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Alkaline phosphatase in human gingival fluid and its relation to periodontitis
Archr oralNO/.Vol.15,pp.1401-1404, 1970. Pergamon Press. Printed inGreat Britain.
ALKALINE PHOSPHATASE IN HUMAN GINGIVAL FLUID AND ITS RELATION TO PERIODONTITIS 1. ISHIKAWAand G. CIMA~ONI Dental School, University of Geneva, Switzerland
PERIODONTAL inflammation
is accompanied by the presence of fluid in the gingival sulcus, and the amount of fluid varies according to the severity of inflammation (MANN, 1963). This gingival or crevicular fluid contains lysosomal and bacterial acid phosphatase (SUEDA, CIMA~ONIand HELD, 1967; SUEDAand CIMASONI,1968), as well as very high levels of beta-glucuronidase. The concentration of this second enzyme has been shown to be positively correlated with the severity of periodontitis (BANG, CIMASONI,HELD, 1970).
We have now found that the activity of alkaline phosphatase is significantly higher in gingival fluid than in serum, and we have in the present work attempted to investigate the correlation between its level of activity and various clinical parameters. By using a recently standardized technique (SIJEDA,BANG and CIMASONI,1969), gingival exudate was collected from the upper incisor and canine region in 21 patients, aged 18-65 years and showing extensive gingival inflammation and periodontitis. The presence of gingivitis was established by the PMA index (SCHOUR and MASSLER, 1948) and the gingival index of LOB and SILNESS(1963). The depths of the periodontal pockets were measured with a blunt explorer along the mesial and distal surfaces of each of the upper incisors, so that for each patient the mean of eight measurements was used in the correlation studies. Radiographs were available for all the patients, and the amount of bone loss in the upper incisor area was measured by the technique of MARSHALL-DAY and SHOURIE(1949) as described in detail by SCHEI et al., (1959). A specially designed transparent ruler, placed on top of the radiograph, enabled the reader to estimate the height of the bone loss as a percentage of root length. The average of the measurements taken for each patient, along the mesial and distal surfaces of the upper incisors, was used in the statistical comparisons. The amount of supragingival calculus was evaluated in the same area by using the calculus index of MUHLEMANNand VILI.A (1967). Alkaline phophatase was measured in 10 ,uI of fluid and 20 ~1 of serum by the calorimetric technique originally described by BESSEY,LOWRY and BROCK (1946), using p-nitrophenyl phosphate as substrate and 0.2 M 2-amino-2-methyl-I-propanol, HCl buffer, pH 10. A blank and a standard were run for each determination. The results of the analysis of alkaline phosphatase activity indicated a mean of 99 ~0 pmoles p-nitrophenol liberated l/min with a standard deviation of 53 *6 in gingival fluid, against a mean of 33 *7 f 9 *6 S.D. in serum. 1401
I.
1402
hIKAWA
AND
G.
c?MASONl
The correlation between the activity of the alkaline phosphatase in the exudate and the mean depth of the periodontal pockets was statistically significant, with a correlation coefficient of O-49 (p c O-05). This correlation is illustrated in Fig. 1.
.
. . . . . .
. .
.
.
. . .
.
l
t* .
I
I
I
2
Mean Ro.
1. Correlation
I 3
I 4
depth of pockets,
I 5
I 6
I
mm
between the activity of alkaline phosphatase depth of periodontal pockets.
in gingival exudate and
A positive correlation was also found between the enzyme concentration in gingival fluid and the mean percentage of bone loss, but this relationship was not significant at the 5 per cent level of probability. Since it is known that marginal bone resorption tends to increase in older individuals (SCHEIet al., 1959), the possible influence of age was further analyzed by studying the correlation between the concentration of alkaline phosphatase in gingival fluid and the percentage of bone loss divided by the age of the patient. The correlation found between these two parameters (enzyme concentration and ratio of bone loss to age) was positive but again not significant at the 5 per cent level of probability. A very low coefficient was found when studying the correlation between the alkaline phosphatase activity in gingival exudate and the calculus index, nor was it possible to show any noticeable degree of correlation between the enzyme concentration in the fluid and either the PMA or the gingival index of inflammation. No correlation could be found between the concentration of alkaline phosphatase in serum and that of gingival fluid. Finally, it is of interest to note that a very high degree of correlation was found between the mean value of pocket depth, measured clinically, and the percentage of bone loss evaluated on radiographs. The same goes, as one could expect, for the correlation between the values of PMA and those of the gingival index of inflammation. The finding that human gingival fluid contains 3 to 4 times more alkaline phosphatase than serum raises the question of the possible origins of this enzyme. Alkaline phosphatase is known to be present in the periodontal tissues, normally
PALKALINEPHOSPHATASE IN HUMAN GINGIVAL.FLUIDAND PERIODONTITIS
1403
confined to a few sites, namely the endothelium of smaller vessels, fibroblasts and the collagen fibres (ZANDFX,1941; VALLOITON,1942 ; CABRINIand CARRANZA,1951). Leucocytes are also rich in alkaline phosphatase (COHN and HIRSCH, 1960), and it is noteworthy that LANGE(1967) found that leucocytes, in smears of gingival fluid, are devoid of alkaline phosphatase. An increase of alkaline phosphatase activity has been shown by histochemical methods in inflamed gingiva, both in humans (-KY, GLICKMANand LITWIN, 1951; DONATHand SZABO,1963 ; MIELERand MIELER,1966) and in ferrets (STAPLE,1957). In addition to tissue origin, a second and probably major source of alkaline phosphatase in gingival fluid could be of bacterial origin (ENNEVERand WARNER, 1952; BOWEN, 1961). NARRODand BRAUNBERG(1966) have attempted to follow changes in specific activity of aminopeptidase as well as acid and alkaline phosphatase in the ante-molar region of the palate of the rat, after injuring this region by an electric probe; both phosphatases rose to maximal values within three days and then dropped slowly. As pointed out by the authors, it is difficult to be certain whether the increased concentration of enzymes is of tissue origin or is instead brought in by invading microorganisms. That the bacteria are probably the major source of alkaline phosphatase in gingival fluid can be inferred from studies on sterile inflammatory exudates such as cantharidin-blister fluid and synovial fluid from inflammatory joint disease; in both these exudates the concentration of alkaline phosphatase is similar or even lower than in serum (SMITH, BURK, ROSEN and CHURCH, 1967; LEHMAN,KREAM and BROGNA,1964). The significant positive correlation found between the alkaline phosphatase in crevicular fluid and the depth of the periodontal pockets indicates a third possible source of the enzyme, i.e. the periodontal bone. It is indeed known that alkaline phosphatase is highly active in this tissue (NAKAMURA et al., 1965). Finally, the relationship between enzyme activity in the fluid and calculus index calls for the following remarks. The action of alkaline phosphatase in releasing phosphate ions from organic phosphates (ROBINSON,1923) has been considered as a possible mechanism for a calculus formation (JENKINS,1966). It is important to stress that, in spite of the high concentration of alkaline phosphatase present in the fluid, the positive correlation found between the enzyme activity and the amount of supragingival calculus was not statistically significant. As for the possible role of alkaline phosphatase in the formation of subgingival calculus, this problem remains open to further investigation. REFERENCES BANG, J., CIMASONI,G. and HELD, A. J. 1970. Beta-glucuronidase correlated with inflammation in the exudate from human gingiva. A&s oral Biof. 15,445-451. BESSEY, 0. A., LOWRY, 0. H. and BROCK, M. J. 1946. A method for the rapid determination of alkaline phosphatase with 5 mm3 of serum. J. biol. Chem. 164,321-329. BOWEN, W. H. 1961. Phosphatase in micro-organisms cultured from carious dentin and calculus.
J. dent. Res. 40, 571-577. CABRINI, R. L. and CARRANZA, F. A. Jr. 1951. Histochemical study on alkaline phosphatase in normal gingivae, varying the pH and the substrate. J. dent. Res. 30,28-32.
1404
I. I~H~KAWAAND G.
CM~NI
COHN, Z. A. and HIRSCH,J. G. 1960. The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. J. exp. Med. 112, 983-1004. DONATH, I. and SZABO,I. 1963. Die Phosphatase-AktivitSt des Parodontiums beim Menschen unter norrnalen und pathologischen VerhBltnissen. Dr. Zahndrztl. Z. 18, 303-311. ENNEVER,J. J. and WARNER, B. W. 1952. Phosphatase and the oral actinomyces. J. dent. Res. 31, 25-26.
JENKINS,G. N. 1966. The Physiology of the Mouth, 3rd edn, p. 108. Blackwells, Oxford. LANGE,D. 1967. Uber das Vorkommen von Zellen in der menschlichen Zahnfleischtaschenlhissigkeit und ihre zytochemischen Reaktionen. Dt. Zahndrztl. Z. 22,838-847. LEHMAN,M. A., KREAM,J. and BROGNA,D. 1964. Acid and alkaline phosphatase activity in the serum and synovial fluid of patients with arthritis. J. Bone. Jt Surg. 464, 1732-1738. Lh, H. and SILNESS,J. 1963. Periodontal disease in pregnancy. 1. Prevalence and severity. Actu odont. stand.
21,535-551.
MANN, W. V. 1963. The correlation J. Periodont.
of gingivitis, pocket depth and exudate from the gingival crevice.
34, 379-387.
MARSHALL-DAY,C. D. and SHOURIE,K. L. 1949. A roentgenographic survey of periodontal disease in India. J. Am. dent. Ass. 39,573-588. MIELBR,I. and MIELER, W. 1966. Die Aktivitat der alkalischen Phosphatase bei Parodontopathien. Dt. Zahn-, Mund- Kieferheilk.
47, 12-20.
M&ILEMANN, H. R. and VILLA, P. R. 1967. The marginal line calculus index. Helv. odont. Acta 11, 175-179. NAKAMURA,R., TSUKAMOTO,Y., HONJO, K., TSUNEMITSU,A. and MATSUMURA,T. 1965. Alkaline and acid phosphatase activity in the gingiva and alveolar bone in scurvy. Archs oral Biol. 10,
765-773. NARROD, S. A. and BRAUNBERG,R. C. 1966. Enzymatic mechanisms related to oral disease. In: Environmental Variables in Oral Disease (edited by KRESHOVER,S. J. and MCCLURE, F. J.) pp. 201-222. American Association for the Advancement of Science, Washington, D.C. ROBISON,R. 1923. The possible significance of hexosephosphoric esters in ossification. Biochem. J. 17, 286-293.
SCHEI,O., WAERHAUG,J., LOVDAL,A. and ARNO, A. 1959. Alveolar bone loss as related to oral hygiene and age. J. Periodont. 30,7-16. SCHOUR, I. and MASSLER,M. 1948. Survey of gingival disease using PMA index. J. dent. Res. 27, 733-734
SMITH,J. G., BURK, P. G., ROSE~T,T. and CHURCH, C. F. 1967. Enzymes in blister fluid. Lab. Invest. 16, 247-253.
STAPLE,P. H. 1957. Alkaline phosphatase activity in gingiva of the ferret. Br. dent. J. 102,305-310. SUEDA, T., C~MASONI,G. and HELD, A. 1967. High levels of acid phosphatase in human gingival fluid. Archs oral Biol. 12, 1205-1207. SUEDA, T. and C~MASONI,G. 1968. The origins of acid phosphatase in human gingival fluid. Archs oral Biol. 13, 553-558.
SUEDA, T., BANG, J. and CIMASONI,G. 1969. Collection
of gingival fluid for quantitative
analysis.
J. dent. Rex 48, 159.
TURESKY,S., GLICKMAN,I. and LITWIN,T. 1951. A histochemical evaluation of normal and inflamed human gingivae. J. dent. Res. 30,792-798. VALLO~~ON,C. E. 1942. Etude bio-histologique de la phosphatase dans la gencive humaine normale et dans les gingivites. Schweiz. Mschr. Zuhnheilk. 52, 512-526. ZANDER,H. A. 1941. The distribution of phosphatase in gingival tissue. J. dent. Res. 20, 347-353.
ALKALINE PHOSPHATASE IN HUMAN GINGIVAL FLUID AND ITS RELATION TO PERIODONTITIS 1. ISHIKAWAand G. CIMA~ONI Dental School, University of Geneva, Switzerland
PERIODONTAL inflammation
is accompanied by the presence of fluid in the gingival sulcus, and the amount of fluid varies according to the severity of inflammation (MANN, 1963). This gingival or crevicular fluid contains lysosomal and bacterial acid phosphatase (SUEDA, CIMA~ONIand HELD, 1967; SUEDAand CIMASONI,1968), as well as very high levels of beta-glucuronidase. The concentration of this second enzyme has been shown to be positively correlated with the severity of periodontitis (BANG, CIMASONI,HELD, 1970).
We have now found that the activity of alkaline phosphatase is significantly higher in gingival fluid than in serum, and we have in the present work attempted to investigate the correlation between its level of activity and various clinical parameters. By using a recently standardized technique (SIJEDA,BANG and CIMASONI,1969), gingival exudate was collected from the upper incisor and canine region in 21 patients, aged 18-65 years and showing extensive gingival inflammation and periodontitis. The presence of gingivitis was established by the PMA index (SCHOUR and MASSLER, 1948) and the gingival index of LOB and SILNESS(1963). The depths of the periodontal pockets were measured with a blunt explorer along the mesial and distal surfaces of each of the upper incisors, so that for each patient the mean of eight measurements was used in the correlation studies. Radiographs were available for all the patients, and the amount of bone loss in the upper incisor area was measured by the technique of MARSHALL-DAY and SHOURIE(1949) as described in detail by SCHEI et al., (1959). A specially designed transparent ruler, placed on top of the radiograph, enabled the reader to estimate the height of the bone loss as a percentage of root length. The average of the measurements taken for each patient, along the mesial and distal surfaces of the upper incisors, was used in the statistical comparisons. The amount of supragingival calculus was evaluated in the same area by using the calculus index of MUHLEMANNand VILI.A (1967). Alkaline phophatase was measured in 10 ,uI of fluid and 20 ~1 of serum by the calorimetric technique originally described by BESSEY,LOWRY and BROCK (1946), using p-nitrophenyl phosphate as substrate and 0.2 M 2-amino-2-methyl-I-propanol, HCl buffer, pH 10. A blank and a standard were run for each determination. The results of the analysis of alkaline phosphatase activity indicated a mean of 99 ~0 pmoles p-nitrophenol liberated l/min with a standard deviation of 53 *6 in gingival fluid, against a mean of 33 *7 f 9 *6 S.D. in serum. 1401
I.
1402
hIKAWA
AND
G.
c?MASONl
The correlation between the activity of the alkaline phosphatase in the exudate and the mean depth of the periodontal pockets was statistically significant, with a correlation coefficient of O-49 (p c O-05). This correlation is illustrated in Fig. 1.
.
. . . . . .
. .
.
.
. . .
.
l
t* .
I
I
I
2
Mean Ro.
1. Correlation
I 3
I 4
depth of pockets,
I 5
I 6
I
mm
between the activity of alkaline phosphatase depth of periodontal pockets.
in gingival exudate and
A positive correlation was also found between the enzyme concentration in gingival fluid and the mean percentage of bone loss, but this relationship was not significant at the 5 per cent level of probability. Since it is known that marginal bone resorption tends to increase in older individuals (SCHEIet al., 1959), the possible influence of age was further analyzed by studying the correlation between the concentration of alkaline phosphatase in gingival fluid and the percentage of bone loss divided by the age of the patient. The correlation found between these two parameters (enzyme concentration and ratio of bone loss to age) was positive but again not significant at the 5 per cent level of probability. A very low coefficient was found when studying the correlation between the alkaline phosphatase activity in gingival exudate and the calculus index, nor was it possible to show any noticeable degree of correlation between the enzyme concentration in the fluid and either the PMA or the gingival index of inflammation. No correlation could be found between the concentration of alkaline phosphatase in serum and that of gingival fluid. Finally, it is of interest to note that a very high degree of correlation was found between the mean value of pocket depth, measured clinically, and the percentage of bone loss evaluated on radiographs. The same goes, as one could expect, for the correlation between the values of PMA and those of the gingival index of inflammation. The finding that human gingival fluid contains 3 to 4 times more alkaline phosphatase than serum raises the question of the possible origins of this enzyme. Alkaline phosphatase is known to be present in the periodontal tissues, normally
PALKALINEPHOSPHATASE IN HUMAN GINGIVAL.FLUIDAND PERIODONTITIS
1403
confined to a few sites, namely the endothelium of smaller vessels, fibroblasts and the collagen fibres (ZANDFX,1941; VALLOITON,1942 ; CABRINIand CARRANZA,1951). Leucocytes are also rich in alkaline phosphatase (COHN and HIRSCH, 1960), and it is noteworthy that LANGE(1967) found that leucocytes, in smears of gingival fluid, are devoid of alkaline phosphatase. An increase of alkaline phosphatase activity has been shown by histochemical methods in inflamed gingiva, both in humans (-KY, GLICKMANand LITWIN, 1951; DONATHand SZABO,1963 ; MIELERand MIELER,1966) and in ferrets (STAPLE,1957). In addition to tissue origin, a second and probably major source of alkaline phosphatase in gingival fluid could be of bacterial origin (ENNEVERand WARNER, 1952; BOWEN, 1961). NARRODand BRAUNBERG(1966) have attempted to follow changes in specific activity of aminopeptidase as well as acid and alkaline phosphatase in the ante-molar region of the palate of the rat, after injuring this region by an electric probe; both phosphatases rose to maximal values within three days and then dropped slowly. As pointed out by the authors, it is difficult to be certain whether the increased concentration of enzymes is of tissue origin or is instead brought in by invading microorganisms. That the bacteria are probably the major source of alkaline phosphatase in gingival fluid can be inferred from studies on sterile inflammatory exudates such as cantharidin-blister fluid and synovial fluid from inflammatory joint disease; in both these exudates the concentration of alkaline phosphatase is similar or even lower than in serum (SMITH, BURK, ROSEN and CHURCH, 1967; LEHMAN,KREAM and BROGNA,1964). The significant positive correlation found between the alkaline phosphatase in crevicular fluid and the depth of the periodontal pockets indicates a third possible source of the enzyme, i.e. the periodontal bone. It is indeed known that alkaline phosphatase is highly active in this tissue (NAKAMURA et al., 1965). Finally, the relationship between enzyme activity in the fluid and calculus index calls for the following remarks. The action of alkaline phosphatase in releasing phosphate ions from organic phosphates (ROBINSON,1923) has been considered as a possible mechanism for a calculus formation (JENKINS,1966). It is important to stress that, in spite of the high concentration of alkaline phosphatase present in the fluid, the positive correlation found between the enzyme activity and the amount of supragingival calculus was not statistically significant. As for the possible role of alkaline phosphatase in the formation of subgingival calculus, this problem remains open to further investigation. REFERENCES BANG, J., CIMASONI,G. and HELD, A. J. 1970. Beta-glucuronidase correlated with inflammation in the exudate from human gingiva. A&s oral Biof. 15,445-451. BESSEY, 0. A., LOWRY, 0. H. and BROCK, M. J. 1946. A method for the rapid determination of alkaline phosphatase with 5 mm3 of serum. J. biol. Chem. 164,321-329. BOWEN, W. H. 1961. Phosphatase in micro-organisms cultured from carious dentin and calculus.
J. dent. Res. 40, 571-577. CABRINI, R. L. and CARRANZA, F. A. Jr. 1951. Histochemical study on alkaline phosphatase in normal gingivae, varying the pH and the substrate. J. dent. Res. 30,28-32.
1404
I. I~H~KAWAAND G.
CM~NI
COHN, Z. A. and HIRSCH,J. G. 1960. The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. J. exp. Med. 112, 983-1004. DONATH, I. and SZABO,I. 1963. Die Phosphatase-AktivitSt des Parodontiums beim Menschen unter norrnalen und pathologischen VerhBltnissen. Dr. Zahndrztl. Z. 18, 303-311. ENNEVER,J. J. and WARNER, B. W. 1952. Phosphatase and the oral actinomyces. J. dent. Res. 31, 25-26.
JENKINS,G. N. 1966. The Physiology of the Mouth, 3rd edn, p. 108. Blackwells, Oxford. LANGE,D. 1967. Uber das Vorkommen von Zellen in der menschlichen Zahnfleischtaschenlhissigkeit und ihre zytochemischen Reaktionen. Dt. Zahndrztl. Z. 22,838-847. LEHMAN,M. A., KREAM,J. and BROGNA,D. 1964. Acid and alkaline phosphatase activity in the serum and synovial fluid of patients with arthritis. J. Bone. Jt Surg. 464, 1732-1738. Lh, H. and SILNESS,J. 1963. Periodontal disease in pregnancy. 1. Prevalence and severity. Actu odont. stand.
21,535-551.
MANN, W. V. 1963. The correlation J. Periodont.
of gingivitis, pocket depth and exudate from the gingival crevice.
34, 379-387.
MARSHALL-DAY,C. D. and SHOURIE,K. L. 1949. A roentgenographic survey of periodontal disease in India. J. Am. dent. Ass. 39,573-588. MIELBR,I. and MIELER, W. 1966. Die Aktivitat der alkalischen Phosphatase bei Parodontopathien. Dt. Zahn-, Mund- Kieferheilk.
47, 12-20.
M&ILEMANN, H. R. and VILLA, P. R. 1967. The marginal line calculus index. Helv. odont. Acta 11, 175-179. NAKAMURA,R., TSUKAMOTO,Y., HONJO, K., TSUNEMITSU,A. and MATSUMURA,T. 1965. Alkaline and acid phosphatase activity in the gingiva and alveolar bone in scurvy. Archs oral Biol. 10,
765-773. NARROD, S. A. and BRAUNBERG,R. C. 1966. Enzymatic mechanisms related to oral disease. In: Environmental Variables in Oral Disease (edited by KRESHOVER,S. J. and MCCLURE, F. J.) pp. 201-222. American Association for the Advancement of Science, Washington, D.C. ROBISON,R. 1923. The possible significance of hexosephosphoric esters in ossification. Biochem. J. 17, 286-293.
SCHEI,O., WAERHAUG,J., LOVDAL,A. and ARNO, A. 1959. Alveolar bone loss as related to oral hygiene and age. J. Periodont. 30,7-16. SCHOUR, I. and MASSLER,M. 1948. Survey of gingival disease using PMA index. J. dent. Res. 27, 733-734
SMITH,J. G., BURK, P. G., ROSE~T,T. and CHURCH, C. F. 1967. Enzymes in blister fluid. Lab. Invest. 16, 247-253.
STAPLE,P. H. 1957. Alkaline phosphatase activity in gingiva of the ferret. Br. dent. J. 102,305-310. SUEDA, T., C~MASONI,G. and HELD, A. 1967. High levels of acid phosphatase in human gingival fluid. Archs oral Biol. 12, 1205-1207. SUEDA, T. and C~MASONI,G. 1968. The origins of acid phosphatase in human gingival fluid. Archs oral Biol. 13, 553-558.
SUEDA, T., BANG, J. and CIMASONI,G. 1969. Collection
of gingival fluid for quantitative
analysis.
J. dent. Rex 48, 159.
TURESKY,S., GLICKMAN,I. and LITWIN,T. 1951. A histochemical evaluation of normal and inflamed human gingivae. J. dent. Res. 30,792-798. VALLO~~ON,C. E. 1942. Etude bio-histologique de la phosphatase dans la gencive humaine normale et dans les gingivites. Schweiz. Mschr. Zuhnheilk. 52, 512-526. ZANDER,H. A. 1941. The distribution of phosphatase in gingival tissue. J. dent. Res. 20, 347-353.