Australian Dental Journal 1998;43:(1):40-4

Gingival crevicular fluid lactoferrin levels in adult per iodontitis patients

C-C. Tsai, DMD, PhD* C. C. Kao, DDS, MDSc* C. C. Chen, DDS, MDSc*

Abstract The present study was designed to determine in a cross-sectional study whether there was any relationship between the levels of lactoferrin in gingival crevicular fluid and clinical periodontal parameters. Crevicular fluid was collected from individual sites using standardized filter paper strips (clinically healthy sites, N=23; periodontitis sites, n=66) and evaluated for lactoferrin by enzyme-linked immunosorbent assay. The data showed that: (1) the total amounts of lactoferrin were 0.003-0.021 ng (30 second sample) (average 0.0090.005 ng) in a clinically healthy periodontium group and 0.0163.847 ng (30 second sample) (average 0.5750.069 ng) in adult periodontitis patients (statistically significantly higher in adult periodontitis patients); and (2) the total amounts of lactoferrin were significantly correlated with clinical parameters, especially a strong positive correlation with gingival crevicular fluid volume (r=0.85, p<0.01) and with probing depth (r=0.71, p<0.01). These results indicated that quantification of lactoferrin in gingival crevicular fluid may be a more sensitive indicator of periodontal pathology than traditional clinical indices.
Key words: Gingival crevicular fluid, periodontitis. lactoferrin,

(Received for publication March 1995. Revised June 1995. Accepted June 1995.)

Introduction From as early as 1958, it has been reported that the increase in volume and flow rate of gingival crevicular fluid (GCF) was associated with the inflammation of gingival tissue.1-3 Recent studies on changes in the constituents of GCF have pointed out that certain biological mediators can be used as diagnostic markers, for example, collagenase,4,5

*Kaohsiung Medical College, School of Dentistry, Kaohsiung City, Taiwan.

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lactate dehydrogenase (LDH),6 aspartate aminotransferase (AST),7 and prostaglandin E (PGE). 8,9 The fundamental pathological processes of periodontal disease are inflammatory responses associated with host-bacterial interaction plus the results of immunopathological changes. From the histopathological point of view, Page and Schr oeder10,11 divided gingivitis/periodontitis into four stages. However, areas of acute inflammation or polymorphonuclear leukocyte (PMNs) infiltration can be seen in each stage. Large accumulations of PMN in the tissues during gingival inflammation have been reported.12-14 These PMNs can release lysosomal substances which not only may have significant effects on microbiota and host tissues but also provide important marker(s) for inflammation of the periodontal tissue. Lysosomes from PMNs contain proteases, carbohydrases, and antimicrobial substances, for example, lactoferrin, lysoszyme, myeloperoxidase, and other strongly biologically active mediators. The interactions of dental plaque microbiota and PMNs and the lysis of PMNs could result in lysosomal release in the sulcus area. 15,16 It has been reported that the GCF levels of PMN lysosomal enzymes, for example, arylsulphatase and -glucuronidase, have a stronger association with bleeding upon probing than the cytoplasmic enzyme lactate dehydrogenase.6 -glucuronidase has currently been suggested as the more promising GCF component in the diagnosis of active periodontal disease.17 Lactoferrin is one of the PMN specific granules18 and could be a useful marker of PMN activity.19 Increased lactoferrin levels have been reported in severe infections, for example, meningitis,20 in burns patients,21 rheumatoid arthritis,22 and chronic salivary gland diseases.23 There are few reports about the quantification of lactoferrin in GCF. Friedman et
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al.24 found that GCF lactoferrin levels in gingivitis, periodontitis, and localized juvenile periodontitis patients were two-fold higher than in periodontally healthy individuals. However, the relationship between the severity of gingival and periodontal inflammation and the levels of GCF lactoferrin was not mentioned. The purpose of this study was to quantify and test whether GCF lactoferrin could be used as an indicator of periodontal inflammation. Materials and methods 1. Subjects The subjects were divided into two groups: experimental and control; all were free of systemic disease and had not taken medication (such as anti-inflammatory agents, antibiotics, immunosuppressants, or contraceptives) that could affect their periodontal status for at least six months prior to the study. Informed consent was obtained from the subjects. Radiographic examination and clinical periodontal assessment including plaque index (PLI), gingival index (GI), probing pocket depth (PD), and attachment levels (AL) were performed. Based on the results the subjects were divided into two groups: experimental (adult periodontitis: 8 males and 5 females, aged 26 to 50 years), and control (clinically healthy: 3 males and 2 females, aged 24 to 27 years). 2. Gingival crevicular fluid (GCF) samples After isolating each tooth with a cotton roll, the plaque index was recorded and the supragingival plaque was carefully removed without touching the marginal gingiva. The gingiva was then gently dried with an air syringe. Gingival crevicular fluid was collected with a Periopaper strip at the orifice of the sulcus 1 mm subgingivally for 30 seconds and then quantified with the Periotron 6000. Each strip was then eluted in 300 L 0.1 mmol/L phosphate buffered saline (pH 7.4) at 25C for one hour while being agitated constantly. The elute was then stored at -70C until assayed. No attempt was made to evaluate the recovery of lactofer rin from the strip. 3. Quantification of GCF lactofer rin A double antibody sandwich enzyme linked immunosorbent assay was used for the quantification of GCF lactoferrin in this study. Ninety six-well flexible microtitration plates were coated with 100 L rabbit anti-human lactoferrin antibody (2.34 g/mL); 2% bovine serum albumin in PBS was used to block free binding sites. After a thorough washing
Harco Electronics Ltd, Manitoba, Canada. DAKO Immunoglobulins Ltd, Glostrup, Denmark. Sigma, Missouri, USA. Australian Dental Journal 1998;43:1.

Table 1. Volumes and lactoferrin levels of gingival crevicular fluid collected for 30 s and clinical parameters at healthy and periodontitis sites
Site Parameters PLI GI PD AL GCF (L) Lactoferrin ng/site-30 s Lactoferrin ng/mL GCF-30 s Healthy (n=23) 0.430.59* 0.220.42 2.390.50 0.120.03 0.0090.005 69.322.2 Periodontitis (n=66) 2.780.45 2.400.61 5.691.85 7.070.33 0.82 0.46 0.575 0.069 552.1419.4

*Data presented as mean SD. Total amount. Concentration. Statistically significant using Mann-Whitney U test (p0.001).

with PBS-Tween 20, the plates were ready for assay for lactoferrin: 100 L rabbit anti-human lactoferrin peroxidase conjugate, (2.03 g/mL) and O-phenylene diamine (OPD) were used as antibody enzyme conjugate and enzyme-substrate, respectively; 100 L of four different concentrations of standard lactoferrin (ranging from 0.0078-0.0625 g/mL), and dilutions (ranging from 2-30 times) of sample elute in triplicate were included in each assay plate. The concentration (ng/mL GCF-30s) of GCF lactoferrin in the sample was calculated by comparison of the sample dilution curves with that of lactoferrin standards. The total amounts (ng/site-30s) of lactoferrin were obtained by multiplying concentrations and GCF volumes. 4. Statistical analysis Data analysis was performed using the SAS statistical package.** Differences in GCF lactoferrin levels were sought using the Mann-Whitney U-test and the Kruskal-Wallis one-way ANOVA. The correlation among the levels of GCF lactoferrin and clinical parameters were assessed using the Pearsons Correlation Coefficient. Results (1) GCF lactoferrin (LF) levels and clinical parameters (Table 1) The LF concentrations (mean SD) of healthy and periodontitis sites were 69.322.2 ng/mL and 552.1419.4 ng/mL, respectively. The total amounts of LF of healthy and periodontitis sites were 0.0090.005 ng/site and 0.5750.069 ng/site, respectively. Both the concentrations and total amounts of LF in periodontitis sites were
,Cappel, North Carolina, USA. Zymed Laboratory Inc, California, USA. **SASInc, North Carolina, USA. 41

Table 2. Comparison of volumes and lactoferrin levels of gingival crevicular fluid with different gingival indices
GI 0, n=18 1, n=9 2, n=31 3, n=31 ANOVA, F-value GI 0-1 GI 0-2 GI 0-3 GI 1-2 GI 1-3 GI 2-3 GCF (L) 0.110.02* 0.210.06 0.600.29 1.110.45 43.96 NS S S S S S Total amount lactoferrin ng/site 0.00680.0079 0.02130.0091 0.1622 0.1059 1.0578 0.0022 31.62 NS NS S NS S S Concentration lactoferrin ng/mL 60.9 8.6 100.8 29.2 263.7 112.6 898.5 362.6 72.5 NS S S NS S S

Table 4. Pearsons correlation coefficients between the clinical parameters and the volumes and lactoferrin levels in gingival crevicular fluid
GCF (L) Lactoferrin ng/site-30 s Lactoferrin ng/mL GCF-30 s GCF (L) 0.85 0.74 PD 0.71 0.71 0.82 GI 0.61 0.74 0.75 PLI 0.45 0.57 0.66 AL 0.62 0.67 0.78

Studied sites, n=89. All values had significant correlations at p 0.01.

*Data presented as mean SD. p<0.05 (F probability). Not significant. Statistically significant at p<0.05 by Scheffes test.

were significantly different, except for those between GI=0 and GI=1. (3) GCF lactoferrin (LF) levels and different plaque indices (PLI) (Table 3) The concentrations and total amounts of LF and GCF volumes from sites of PLI=3 were significantly higher than from the sites of PLI=0, 1, and 2. No significant statistical difference was observed between the sites of PLI=0 and 1, PLI=0 and 2, nor between sites of PLI=1 and 2. (4) The correlations of lactoferrin (LF) levels with clinical parameters (Table 4) The Pearsons product moment correlation (r) analyses indicated that: (a) the total amounts of LF (ng/site) were most strongly correlated with the volumes of GCF (r=0.85), followed by PD (r=0.71), AL (r=0.62), GI (r=0.61) and PLI (r=0.45); and (b) the concentrations (ng/mL) were most strongly correlated with PD (r=0.77), followed by GI, volume of GCF (r=0.74), AL (r=0.67), and PLI (r=0.57). The data also showed that the volume of GCF was most strongly correlated with PD (r=0.82), followed by AL (r=0.78), GI (r=0.75), and PLI (r=0.66). Discussion The histological evidence of experimental periodontitis in animals indicated that progressive disease is associated with the presence and persistence of strong acute inflammatory reactions.25-27 The continuous influx of ulcerative sulcular epithelium and PMNs into the gingival sulcular areas was seen at the time of loss of periodontal attachment and alveolar bone. Also, an increase of lysosomal collagenase in GCF had been reported during the active stage of periodontal disease.27 G i n gi va l crevicular fluid collagenase was mainly from PMNs.28 These findings indicated that marked PMN responses were accompanied with the active stage of destructive periodontitis. Tissue destruction associated with PMNs may be an important mechanism of many human diseases.29 Beside the
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significantly higher than the healthy sites (U-test, p<0.001). The clinical parameters of healthy and periodontitis sites were also significantly different. (2) GCF lactoferrin (LF) levels and different gingival indices (Table 2) The total amounts of LF from sites of GI=3 were significantly higher than from sites of GI=0, GI=1, and GI=2, but not significantly different between the individual sites of GI=0 and GI=1, GI=0 and GI=2, nor between sites of GI=1 and GI=2. The concentrations of LF from sites of GI=3 were significantly higher than from sites of GI=0, GI=1, and GI=2. The concentrations of LF between the sites of GI=2 and GI=0 were also significantly different. No difference in the concentrations of LF between the sites of GI=0 and GI=1 or between the sites of GI=1 and GI=2 were observed. The GCF volumes between sites of varying gingival indices

Table 3. Comparison of volumes and lactoferrin levels of gingival crevicular fluid with different plaque indices
PLI 0, n=14 1, n=9 2, n=13 3, n=53 ANOVA, F-value PLI 0-1 PLI 0-2 PLI 0-3 PLI 1-2 PLI 1-3 PLI 2-3 GCF (L) Total amount lactoferrin ng/site Concentration lactoferrin ng/mL 60.9 8.8 94.343.9 198.2152.2 636.9 421.5 17.49 NS NS S NS S S

0.110.03* 0.00680.002 0.160.07 0.0171 0.0161 0.44 0.29 0.12340.2035 0.91 10.46 0.6846 0.7225 24.82 9.61 NS NS NS NS S S NS NS S S S S

*Data presented as mean SD. p<0.05 (F probability). Not significant. Statistically significant at p<0.05 by Scheffes test.
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release of lysosomal enzymes, the formation of active oxygen metabolites (O2-, H 2O2, HOCl, and so on) can also cause tissue destruction. The oxygen metabolites can also inactivate protease inhibitors leading to the activation of protease. This synergic effect causes further tissue destruction and may be a reason for the intermittent feature of some inflammatory diseases. Periodontitis is a good example. Furthermore, endotoxins from Gram (-) bacteria and/or cytokines (IL-1 and TNF)30 can adjust the functions of PMNs and monocytes in the periodontal tissues. These cellular actions play an important role in the progression of periodontal disease. In 1983, Friedman et al.24 evaluated the levels of lysozyme and lactoferrin in GCF by rocket immunoelectrophoresis and reported that levels of lysozyme, but not lactoferrin, were significantly higher in localized juvenile periodontitis patients as compared with gingivitis and adult periodontitis patients. However, the normal group had a significantly lower level of lactoferrin as compared with the other three disease groups. In this study, both the concentrations and total amounts of lactoferrin were significantly higher in periodontitis patients as compared with the control group. From the analyses of the relationships of lactoferrin levels and GI/PLI (Tables 2, 3), significantly higher LF levels were seen only in higher GI/PLI sites and there was no significant statistical difference between lower GI/PLI sites. This implies that GI/PLI may not be adequate for assessing degrees of periodontal (gingival) inflammation. Significant difference in GCF volumes seen between different GIs (except between GI 0 and 1) plus a high correlation between GCF volumes and GI (r=0.75) in the present study further support the hypothesis that changes in GCF volumes remain a good indicator of gingival inflammation. The total amounts and concentrations of GCF lactoferrin were highly correlated with the periodontal clinical parameters (r=0.45 to 0.85). Similar results have been reported indicating that PMNderived myeloperoxidase31 was highly correlated with GCF volumes and that -glucuronidase was positively correlated with pocket probing depth. Gingival crevicular fluid production is a result of the increase in permeability of microvasculatures of the periodontal tissue due to inflammatory reactions. Lactoferrin released from PMNs into the GCF is a good indicator of periodontal inflammation. The strong correlation of the clinical periodontal parameters and the levels of LF (ng/site) suggests that increasing severity of periodontal inflammation was accompanied by an increase of GCF lactoferrin. Therefore, the quantification of GCF LF can be a sensitive and objective method of detecting the degree of perioAustralian Dental Journal 1998;43:1.

dontal inflammation. Since the present study was one of the cross-sectional investigations for the understanding of the relationship between GCF lactoferrin and the progression or disease activity of periodontal disease, a longitudinal study will be required. Acknowledgements The authors gratefully acknowledge the help of Miss I-Chu Li, Dr Yi-Min Wu, and the financial support provided by the National Science Council Grant, NSC 79-0412-B-037-45, in Taiwan, Republic of China. References
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17. Lamster IB, Oshrain RL, Harper DS, Celenti RS, Hovliaras CA, Gordon JM. Enzyme activity in crevicular fluid for detection and prediction of clinical attachment loss in patients with chronic adult periodontitis 6-month results. J Periodontol 1988;59:51623. 18. Baggiolini M, De Duve C, Masson PL, Heremans JF. Association of lactoferrin with specific granules in rabbit heterophil leucocytes. J Exp Med 1970;131:559-70. 19. Lefell MS, Spitznagel JK. Association of lysozyme and lactoferrin in granules of human polymorphonuclear leucocytes. Infect Immun 1972;6:761-5. 20. Gutteberg TJ, Haneberg B, Jorgensen T. The latency of serum acute phase protein in meningococcal septicemia, with special emphasis on lactoferrin. Clinica Chimica Acta 1984;136:173-8. 21. Wolach B, Coates TD, Hugli TE, Boxer LA. Plasma lactoferrin reflects granulocyte activation via complement in burn patients. J Lab Clin Med 1984;103:284-93. 22. Tsai WJ, Liu HW, Yen JH, Chen JR, Lin SF, Chen TP. Lactoferrin in rheumatoid arthritis and systemic lupus erythematosus. Kaohsiung J Med Sci 1991;9:22-6. 23. Tabak L, Mandel I, Karla D, Baumarsh H. Alteration in lactoferrin in salivary gland disease. J Dent Res 1978;57:43-7. 24. Friedman SA, Mandel ID, Herrera MS. Lysozyme and lactoferrin quantitation in the crevicular fluid. J Periodontol 1983;54:347-50. 25. Heijl L, Rifkin BR, Zander HA. Conversion of chronic gingivitis to periodontitis in squirrel monkeys. J Periodontol 1976;47:71016. 26. Schr oeder HE, Lindhe J. Conditions and pathologic features of rapidly destructive, experimental periodontitis in dogs. J Periodontol 1980;51:6-19.

27. Kryshtalskyj E, Sodek J, Ferrier JM. Correlation of collagenolytic enzymes and inhibitors in gingival crevicular fluid with clinical and microscopic changes in experimental periodontitis in the dog. Arch Oral Biol 1986;31:21-31. 28. Sorsa T, Uitto VJ, Suomalainen K, Vauhkonen M, Lindy S. Comparison of interstitial collagenases from human gingiva, sulcular fluid and polymorphonuclear leukocytes. J Periodont Res 1988;23:386-93. 29. Weiss JJ. Tissue destruction by neutrophils. N Engl J Med 1989;320:365-76. 30. Movat HZ, Cybulsky MI, Colditz IG, Chan MKW, Dinarello CA. Acute inflammation in gram-negative infection: endotoxin, interleukin 1, tumor necrosis factor, and neutrophils. Fed Proc 1987;46:97-104. 31. Wolff LF, Smith QT, Snyder WK, Bedrick JA, Liljemark WF, Aeppli DA, Bandt CL. Relationship between lactate dehydrogenase and myeloperoxidase levels in human gingival crevicular fluid and clinical and microbial measurements. J Clin Periodontol 1988;15:110-5.

Address for correspondence/reprints: Professor C-C Tsai, Kaohsiung Medical College, School of Dentistr y, No. 100, Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan, Republic of China.

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Australian Dental Journal 1998;43:1.