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The oxygen consumption of gingival crevicular epithelium
The oxygen consumption of gingival crevicular epithelium Bobert E. Morgan, D.D.S., X.8.,* and W. J. Wingo, Ph.D., Birmingham, Ala.
I
nvestigations of the oxygen consumption of human gingivalm4 have revealed that this tissue has a very low respiratory rate and that the presence of inflammation causes this rate to increm, except possibly in those tissues in which degeneration predominates. However, there has been little information regarding alterations in the respiratory rate of the gingival epithelium. Schrader and Schrader,2 using epithelium mechanically separated from subepithelial tissues, found that epithelium from inflamed tissue respired at a rate two to three times that of epithclium secured from normal tissue. Burstone, using histochemical techniques, found that the epithelium in inflamed tissue had an eleva.ted activity of cytochrome oxidase, an enzyme which activates oxygen at the terminal point of the electron transport chain. Since any knowledge of metabolic dera.ngements should have clinic.al significance, it was deemed important to attempt to correlate the oxygen consumption of the crcvicular epithelium with the clinical appearance of the tissue. MATERIALS
AND METHODS
Patients were selected from the periodont,al clinics of the Veterans Administration Hospital in Birmingham, Alabama, and the School of Dentistry, University of Alabama, a.nd were classified according t,o the rriberia of Parfitt.” Only those classified as Group l-t and Group 2-t. were used in this study. The biopsy spccimcns wcrc rcmo\-ed nndcr block :ulcwt.hcsin. nncf plac4 in ~olrl Krebs-Ringer phosphate solution. Each specimen was rinsed sevrral times to remove surface bact.eria and, with the aid of a dissecting microscope, slices were made parallel to the crevicular surface. Several microscopic controls revealed that while the slices were not pure epithelium, they cont,ained only very small This work, carried out while the author was a Clinical Investigator, Veterans Administration Hospital, Birmingham, Ala., was submitted in partial fulfillment of the requirements for the M.S. degree at the Graduate School of the University of Alabama. *Present address: School of Dentistry, University of Alabama.
257
258
Illorgm
md
Wingo
O.S., 0.x & O.P. August, 1966
amounts of nonepithclial tissue elements. A standard time of one hour elapsed between actual biopsy and the determination of oxygen consumption. In order to permit the use of such small tissue samples, a method was devised which utilized the polarographic determination of oxygen.? The method was based upon the fact that dissolved oxygen will be reduced at a platinum electrode at an applied voltage of 0.6 volts and that the resulting current flow is directly proportional to the amount of dissolved oxygen if the surface area of the platinum electrode is held constant. A constant volume cell was constructed from a tuberculin syringe, aa shown in Fig. 1. The tissue was placed in the space below the silver wire screen, and the stirrer wax activated by an external rotating magnet. This prevented polarization of the platinum electrode. The current flow was determined by attaching the cell to an Ampot (E. H. Sargent and Co.), which acted as power supply, microammeter, voltmeter, and potentiometer. The current flow at 0.6 volt could be read to the nearest 0.1 microampere. The cell was calibrated by measuring the current when the cell was filled with solutions containing different amounts of oxygen. There was found to be a linear relat.ionship between oxygen content and resulting current. A calibration curve was run at both the beginning and the end of each experiment. Dur-
-C
E
-B
-A
D.
Fig. 1. Construction of cell. 8, Tuberculin syringe; B, silver wire screen; electrode: P, svrinee &mcrer adiustahle to rnnntaat denth : 73. stirrer.
C, platinum
wire
Oxygen
consumption
of
crericulur
epithelium
259
ing each experiment the current flow was measured a.t 0, 30, and 60 minutes. Thus, since the volume of the ccl1 was known, t.he oxygen consumption could be calculated for each interval. We quantitated the tissue at the end of each experiment by determining its nitrogen content according to the ultramicromethod of Thompkins and Kirk.B The rate of oxygen utilization was expressed as microliters of oxygen consumed per microgram of nitrogen per hour. RESULTS AND DISCUSSION The results, which arc summarized ii1 Table I, indicate that gingival. crevieular epithelium respires with a mean Qo2 of 0.010 microliter of oxygen per microgram of nitrogen per hour and that there was no difference between the two groups on the basis of clinical classification. There are several possible explanations for this. First, the separation of specimens into either Group l+ or Group 2+ WBS based on purely clinical criteria and was quite subject to human error. Perhaps if the tissue had been classified according to microscopic appearance, a different correlation might have been obtained. Second, the method of expressing the respiratory rate may have introduced error. Generally, the respiratory rate is expremed as the amount of oxygen consumed per unit of either wet or dry weight of tissue or per unit of total nitrogen. However, Berenblum and associate9 have shown that tissues have totally different respiratory rates if expressed in terms of nucleic acid content rather than dry weight. Since many of the chemical changes associated with inflammation arc still unknown, the values reported in this article might have had a better correlation wit.h the clinical state if a nucleic acid standard had been used rather than t.he total-nitrogen standard. Another possibility lies in the fact t.hat, since Burstone has shown that there is an increase in cyt.ochrome oxidase activity in gingival epithelium associated with inflammation, one of the earliest inflammatory changes in gingival epithelium might well be an increase in oxygen utilization. If we assume a certain moisture and nitrogen content of epithelial tissue,3 it is possible t.o calculate a respiratory rate based on dry weight and compare it with values reported for whole gingiva.. This reveals that gingival epit.helium respires at a rate a.pproxima.tely twice that of whole gimgiva, which essentially agrees with the findings of Schrader and Schrader.* IJitt.h!
il~~Orl~lahll
is
il~ilihlJlC
rC'&dillg
thy
1ldZL~J&
t-liiwidc~iatics
of
human gingira, and ~nuch work remains to bc: done before we can underst.and the metabolic. events underlying t.hc morphologic changes in gingival disease. Table I Group l+ 2+ “Microliters
A-umber of aamylev 10 9 of oxygen
per microgram
Standard Mean Q.,” 0.010 0.010 of nitrogen
Xange 0.009-0.012 0.008-0.012 per hour.
deviation
0.0011 0.0014
260
Morgm
rind Wing0
O.S., ox & O.P. August, 1966
SUMMARY A method of determining the oxygen consumption of small tissue samples has bc!cn dascribcd. This method was applied to gingival crevicular epithelium and revealed that this tissue has a mean Qo2 of 0.010 microliter of oxygen per microgram of nitrogen per hour, which is approximately twice that of whole gingiva. There was no correlation between Qoz and the clinical state of the tissue. Possible explanations for these results have been discussed. REFERENCES
of Oxygen Consumption in Normal I., Turesky, S., and Hill, R.: Determination and Inflamed Gingiva Using the Warburg Manometric Technique, J. D. Rec. 28: 83-94, 1949. Schrader, H. K., and Schrader, R.: Oxygen Uptake by Normal and Inflamed Gingiva and Saliva, Helvet. odont. acta 1: 13-16, 1957. Senter, A. D., Eiler, J. J., and Lee, K. H.: Endogenous Respiration of Human Gingival Tissue, Proc. Sot. Exper. Biol. & Med. 100: 323-324, 1959. Technique for Study of Human Volpe, A. K., and Manhold, J. H.: Microrespirometer Gingival Tissue, J. D. Res. 41: 420-426, 1962. Study of Cytochrome Oxidase in Normal and Inflamed Burstone, M. 8.: Histochemical Gingiva, ORAL &JR&, ORAL MED. & ORAL PATH. 13: 1501-1505, 1960. Par%, G. J.: Five Year Longitudinal Study of the Gingival Condition of a Group of Children in England, J. Periodont. 28: 26-32, 1957. Connelly, C. Y.: Methods for Measuring Tissue Oxygen Tension; Theory and Evaluation: The Oxvnen Electrode. Fed. Proc. 16: 681-684. 1957. Thompk&, E. R., a& Kirk, P. -L.: An Improved Diffusion Method for Total Nitrogen, J. Biol. Chem. 142: 477-485, 1942. Berenblum, I., Chain, E., and Heatley, N. G.: The Study of Metabolic Activities of Small Amounts of Surviving Tissues, Bicxhem. J. 33: 68-74, 1939.
1. Glickman,
2. 3. 4. 5. 0. 7. 8. 9.
I
nvestigations of the oxygen consumption of human gingivalm4 have revealed that this tissue has a very low respiratory rate and that the presence of inflammation causes this rate to increm, except possibly in those tissues in which degeneration predominates. However, there has been little information regarding alterations in the respiratory rate of the gingival epithelium. Schrader and Schrader,2 using epithelium mechanically separated from subepithelial tissues, found that epithelium from inflamed tissue respired at a rate two to three times that of epithclium secured from normal tissue. Burstone, using histochemical techniques, found that the epithelium in inflamed tissue had an eleva.ted activity of cytochrome oxidase, an enzyme which activates oxygen at the terminal point of the electron transport chain. Since any knowledge of metabolic dera.ngements should have clinic.al significance, it was deemed important to attempt to correlate the oxygen consumption of the crcvicular epithelium with the clinical appearance of the tissue. MATERIALS
AND METHODS
Patients were selected from the periodont,al clinics of the Veterans Administration Hospital in Birmingham, Alabama, and the School of Dentistry, University of Alabama, a.nd were classified according t,o the rriberia of Parfitt.” Only those classified as Group l-t and Group 2-t. were used in this study. The biopsy spccimcns wcrc rcmo\-ed nndcr block :ulcwt.hcsin. nncf plac4 in ~olrl Krebs-Ringer phosphate solution. Each specimen was rinsed sevrral times to remove surface bact.eria and, with the aid of a dissecting microscope, slices were made parallel to the crevicular surface. Several microscopic controls revealed that while the slices were not pure epithelium, they cont,ained only very small This work, carried out while the author was a Clinical Investigator, Veterans Administration Hospital, Birmingham, Ala., was submitted in partial fulfillment of the requirements for the M.S. degree at the Graduate School of the University of Alabama. *Present address: School of Dentistry, University of Alabama.
257
258
Illorgm
md
Wingo
O.S., 0.x & O.P. August, 1966
amounts of nonepithclial tissue elements. A standard time of one hour elapsed between actual biopsy and the determination of oxygen consumption. In order to permit the use of such small tissue samples, a method was devised which utilized the polarographic determination of oxygen.? The method was based upon the fact that dissolved oxygen will be reduced at a platinum electrode at an applied voltage of 0.6 volts and that the resulting current flow is directly proportional to the amount of dissolved oxygen if the surface area of the platinum electrode is held constant. A constant volume cell was constructed from a tuberculin syringe, aa shown in Fig. 1. The tissue was placed in the space below the silver wire screen, and the stirrer wax activated by an external rotating magnet. This prevented polarization of the platinum electrode. The current flow was determined by attaching the cell to an Ampot (E. H. Sargent and Co.), which acted as power supply, microammeter, voltmeter, and potentiometer. The current flow at 0.6 volt could be read to the nearest 0.1 microampere. The cell was calibrated by measuring the current when the cell was filled with solutions containing different amounts of oxygen. There was found to be a linear relat.ionship between oxygen content and resulting current. A calibration curve was run at both the beginning and the end of each experiment. Dur-
-C
E
-B
-A
D.
Fig. 1. Construction of cell. 8, Tuberculin syringe; B, silver wire screen; electrode: P, svrinee &mcrer adiustahle to rnnntaat denth : 73. stirrer.
C, platinum
wire
Oxygen
consumption
of
crericulur
epithelium
259
ing each experiment the current flow was measured a.t 0, 30, and 60 minutes. Thus, since the volume of the ccl1 was known, t.he oxygen consumption could be calculated for each interval. We quantitated the tissue at the end of each experiment by determining its nitrogen content according to the ultramicromethod of Thompkins and Kirk.B The rate of oxygen utilization was expressed as microliters of oxygen consumed per microgram of nitrogen per hour. RESULTS AND DISCUSSION The results, which arc summarized ii1 Table I, indicate that gingival. crevieular epithelium respires with a mean Qo2 of 0.010 microliter of oxygen per microgram of nitrogen per hour and that there was no difference between the two groups on the basis of clinical classification. There are several possible explanations for this. First, the separation of specimens into either Group l+ or Group 2+ WBS based on purely clinical criteria and was quite subject to human error. Perhaps if the tissue had been classified according to microscopic appearance, a different correlation might have been obtained. Second, the method of expressing the respiratory rate may have introduced error. Generally, the respiratory rate is expremed as the amount of oxygen consumed per unit of either wet or dry weight of tissue or per unit of total nitrogen. However, Berenblum and associate9 have shown that tissues have totally different respiratory rates if expressed in terms of nucleic acid content rather than dry weight. Since many of the chemical changes associated with inflammation arc still unknown, the values reported in this article might have had a better correlation wit.h the clinical state if a nucleic acid standard had been used rather than t.he total-nitrogen standard. Another possibility lies in the fact t.hat, since Burstone has shown that there is an increase in cyt.ochrome oxidase activity in gingival epithelium associated with inflammation, one of the earliest inflammatory changes in gingival epithelium might well be an increase in oxygen utilization. If we assume a certain moisture and nitrogen content of epithelial tissue,3 it is possible t.o calculate a respiratory rate based on dry weight and compare it with values reported for whole gingiva.. This reveals that gingival epit.helium respires at a rate a.pproxima.tely twice that of whole gimgiva, which essentially agrees with the findings of Schrader and Schrader.* IJitt.h!
il~~Orl~lahll
is
il~ilihlJlC
rC'&dillg
thy
1ldZL~J&
t-liiwidc~iatics
of
human gingira, and ~nuch work remains to bc: done before we can underst.and the metabolic. events underlying t.hc morphologic changes in gingival disease. Table I Group l+ 2+ “Microliters
A-umber of aamylev 10 9 of oxygen
per microgram
Standard Mean Q.,” 0.010 0.010 of nitrogen
Xange 0.009-0.012 0.008-0.012 per hour.
deviation
0.0011 0.0014
260
Morgm
rind Wing0
O.S., ox & O.P. August, 1966
SUMMARY A method of determining the oxygen consumption of small tissue samples has bc!cn dascribcd. This method was applied to gingival crevicular epithelium and revealed that this tissue has a mean Qo2 of 0.010 microliter of oxygen per microgram of nitrogen per hour, which is approximately twice that of whole gingiva. There was no correlation between Qoz and the clinical state of the tissue. Possible explanations for these results have been discussed. REFERENCES
of Oxygen Consumption in Normal I., Turesky, S., and Hill, R.: Determination and Inflamed Gingiva Using the Warburg Manometric Technique, J. D. Rec. 28: 83-94, 1949. Schrader, H. K., and Schrader, R.: Oxygen Uptake by Normal and Inflamed Gingiva and Saliva, Helvet. odont. acta 1: 13-16, 1957. Senter, A. D., Eiler, J. J., and Lee, K. H.: Endogenous Respiration of Human Gingival Tissue, Proc. Sot. Exper. Biol. & Med. 100: 323-324, 1959. Technique for Study of Human Volpe, A. K., and Manhold, J. H.: Microrespirometer Gingival Tissue, J. D. Res. 41: 420-426, 1962. Study of Cytochrome Oxidase in Normal and Inflamed Burstone, M. 8.: Histochemical Gingiva, ORAL &JR&, ORAL MED. & ORAL PATH. 13: 1501-1505, 1960. Par%, G. J.: Five Year Longitudinal Study of the Gingival Condition of a Group of Children in England, J. Periodont. 28: 26-32, 1957. Connelly, C. Y.: Methods for Measuring Tissue Oxygen Tension; Theory and Evaluation: The Oxvnen Electrode. Fed. Proc. 16: 681-684. 1957. Thompk&, E. R., a& Kirk, P. -L.: An Improved Diffusion Method for Total Nitrogen, J. Biol. Chem. 142: 477-485, 1942. Berenblum, I., Chain, E., and Heatley, N. G.: The Study of Metabolic Activities of Small Amounts of Surviving Tissues, Bicxhem. J. 33: 68-74, 1939.
1. Glickman,
2. 3. 4. 5. 0. 7. 8. 9.