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Analysis of Arylsulfatases A and B, Acid Phosphatase, Lactate Dehydrogenase, and Aspartate Transaminase in Chronic Periapical Lesions of Endodontic Origin
JOURNAL OF ENDODONTICS Copyright © 2001 by The American Association of Endodontists
Printed in U.S.A. VOL. 27, NO. 4, APRIL 2001
Analysis of Arylsulfatases A and B, Acid Phosphatase, Lactate Dehydrogenase, and Aspartate Transaminase in Chronic Periapical Lesions of Endodontic Origin Akbar Khayat, DDS, MScD, Nahal Vesal, DDS, and Mojgan Rasti, MSc
Aqrabawi et al. (4) demonstrated higher levels of arylsulfatase A (ASA) and marked activity of arylsulfatase B (ASB) (both of lysosomal origin) in periapical lesions. They also stated that arylsulfatases present in periapical lesions are of human rather than bacterial origin. Another lysosomal enzyme that may be involved in periapical pathosis is acid phosphatase (ACP). This enzyme is an acid hydrolase and has been postulated to be involved in bone resorption (5) and intracellular digestion (6). Lactate dehydrogenase (LDH), a cytoplasmic enzyme of anaerobic glycolysis, also increases in activity when the pulp becomes necrotic and oxidative metabolism and ATP production diminish (7). Due to the presence of insufficient data on the activities of some lysosomal enzymes, such as acid hydrolases and other cytoplasmic enzymes involved in the pathogenesis of periapical lesions, a study was undertaken to measure the activities of ASA and ASB, ACP, LDH, and glutamate oxalacetate transaminase (aspartate transaminase) (GOT) in periapical lesions and compare their values with those found in normal periapical tissues.
Attempts were made to detect and measure the activities of arylsulfatases. A&B acid phosphatase, lactate dehydrogenase, and glutamate oxaloacetate transaminase (aspartate transaminase) enzymes in human chronic lesions of endodontic origin. Thirteen periapical lesions of endodontic origin and 11 noninflamed control periapical tissues were obtained. The specimens were carried to the laboratory on liquid nitrogen and kept at ⴚ70°C. Samples were thawed, homogenized, and then assayed for enzyme activities. The specific activities of arylsulfatase A (nmol/hr/mg protein) were 55.0 ⴞ 10.7 (chronic lesions) vs. 3.4 ⴞ 2.2 (controls) (p < 0.01). Arylsulfatase B specific activities (nmol/hr/mg protein) were 50.3 ⴞ 6.4 (chronic lesions) vs 91.8 ⴞ 18.4 (controls). Total acid phosphatase activities (mU/mg protein) were 45.8 ⴞ 6.6 (chronic lesions) vs. 26.8 ⴞ 3.1 (controls). Lactate dehydrogenase activities (Berger-Broida units/mg protein) of the chronic periapical lesions were significantly higher than the control group (362 ⴞ 63.2) vs. (140 ⴞ 46.0) (p < 0.05). There was no significant difference between the specific activities of aspartate transaminase in chronic lesions and the control group (68.0 ⴞ 14.5) vs. (53.0 ⴞ 10.4) mU/mg protein).
MATERIALS AND METHODS Tissue from chronic periapical lesions of endodontic origin was obtained at the time of endodontic surgery from patients who were referred to the Department of Endodontics, School of Dentistry, Shiraz University, Iran. Patients were evaluated for systemic diseases and periodontal problems, and such patients were excluded from the study. The noninflamed control periapical tissues were obtained after extraction of healthy teeth for orthodontic reasons. Specimens were carried to the laboratory on liquid nitrogen and kept at ⫺70°C. Frozen samples were thawed and homogenized at 4°C in 4 ml of 0.9% NaCl in a hand-operated ground glass Potter homogenizer with 20 up-and-down strokes. The homogenate was centrifuged for 10 min at 11,000 ⫻ g, and the supernatants were kept at ⫺70°C before enzymatic analysis.
It is known that one of the most important causes of periapical lesions is bacteria from infected root canals. Bacteria as a living irritant can release enzymes or can affect cells to elaborate enzymes. Dahlen et al. (1) reported that production of various enzymes may be related to the bacterial invasion of the tissue. Hashioka et al. (2) determined the correlation between clinical symptoms and the activity of enzymes produced by bacteria isolated from the infected root canals. Some of the enzymes derived from cells are contained in cell organelles called lysosomes. Release of lysosomal enzymes after phagocytosis may result in damage to the surrounding cells and tissues (3).
Enzyme Analysis ASA and ASB activities were determined according to the method described by Baum et al. (8), except that the dialysis step was omitted. Activity of ASA was determined upon addition of 0.3 ml of reagent A (10 mM nitrocatechol sulfate, 0.5 mM sodium 285
286
Khayat et al.
Journal of Endodontics
TABLE 1. Specific activities of ASA, ASB, ACP, LDH, and GOT in chronic periapical lesions and their comparison with those of the control groups Enzyme Activities
No. of Samples
ASA (nmol/hr/mg protein) ASB (nmol/hr/mg protein) ACP (mU/mg protein) LDH (Berger-Broida units/mg protein) GOT (U/mg protein)
Range
Mean ⫾ SEM
11 Lesions 7 Controls 11 Lesions 7 Controls 13 Lesions 11 Controls 13 Lesions 11 Controls
18.8–119.5 0–16.3 23.6–81.4 36.6–147.1 16.6–80.8 12.3–42.5 0–758.1 0–454.5
55.0 ⫾ 10.7 3.4 ⫾ 2.2 50.3 ⫾ 6.4 91.8 ⫾ 18.4 45.8 ⫾ 6.6 26.8 ⫾ 3.1 362 ⫾ 63.2 140 ⫾ 46.0
10 Lesions 7 Controls
21.0–175.2 23.3–98.9
68.0 ⫾ 14.5 53.0 ⫾ 10.4
p Values p ⬍0.01 p ⬍0.05 p ⬍0.05 p ⬍0.05
Not significant
Values are the means ⫾ SEM.
pyrophosphate, 1.7 M NaCl in 0.5 M sodium acetate buffer, pH 5.0) to 0.3 ml of the 11,000 ⫻ g supernatant and incubation at 37°C for 1 hr. The reaction was terminated on addition of 0.3 ml of 1 N NaOH. The absorbance of 4-nitrocatechol produced was measured in a Shimatzu spectrophotometer at 515 nm. ASB activity was determined by adding 0.3 ml of the 11,000 ⫻ g supernatant to each of two sets of assay tubes containing 0.3 ml of reagent B (50 mM nitrocatechol sulfate, 10 mM barium acetate in 0.5 M sodium acetate buffer, pH 6.0). The mixtures were incubated at 37°C. The reaction in one set of the tubes was terminated after 30 min and in another set after 90 min by adding 0.3 ml of 1.0 N NaOH. The absorbance of the liberated 4-nitrocatechol was measured spectrophotometrically at 515 nm. Control tubes in which the enzyme was inactivated by 1 N NaOH before the addition of substrates were used to set the spectrophotometer to zero absorbance. Preparation of standard curves and calculation of enzyme activities were as described by Baum et al (8). LDH in experimental and control specimens was measured by a kit produced by Darman Kave (Isfahan, Iran). The procedure is based on the method of Cabaud and Wroblewski (9), in which the enzyme source is incubated at 37°C with a given amount of pyruvate and NADH, as substrates. At the end of the incubation period, the remaining pyruvate is reacted with 2,4-dinitrophenylhydrazine and produces a brown color in an alkaline solution. The enzyme activity is calculated by measuring the absorbance of the colored solution at 450 nm. ACP was determined by a kit produced by Zist Chemie (Tehran, Iran) based on a procedure described by the Enza-trol manual (10), in which the enzyme catalyzes the hydrolysis of p-nitrophenyl phosphate in an acidic pH. p-Nitrophenol produced per unit time is proportional to enzyme activity that is measured in an alkaline medium at 405 nm. The activity of GOT in specimens was determined by a kit produced by Zist Chemie (Tehran, Iran) based on the procedure of Reitman and Frankel (11). In this procedure, liberated oxalacetate is decarboxylated to pyruvate and is converted to a hydrazone. The hydrazone produces a brown color in an alkaline medium that is measured at 505 nm. RESULTS Protein in 11,000 ⫻ g supernatants was measured by a method described by Bradford (12). Specific activities of various enzymes were expressed in terms of their respective activity units per mg protein. Student’s t test was used for the statistical analysis of data.
Activities and specific activities of the enzymes ASA, ASB, ACP, LDH, and GOT in chronic periapical lesions and the various control samples were determined. The ranges, mean values, and level of significance of various enzyme specific activities of the chronic periapical lesions and normal controls were calculated and shown in Table 1. As shown in Table 1, the specific activity of ASA in chronic periapical lesions was statistically higher than that of the control group (p ⬍ 0.01), whereas that of ASB was statistically lower than the control group (p ⬍ 0.05). As indicated in Table 1, the specific activity of ACP and LDH in the chronic periapical lesions were statistically higher than those of their control groups (p ⬍ 0.05). There was, however, no statistically significant difference in the specific activities of GOTs of the two groups.
DISCUSSION The results of this study show a pattern of multiple enzymatic activities in the pathogenesis of human lesions of endodontic origin. In this study, the specific activity of ASA is almost 17 times higher than that of the control specimens. Aqrabawi et al. (4) showed that ASA activity in chronic periapical lesions increases 2 to 5 times more than the control groups. These authors express enzyme units in terms of n-molar/hr instead of n-moles/hr/ml, which is the conventional enzyme unit. Our data (Table 1) also show significantly higher specific activity of ASB in the control group. Because our control group was of a much younger age than the experimental group (10 to 13 yr vs. 13 to 54 yr), it may be possible that ASB was actually acting as a sulfotransferase for the synthesis of sulfated proteoglycans in the periodontal ligament space (13). Arylsulfatase is a lysosomal enzyme and is found not only in polymorphonuclear cells, but in other cells such as fibroblasts (14), mast cells (15), macrophages and osteoclasts (16). The specific activity of LDH was more than twice that of the control groups (Table 1). LDH is an enzyme of anaerobic glycolysis and is released on cell death and may be used as a marked for cellular necrosis. Lamster et al. (17) measured the activity of LDH in gingival crevicular fluid and demonstrated that, after scaling and root planing, the activity of LDH in healthy or mildly inflamed gingival sulcus is approximately 700 to 1000 times higher. It has been postulated that the sources of LDH include polymorphonuclear cells, epithelial cells (18), and fibroblasts (19). Because lesions of endodontic origin are defensive and are healthy tissue,
Vol. 27, No. 4, April 2001
Enzymes in Periapex
increased specific activity of LDH in lesions of endodontic origin is consistent with the findings of the literature. In this study the much higher specific activity of ACP in chronic lesions, compared with controls, is probably indicative of bone resorption, because the increased activity of ACP reflects osteoclastic activity (5). There was no significant difference between the GOT specific activities of the chronic lesions and the normal group. GOT has been used as a promising marker of tissue breakdown in periodontitis (20). So far no other report on the significance of GOT in dental medicine is found in the literature. This research was supported by a grant from the Medical Research Council of Shiraz University of Medical Sciences. Dr. Khayat is associate professor and chairman, Department of Endodontics, School of Dental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Dr. Vesal is a former endodontic resident, School of Dental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Ms. Rasti is an instructor, Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran. Address requests for reprints to Dr. Akbar Khayat, School of Dental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
References 1. Dahlen G, Wikstrom M, Moller A. Production of histologic enzymes by a combination of oral bacteria with known pathogenicity. J Dent Res 1983; 62:1041– 4. 2. Hashioka K, Zuzuki K, et al. Relationship between clinical symptoms and enzyme-producing bacteria isolated from infected root canal. J Endodon 1994;20:75–7. 3. Seltzer S, Bender IB. The dental pulp, biologic considerations in dental procedures. 3rd ed. Tokyo: Ishiyaku Euro America, Inc., 1990:155. 4. Aqrabawi J, Schilder H, Toselli P, Franzblau C. Biochemical and histochemical analysis of the enzyme arylsulfatase in human lesions of endodontic origin. J Endodon 1993;19:335– 8.
287
5. De Duve C. Lysosomes, new groups of cytoplasmic particles. In: Hayashi T, ed. Subcellular particles. New York: Ronald Press, 1959. 6. Woessner JF. Acid hydrolases of connective tissue. In: Hall DA, ed. International review of connective tissue research. Vol. 3. New York: Academic Press, 1965:201– 60. 7. Le Bell YL, Larmas M. Adenosine-5-triphosphate levels of the human tooth pulp during health and disease. Arch Oral Biol 1979;24:313. 8. Baum H, Dodgson KS, Spencer B. The assay of arylsulfatases A and B in human urine. Clin Chim Acta 1959;4:453–5. 9. Cabaud PC, Wroblewski F. Colorimetric measurement of lactate dehydrogenase in biological fluids. Am J Clin Pathol 1958;30:234 – 6. 10. Dade Reagents, Inc. Determination of alkaline and acid phosphatases. In: Enza-trol. Manual of clinical methods. Miami: Dade Reagents, Inc., 1963: 41. 11. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic-oxaloacetic and glutamic pyruvic transaminase. Am J Clin Pathol 1957;28:56. 12. Bradford MA. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248 –54. 13. Gibson WA, Fullmer HM. Histochemistry of the periodontal ligament. V. The arylsulfatases. J Periodontol 1970;41:102– 4. 14. Mercelis R, Van Elsen AF, Leroy JG. Arylsulfatases A and B in human diploid fibroblasts: differential assay with 4-methylumbelliferylsulfate and AgNO3. Clin Chim Acta 1979;93:85–92. 15. Lynch SM, Austen KF, Wasseerman SI. Release of arylsulfatase A but not B from rat mast cells by noncytolytic secretory stimuli. J Immunol 1978; 121:1394 –9. 16. Dorey CK, Bick KL. Ultrahistochemical analysis of glycosaminoglycan hydrolysis in the rat periodontal ligament. Calcif Tissue Res 1977;24:143–9. 17. Lamster IB, Mandella RG, Gordon JM. Lactate dehydrogenase activity in gingival crevicular fluid collected with filter paper strips: analysis in subjects with noninflamed and mildly inflamed gingiva. J Clin Periodontol 1985;12:153– 61. 18. Lamster IB, Oshrain RL, Gordon JM. Enzyme activity in human gingival crevicular fluid: considerations in data reporting based on analysis of individual crevicular sites. J Clin Periodontol 1986;13:799 – 804. 19. Page RC, Schroeder HE. The pathogenesis of chronic inflammatory periodontal disease: a summary of current work. Lab Investig 1976;33:235– 49. 20. Fine DH, Mandel ID. Indicators of periodontal disease activity: an evaluation. J Clin Periodontol 1986;13:533– 46.
You Might Be Interested Devotees of popular press accounts of motoring tragedies and mystery novel aficionados are familiar with blood-alcohol level findings and their implications for pre-death behavior. It turns out that blood-alcohol levels may be significantly increased by the post-mortem invasion of the blood stream by gut bacteria which convert glucose to alcohol (BMJ 316:87). Samples taken from the vitreous humor are far more accurate because it is well protected from bacteria after death. So we may be reading, “His eye-alcohol level exceeded. . . .” William Cornelius
Printed in U.S.A. VOL. 27, NO. 4, APRIL 2001
Analysis of Arylsulfatases A and B, Acid Phosphatase, Lactate Dehydrogenase, and Aspartate Transaminase in Chronic Periapical Lesions of Endodontic Origin Akbar Khayat, DDS, MScD, Nahal Vesal, DDS, and Mojgan Rasti, MSc
Aqrabawi et al. (4) demonstrated higher levels of arylsulfatase A (ASA) and marked activity of arylsulfatase B (ASB) (both of lysosomal origin) in periapical lesions. They also stated that arylsulfatases present in periapical lesions are of human rather than bacterial origin. Another lysosomal enzyme that may be involved in periapical pathosis is acid phosphatase (ACP). This enzyme is an acid hydrolase and has been postulated to be involved in bone resorption (5) and intracellular digestion (6). Lactate dehydrogenase (LDH), a cytoplasmic enzyme of anaerobic glycolysis, also increases in activity when the pulp becomes necrotic and oxidative metabolism and ATP production diminish (7). Due to the presence of insufficient data on the activities of some lysosomal enzymes, such as acid hydrolases and other cytoplasmic enzymes involved in the pathogenesis of periapical lesions, a study was undertaken to measure the activities of ASA and ASB, ACP, LDH, and glutamate oxalacetate transaminase (aspartate transaminase) (GOT) in periapical lesions and compare their values with those found in normal periapical tissues.
Attempts were made to detect and measure the activities of arylsulfatases. A&B acid phosphatase, lactate dehydrogenase, and glutamate oxaloacetate transaminase (aspartate transaminase) enzymes in human chronic lesions of endodontic origin. Thirteen periapical lesions of endodontic origin and 11 noninflamed control periapical tissues were obtained. The specimens were carried to the laboratory on liquid nitrogen and kept at ⴚ70°C. Samples were thawed, homogenized, and then assayed for enzyme activities. The specific activities of arylsulfatase A (nmol/hr/mg protein) were 55.0 ⴞ 10.7 (chronic lesions) vs. 3.4 ⴞ 2.2 (controls) (p < 0.01). Arylsulfatase B specific activities (nmol/hr/mg protein) were 50.3 ⴞ 6.4 (chronic lesions) vs 91.8 ⴞ 18.4 (controls). Total acid phosphatase activities (mU/mg protein) were 45.8 ⴞ 6.6 (chronic lesions) vs. 26.8 ⴞ 3.1 (controls). Lactate dehydrogenase activities (Berger-Broida units/mg protein) of the chronic periapical lesions were significantly higher than the control group (362 ⴞ 63.2) vs. (140 ⴞ 46.0) (p < 0.05). There was no significant difference between the specific activities of aspartate transaminase in chronic lesions and the control group (68.0 ⴞ 14.5) vs. (53.0 ⴞ 10.4) mU/mg protein).
MATERIALS AND METHODS Tissue from chronic periapical lesions of endodontic origin was obtained at the time of endodontic surgery from patients who were referred to the Department of Endodontics, School of Dentistry, Shiraz University, Iran. Patients were evaluated for systemic diseases and periodontal problems, and such patients were excluded from the study. The noninflamed control periapical tissues were obtained after extraction of healthy teeth for orthodontic reasons. Specimens were carried to the laboratory on liquid nitrogen and kept at ⫺70°C. Frozen samples were thawed and homogenized at 4°C in 4 ml of 0.9% NaCl in a hand-operated ground glass Potter homogenizer with 20 up-and-down strokes. The homogenate was centrifuged for 10 min at 11,000 ⫻ g, and the supernatants were kept at ⫺70°C before enzymatic analysis.
It is known that one of the most important causes of periapical lesions is bacteria from infected root canals. Bacteria as a living irritant can release enzymes or can affect cells to elaborate enzymes. Dahlen et al. (1) reported that production of various enzymes may be related to the bacterial invasion of the tissue. Hashioka et al. (2) determined the correlation between clinical symptoms and the activity of enzymes produced by bacteria isolated from the infected root canals. Some of the enzymes derived from cells are contained in cell organelles called lysosomes. Release of lysosomal enzymes after phagocytosis may result in damage to the surrounding cells and tissues (3).
Enzyme Analysis ASA and ASB activities were determined according to the method described by Baum et al. (8), except that the dialysis step was omitted. Activity of ASA was determined upon addition of 0.3 ml of reagent A (10 mM nitrocatechol sulfate, 0.5 mM sodium 285
286
Khayat et al.
Journal of Endodontics
TABLE 1. Specific activities of ASA, ASB, ACP, LDH, and GOT in chronic periapical lesions and their comparison with those of the control groups Enzyme Activities
No. of Samples
ASA (nmol/hr/mg protein) ASB (nmol/hr/mg protein) ACP (mU/mg protein) LDH (Berger-Broida units/mg protein) GOT (U/mg protein)
Range
Mean ⫾ SEM
11 Lesions 7 Controls 11 Lesions 7 Controls 13 Lesions 11 Controls 13 Lesions 11 Controls
18.8–119.5 0–16.3 23.6–81.4 36.6–147.1 16.6–80.8 12.3–42.5 0–758.1 0–454.5
55.0 ⫾ 10.7 3.4 ⫾ 2.2 50.3 ⫾ 6.4 91.8 ⫾ 18.4 45.8 ⫾ 6.6 26.8 ⫾ 3.1 362 ⫾ 63.2 140 ⫾ 46.0
10 Lesions 7 Controls
21.0–175.2 23.3–98.9
68.0 ⫾ 14.5 53.0 ⫾ 10.4
p Values p ⬍0.01 p ⬍0.05 p ⬍0.05 p ⬍0.05
Not significant
Values are the means ⫾ SEM.
pyrophosphate, 1.7 M NaCl in 0.5 M sodium acetate buffer, pH 5.0) to 0.3 ml of the 11,000 ⫻ g supernatant and incubation at 37°C for 1 hr. The reaction was terminated on addition of 0.3 ml of 1 N NaOH. The absorbance of 4-nitrocatechol produced was measured in a Shimatzu spectrophotometer at 515 nm. ASB activity was determined by adding 0.3 ml of the 11,000 ⫻ g supernatant to each of two sets of assay tubes containing 0.3 ml of reagent B (50 mM nitrocatechol sulfate, 10 mM barium acetate in 0.5 M sodium acetate buffer, pH 6.0). The mixtures were incubated at 37°C. The reaction in one set of the tubes was terminated after 30 min and in another set after 90 min by adding 0.3 ml of 1.0 N NaOH. The absorbance of the liberated 4-nitrocatechol was measured spectrophotometrically at 515 nm. Control tubes in which the enzyme was inactivated by 1 N NaOH before the addition of substrates were used to set the spectrophotometer to zero absorbance. Preparation of standard curves and calculation of enzyme activities were as described by Baum et al (8). LDH in experimental and control specimens was measured by a kit produced by Darman Kave (Isfahan, Iran). The procedure is based on the method of Cabaud and Wroblewski (9), in which the enzyme source is incubated at 37°C with a given amount of pyruvate and NADH, as substrates. At the end of the incubation period, the remaining pyruvate is reacted with 2,4-dinitrophenylhydrazine and produces a brown color in an alkaline solution. The enzyme activity is calculated by measuring the absorbance of the colored solution at 450 nm. ACP was determined by a kit produced by Zist Chemie (Tehran, Iran) based on a procedure described by the Enza-trol manual (10), in which the enzyme catalyzes the hydrolysis of p-nitrophenyl phosphate in an acidic pH. p-Nitrophenol produced per unit time is proportional to enzyme activity that is measured in an alkaline medium at 405 nm. The activity of GOT in specimens was determined by a kit produced by Zist Chemie (Tehran, Iran) based on the procedure of Reitman and Frankel (11). In this procedure, liberated oxalacetate is decarboxylated to pyruvate and is converted to a hydrazone. The hydrazone produces a brown color in an alkaline medium that is measured at 505 nm. RESULTS Protein in 11,000 ⫻ g supernatants was measured by a method described by Bradford (12). Specific activities of various enzymes were expressed in terms of their respective activity units per mg protein. Student’s t test was used for the statistical analysis of data.
Activities and specific activities of the enzymes ASA, ASB, ACP, LDH, and GOT in chronic periapical lesions and the various control samples were determined. The ranges, mean values, and level of significance of various enzyme specific activities of the chronic periapical lesions and normal controls were calculated and shown in Table 1. As shown in Table 1, the specific activity of ASA in chronic periapical lesions was statistically higher than that of the control group (p ⬍ 0.01), whereas that of ASB was statistically lower than the control group (p ⬍ 0.05). As indicated in Table 1, the specific activity of ACP and LDH in the chronic periapical lesions were statistically higher than those of their control groups (p ⬍ 0.05). There was, however, no statistically significant difference in the specific activities of GOTs of the two groups.
DISCUSSION The results of this study show a pattern of multiple enzymatic activities in the pathogenesis of human lesions of endodontic origin. In this study, the specific activity of ASA is almost 17 times higher than that of the control specimens. Aqrabawi et al. (4) showed that ASA activity in chronic periapical lesions increases 2 to 5 times more than the control groups. These authors express enzyme units in terms of n-molar/hr instead of n-moles/hr/ml, which is the conventional enzyme unit. Our data (Table 1) also show significantly higher specific activity of ASB in the control group. Because our control group was of a much younger age than the experimental group (10 to 13 yr vs. 13 to 54 yr), it may be possible that ASB was actually acting as a sulfotransferase for the synthesis of sulfated proteoglycans in the periodontal ligament space (13). Arylsulfatase is a lysosomal enzyme and is found not only in polymorphonuclear cells, but in other cells such as fibroblasts (14), mast cells (15), macrophages and osteoclasts (16). The specific activity of LDH was more than twice that of the control groups (Table 1). LDH is an enzyme of anaerobic glycolysis and is released on cell death and may be used as a marked for cellular necrosis. Lamster et al. (17) measured the activity of LDH in gingival crevicular fluid and demonstrated that, after scaling and root planing, the activity of LDH in healthy or mildly inflamed gingival sulcus is approximately 700 to 1000 times higher. It has been postulated that the sources of LDH include polymorphonuclear cells, epithelial cells (18), and fibroblasts (19). Because lesions of endodontic origin are defensive and are healthy tissue,
Vol. 27, No. 4, April 2001
Enzymes in Periapex
increased specific activity of LDH in lesions of endodontic origin is consistent with the findings of the literature. In this study the much higher specific activity of ACP in chronic lesions, compared with controls, is probably indicative of bone resorption, because the increased activity of ACP reflects osteoclastic activity (5). There was no significant difference between the GOT specific activities of the chronic lesions and the normal group. GOT has been used as a promising marker of tissue breakdown in periodontitis (20). So far no other report on the significance of GOT in dental medicine is found in the literature. This research was supported by a grant from the Medical Research Council of Shiraz University of Medical Sciences. Dr. Khayat is associate professor and chairman, Department of Endodontics, School of Dental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Dr. Vesal is a former endodontic resident, School of Dental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Ms. Rasti is an instructor, Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran. Address requests for reprints to Dr. Akbar Khayat, School of Dental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
References 1. Dahlen G, Wikstrom M, Moller A. Production of histologic enzymes by a combination of oral bacteria with known pathogenicity. J Dent Res 1983; 62:1041– 4. 2. Hashioka K, Zuzuki K, et al. Relationship between clinical symptoms and enzyme-producing bacteria isolated from infected root canal. J Endodon 1994;20:75–7. 3. Seltzer S, Bender IB. The dental pulp, biologic considerations in dental procedures. 3rd ed. Tokyo: Ishiyaku Euro America, Inc., 1990:155. 4. Aqrabawi J, Schilder H, Toselli P, Franzblau C. Biochemical and histochemical analysis of the enzyme arylsulfatase in human lesions of endodontic origin. J Endodon 1993;19:335– 8.
287
5. De Duve C. Lysosomes, new groups of cytoplasmic particles. In: Hayashi T, ed. Subcellular particles. New York: Ronald Press, 1959. 6. Woessner JF. Acid hydrolases of connective tissue. In: Hall DA, ed. International review of connective tissue research. Vol. 3. New York: Academic Press, 1965:201– 60. 7. Le Bell YL, Larmas M. Adenosine-5-triphosphate levels of the human tooth pulp during health and disease. Arch Oral Biol 1979;24:313. 8. Baum H, Dodgson KS, Spencer B. The assay of arylsulfatases A and B in human urine. Clin Chim Acta 1959;4:453–5. 9. Cabaud PC, Wroblewski F. Colorimetric measurement of lactate dehydrogenase in biological fluids. Am J Clin Pathol 1958;30:234 – 6. 10. Dade Reagents, Inc. Determination of alkaline and acid phosphatases. In: Enza-trol. Manual of clinical methods. Miami: Dade Reagents, Inc., 1963: 41. 11. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic-oxaloacetic and glutamic pyruvic transaminase. Am J Clin Pathol 1957;28:56. 12. Bradford MA. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248 –54. 13. Gibson WA, Fullmer HM. Histochemistry of the periodontal ligament. V. The arylsulfatases. J Periodontol 1970;41:102– 4. 14. Mercelis R, Van Elsen AF, Leroy JG. Arylsulfatases A and B in human diploid fibroblasts: differential assay with 4-methylumbelliferylsulfate and AgNO3. Clin Chim Acta 1979;93:85–92. 15. Lynch SM, Austen KF, Wasseerman SI. Release of arylsulfatase A but not B from rat mast cells by noncytolytic secretory stimuli. J Immunol 1978; 121:1394 –9. 16. Dorey CK, Bick KL. Ultrahistochemical analysis of glycosaminoglycan hydrolysis in the rat periodontal ligament. Calcif Tissue Res 1977;24:143–9. 17. Lamster IB, Mandella RG, Gordon JM. Lactate dehydrogenase activity in gingival crevicular fluid collected with filter paper strips: analysis in subjects with noninflamed and mildly inflamed gingiva. J Clin Periodontol 1985;12:153– 61. 18. Lamster IB, Oshrain RL, Gordon JM. Enzyme activity in human gingival crevicular fluid: considerations in data reporting based on analysis of individual crevicular sites. J Clin Periodontol 1986;13:799 – 804. 19. Page RC, Schroeder HE. The pathogenesis of chronic inflammatory periodontal disease: a summary of current work. Lab Investig 1976;33:235– 49. 20. Fine DH, Mandel ID. Indicators of periodontal disease activity: an evaluation. J Clin Periodontol 1986;13:533– 46.
You Might Be Interested Devotees of popular press accounts of motoring tragedies and mystery novel aficionados are familiar with blood-alcohol level findings and their implications for pre-death behavior. It turns out that blood-alcohol levels may be significantly increased by the post-mortem invasion of the blood stream by gut bacteria which convert glucose to alcohol (BMJ 316:87). Samples taken from the vitreous humor are far more accurate because it is well protected from bacteria after death. So we may be reading, “His eye-alcohol level exceeded. . . .” William Cornelius