Phospholipase A2 Activity in Gingival Crevicular Fluid from Patients with Periodontal Disease: A possible Marker of Disease Activity

The activity of phospholipase A2 in human gingival crevicular fluid (GCF) associated with periodontal disease was demonstrated. Based upon the presence or absence of bleeding on probing (BOP), which is a marker for the disease activity, there were higher levels of the enzyme activity in BOP positive, than in negative sites. When the BOP positive sites became negative after periodontal therapy, the enzyme activity decreased dramatically to almost undetectable levels. There were no significant differences between the activity before and after treatment when the BOP positive sites remained unchanged. These results suggest that the activity in GCF reflects periodontal disease conditions and that it can be used as a marker for disease activity.


Introduction
Phospholipase A 2 (PLA2) is a key enzyme in the production of potent inflammatory mediators, namely prostaglandins, leukotrienes and platelet activating factor. In fact, high levels of PLA2 activity have been found in either the sites and/or sera of patients with various inflammatory diseases such as rheumatoid arthritis, 2'3 acute pancreatitis, 4,s inflammatory bowel disease, 6 and septic shock. 7 Furthermore, PLA2 activity in human synovial cells, rat astrocytes, 9 and rabbit articular chondrocytes 1 was markedly induced in response to inflammatory cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF). The serum PLA2 activity in some of those conditions correlates with disease activity. 6'11'12 Collectively, these results suggest that PLA2 plays an important role in the process of inflammatory disease and that the determination of the activity can be a useful tool for diagnosis. Periodontitis CaC12. The samples were stored at -20C until the assay for PLA2 activity.
Gingival tissues, blood samples and saliva: Inflammatory gingival tissues were taken from the seven sites of seven patients immediately after collection of GCF upon pocket curettage (a periodontal therapy). The tissues were rinsed thoroughly with ice-cold isotonic solutions, weighed, then minced and homogenized in 50 mM Tris-HC1 (pH 7.4) using a glass homogenizer immersed in ice-cold water, followed by centrifugation at 1 500 x g for 30 min.
The supernatant was used as the enzyme source (the supernatant). Blood samples were collected after GCF sampling, at six sites where BOP was observed and saliva was collected from the mouths of six patients with a micro capillary of the same size as that described above.
Enwme assay: The assay for PLA2 activity was performed using the modified methods of Nishijima et al. 23    with a mean_-FS.E. (n=108) of 5.6_+0.2mm, respectively. No relationship was found between the enzyme activity and either the volume or the probing depth.
Although clinical parameters such as probing depth and the GCF volume have some limitations, bleeding upon probing (BOP) is a fairly accurate clinical sign of active disease. 21 Thus the sites were assigned to two groups by using the marker, BOP. Table 1 shows that 67 and 41 sites were in the BOP positive and negative groups respectively. The enzyme level in the BOP positive group was about 2.5-fold higher than that in the negative group.
It is possible that the high levels of the enzyme activity detected in GCF partially resulted from blood and/or saliva that might have entered the GCF at the sampling sites. PLAz levels in both saliva and sampled blood were determined as shown in Fig. 3. The mean -+-S.E. of PLA2 activity in GCF was 110.6 41.2 pmol/h/l (n 18), whereas that in blood was 3.6 _-+ 3.6 pmol/h/#l. No PLA2 activity was detected in saliva. These results suggested that the enzyme activity detected in GCF was only from gingival tissue around the crevice. To confirm this further, the PLA2 activity in gingival tissue taken on periodontal therapy (curettage) from the seven patients was measured and compared with that in GCF of the same sites. As shown in Fig. 4, there was a highly significant correlation (r-0.93) between PLA2 activity in GCF and in the gingival tissue. These data indicated that the PLA2 activity detected in GCF was mainly from the periodontal tissue around the sites. The PLA2 activity in GCF was determined before and after the periodontal treatment at 19 sites. The levels of PLA2 activity were significantly decreased after treatment, suggesting a relationship between the enzyme activity and periodontal disease (Fig. 5).
We therefore examined more precisely whether the PLAz activity in GCF reflects periodontal conditions (active phase or inactive phase). Thus, among the 19 sites, 14 where BOP was positive were selected and the activity was measured before and  However, in five sites where BOP remained positive despite treatment, there were no significant changes in the enzyme activity.

Discussion
The PLA2 activity in GCF from patients with periodontal disease was determined. About half the GCF samples from the sites examined had detectable PLA2 activity, whereas the other half had none under the present assay conditions (Fig. 1). These data suggest that there is no relationship between the enzyme activity and the disease.
However, all 108 sites were divided into two groups by means of a marker for the active stage of periodontal inflammation, BOP, then the activity between the two groups was compared. The level of activity in the positive group was about 2.5 times higher than that in the negative group (Table 1).
Furthermore, when 19 sites were selected from among those where PLA2 activity was detected, and the activity in GCF was measured both before and after periodontal therapy, there was a significant decrease in the enzyme activity of GCF after treatment (Fig. 5). Though the total number of sites examined (five and nine) were not necessarily enough to allow interpretation of the results from Table 2, it may be of more interest that when BOP positive sites became negative after periodontal therapy, the activity markedly decreased to about 0.8% of that before therapy. In contrast, when the BOP positive sites remained unchanged even after treatment, the activity decreased to about 10%, but it was not statistically different from that before therapy (Table 2). Since periodontal inflammation typically has a relatively short active phase where tissue destruction occurs, then reaches a prolonged resting stage where apparent changes (destruction) are not observed, 2s the results in this study indicate that the activity reflects the periodontal disease stage and that it can be a marker for the active phase of this disease.
A trace of activity was found in peripheral blood that flowed from periodontal tissue into the gingival crevice (periodontal pocket) upon periodontal examination (probing) and no activity in saliva from the oral cavity was found (Fig. 3). There was a close relationship between the activity in both GCF and the periodontal tissue taken from the same sites (Fig. 4). These results indicated that the activity detected in GCF was from that produced in the tissues then secreted into the crevice.
In inflammatory disorders, such as rheumatoid arthritis, 11 acute pancreatitis 12 and Crohn's disease, 6 the activity of extracellular PLA2 in the serum is correlated with disease activity and the measurement of PLA2 activity has been proved to be a useful means of diagnosis. High levels of IL-1/ 26 and/or TNF 7 in synovial fluid from patients with arthritis and in GCF from patients with periodontal disease have been reported. 14'28 Since our previous studies demonstrated that these cytokines induce PLA2 activity in rat gingival fibroblasts, PLA2 activity detected in GCF may result from similar events in periodontal tissue.
To the best of our knowledge, this study is the first to demonstrate the presence of PEA2 activity in GCF from patients with periodontal disease. The results suggest that measuring PLA2 activity in GCF will be a quantifiable marker of periodontal disease activity.