitivity: 80%, specificity: 91%). These findings suggest that the combination of several biochemical parameters in crevicular fluid could give more information to predict future clinical ALOSS.

Samples of gingival crevicular fluid (GCF) were collected in 30 volunteers with inflamed gingiva, using either capillary tubes (cGCF) or Durapore strips (sGCF). They were examined, together with samples of serum from the same patients, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and/or by two-dimensional electrophoresis, followed by silver staining. The results confirmed that the distribution of the major proteins in GCF is similar to that found in serum. However, an 8.5 kDa protein was found in gingival fluid but not in serum. The low molecular-weight protein appears to decrease with time of fluid sampling with both techniques, and does not originate either from blood or from the cellular fraction of GCF. Two-dimensional electrophoretic analysis suggested that it may consist of several polypeptides.

The peripheral distribution of spiramycin and metronidazole, which are combined in the proprietary drug "Rodogyl", has been studied in gingival fluid, saliva and blood after a single administration to 12 healthy volunteers and after repeated administration to 4 patients with recurring severe periodontitis. Analysis of the 2 antibiotics have been performed at regular intervals during the 24-h period immediately following the administration to the volunteers and after the 1st and the 15th days of repeated administration to the patients. The results show that gingival fluid contains concentrations of spiramycin and metronidazole higher than those needed to inhibit the growth of periodontopathic bacteria. Spiramycin was found at higher concentrations in GCF than in blood, although this feature was not found for metronidazole, which was administered simultaneously and showed similar concentrations in both fluid and serum. Such high concentrations persist for a long time, and suggest the potential of this compound in the treatment of severe cases of periodontitis.

The present investigation was designed to assess the effects of strips made of different materials on the recovery of enzymes in gingival crevicular fluid (GCF) (Experiment 1), and to examine a possible relationship between lysosomal enzyme activities and number of crevicular polymorphonuclear leukocytes (PMNs) (Experiment 2). In Experiment 1, GCF was collected with capillaries from 14 patients with periodontal disease, and applied on various test strips in microcentrifuged tubes or directly into tubes (controls). Strips were then shaken and centrifuged to elute GCF enzymes. The supernate was used for determinations of alkaline phosphatase (ALP), beta-glucuronidase (BG) and aspartate aminotransferase (AST) activities. The % recoveries were calculated as relative percentages to controls. When using saline as eluent, 90% or more ALP, BG and AST were found to be recovered from strips of durapore and papers. More than 35% of ALP and BG was found to remain on paper points. However, the % recoveries from paper points were improved using eluent with 0.1% (w/v) cetyl pyridinium chloride. In Experiment 2, 54 GCF samples were collected from 3 periodontitis patients by using durapore strips, in order to measure both PMN numbers and BG activities in the same samples. The 2 parameters showed strong and positive correlation with 0.847 (p < 0.001) of the Pearson's correlation coefficient. These findings suggest that durapore is a useful material not only for counting PMNs but also for measuring activities of GCF enzymes. Elevation of BG activities in GCF can be due to increasing numbers of PMNs.

Prostaglandin E2 (PGE2) and alkaline phosphatase (ALP) have often been measured in gingival crevicular fluid (GCF) as possible indicators of gingival inflammation and bone metabolism. Osteocalcin (OC), a major component of bone matrix, is mainly produced by osteoblasts, and could also be considered as a marker of bone turnover. The aims of this preliminary study were to examine if OC was present in GCF and to assess the relationships of OC, PGE2 and ALP in GCF to periodontal conditions. GCF samples were collected with durapore strips from 34 healthy, 72 gingivitis and 118 periodontitis sites in 17 subjects. ELISA techniques were used for the determinations of OC and PGE2. ALP was measured spectrophotometrically by using p-nitrophenyl phosphate as substrate. Total amounts and concentrations of PGE2 and ALP were significantly higher in periodontitis as compared to healthy and gingivitis sites, and were significantly and positively correlated with probing depth (PD) and gingival index (GI). OC was present in GCF from both healthy and diseased sites with mean concentrations more than ten times greater than normal serum levels. Total OC amounts from strips soaked with GCF from periodontitis sites were significantly higher than those found in healthy and gingivitis sites. When the data were expressed as concentrations, OC showed significantly positive correlations with GI, but not with PD. However, total amounts of OC significantly correlated with both clinical parameters. OC, PGE2 and ALP were found to have significantly positive correlations with each other, both when expressed as total amounts and concentrations. These data suggest that a significant amount of OC present in GCF is produced locally by periodontal tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Many antibiotics are utilized in dentistry, particularly in periodontology, to treat aggressive forms of periodontitis. In order to be efficient, both on a clinical and on a microbiological point of view, these substances must be present in sufficient concentrations in the gingival sulcus. The aim of the present literature review was to summarize studies concerning the passage of the various families of antibiotics into the gingival fluid, together with their spectrum and clinical efficacy. Whereas certain antibiotics, such as natural or semisynthetic tetracyclines or spiramycin, are able to accumulate in gingival fluid comparatively to serum, others, like metronidazole, tinidazole and rifampicin, have been found in similar concentrations in the fluid as in serum. Some other antibiotics, such as erythromycin and ampicillin, are, on the contrary, less concentrated in crevicular fluid as compared to blood. One has to remember that any systemic antibiotic treatment will be useful, clinically and microbiologically speaking, only when a mechanical therapy is also applied.

The relative concentrations and absolute amounts of neutrophil elastase and its two inhibitors, alpha 2-macroglobulin (alpha 2-M) and alpha 1-antitrypsin (alpha 1-AT), were determined in gingival crevicular fluid (GCF) collected from six dental students who refrained from brushing the upper left or right quadrant during three weeks. Plaque and gingival indices and flow of GCF were measured before, during, and after the three weeks of no brushing. Functional elastase, representing the enzyme complexed with alpha 2-M, was measured by use of a low-molecular-weight fluorogenic substrate. Elastolytic activity in GCF was also assayed by use of elastin as substrate. Antigenic elastase, representing the enzyme complexed with alpha 1-AT, as well as the inhibitors alpha 2-M and alpha 1-AT were measured by ELISA. After three weeks of plaque accumulation, the concentrations of both functional and antigenic elastase increased by a factor of about 3, whereas the concentrations of the inhibitors increased in a much higher proportion. No free elastase could be detected in GCF, as evidenced by the Sephadex G-75 elution profile of GCF, by the negative results obtained when elastin was used as substrate, and by the demonstration that pure enzyme kept its activity against the low-molecular-weight substrate after being saturated by alpha 2-M.

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.

Albumin and alpha 2-macroglobulin were analysed in gingival fluid collected by filter paper strips from 6 volunteers before, during and after 10 days of dental plaque accumulation. The two proteins were also determined in samples of venous blood at the beginning of abstention from tooth brushing and 10 days later. A nephelometric technique was used for the analysis of albumin, but alpha 2-macroglobulin was determined by ELISA. The plaque and gingival indices, as well as the flow of gingival fluid, increased significantly during the period of plaque accumulation and then returned to baseline values. The concentrations of the two proteins in blood remained within normal limits. In gingival fluid, however, the concentration of albumin increased about two-fold at the peak of inflammation, while that of alpha 2-macroglobulin increased as much as 6 times. The enhanced passage of alpha 2-macroglobulin into the gingival sulcus in comparison to the entirely plasma-derived albumin was consistent with the concept that the greater excretion of globulin during inflammation must be due to an increased local synthesis.

Gingival fluid was collected with glass capillaries tubing from the upper premolar area in a group of 7 volunteers, after allowing dental plaque to accumulate for 12-36 h, and in a group of patients with gingivitis. Whereas no fluid could be collected in the absence of plaque, increasing amounts were recovered during plaque accumulation, in the absence of clinical signs of gingival inflammation. The ratios of albumin concentrations in gingival fluid and plasma also increased significantly with increasing time of plaque accumulation. These fluid:plasma ratios of albumin concentrations were significantly lower than the ratios found for the inflamed sites in the second group of patients. These results support the hypothesis that, in an early inflammatory response, the fluid is not a typical inflammatory exudate and is probably modulated by an osmotic gradient.