found to increase with increasing flow rate [Anderson, 1949; Thaysen, Thorn

Transcription

1 Quart. J. exp. Phy8iol. (1966) 51, CARBONIC ANHYDRASE ACTIVITY IN SALIVA IN MAN. By I. SZABO,* D. K. MASON and R. McG. HARDEN. From the Dental Hospital and School, and University Department of Medicine, Western Infirmary, Glasgow. (Received for publication 7th January 1966) The carbonic anhydrase activity has been measured in human parotid, submandibular and mixed saliva. In parotid saliva the activity was related to salivary flow rate, rising from less than 1'6 K/ml. at low flow rates to a maximlum of about 2.2 K/ml. at higher rates of flow. The enzyme activity remained constant at flow rates greater than 0-8 ml./min. The concentration in submandibular saliva tended to be less than the concentration in parotid saliva. The enzyme activity in the blood was found to be 1*9 K/ml. Enzyme activity was not affected by storage at 00 C., 370 C. and room temperature for up to 48 hr. Similar values were found when saliva was collected under oil and when saliva was collected without oil. THE enzyme carbonic anhydrase is present in the salivary glands in animals [Yoshimura et al., 1959; Draus, Miklos and Leung, 1962] and in man [Sand, 1949]. Rapp [1946] first demonstrated carbonic anhydrase activity in human saliva, and suggested that the enzyme accelerated the removal of carbon dioxide, resulting in alkalinization of the saliva. Szabo [1965] later confirmed that there was a relationship between enzyme activity and salivary ph. The ph and concentration of bicarbonate in saliva have been found to increase with increasing flow rate [Anderson, 1949; Thaysen, Thorn and Schwartz, 1954; Dawes and Jenkins, 1964]. It seemed of interest, therefore, to study the effect of increasing flow rate on the carbonic anhydrase activity in saliva. The ph of saliva and carbonic anhydrase activity vary with temperature and method of collection [Szabo and Hattyasy, 1965; Rapp, 1946; Altschule and Lewis, 1949]. The effect of collection methods and of storage at different temperatures on the salivary carbonic anhydrase activity has therefore also been studied. METHODS Subjects Studied. - The subjects studied were normal volunteers and included members of the hospital staff. Their ages ranged from 33 to 60 years and all but one were males. None had clinical evidence of metabolic or salivary gland disorders. Saliva Collection. - Parotid saliva was collected using a modified Carlson-Crittenden device constructed of nylon with an external diameter of 20 mm. and an inner chamber diameter of 10 mm. Submandibular saliva was collected by cannulation of the duct [Kerr, 1961], or by a segregator appliance where the subjects had lower anterior teeth [Schneyer, 1955]. Mixed saliva was obtained by spitting at a controlled rate * At present Senior Lecturer Department Prosthetics, II Clinic for Dental and Oral Diseases, Szeged, Hungary. 202

2 Carbonic Anhydrase Activity in Saliva in Man 203 of once per min. [Kerr, 1961]. The parotid and submandibular saliva was passed through a photoelectric drop counter to ensure that the pattern of salivary flow during each collection period was regular [Mason, Harden, Rowan and Alexander, 1966a]. It was then collected in a bottle and its volume measured by weight. In six subjects saliva was also collected under oil. In a further six subjects saliva was collected under resting conditions and after stimulation with salt, chewing paraffin wax, sucking a fruit gum, oxo and lemon juice. In this way saliva secreted at flow rates varying from 0 05 to 1*75 ml./min. was obtained. Parotid, submandibular and mixed saliva was collected after lemon juice stimulation in two subjects on each of three consecutive days. Estimation of Carbonic Anhydrase Activity. -The Gloster [1955] modifications of the method of Krebs and Roughton was used. The estimation was conducted at Carbonic Anhydrose (K/ml) 0A /x flow rate (ml/min) FIG. 1. Carbonic anhydrase activity in parotid saliva secreted at different flow rates in five subjects. a temperature of 25 C. 0 5 ml. of 0-02 M NaHCO3 solution was placed in the side arm of a Warburg flask and 0-2 ml. of the saliva sample in 2-3 ml. 0.1 M phosphate buffer was placed in the flask itself. In a control flask 0-2 ml. distilled water was used in place of the saliva. After 10 min. equilibration the solutions were mixed and the amount of CO2 produced was measured at intervals of 1 min. on the manometer. Each test was carried out in duplicate, the mean value at the fifth min. being taken as the enzyme activity. This was expressed in K/ml. saliva [Mitchell, Pozzani and Fessender, 1945; Altschule and Lewis, 1949]. In seventeen samples the enzyme activity was measured immediately after collection and 24 and 48 hr. later Four samples were kept at 0 C., seven at room temperature and six at 370 C. RESULTS The carbonic anhydrase activity in saliva estimated immediately after collection and 24 and 48 hr. later is shown in Tables I-Ill. There is no significant change in enzyme activity in any sample. Furthermore, the results in the saliva collected under oil are similar to the values obtained when no oil was used (Table III). The carbonic anhydrase activity in saliva increased with increasing flow in the five patients from resting values under 1.6 K/ml. to a maximum of about 2.2 K/ml. As there were no significant differences between the

3 204 Szabo, Mason and Harden regression coefficients, the results from the five patients were considered together (fig. 1). Up to flow rates of 0.80 ml./min. there was a straight line relation between flow rate and enzyme concentration, y = 1.00lx where x = flow rate (ml./min.) and y = enzyme concentration (K/ml.) (r = +0.89, p < 0.01.) At rates greater than 0.80 ml./min. the enzyme TABLE I. CARBONIC ANHYDRASE ACTIVITY IN SALIVA (K/ml.) IMMEDIATELY AFTER COLLECTION AND AFTER STORING FOR 24 AND 48 HR. AT 00 C. Sample No. On collection After 24 hr. After 48 hr concentration remained constant, y = x Since r = -0.04, which is not significantly different from 0, we can take y,= as the regression of y on x. The standard deviation of y is The carbonic anhydrase activity in saliva remained relatively constant from day to day in parotid, mixed and submandibular saliva (Table IV). TABLE II. CARBONIC ANHYDRASE ACTIVITY IN SALIVA (K/ml.) IMMEDIATELY AFTER COLLECTION AND AFTER STORING FOR 24 AND 48 HR. AT ROOM TEMPERATURE. Sample No. On collection After 24 hr. After 48 hr The enzyme activity in the blood was greater than that found in resting saliva, but less than that found in saliva collected after stimulation. DIsCUSSION The concentration of many salivary constituents varies with flow rate [Dawes and Jenkins, 1964; Mason, Harden and Alexander, 1966 b]. In this study we have shown that the carbonic anhydrase activity increases with flow rate to a maximum value at flow rates of about 0.75 ml./min., the concentration remaining constant at rates above this level. The secretion of saliva is a complex process, the composition of the precursor fluid secreted in the acini being modified in the ducts due to secretion and reabsorption of constituents. The changes in carbonic anhydrase activity with increasing flow rate could be accounted for in a number of ways and further work is required to elucidate this problem. One explanation would be that the enzyme secreted in the acini is reabsorbed in the ducts and that the reabsorptive process has a limited capacity. A second contributory factor might be that the permeability of the acinar and/or duct cells increases with increased glandular secretion [Burgen and Seeman, 1958]. If the gland were

4 Carbonic Anhydrase Activity in Saliva in Man to secrete a small constant secretion free from carbonic anhydrase (v ml./min.) and a variable secretion containing a constant concentration of this enzyme (K), then the relation between the concentration of carbonic anhydrase in saliva (c) and the salivary flow rate (V) would be vk c=k- V However, when the observed c is plotted against the reciprocal of the observed V, the experimental data does not lie around a straight line, suggesting that the above relationship does not hold for carbonic anhydrase. TABLE III. CARBONIC ANHYDRASE ACTIVITY IN SALIVA (K/ml.) COLLECTED WITHOUT OIL AND UNDER OIL, IMMEDIATELY AFTER COLLECTION AND AFTER 48 HR. AT 370 C. Sample No Without oil On collection After 48 hr. 2' *06 1* '04 2*04 1*97 1* On collection ^06 Under oil After 48 hr *09 2*00 2*04 2'02 2'02 It has been proposed that the presence of carbonic anhydrase in human saliva can speed the removal of carbon dioxide from the saliva and that this results in a more alkaline saliva with an increased tendency to precipitation TABLE IV. CARBONIC ANHYDRASE ACTIVITY IN PAROTID, SUBMANDIBULAR AND MIXED SALIVA OF Two SUBJECTS ON THREE CONSECUTIVE DAYS, COLLECTEI} AFTER LEMON JUICE STIMULATION. Parotid saliva Submandibular saliva Mixed saliva Blood {Day 1 {Day 1 Day 1 Carbonic anhydrase activity (K/ml.) r A I Subject 1 Subject 2 2*18 2*07 2*19 2*03 2*18 2*13 2*18 2*08 2*00 2* O *00 2*06 2* *11 2* * *10 1*87 1* of calcium salts [Rapp, 1946]. More recently McConnell, Frajola and Deamer [1961] have supported the view that carbonic anhydrase plays a role in the formation of oral calculus. This enzyme accelerates the conversion of carbon dioxide to bicarbonate, and Sand [1951] and Wechsler

5 206 Szabo, Mason and Harden [1959] have demonstrated that in addition to bicarbonate obtained from other sources, an appreciable proportion of the salivary bicarbonate is derived from metabolism in the salivary glands. Following the administration of a carbonic anhydrase inhibitor, the bicarbonate concentration in saliva decreases [Neidermeier et. at., 1955]. Like carbonic anhydrase, the concentration of bicarbonate in saliva and the salivary ph increase with increasing flow rate and remain relatively constant at high flow rates [Anderson, 1949; Thaysen, Thorn and Schwartz, 1954; Dawes and Jenkins, 1964]. In any study of carbonic anhydrase activity in saliva it is desirable to collect the samples at flow rates greater than 0.8 ml./min., at which levels the concentration appears to be independent of flow rate. Collection of the saliva under oil is unnecessary. ACKNOWLEDGMENTS We are grateful to Dr. Robb, Department of Statistics, and to the staff of the Biochemistry Department, University of Glasgow, where part of these investigations was undertaken. Sister B. Muirhead gave technical assistance. REFERENCES ALTSCHULE, M. D. and LEWIS, H. D. (1949). J. Biol. Chem. 180, 557. ANDERSON, D. J. (1949). J. Dent. Re8. 28, 583. BURGEN, A. S. V. and SEEMAN, P. (1958). Can. J. Biochem. Physiol. 36, 119. DAWES, C. and JENKINS, G. N. (1964). J. Physiol. 170, 86. DRAUJS, F. J., MiKLos, F. L. and LEUNG, S. W. (1962). J. Dent. Res. 41, 497. GLOSTER, J. (1955). Brit. J. Ophthal. 39, 743. KERR, A. C. (1961). The Physiological Regulation of Salivary Secretions in Man. Oxford: Pergamon Press. MASON, D. K., HARDEN, R. McG., RowAN, D. and ALEXANDER, W. D. (1966 a). J. Dent. Res. [In the press.] MASON, D. K., HARDEN, R. MCG. and ALEXANDER, W. D. (1966 b). Arch. Oral. Biol. 11, 235. MCCONNELL, D., FRAJOLA, W. and DEAMER, D. (1961). Science, 133, 281. NEIDERMEIER, W., STONE, R. E., DREIZEN, S. and SPIES, T. D. (1955). Proc. Soc. Exper. Biol. & Med. 88, 273. MlTCHELL, C. A., POZZANI, U. C. and FESSENDEN, R. W. (1945). J. Biol. Chem. 160, 283. RAPP, G. W. (1946). J.A.D.A. 33, 191 SAND, H. F. (1949). Dr. Philos. Thesis, University of Oslo. SAND, H. F. (1951). J. Appl. Physiol. 4, 66. SCHNEYER, L. H. (1955). J. Dent. Res. 34, 257. SZABO, I. (1965). Ki8erl Orvostud. 17, 210. SZABO, I. and HATTYASY, D. (1965). Acta. Physiol. 27, 141. THAYSEN, J. H., THORN, N. A. and SCHWARTZ, I. L. (1954). Amer. J. Physiol. 178, 155. WECHSLER, A. (1959). M.Sc. Thesis, McGi1 University. YOSHIMURA, H., IWASAKI, H., NISHIKAWA, T. and MATSUMOTO, S. (1959). Jap. J. Physiol. 9, 106.