The relationship between gingival crevicular fluid cathepsin B activity and periodontal attachment loss in chronic periodontitis patients: a 2-year longitudinal study

This study aims to determine whether gingival crevicular fluid (GCF) cathepsin B levels, total activity (TA) and concentration (EC) predict progressive attachment loss (AL). Seventy-five previously untreated patients with moderate periodontitis were recruited. GCF was collected from 16 molar and premolar mesiobuccal sites and probing attachment level (PAL) and probing depth (PPD) were measured with an electronic probe. Gingival, gingival bleeding and plaque indices were then scored. Prior to baseline patients were given basic periodontal treatment after which the above procedures were repeated. Carefully localized radiographs were taken of the test teeth and repeated annually. Patients were seen 3-monthly for 2 yr and the procedures were repeated. One hundred and twenty-one AL sites, 90 rapid AL (RAL) and 31 gradual AL (GAL), in 49 patients were detected. Cathepsin B levels (TA & EC) at RAL sites were significantly higher (p<0.0001) than paired control sites at the attachment loss time (ALT) and prediction time (PT). Mean levels (TA & EC) over the study period at GAL sites were significantly higher (p<0.0001) than paired control sites. Using a critical value (CV) of 7.5 μU/30 s (TA) and 30 μU /μL (EC) showed a sensitivity of 100% and specificity of 99.83% (TA) and 100% and 99.75%(EC) at both ALT & PT. Mean cathepsin B levels (TA & EC) were significantly higher (p<0.0001) at RAL and GAL sites than nonattachment loss (NAL) sites in AL patients in intrapatient comparisons and mean patient levels were significantly higher (p<0.0001) in AL patients than NAL patients in interpatient comparisons. These results indicate that GCF cathepsin B may serve as a predictor of attachment loss.     

龈沟液弹性蛋白酶洗提和保存方法的初步研究

目的 :观察不同保存温度、时间和不同缓冲液以及不同洗提方法对龈沟液弹性蛋白酶 (GCF-EA)活性的影响。方法 :采用底物反应法分别于取样即刻和 1、2、3、4周时检测 - 2 0℃和 - 70℃保存的PBS组和Tris-HCl组以震荡法和震荡 +离心法洗提的GCF样本中EA的活性。结果 :不同洗提方法间洗提效果的差异具有显著性 (0 .0 1

0 .5 )。在相同时间内 ,PBS组或Tris -HCl组 - 2 0℃保存的GCF样本EA活性与- 70℃者间的差异无显著性 (P >0 .0 5 ) ,但 - 2 0℃保存者有略高于 - 70℃者的倾向 ,且 - 2 0℃比 - 70℃条件下保持EA活性的时间略长。结论 :将滤纸条上的GCF样本进行洗提时 ,采用“震荡 +离心法”优于“震荡法”。GCF样本的保存过程中 ,缓冲液的种类以及保存温度可影响GCF -EA的活性。以PBS作为缓冲液、- 2 0℃保存可能更利于其中EA活性的稳定。     

Correlation between gingival crevicular fluid dipeptidyl peptidase II and IV activity and periodontal attachment loss. A 2–year longitudinal study in chronic periodontitis patients

OBJECTIVE: The aim of this study is to determine whether gingival crevicular fluid (GCF) dipeptidyl peptidase (DPP) II or IV levels, total activity (TA) and concentration (EC), predict progressive attachment loss (AL). SUBJECTS AND METHODS: Seventy five patients with moderate periodontitis were recruited. GCF was first collected from 16 molar and premolar mesiobuccal sites and then probing attachment level (PAL) and probing pocket depth (PPD) were measured with an electronic probe. Finally, gingival index, gingival bleeding and plaque indices were scored. Patients were given basic periodontal treatment prior to baseline after which the above procedures were repeated. Patients were seen 3 monthly for 2 years and the procedures were repeated. Carefully localised radiographs were taken of the test teeth annually. RESULTS: One hundred and twenty AL sites, 88 rapid AL (RAL) and 32 gradual AL (GrAL), in 48 patients were detected. DPP It and IV levels (TA and EC) at RAL sites were significantly higher (P ≤ 0.0001) than paired control sites at the attachment loss time (ALT) and prediction time (PT). Mean levels over the study period of both proteases (TA and EC) at GrAL sites were significantly higher (P≤ 0.0001) than paired control sites. The GCF levels of DPP IV were always slightly higher than those of DPP II. Critical values (CV) of 5 μU per 30 s (TA) and 25 μU μPI^-1 (EC) for both proteases showed high sensitivity and specificity values for TA and EC and these were the same at both ALT and PT. The positive predictive values were slightly higher for DPP II. Mean site DPP II and IV levels (TA and EC) in intra-patient comparisons were significantly higher (P ≤ 0.0001) at RAL and GrAL sites than non-attachment loss (NAL) sites in AL patients and mean patient levels were significantly higher (P ≤ 0.0001) in AL patients than NAL patients in inter-patient comparisons. CONCLUSIONS: These results indicate that both GCF DPP II and IV may be predictors of periodontal attachment loss.     

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.     

吸烟对牙龈炎治疗前后龈沟液中弹性蛋白酶水平的影响

目的:研究吸烟和不吸烟牙龈炎患者治疗前后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:慢性龈缘炎患者37例,共119个探诊出血位点,GI≥2,PD≤3 mm,AL≤1 mm的牙龈炎位点,吸烟组17例59个位点,非吸烟组20例60个位点.观察牙周治疗前后PLI,GI,BOP和龈沟液中EA-s,EA-p水平的变化.结果:治疗前,吸烟组GI低于非吸烟组(P＜0.05),两组间EA-s,EA-p水平无显著性差异(p＞0.05).治疗后,两组各临床指标和EA-s水平均有不同程度下降.两组间各临床指标和EA-s,EA-p水平无显著性差异(p＞0.05).结论:吸烟对牙龈炎患者治疗前后龈沟液中EA-s,EA-p水平无显著影响.     

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.     

Influence of oral hygiene on elastase concentration of gingival crevicular fluid

PMN elastase concentration of gingival crevicular fluid (GCF) was investigated in 11 healthy volunteers before professional tooth cleaning and after a 5-week period of intensive oral hygiene. GCF was collected using filter paper strips and the enzyme concentration was evaluated by the ELISA-technique. Intensive daily oral hygiene led to a considerable improvement in the clinical indices and to a reduction in the concentration of elastase in GCF. Despite the changes in the oral hygiene status, functional elastase was still present in the samples at the end of the experiment. This could mean that even at clinically healthy sites there is a lack of alpha-1-proteinase inhibitor, the major serum protein inactivating PMN elastase.     

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。     

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.     

Calprotectin in gingival crevicular fluid correlates with clinical and biochemical markers of periodontal disease

Clinical and biochemical markers of periodontal disease have been used for precise objective diagnosis of periodontal inflammation. Interleukin 1beta (IL-1beta) and prostaglandin E2 (PGE2), inflammatory factors, levels in gingival crevicular fluid (GCF) of patients with periodontal disease are elevated and have been studied as biochemical markers. The levels of calprotectin, a leukocyte protein, in body fluids of patients with some inflammatory diseases are raised. Recently, we detected calprotectin in GCF and its concentrations in periodontal pockets were higher than those in healthy gingival crevices. In this study, we investigated the correlations between GCF calprotectin levels and clinical indicators (probing depth and bleeding on probing, BOP), and the IL-1beta or PGE2 levels in GCE Probing depth and BOP at 130 sites of 110 subjects with periodontal or other oral diseases were examined, then GCF samples were collected and their calprotectin, IL-1beta and PGE2 were determined by ELISA. The calprotectin level correlated positively with the probing depth and was significantly higher at BOP-positive than BOP-negative sites. There were significant, positive correlations between the calprotectin and IL-1beta or PGE2 concentrations. These results indicate that the calprotectin level in GCF correlates well with clinical and biochemical markers of periodontal disease and suggest that calprotectin may be useful for evaluating the extent of periodontal inflammation.     

吸烟对牙龈炎患者和健康人龈沟液中弹性蛋白酶水平的影响

目的比较牙龈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。方法选取慢性牙龈炎患者37人,共119个探诊出血、牙龈指数(gingival index,GI)≥2、牙周袋探诊深度(probing depth,PD)≤3mm、附着丧失(attachment loss,AL)≤1mm的牙龈炎位点,将其分为吸烟组59个,非吸烟组60个。同时选取牙周健康者31人作为对照,共85个探诊不出血、GI≤1、PD≤3mm、AL≤1mm的位点,同样分为两组,吸烟组45个,非吸烟组40个。观察牙周临床指标菌斑指数(plaque index,PLI)、GI、探诊出血指数(bleeding on probing,BOP)和龈沟液中EA-S、EA-p水平的变化。结果牙龈炎患者中,吸烟组的PLI明显高于非吸烟组(P<0.05),GI低于非吸烟组(P<0.05),健康者的各临床指标均无显著差异(P>0.05)。无论是牙龈炎患者还是健康者,吸烟组与非吸烟组间EA-s,EA-p水平均无显著差异(P>0.05)。结论吸烟对牙龈炎患者和健康人龈沟液EA水平无显著影响。     

龈沟液中与牙周病有关的细胞因子的研究进展

牙周病时龈沟液内含有多种可作为诊断指标的细胞因子,由于取样简单无创,且能重复取样,易为患者所接受。因此,近年来学者们对龈沟液内的细胞因子在牙周炎活动期的诊断﹑治疗及疗效评价中作用的研究很多。本文对龈沟液中与牙周病有关的细胞因子的研究进展作一综述。     

Elastase in gingival crevicular fluid from smokers and non-smokers with chronic inflammatory periodontal disease

OBJECTIVE: To compare elastase concentrations in gingival crevicular fluid (GCF) from individual sites of smokers and non-smokers.
MATERIALS AND METHODS: Twelve pairs of smokers and non-smokers with untreated, moderate to advanced chronic inflammatory periodontal disease were matched for gender, age, ethnicity and the clinical and radio-graphic extent of disease. Durapore filter strip samples were collected over 30 s from two mesiopalatal sites on upper left posterior teeth. Samples were analysed for: I) polymorphonuclear neutrophil leucocyte (PMNL) cell counts; 2) PMNL elastase-αI-antitrypsin complex in the GCF supernatant by ELISA; and 3) functional elastase, free or bound to α2-macroglobulin, estimated from activity against N-tert-butoxycarbonyI-alanyl-prolyl-nor-valylg-chlorothiobenzyl ester in supernatant and lysates of GCF PMNLs.
RESULTS: There were no differences in disease parameters between groups except that bleeding on probing was less extensive in smokers (P< 0.001). Cell counts and elastase content of crevicular PMNLs showed no differences between groups. Lower concentrations of elastase were found in GCF supernatants from smokers than non-smokers. This difference was observed for functional elastase (mean [s.d.] = 30.21 [17.60] against 73.77 [75.26] ng μI^-1, P <0.05) and elastase complexed with αl-antitrypsin (8.97 [6.54] ng μl^-1 against 25.71 [22.07] ng μI^-1, P < 0.001).
CONCLUSIONS: Smokers have lower elastase concentrations in GCF than non-smokers. Further investigation is required to elucidate the underlying cause and its relationship with periodontal disease.     

Changes in gingival crevicular fluid matrix metalloproteinase-8 levels during periodontal treatment and maintenance

OBJECTIVES: The aim of this study was to investigate the effect of scaling and root planing (SRP) and the maintenance phase of treatment on the gingival crevicular fluid (GCF) matrix metalloproteinase-8 (MMP-8) levels. MATERIALS AND METHODS: Clinical measurements and GCF samples were taken from four sites in 20 adult periodontitis patients before and after SRP and during a 3-month maintenance phase of treatment. MMP-8 levels were measured from GCF samples by time-resolved immunofluorometric assay (IFMA) with monoclonal antibodies. RESULTS: SRP improved the clinical indices as would be predicted, 6.1 mm (SD = 1.4) at baseline compared with 4.3 mm (SD = 1.6) post-treatment (P < 0.001). Attachment level (AL) reduced but not significantly between these two visits 13.4 mm (SD = 2.4) compared with 12.8 mm (SD = 2.4) (P < 0.08) post therapy. GCF MMP-8 levels reduced after initial treatment from 33.8 micro g/30 s sample to 23.5 micro g/30 s, which just failed to reach statistical significance (P = 0.07). However, when MMP-8 levels were expressed as a concentration, the differences following initial therapy were significant (54.1 ng/ micro L at baseline compared with 34.2 ng/micro L post treatment; P < 0.005). The difference, however, between the baseline MMP-8 levels (33.8 ng/30 s) and the final visit (16 ng/30 s) following maintenance was markedly significant (P < 0.001) for both absolute amounts and on a concentration basis. CONCLUSION: In conclusion, clinical improvement following SRP was associated with significant reductions in MMP-8 levels. The GCF concentration of MMP-8 decreased after initial therapy but reduced even more dramatically (approximately 50%) following a 3-month period of maintenance (P < 0.001).     

Altered relation between granulocyte elastase and α-2-macroglobulin in gingival crevicular fluid from sites with periodontal destruction

Abstract Granulocyte elastase activity and α-2-macroglobulin (α-2-MG) were studied in the gingival crevicular fluid (GCF) from 3 categories of sites in 6 patients with gingivitis and 6 patients with periodontitis. 6 inflamed sites in each gingivitis patient were sampled on paper strips and 12 sites, 6 with and 6 without attachment loss and periodontal pockets, were selected in each periodontitis patient. To avoid the influence of increase GCF volume from deep pockets, the elastase activity and the α-2-MG were calculated per μl of GCF. The proteolytic activity of elastase was measured with a low molecular weight substrate and the antiprotease, α-2-MG, with ELISA. The measured activity could be ascribed to elastase that had been released into the gingival tissues and into the GCF prior to sampling. In the periodontitis patients, the sites with tissue destruction had a significantly higher elastase activity per site and per μl GCF and a significantly lower α-2-MG per μl than the 2 other categories of sites without tissue destruction. The destructive inflammation seems to be associated with increased release of elastase, either from more numerous or from more active granulocytes and with an increased proteolytic consumption of the inhibitor accompanied by the fast elimination of the protease-inhibitor-complex. In conclusion, the study shows a strong relationship between elastase activity and tissue destruction, a finding that supports the pathogenic theory of an involvement of granulocytes and their proteolytic enzymes in the mechanism of periodontal destruction.     

Collagen I carboxyterminal propeptide in human gingival crevicular fluid before and after periodontal treatment

The amount of procollagen I carboxyterminal propeptide (PICP) in crevicular fluid (CF) was measured in three periodontitis patients. Samples were collected from 29 sites before treatment (scaling, root planing, and curettage) and 2, 5, 10, 20, and 40 days after treatment, by placing two paper strips in periodontal pockets for 5 s. The amount of fluid in strips was measured by the Periotron device. Control samples were collected from subjects with minimal gingival inflammation. PICP was extracted into saline solution and determined by a radioimmunologic method. Plaque index, papilla bleeding index, and pocket depth were recorded before and 40 days after treatment. The CF PICP mean concentration was 4.2 mg/1 in the pretreatment samples. Five days after treatment a statistically significant increase in PICP concentration was seen in all subjects. The peak appeared on days 5 or 10 in 27 sites. The mean peak PICP concentrations of the subjects were 5-10 times higher than the pretreatment values. Twenty days after treatment, mean PICP concentration decreased to pretreatment level. PICP concentrations did not correlate with the clinical parameters. In control samples PICP amounts were below the detection limit. CF PICP is a new marker of type 1 collagen metabolism in periodontal tissues. It was concluded that elevated PICP concentrations in CF after periodontal treatment reflected increased type I collagen synthesis in periodontal tissues and that the peak in type I collagen synthesis takes place 5-10 days after treatment.     

Matrix metalloproteinase-8 levels and elastase activities in gingival crevicular fluid from chronic adult periodontitis patients

AIM: To make an initial assessment of the periodontal diagnostic potential of immunoreactive matrix metalloproteinase-8 (MMP-8) in gingival crevicular fluid (GCF) by comparison with elastase activity which has previously been associated with disease severity and progression. METHODS: GCF was collected from molar and premolar sites of 16 chronic adult periodontitis patients before treatment and 13 of this group 2 weeks after scaling and root planing. Samples were analysed for MMP-8 by immunofluorometric assay and for elastase activity with a fluorogenic substrate. RESULTS: Mean patient clinical parameters and GCF enzyme totals both decreased significantly after treatment. Total MMP-8 levels and elastase activities generally correlated significantly with gingival and bleeding indices. For GCF concentrations, only MMP-8 showed a significant fall after treatment, and some significant correlations with clinical parameters. Amounts of the 2 enzymes correlated significantly with each other. CONCLUSIONS: Similarities between MMP-8 and elastase probably reflect the fact that both enzymes are associated mainly with neutrophils: MMP-8 levels may have fallen more after treatment because the assay, unlike that for elastase, would most likely not have detected much enzyme bound to alpha-macroglobulin. The immunoassay for MMP-8 is more specific and convenient than functional collagenase assays, and might be suitable for monitoring the periodontal condition.     

Changes in substance P and neurokinin A in gingival crevicular fluid in response to periodontal treatment

BACKGROUND: The study of periodontitis provides a unique model for assessing the involvement of neuropeptides in inflammatory disease. AIM: To investigate the effects of periodontal treatment, resulting in a return to periodontal health, on the levels of substance P (SP) and neurokinin A (NKA) in gingival crevicular fluid (GCF). METHOD: We completed a cause of non-surgical treatment for 8 subjects with periodontitis (6 females 2 males, mean age 45.1, range 38-67 years) started a course of non-surgical periodontal treatment. Clinical indices were measured at 2 periodontitis sites at the initial visit and at 8 weeks after the completion of treatment in each subject. A 30-s sample of GCF was collected from each test site using perio paper strips. Each strip was placed into 500 microl of ice cold 0.1 M PBS, pH 7.4, vortex mixed for 30 s, and then stored at -70 degrees C until analysed by radioimmunoassay. RESULTS: The clinical condition of all test sites improved as a result of the periodontal treatment. The levels (pg/30 s sample) of SP fell from 56.3 (SD 66.0) at the initial visit to 4.2 (3.1) after treatment, p=0.017. The concentration (pg/microl) of SP in GCF fell from 140.6 (175.6) to 24.2 (11.1), p=0.036. The levels of NKA fell from 30.5 (17.1) to 10.6 (4.9), p=0.012 whereas the concentration changed little from 85.4 (43.5) to 61.6 (15.1), p=0.41. CONCLUSION: The reduction in inflammation resulting from effective periodontal treatment is associated with a reduction in the levels of tachykinins in gingival crevicular fluid.     

A rapid spectrophotometric assay for tetracycline in gingival crevicular fluid

AIM: The aim of this study was to investigate a rapid spectrophotometric assay for its potential to measure tetracycline levels in gingival crevicular fluid (GCF). MATERIALS AND METHODS: The technique involves complexation of tetracycline with molybdenum in order to shift the absorbance spectrum away from that region where interference with plasma proteins is a problem. The sensitivity of the assay and reproducibility of elution were examined together with an assessment of the effect of plasma proteins. The assay was also tested in a small pilot clinical project, measuring tetracycline levels in GCF following placement of a test gel formulation in 25 periodontal pockets in 5 patients. RESULTS: The in vitro results showed good sensitivity of the assay over the concentration range tested (0.5-200 microg tetracycline) and with little effect of plasma proteins. Elution from the paper strips was reproducible with a good linear correlation between direct and filter absorbed assays (r=0.9989, p<0.01). The pilot clinical study indicated a mean half-time of tetracycline in GCF of 28 min with confidence intervals of 21 to 34 min, although wide variation between the drug levels of individual periodontal pockets was seen. CONCLUSIONS: The results indicate good sensitivity for this assay to measure tetracycline hydrochloride in vivo. The potential for rapidly processing large numbers of samples contrasts with the assay time and limited sample throughput of other methods such as high pressure liquid chromatography (HPLC) and suggests that the technique may be a useful addition to current techniques for measuring tetracycline hydrochloride in vivo.