Quantitative analysis of MRP-8 in gingival crevicular fluid in periodontalhealth and disease using microbore HPLC

Background: The protein components of GCF can be separated by reverse-phase microbore HPLC on a C18 column with detection on the basis of 214 run abso rbance. A single major symmetrical protein peak eluting with a retention ti me of 26 min (50% acetonitrile) was evident in gingival crevicular fluid (G CF) from periodontitis patients but not in healthy GCF This protein was ide ntified as human MRP-8 by N-terminal amino acid sequencing and liquid chrom otography quadropole mass spectrometry. Aims: To quantify the amount of MRP-8 detectable in GCF from individual hea lthy, gingivitis and periodontitis affected sites and to study the relation ship, if any, between the levels of this responsive protein and periodontal health and disease. Methods. GCF was sampled (30 s) from healthy, gingivitis, and periodontitis sites in peridontitis subjects (n=15) and from controls (n=5) with clinica lly healthy gingiva and no periodontitis. Purified MRP-8 was sequenced by E dmann degradation and the phenylthiohydantoin (PTH) amino acid yield determ ined (by comparison of peak area with external PTH amino acid standards). T his value was subsequently used to calculate the relative amount of protein in the peak eluting with a retention time of 26.0 min (MRP-8) in individua l GCF chromatograms. Results: Higher levels of MRP-8 were detected in inflammatory sites: period ontitis 457.0 (281.0) ng; gingivitis 413.5 (394.5) ng compared with periodo ntally healthy sites in diseased subjects 14.6 (14.3) ng and in controls 18 .6 (18.5) ng, p = 0.003. There was at least 20-fold more MRP-8 in the infla mmatory compared with the healthy sites studied. Conclusions: The preliminary data indicate that MRP-8 is present in GCF, wi th significantly greater amounts present at diseased than healthy sites. A systematic study of the relationship of this protein to periodontal disease could prove useful in further clarifying whether MRP-8 could be a reliable GCF biomarker of gingivitis and periodontitis.