Effect Of Inflammation Upon Human Gingival Oxidative Metabolism
Introduction
Cytochrome oxidase (E.C1.9.3.1.) and NADH cytochrome C reductase (E.C.I, 6. 2. I.) were determined in essentially normal human gingiva (Eichel & Sharhrik 1964), however these parameters have not been previously analyzed in relation to the effects imposed by gingival inflammation. Endogenous respiration studies revealed that the QOj is stimulated in mild gingival inflammation, and depressed in highly inflamed gingiva (Glickman el al. 1949, Manhold & Volpe 196.1). On the other hand recent polarographie studies (Zajieck & Kindlova 1972) revealed that initial QO_. values remained constant in human gingiva and were not significantly altered by the degree of inflammation. In light of these divergent views, we wish to report on the effect of inflammation upon cytochrome oxidase and NADH cytochrome c reductase activities in total homogenates of human gingiva.
Methods and Materials
Tissue Preparation
Human gingival biopsy specimens were obtained irom 22 male patients (age 23-72), undergoing periodontal treatment. Regional block anesthesia was administered to avoid infiltration of the tissue. The tissue was surgically removed and cut into two portions one of which was prepared for histologic study. The other was washed in cold 0.075 M phosphate buffer pH 7.0. to remove adhering blood and quickly frozen at - 70°C on dry ice. The fro/en gingival samples were analyzed for their enzymatic activity within one day after their removal. Gingival homogenates were prepared by a modification of the methods (Hoober & Bernstein 1966) utilized tor the homogenization of rat skin. The frozen gingival tissue (25 -5U mg wet wt.) was immersed in liquid N, (-270'C) for 3 minutes, and pulverized to small granular fragments. The gingival fragments were homogenized in cold buffer in Ten Broeck ground glass homogenizers which were ground to fit with a clearance tolerance o( (1.1-0.15 mm. The enzymatic activities were determined immediately after tissue homogenization.
Enzyme Analysis
Cytochrome oxidase (E. C. 1. 9. 3. 1.) (Wainio et al. 1951) was determined spectrophotometrically hy following the oxidation of dithionite reduced cytochrome c* at I al 25°C in a Gilford Model 2400 spectrophotometer. In nil experiments the auto-oxidation rate of lerrocytoehrome c before the addition of the enzyme was negligible. The E.VHI "", absorbance values for homogenate settling were for the most part negligible (0.0 % in 16 samples), and where detected they ranged from 0,(12-13.0'; of the enzymatic value. The average settling value was 2.5 ± 0.9 ci for the 22 samples which were analyzed. Settling values were determined by re-reducing the reaction media with excess dithionite, resuspending the homogenate in the reaction media, and following the change in optical absorbance at Ejaonni vs- time graphically. The settling values were subtracted from the total observed absorbance value obtained in the affected enzyme assays, and the specific activities were determined from the corrected values. The reaction mixture contained: 75 mM KH..PO|-Na,.HPO4 buffer pH 6.75. 5.6 uM cytochrome c and (25-150) jig protein (homogenale) in a final volume of 300 u. Cytochrome c concentration was determined from the millimolar extinction coefficient of cytochrome c H-,,-,;, t]ll = 18.5 mm ' cm ' for the reduced minus oxidized cvtoehrome c.
NADH Cytochrome c Reductase. NADH cytochrome c reductase (E. C. 1. 6. 2. 1.) (Jeng et al. 1968) was determined by following the reduction of cytochrome c at Er,,Tnm at 25'C. The millimolar extinction coefficient utilized for cytochrome c in the cytochrome oxidase assay was utilized for this assay. The assay system contained: 33 mM KHiPOi-NaoHPO., pH 7.75 buffer, S.2 ftM cytochrome c, 3.0mM KCN, 85 /LIM NADH.** and (25-150) ^g protein (homogenate) in final volume of 300 II.
Specific Activities
Specific activities are expressed as nanomoies of cytochrome c oxidized (cytochrome oxidase) or reduced (NADH cytochrome c reductase) per mg protein per min.
Protein Determination
Tissue homogenates (10-20 /A) were digested in 0.05 N NaOH (final concentration) for one hour at 25°C, and analyzed for protein by the method of Lowry et al.. (1951). Crystalline albumin*** was used as a standard.
Statistics
The mean, standard deviation and standard error were determined for each group. Values exceeding the limits ± 1.96 SD were discarded. The difference between each group was analyzed by the "student t" test and all values p < .05 or less were considered to be significant.
Reagents
NADH** (Grade 111!, and cytochrome e* (type I 11 Horse Heart I, were obtained from Sigma Chemical Corp., St. Louis, Mo. Crystalline albumin*** was obtained from Pentex Corp. Kankakee, 111.
Histologic Evaluation
The biopsy specimens were cut at 7 /i and stained with hematoxylin and eosin. Each section was diagnosed as mild, moderate or
224 FINE. EGNOR, F O M T E C C H I O , FROUM, SCOPP AND STAHL
severely inflamed depending on (he extent of inflammatory infiltrate found in the represenlativc sections of the specimens.
Results
Histological ami Clinical Evaluations
Histologic evaluation of the biopsy specimens revealed that 13 gingival samples fell within the mildly inflamed category, mid 9 samples were designated as severely inflamed. The specific activities of cytochrome oxidase, and NADH cytochrome c reductase activities were correlated with the observed degree of gingival inflammation and the data was statistically analyzed.
C 'ytochrome Oxidase
(Cytochrome oxidase specific activity) was found to decline from 5.4 ± 0.7 in mildly inflamed to 3.0 ± 0.3 in severely inflamed gingiva (Table I). The specific activity of (he mildly inflamed was significantly greater (p < .001) that that of the severely inflamed gingival samples.
NADH Cytochrome c Reductase
NADH cytochrome c reductase activity was not altered significantly by the degree of gingival inflammation. The specific activities were 16.5 ± 1.6, and 15.3 ± 2,1 respectively in mildly and severely inflamed gingiva
Discussion
Although the consistency of inflamed gingiva may vary considerably from area to area, and thereby effect the degree of fragmentation, this does not appear to be the case in our preparations. The exposure of our gingival tissues to liquid N.>. pulverization of the (issue, thawing and subsequent homogenization appears to adequately disrupt the cells. Evidence for this interpretation lies in the observation that the distribution of NADH cytochrome c reductase in both mildly inflamed and markedly inflamed gingiva did not vary significantly. If the decline in cytochrome oxidase was influenced by our tissue preparation techniques, similar results should be observed in the NADH cytochrome c reductase distributions. The amount of settling in our assay system was found to be negligible for the most part and it could not account for the marked decline in cytochrome oxidase activity observed in markedly inflamed gingiva.
The elevated cylochiomc oxidase activity observed in our study coincides with the observations made by Manhold and Volpj (1963) in their endogenous QCK respiration studies of mildly inflamed and proliferating human gingiva. The increase in QOL> and cytochrome oxidase in gingiva may reflect increased mitochondrial high energy bond (ATP) synthesis, which is required to sustain the highly synthelic processes associated with DNA synthesis, mitosis, as well as cellular proliferation. Further evidence for the increased bioencrgelic requirements in the pre-proliferating and proliferative stages of cell development is the observation that increased QOL. (endogenous respiration) (Glickman el al. 1949), and cytochrome oxidase (Fine 197(1) reaches peaks in activity at a time when major increases in the mitolic index and proliferation (epitheiialization) occur in regenerating gingival and skin wounds. On the other hand ihe sharp decline in cytochrome oxidase and endogenous QOo observed in markedly inflamed (Manhold & Volpe 1963, Giickman et al. 1949) gtngiva appear to be reflections of changes occurring within the mitochondria as well as other subcellular components during significant gingival tissue destruction. We were unable to delect an increase in cytochrome oxidase in markedly inflamed and proliferating gingiva. although increases in the endogenous QO. was detected by Giickman et al. (1949) in this type of gingival pathology. However, it is conceivable lhal as inflammation subsides, a preproliferative phase (S phase of mitosis) occurs within the migrating cells at the wound edge which requires increased ATP synthesis. Subsequently an increase in endogenous QOL. is also observed. Our cytochrome oxidase data, and the data reported for endogenous respiration QOj do not coincide with the polarographic QO. observations made by Zajieek and Kindlova (1972), who reported thai the initial QO_> values remained constant in human gingiva, and were not significantly altered by the degree of inflammation.
NADH cytochrome c reductase activity was not markedly altered by the degree of inflammation in human gingiva. Our data for human gingiva parallel the observation made by Eichel and Shahrik (1964), for human non-inflamed gingiva. NADH cylochrome c reductase activity was found to be significantly greater than cytohrome oxidase in our studies which correlates with the observation made by Eiehcl and Shahrik
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References
Eichel, B. & Shahrik, S. 1964. Cytochemicul aspects of oidative enzyme metabolism in gingiva. Adv. Oral Biol. 1: 131-174. Fine, A. S. 1970. The biochemical determination of cytochrome oxidase and NADH cyto chrome c rcductase activity in gingiva and skin during wound healing in rats of various ages. Thesis New York University. Fine, A. S., Scopp, I. W. & Egnor. R. 1973a. Subcellular distribution of NADH cytochrome c reductase in rat gingiva. J. Dent. Res. 52: 387. Fine, A. S., Scopp, 1. W., Egnor, R., Froum, S., Thaler, R. & Stahl, S. S. 1973b. Subcellular distribution of oxidative enzymes in human inflamed and dilantin hyperplastic gingiva. J. Dem. Res. 52: Abst. 147. Giickman, I., Turesky, S. & Hill, R. 1949. Determination of oxygen consumption in normal and inflamed gingiva using the Warburg manometric technique. J. Dent. Res. 28: 83-94. Hoober, J. K. & Bernslein, I. A. 1966. Protein synthesis related (o epidermal differentiation. Proe. Nut. Acud. Sci. 56: 594-601. Jeng, M., Hall, C, Crane. F. L., Takahashi, N., Tamura, S. & Folkers, K. 1968.
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