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Observations on the Effects of Longtime Administration of Aspirin and Cortisone on the Secretion rate and Chemical Composition of Saliva
Vol. 61, No. I
G ASTROENTEROLOG Y
Copyri ght© l97l by The Williams & Wil kins Co.
Printed in U. S .A.
OBSERVATIONS ON THE EFFECTS OF LONGTIME ADMINISTRATION OF ASPIRIN AND CORTISONE ON THE SECRETION RATE AND CHEMICAL COMPOSITION OF SALIVA TORU TAKEUCHI , M .D. ,
F. R.
STEGGERDA, PH . D . , AND E.
R.
ENSRUD, M.D .
Department of Physiology and Biophysics, University of Illinois , Urbana , fllinois
Concentration of mucin was changed in proportion to the frequency of electrical stimulation until 20 cycles per sec was reached. Injections of small amounts of pilocarpine evoked saliva consisting of quite different composition of carbohydrate fractions compared with that produced by nerve stimulation. Administration of aspirin and cortisone caused significant decreases in most of the components of protein-bound carbohydrates. The most marked difference observed in the effects of aspirin and cortisone injections on the carbohydrate components was that observed with sialic acid. With aspirin the sialic acid fraction showed the greatest decrease while with cortisone this fraction gave the least change. Aspirin and cortisone caused no significant change in the total protein content and rate of secretion of saliva. In recent years there has been considerable interest in the effects of antiarthritic drugs (especially aspirin) on the rate and chemical composition of mucin secretion in the stomach. ,_ 3 It will be the purpose of this report to describe the effects of longtime administration of aspirin and cortisone on the secretion rate and chemical composition of saliva from the submaxillary gland of the dog when produced via nerve (chorda tympani) and humoral (pilocarpine) stimulation.
into the duct. In all the experiments, the same size catheter was used in order to control as much as possible the size of the droplet of saliva collected. To make quantitative salivary secretion studies by means of nerve stimulation, the ramus communicans (chorda tympani) branch of the lingual nerve to the submaxillary gland was separated from its surrounding connective tissue. The lingual nerve was then ligated and sectioned proximally to where the chorda tympani branch left the nerve. Nerve stimulation was applied through a bipolar platinum electrode (diameter of the platinum wire, 0.6 mm) connected to a square wave electrical stimulator. In establishing a calibration for a maximum rate of secretion, the voltage strength was always kept constant and the frequency, or cycles per sec , was varied. The procedure was to collect first a total of 5 ml of saliva with continuous stimulation of the chorda tympani at a strength of stimulus of 7 v and a frequency of 2 cycles per sec. Then, after 2 min, this was repeated at a frequency of 6 cycles, and thereafter consecutively at frequencies of 10, 20, 40, and 60 cycles per sec with the maximum rate of secretion found to be at 20 cycles per sec. To standardize the rate of secretion, saliva was recorded as the number of cubic centimeters secreted per min per g of wet weight of the submaxillary gland. When pilocarpine hydrochloride in saline was
Experimental Method In these experiments, healthy mongrel dogs, both male and female , weighing between 10.5 and 18.5 kg were used. At the time of making salivary collections, the dogs were anesthetized with intravenous injections of sodium pentobarbital (Nembutal, 30 mg per kg of body weight). For the purpose of obtaining normal control data, one of the submaxillary glands and its duct was dissected free and a polyethylene catheter (inner diameter, 1.1 mm) was inserted Received December 29, 1970. Accepted February 18, 1971. Address requests for reprints to: Dr. E . R. Ensrud, Carl e Clinic, Urbana, lllinois 61801. This work was supported by a grant in aid from the Carle Hospital Foundation , Urbana , lllinois. 69
70
TAKEUCHI ET AL .
used as a salivary flow stimulant, the drug was injected into the external jugular vein. To record a flow similar to that observed when the chorda tympani was stimulated, a number of intravenous injections of different concentrations of pilocarpine had to be made . The range in concentration used was from 0.1 to 0.4 mg per kg of body weight. The time it took to collect the first 5 ml of saliva with different dosages of pilocarpine was recorded, starting with 0.10 mg per kg of body weight, followed later with a dosage of 0.15 mg per kg, another dose of 0.20 mg per kg, and, finally, a dose of 0.40 mg per kg. From this data the rate of secretion per min per g of wet weight of salivary tissue could be similarly calculated. It was necessary to wait nearly 1 1/z hr between injections for the effects of each injection to wear off. When the rate of secretion was found to parallel closely that of the maximum rate of secretion by nerve stimulation, the dosage was recorded and this amount was used in all subsequent experimental studies. During an experiment, two consecutive 5-ml volumes of saliva were collected with either nerve or drug stimulation for future chemical analysis. In collecting the saliva samples, to make sure the secretions were from the gland cells and not lodged in the ducts, the first 60 drops of saliva were discarded. After the saliva collection was completed, the duct and its attached submaxillary gland were removed, weighed, and preserved for further histological studies. The saliva was collected in 10-ml graduated centrifuge tubes submerged in crushed ice to suppress any catalytic reactions. During the salivary collection periods, the animals were infused with intravenous saline to avoid any significant change in electrolyte and water balance. When the salivary flow was collected after aspirin or cortisone administration, the stimulation via the nerve or pilocarpine was always the same as that used in making the original or control collection. The chemical analysis made on the saliva samples was for total protein and four different carbohydrate fractions. The protein analysis was performed immediately after collection, using the Folin phenol reagent of Lowry et a!., 4 with bovine crystalline albumin used as a standard. The carbohydrate fractions studied were determined as two separate entities: that portion which was protein-bound and that existing as free carbohydrate. To determine the proteinbound portion of each of the carbohydrate fractions present, a measured sample of saliva was diluted fifty times with 95 % alcohol and, after thorough mixing, was stored overnight at 2 to 4 C. The resulting precipitate was then sepa-
Vol . 61, No . I
rated by centrifuging for 15 min at 3000 rpm and the alcohol was decanted. The precipitate was then mixed with alcohol as above, and again the precipitate was separated by centrifugation. The different protein-bound carbohydrate fractions determined were the sialic acid , fucose, hexoses, and hexosamines. The sialic acid was determined by the thiobarbituric acid assay of Warren ' after hydrolysis of precipitate with 0.1 N H 2 SO, at 80 C for 1 hr. The fucose fraction was measured by a procedure described by Dische and Shettles, 6 and the hexose fraction by the simplified phenol method of Dubois et al. 7 In these determinations it was first necessary to dissolve a measured amount of the dried precipitate in 0.1 N NaOH. For the determination of the hexosamine fraction, the dried saliva was first hydrolyzed with 3 N HCl in stoppered tubes. It was then heated for 3 hr, after which the saliva was analyzed according to a modification of the Elson-Morgan method, employed by Remington. 8 All measurements were recorded with the Beckman spectrophotometer DB equipment. To determine the amounts of sialic acid, fucose, hexose, and hexosamines present as free carbohydrate fractions, the procedures just described were used, except that the saliva samples were not first precipitated with 95 % alcohol. The results obtained gave the total amount of each carbohydrate present, and all that was necessary to get the amount of the free carbohydrate fraction present was to subtrace the result obtained for the protein-bound fraction from the total amount of each fraction. The actual amounts of the free carbohydrate fractions were practically insignificant and therefore are not dealt with further in this report.
Results Establishment of uniform rates of secretion with nerve and pilocarpine stimulation. Table 1 shows the results of a number of calibrations recorded in different animals in the establishment of the maximum flow of saliva with nerve stimulation and the corresponding response of flows recorded using pilocarpine stimulation. As will be noted, the average strength of electrical stimulation necessary for obtaining the maximum rate of secretion for nerve stimulation was 7 v at 20 cycles per sec, while 0.20 mg of intravenous pilocarpine per kg of body weight was necessary for obtaining a similar maximum secretion rate. The
TABLE
1. Average rates of secretion of saliva with nerve and pilocarpine stimulation Ne rve st imulat ion
Volta ge an d frequenc y
Piloca rpine sti mulation Secretion volume"
ml/11 (wet wt)/m in
7 7 7 7 7 7
v v v v v v
at at at at at at
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EFFECTS OF ASPIRIN AND CORTISONE ON SALIVA
July 1971
(8)' 2 cycles/sec 5 cycles/sec (18) 10 cycles/sec (5) 20 cycles/sec ( 18) 40 cycles/sec ( 13) 60 cycles/sec (5)
0 . 08 0 . 16 0 .31 0.49 0 . 48 0 . 39
± 0 . 02 ± 0.04 ± 0.05 ± 0 .03 ± 0.05 ± 0 . 14
Dose m~J/kg
body wt
S ecreti on volum e" ml/1-{ (w et wt )/min
0.10 (6)' 0 . 15 (6) 0.20 (6)
0.21 ± 0.08 0.31 ± 0.08 0 .51 ± 0 .07
0 . 40 (6)
0.29 ± 0.09
a All values are expressed as mean ± SD . " Figures in parentheses indicate number of determinations used in making up the average.
former gave an average value of 0.49 ± 0.03 ml per g of wet weight per min; the latter gave a reading of 0.51 ± 0.07 ml. The consistency of these results in all animals, as shown by the smallness of the standard error, is surprising. These two levels of stimulation will be used in making the comparisons in salivary responses when the effects of aspirin and cortisone are made. The two levels of stimulation are well within the normal range of physiological responses and should therefore serve as good controls for any increase or decrease that might occur as a result of aspirin or cortisone administration . The data also show that the secretion rate with nerve stimulation increased progressively up to the maximum rate of 7 v, 20 cycles frequency and then stopped increasing in quantity, maintaining a more or less similar output with each succeeding stimulation up to at least 60 cycles per sec. The observed response resulting from pilocarpine stimulation was different from that observed with nerve stimulation in that the secretion rate fell off markedly as the dosage of pilocarpine was increased from 0.2 mg per kg (maximum secretion rate) to 0.4 mg per kg. Chemical composition of saliva following nerve and pilocarpine stimulation. In order to make a satisfactory comparison between the longtime administration of aspirin and cortisone on the chemical composition of saliva , controlled observations must first be made to establish a norm. The quantitative observations on the total protein and
four different protein-bound carbohydrate fractions-sialic acid, fucose, hexoses, and hexosamines-of saliva when collected by nerve stimulation are given in table 2. As will be noted, the observed protein and carbohydrate content of the samples of saliva collected were not significantly different when the frequency of the nerve stimulation was increased from 10 cycles per sec to 40 cycles per sec . The only difference observed was that the protein content of the saliva was less with frequencies of 2 and 5 cycles per sec. This difference does not, however, exist with reference to the concentrations of proteinbound carbohydrates. The amounts of sialic acid, fucose , hexoses, and hexosamines do not vary when the frequency of nerve stimulation is varied from 2 to 60 cycles per sec . The distribution of the carbohydrate fractions is in general agreement with that reported by Dische et al. 9 The results obtained for protein and protein-bound carbohydrate fractions collected after pilocarpine injections are given in table 3. To make a comparison between the protein and protein-bound carbohydrates obtained in saliva collected by pilocarpine stimulation and similar components obtained with nerve stimulation, column 1 under the heading of nerve stimulation is also included in the table. The results are those obtained when the nerve stimulation was at an intensity of 7 v and at a frequency of 20 cycles per sec with a maximum rate of secretion recorded. As will be noted, the protein content of
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Vol. 61, No . 1
TAKEUCHI ET AL. TABLE Component
2. Th e composition of fi ve components of mucin evoked by nerve stimulation 7v. 2c" (8) "
7v. 5c (18)
7v. IOc (5)
7v, 20c (18)
7v. 60c (5)
7v, 40c (13)
mt:/100 ml sa liua
Protein .. ..... .
224 .7 ± 40 . 2'"
1 315 .5 ± 40.0
1425 .9 ± 101. 3
1433. 3 ±32. 0
1468. 1 ± 57 . 2
1429.9 ±90 . 0
mn/100 mg protein
carboProtein-bound hydrate Sialic Acid .... . . . 14 .9 Fucose . ' . . .. . . 16. 8 ... 23 .1 Hexoses Hesosamines 25 .8
± ± ± ±
2.0 13 .1 ± 1.1 3 .2 14.1 ± 2. 5 3.1 26.1 ± 2 .8 5. 1 36.4 ± 4 .5
12.3 14.7 24.6 39.0
± ± ± ±
1.1 1.1 3.8 4.1
12. 1 14 .5 25. 1 38. 7
± ± ± ±
1.9 12. 1 ± 1. 2 13 .0 ± 2.0 23 .7 ± 4 . 9 37 . 7 ±
1. 8 1.8 2 .9 4.1
13 .5 17.0 24.4 41.2
± ± ± ±
2. 8 2. 1 3. 7 5.
" v, volts; c, cycles per sec. h Numbers in parentheses indicate number of dogs used. '" All values are expressed as mean ± s o. TABLE
3. Composition and comparison of fi ve components of saliva evoked by pilocarpine stimulation Nerve stimulationa
Vari ous pilocarpine dosages (mg/kg of body weight )
Component
7 v. 20c" (18)··
0. 10 (61
0. 15 (6)
I
mg/100 ~~sa liva
Protein ... ......
433 . 3 ± 32 . o1 58. 5" ± 14. 61133. 6" ± 50 .5 I 173.9" ± 32. 8 m!f/ /00 mg protein
Protein-bound carhydrate Sialic acid ... . . Fucose . . . . . . . . . Hexose . Hexosamine
12.1 14. 5 25 .1 38 .1
± ± ± ±
1.9 1. 2 2.0 4.9
18 .6' 7 .8" 25.9'' 29.3'
± ± ± ±
4 .3 1. 8 5.1 5 .6
21 .2" 7. 1" 22. 1' 32 . 21
± ± ± ±
1.9 1.1 4.6 3.2
0 .4 (6)
0.2 (6)
18 . 1" 9 .3 1 24 .4' 32 . 7h
± 0. 9 ± 1.7 ± 2 .1 ± 3. 3
I 451. 4' 12 . ge 16. 3h 28 .8• 38 . 4•
± 76 .5
± ± ± ±
1. 9 1.8 1.7 4.1
Nerve stimulation : represents data received for maximum ra te of secretion. v, volts; c, cycles per sec. ' Numbers in parentheses indicate number of dogs used. <1 p < 0.001. ' Not significant. I p < 0.03. • P < 0.005. h p < 0.2. a h
the saliva resulting from the pilocarpine stimulation is significantly less than with nerve stimulation with dosages up to 0.4 mg per kg of body weight. At the dosage of 0.4 mg per kg of body weight, the protein content of saliva is not significantly different from that observed with nerve stimulation . The differences in carbohydrate content in the saliva compared with that found in nerve-stimulated saliva were significant in some areas, but not in others. The fucose concentrations were
less in most cases than any other carbohydrate fraction . As will be noted, the carbohydrate fractions were quite similar to those observed with nerve stimulation when the dosage was at the 0.4 mg per kg level. The consistency with which the differences in chemical concentration of saliva appear with nerve and pilocarpine stimulation, as observed by the small standard error, definitely supports the concept of the presence of a basic response difference between nerve and pilocarpine stimulation at the cellular level.
July 1971
Effects of aspirin administration on salivary secretion and its composition. The 5 dogs used in these experiments were given aspirin orally in doses of 4 grains per kg per day for a period ranging from 29 to 33 days. In all the tests made, the stimulation rate for nerve and pilocarpine effects was made at the previously determined maximum rate of secretion: 7 vat 20 cycles per sec for nerve stimulation and 0.2 mg per kg of body weight for pilocarpine. The average results obtained are presented in table 4. It will be noted that the maximum rate of secretion of the submaxillary gland was not significantly different from that reported in tables 2 and 3. Also, there was no difference in rate of secretion over that observed before aspirin was given. The data also show that the longtime administration of high doses of aspirin had no significant effect on the protein content of the saliva with either nerve or pilocarpine stimulation. In the case of the effects of aspirin on the protein-bound carbohydrates present in the saliva, however, there was a significant reduction found in all four fractions studied. The amounts of each carbohydrate studied are expressed as milligrams of carbohydrate bound per 100 mg of protein present in the collected sample. The actual distribution of the protein-bound carboTABLE 4.
hydrates shows that the sialic acid and fucose were the lowest in concentration with hexosamine the highest. The difference in carbohydrate content in the saliva collected via pilocarpine stimulation was greater than that observed with nerve stimulation. The calculated difference resulting from aspirin administration in terms of percentage gave a decrease for each of the four carbohydrates tested. The decrease with the nervestimulated group ranged from 25 to 44% while the pilocarpine-stimulated group gave a range of 56 to 73%. Of the four proteinbound carbohydrates studied, it appears that the sialic acid fraction is the most sensitive to aspirin administration and hexosamine the least. There seems to be little doubt that aspirin can have a marked effect on the concentration of the protein-bound carbohydrates. In experiments where the free nonprotein carbohydrates were determined, the amounts present were small and not significant. Effects of cortisone administration on salivary secretion and its composition. In a similar experiment, 5 dogs were tested with orally administered cortisone in daily dosages of 100 mg per dog. As indicated in table 5, there was no difference in the maximum secretion rate of saliva before and after 30 consecutive days of cortisone administration either with nerve stimulation
Effects of aspirin on salivary secretion and its composition a
Nerve stimulation P Value
Condition
Change from control
ml/a rvet wt /min
0 . 49 ± 0 . 03 j 0 . 46 ± 0 . 06
Pilocarpine stimulation P Value
Before aspirin
After aspirin
Before aspirin
Secretion rate .
NS•
413 ± 68
I
14.7 15.6 24.6 39.5
a Average of 5 dogs. • NS, not significant.
± ± ± ±
1.3 2.1 3.8 6.3
ml/g tt•et wt/min
0.47 ± 0.09 10.41 ± 0.13
NS
-12
NS
-27
m g/100 ml saliua
411 ± 48
8.2 9 .5 16 . 0 29.8
'/,
Yr
NS
-1
226 ± 116 1164 ± 115 mR/100 mg protein
mp,l /()(} m g protem
Protein-bound carbohydrate Sialic acid .. Fucose Hexose . Hexosa mine
Change from control
After aspirin
- 6
mg/ 100 ml saliua
Protein concentration .
73
EFFECTS OF ASPIRIN AND CORTISONE ON SALIVA
± ± ± ±
1.7 2.3 3.3 7.0
< 0 . 001 < 0.005 < 0.01 < 0 .1
-44 -39 -35 -25
18. 3 11.5 28.1 36 .8
± ± ± ±
8.0 3.1 5.5 4.5
4.9 4.6 10.1 16.3
± ± ± ±
2.2 3.9 3.5 6.7
< 0.001 < 0.05 < 0.01 < 0.001
-73 -60 - 64 -56
Vol. 61 , No . I
TAKEUCHI ET AL.
74 TABLE
5. Effects of cortisone on salivary secretion and its composition" Nerve stim ulation
Condition Before cort isone
ml/g
Secretion rate .
Change from control
P Valu e
Pilocarpine stimulation P Value
wel wt/m in
c·
0 .45 ± 0.1110.44 ± 0.09
' -2
NS '
417 ± 79 m~/100
0 .33 ± 0.091 0.45 ± 0 . 09 mg/ 100 ml
Protein-bound carbohydrate Sialic acid . Fucose Hexose . Hexosamine
14.7 17.6 24.1 42 . 1
± ± ± ±
2.4 2.5 1.7 3 .0
1 377 ± 93
- 10
NS
222 ± 114 mg/ 100
mp protein
15.9 12.2 18 . 2 33.3
± 2.5 ± 3.1 ± 2.0 ± 3.4
NS <0.05 < 0.001 < 0.005
+8 -31 - 24 -21
',
ml/g wet wt/min
mg/ 100 ml saliua
Protein concentration .
After cortisone
Before cortisone
After cortisone
22.1 ± 16.8 ± 28.3 ± 36.4 ±
5 .0 7.5 7.5 4.9
Change from control
I 180 ±50
~ ;:
NS
+36
NS
-19
NS < 0 .2 < 0 .01 < 0.01
+5 - 46 -47 - 26
~aliua
protein
23.2 ·9.1 14.9 27.0
± 5.2 ±7.5 ± 7.5 ± 0 .9
Average of 5 dogs. • NS, not significant.
a
or injection of pilocarpine. As in the case of the aspirin experiments, the protein content showed no significant difference. A depressant effect of cortisone on the composition of the protein-bound carbohydrate was noted in the fucose, hexoses, and hexosamines, but not so great as that observed in the aspirin experiment (see table 4). The decrease in concentration of these fractions was more statistically significant with nerve-produced saliva than in the pilocarpine-stimulated saliva samples, although in percentages, the pilocarpine samples gave greater differences. The one notable difference observed was that cortisone did not depress the sialic acid carbohydrate fraction, while with aspirin administration the sialic acid fraction was inhibited more than any of the other fractions studied. In general, there was less change in the production of carbohydrate than was observed with the aspirin treatment. As indicated in table 5, the percentage differences in concentration of the four carbohydrate fractions gave a range of + 8 to -31 % with the nerve stimulation and +5 to -47% when pilocarpine was used. Discussion In making comparative studies of the effects of longtime administration of drugs on the secretory function of an organ, such
as a salivary gland, it is important to eliminate such factors as the nature of the response to the stimulus, compensatory hypertrophy of the exposed gland, as well as the presence of any dietary differences that might occur during the time of drug administration. The establishment of a fixed response to nerve and pilocarpine stimulation, such as maximum rate of secretion, is important in making these studies so that the gland under these conditions is not overstimulated to the point where structural damage might be induced and the secreting power of the gland cells exceeded. The fact that the remaining salivary gland studied in these experiments, after the longtime administration of aspirin or cortisone, responded in their rate of secretion to either nerve or pilocarpine stimulation with the same degree of efficiency as previously observed supports the fact that the gland was not damaged as far as its responses to nerve and pilocarpine are concerned. Furthermore, to test whether the chemical changes observed in the saliva after the 30 days of drug administration were actually due to the drug and not to some type of compensatory reaction that might have taken place to alter the chemical composition of the salivary secretion, the following experiment was performed. The submaxillary
July 1971
EFFECTS OF ASPIRIN AND CORTISONE ON SALIVA
salivary glands of 5 dogs were exposed and their ducts cannulated. Saliva was then collected at the maximum rate of secretion by nerve stimulation as well as with an established pilocarpine rate of secretion. The samples in each case were tested for protein and carbohydrate composition as outlined above. The dogs were then kept for 60 days (30 days recuperation and 30 days to represent the drug administration period), after which . the salivary samples were similarly collected and tested for protein and carbohydrate composition. Theresults of all 5 dogs showed no significant change between the first and second salivary collection periods. The dogs ate all their provided food every day, and there was no change in total body weight. These experiments support the concept that the operative procedures.used did not alter the normal functioning of the salivary gland. As already mentioned, with the 5 dogs used in the aspirin experiment there was an occasional day when the dogs did not eat all their food, and on one or two occasions 1 of the dogs vomited. Although no significant weight loss occurred in these experimental animals, there still remained the possibility that the dietary upset could be responsible for the changes produced in the protein-bound carbohydrates observed in table 2. To rule out this possibility, the following experimental procedure was used. Saliva was collected from 3 dogs and a chemical analysis was made of the samples. The dogs were then kept on a daily routine of commercial dog biscuit for 30 days, except that the total dietary allotment was reduced by 50%. In this case the dog lost weight, but when the 30-day experimental period was over and the salivary secretion was chemically analyzed, it was noted that all the parameters studied showed no decrease as was observed in the aspirin and cortisone experiment. These findings are supported by the observations of Sreebny et al. 10 They also lend support to the fact that the experimental results observed and reported were not closely associated with any occasional small dietary upset resulting from aspirin administration. Although our observations on nerve and
75
pilocarpine stimulation of the salivary gland and its composition of different proteins and certain protein-bound carbohydrates are somewhat similar to those reported by Dische et al 9 they differ markedly in the strength and frequency of nerve stimulation used; Dische et al. 9 varied the voltage and kept the frequency constant (40 cycles per sec). The voltage variation is less a factor in obtaining quantitative results in physiological stimulations of live tissues than frequency changes. Furthermore, Dische et al. 9 used pilocarpine as a stimulant for salivary secretion by varying the dose from 0.4 mg to 4.0 mg at a continuous rate of infusion. This dosage and manner of administration is far different from that used in the above experiments and does not lend itself to the establishment of a threshold for maximum rate of secretion. Diamant et al., 11 from their experiments on submaxillary secretion in man, reported that the maximum rate of secretion occurred at a frequency of 10 cycles per sec with a voltage variation between 2 and 3 v. This low rate of frequency and voltage may be associated with species difference or the depth of anesthesia used. We believe that our method of calibration of nerve and pilocarpine administration was necessary to establish a standard for comparison of saliva composition before and after the longtime administration of such drugs as aspirin and cortisone. To explain the cause for the differences in salivary secretion and salivary composition that were observed between the aspirin- and·cortisone-treated dogs is not easy. Menguy and Masters, 2 • 3 in their experiments on gastric mucin production in dogs receiving aspirin and cortisone, discuss the possible theoretical cause for the decreases in mucin secretions that they observed. Our observations may be interpreted in a similar manner. An examination of their experimental procedure showed that they administered a total of 80 g of aspirin orally every 3rd day for about 40 days, and administered cortisone intravenously in doses of 150 mg every day for 15 days. In our experiments, much greater daily dosages of aspirin (4 grains per kg of
TAKEUCHI ET AL.
76
body weight) for 30 days were tolerated without any consistent ill effects except for slight losses of body weight. The cortisone dosage of 100 mg per day per dog was an estimate based on the effectiveness of this drug in clinical use and, therefore, it is admitted that any real comparison between the two drugs must be treated with caution.
5. 6.
7.
8. REFERENCES 1. Winkelman EI, Summerskill WHJ: Gastric secretion in relation to gastrointestinal bleeding from salicylate compounds. Gastroenterology 40:5663, 1961 2. Menguy R, Masters YF: Effect of cortisone on mucoprotein secretion by gastric antrum of dogs: pathogenesis of steroid ulcer. Surgery 54:19-28, 1963 3. Men guy R, Masters YF: Effects of aspirin on gastric mucous secretion. Surg Gynec Obstet 120:9298, 1965 4. Lowry OH, Rosenbrough NJ, Farr AL, et al:
9.
10.
11.
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Protein measurement with the Falin phenol reagent. J Bioi Chern 193:265- 275, 1951 Warren L: The thiobarbituric acid assay of sialic acids. J Bioi Chern 234:1971-1975, 1959 Dische Z, Shettles LB: A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J Bioi Chern 175: 595-603, 1948 Dubois M, Gills KA, Hamilton JD, et al : Colorimetric method for determination of sugars and related substances. Anal Chern 28:350-356, 1956 Remington C: Seromucoid and the bound carbohydrate of the serum proteins. Biochem J 34: 931-940, 1940 Dische Z, Pallavicini C, Kanasaki H, et al: Influence of the nature of the secretory stimulus on the composition of the carbohydrate moiety of glycoproteins of the submaxillary saliva. Arch Biochem 97:459-469, 1962 Sreebny LM, Johnson DA: Effect of food consistency and decreased food intake on rat parotid and pancreas. Amer J Physiol 215:455-460, 1968 Diamant H, Enfors B, Holmstedt B: Salivary secretion in man elicited by means of stimulation of the chorda tympani. Acta Physiol Scand 45: 293-299, 1959
G ASTROENTEROLOG Y
Copyri ght© l97l by The Williams & Wil kins Co.
Printed in U. S .A.
OBSERVATIONS ON THE EFFECTS OF LONGTIME ADMINISTRATION OF ASPIRIN AND CORTISONE ON THE SECRETION RATE AND CHEMICAL COMPOSITION OF SALIVA TORU TAKEUCHI , M .D. ,
F. R.
STEGGERDA, PH . D . , AND E.
R.
ENSRUD, M.D .
Department of Physiology and Biophysics, University of Illinois , Urbana , fllinois
Concentration of mucin was changed in proportion to the frequency of electrical stimulation until 20 cycles per sec was reached. Injections of small amounts of pilocarpine evoked saliva consisting of quite different composition of carbohydrate fractions compared with that produced by nerve stimulation. Administration of aspirin and cortisone caused significant decreases in most of the components of protein-bound carbohydrates. The most marked difference observed in the effects of aspirin and cortisone injections on the carbohydrate components was that observed with sialic acid. With aspirin the sialic acid fraction showed the greatest decrease while with cortisone this fraction gave the least change. Aspirin and cortisone caused no significant change in the total protein content and rate of secretion of saliva. In recent years there has been considerable interest in the effects of antiarthritic drugs (especially aspirin) on the rate and chemical composition of mucin secretion in the stomach. ,_ 3 It will be the purpose of this report to describe the effects of longtime administration of aspirin and cortisone on the secretion rate and chemical composition of saliva from the submaxillary gland of the dog when produced via nerve (chorda tympani) and humoral (pilocarpine) stimulation.
into the duct. In all the experiments, the same size catheter was used in order to control as much as possible the size of the droplet of saliva collected. To make quantitative salivary secretion studies by means of nerve stimulation, the ramus communicans (chorda tympani) branch of the lingual nerve to the submaxillary gland was separated from its surrounding connective tissue. The lingual nerve was then ligated and sectioned proximally to where the chorda tympani branch left the nerve. Nerve stimulation was applied through a bipolar platinum electrode (diameter of the platinum wire, 0.6 mm) connected to a square wave electrical stimulator. In establishing a calibration for a maximum rate of secretion, the voltage strength was always kept constant and the frequency, or cycles per sec , was varied. The procedure was to collect first a total of 5 ml of saliva with continuous stimulation of the chorda tympani at a strength of stimulus of 7 v and a frequency of 2 cycles per sec. Then, after 2 min, this was repeated at a frequency of 6 cycles, and thereafter consecutively at frequencies of 10, 20, 40, and 60 cycles per sec with the maximum rate of secretion found to be at 20 cycles per sec. To standardize the rate of secretion, saliva was recorded as the number of cubic centimeters secreted per min per g of wet weight of the submaxillary gland. When pilocarpine hydrochloride in saline was
Experimental Method In these experiments, healthy mongrel dogs, both male and female , weighing between 10.5 and 18.5 kg were used. At the time of making salivary collections, the dogs were anesthetized with intravenous injections of sodium pentobarbital (Nembutal, 30 mg per kg of body weight). For the purpose of obtaining normal control data, one of the submaxillary glands and its duct was dissected free and a polyethylene catheter (inner diameter, 1.1 mm) was inserted Received December 29, 1970. Accepted February 18, 1971. Address requests for reprints to: Dr. E . R. Ensrud, Carl e Clinic, Urbana, lllinois 61801. This work was supported by a grant in aid from the Carle Hospital Foundation , Urbana , lllinois. 69
70
TAKEUCHI ET AL .
used as a salivary flow stimulant, the drug was injected into the external jugular vein. To record a flow similar to that observed when the chorda tympani was stimulated, a number of intravenous injections of different concentrations of pilocarpine had to be made . The range in concentration used was from 0.1 to 0.4 mg per kg of body weight. The time it took to collect the first 5 ml of saliva with different dosages of pilocarpine was recorded, starting with 0.10 mg per kg of body weight, followed later with a dosage of 0.15 mg per kg, another dose of 0.20 mg per kg, and, finally, a dose of 0.40 mg per kg. From this data the rate of secretion per min per g of wet weight of salivary tissue could be similarly calculated. It was necessary to wait nearly 1 1/z hr between injections for the effects of each injection to wear off. When the rate of secretion was found to parallel closely that of the maximum rate of secretion by nerve stimulation, the dosage was recorded and this amount was used in all subsequent experimental studies. During an experiment, two consecutive 5-ml volumes of saliva were collected with either nerve or drug stimulation for future chemical analysis. In collecting the saliva samples, to make sure the secretions were from the gland cells and not lodged in the ducts, the first 60 drops of saliva were discarded. After the saliva collection was completed, the duct and its attached submaxillary gland were removed, weighed, and preserved for further histological studies. The saliva was collected in 10-ml graduated centrifuge tubes submerged in crushed ice to suppress any catalytic reactions. During the salivary collection periods, the animals were infused with intravenous saline to avoid any significant change in electrolyte and water balance. When the salivary flow was collected after aspirin or cortisone administration, the stimulation via the nerve or pilocarpine was always the same as that used in making the original or control collection. The chemical analysis made on the saliva samples was for total protein and four different carbohydrate fractions. The protein analysis was performed immediately after collection, using the Folin phenol reagent of Lowry et a!., 4 with bovine crystalline albumin used as a standard. The carbohydrate fractions studied were determined as two separate entities: that portion which was protein-bound and that existing as free carbohydrate. To determine the proteinbound portion of each of the carbohydrate fractions present, a measured sample of saliva was diluted fifty times with 95 % alcohol and, after thorough mixing, was stored overnight at 2 to 4 C. The resulting precipitate was then sepa-
Vol . 61, No . I
rated by centrifuging for 15 min at 3000 rpm and the alcohol was decanted. The precipitate was then mixed with alcohol as above, and again the precipitate was separated by centrifugation. The different protein-bound carbohydrate fractions determined were the sialic acid , fucose, hexoses, and hexosamines. The sialic acid was determined by the thiobarbituric acid assay of Warren ' after hydrolysis of precipitate with 0.1 N H 2 SO, at 80 C for 1 hr. The fucose fraction was measured by a procedure described by Dische and Shettles, 6 and the hexose fraction by the simplified phenol method of Dubois et al. 7 In these determinations it was first necessary to dissolve a measured amount of the dried precipitate in 0.1 N NaOH. For the determination of the hexosamine fraction, the dried saliva was first hydrolyzed with 3 N HCl in stoppered tubes. It was then heated for 3 hr, after which the saliva was analyzed according to a modification of the Elson-Morgan method, employed by Remington. 8 All measurements were recorded with the Beckman spectrophotometer DB equipment. To determine the amounts of sialic acid, fucose, hexose, and hexosamines present as free carbohydrate fractions, the procedures just described were used, except that the saliva samples were not first precipitated with 95 % alcohol. The results obtained gave the total amount of each carbohydrate present, and all that was necessary to get the amount of the free carbohydrate fraction present was to subtrace the result obtained for the protein-bound fraction from the total amount of each fraction. The actual amounts of the free carbohydrate fractions were practically insignificant and therefore are not dealt with further in this report.
Results Establishment of uniform rates of secretion with nerve and pilocarpine stimulation. Table 1 shows the results of a number of calibrations recorded in different animals in the establishment of the maximum flow of saliva with nerve stimulation and the corresponding response of flows recorded using pilocarpine stimulation. As will be noted, the average strength of electrical stimulation necessary for obtaining the maximum rate of secretion for nerve stimulation was 7 v at 20 cycles per sec, while 0.20 mg of intravenous pilocarpine per kg of body weight was necessary for obtaining a similar maximum secretion rate. The
TABLE
1. Average rates of secretion of saliva with nerve and pilocarpine stimulation Ne rve st imulat ion
Volta ge an d frequenc y
Piloca rpine sti mulation Secretion volume"
ml/11 (wet wt)/m in
7 7 7 7 7 7
v v v v v v
at at at at at at
71
EFFECTS OF ASPIRIN AND CORTISONE ON SALIVA
July 1971
(8)' 2 cycles/sec 5 cycles/sec (18) 10 cycles/sec (5) 20 cycles/sec ( 18) 40 cycles/sec ( 13) 60 cycles/sec (5)
0 . 08 0 . 16 0 .31 0.49 0 . 48 0 . 39
± 0 . 02 ± 0.04 ± 0.05 ± 0 .03 ± 0.05 ± 0 . 14
Dose m~J/kg
body wt
S ecreti on volum e" ml/1-{ (w et wt )/min
0.10 (6)' 0 . 15 (6) 0.20 (6)
0.21 ± 0.08 0.31 ± 0.08 0 .51 ± 0 .07
0 . 40 (6)
0.29 ± 0.09
a All values are expressed as mean ± SD . " Figures in parentheses indicate number of determinations used in making up the average.
former gave an average value of 0.49 ± 0.03 ml per g of wet weight per min; the latter gave a reading of 0.51 ± 0.07 ml. The consistency of these results in all animals, as shown by the smallness of the standard error, is surprising. These two levels of stimulation will be used in making the comparisons in salivary responses when the effects of aspirin and cortisone are made. The two levels of stimulation are well within the normal range of physiological responses and should therefore serve as good controls for any increase or decrease that might occur as a result of aspirin or cortisone administration . The data also show that the secretion rate with nerve stimulation increased progressively up to the maximum rate of 7 v, 20 cycles frequency and then stopped increasing in quantity, maintaining a more or less similar output with each succeeding stimulation up to at least 60 cycles per sec. The observed response resulting from pilocarpine stimulation was different from that observed with nerve stimulation in that the secretion rate fell off markedly as the dosage of pilocarpine was increased from 0.2 mg per kg (maximum secretion rate) to 0.4 mg per kg. Chemical composition of saliva following nerve and pilocarpine stimulation. In order to make a satisfactory comparison between the longtime administration of aspirin and cortisone on the chemical composition of saliva , controlled observations must first be made to establish a norm. The quantitative observations on the total protein and
four different protein-bound carbohydrate fractions-sialic acid, fucose, hexoses, and hexosamines-of saliva when collected by nerve stimulation are given in table 2. As will be noted, the observed protein and carbohydrate content of the samples of saliva collected were not significantly different when the frequency of the nerve stimulation was increased from 10 cycles per sec to 40 cycles per sec . The only difference observed was that the protein content of the saliva was less with frequencies of 2 and 5 cycles per sec. This difference does not, however, exist with reference to the concentrations of proteinbound carbohydrates. The amounts of sialic acid, fucose , hexoses, and hexosamines do not vary when the frequency of nerve stimulation is varied from 2 to 60 cycles per sec . The distribution of the carbohydrate fractions is in general agreement with that reported by Dische et al. 9 The results obtained for protein and protein-bound carbohydrate fractions collected after pilocarpine injections are given in table 3. To make a comparison between the protein and protein-bound carbohydrates obtained in saliva collected by pilocarpine stimulation and similar components obtained with nerve stimulation, column 1 under the heading of nerve stimulation is also included in the table. The results are those obtained when the nerve stimulation was at an intensity of 7 v and at a frequency of 20 cycles per sec with a maximum rate of secretion recorded. As will be noted, the protein content of
72
Vol. 61, No . 1
TAKEUCHI ET AL. TABLE Component
2. Th e composition of fi ve components of mucin evoked by nerve stimulation 7v. 2c" (8) "
7v. 5c (18)
7v. IOc (5)
7v, 20c (18)
7v. 60c (5)
7v, 40c (13)
mt:/100 ml sa liua
Protein .. ..... .
224 .7 ± 40 . 2'"
1 315 .5 ± 40.0
1425 .9 ± 101. 3
1433. 3 ±32. 0
1468. 1 ± 57 . 2
1429.9 ±90 . 0
mn/100 mg protein
carboProtein-bound hydrate Sialic Acid .... . . . 14 .9 Fucose . ' . . .. . . 16. 8 ... 23 .1 Hexoses Hesosamines 25 .8
± ± ± ±
2.0 13 .1 ± 1.1 3 .2 14.1 ± 2. 5 3.1 26.1 ± 2 .8 5. 1 36.4 ± 4 .5
12.3 14.7 24.6 39.0
± ± ± ±
1.1 1.1 3.8 4.1
12. 1 14 .5 25. 1 38. 7
± ± ± ±
1.9 12. 1 ± 1. 2 13 .0 ± 2.0 23 .7 ± 4 . 9 37 . 7 ±
1. 8 1.8 2 .9 4.1
13 .5 17.0 24.4 41.2
± ± ± ±
2. 8 2. 1 3. 7 5.
" v, volts; c, cycles per sec. h Numbers in parentheses indicate number of dogs used. '" All values are expressed as mean ± s o. TABLE
3. Composition and comparison of fi ve components of saliva evoked by pilocarpine stimulation Nerve stimulationa
Vari ous pilocarpine dosages (mg/kg of body weight )
Component
7 v. 20c" (18)··
0. 10 (61
0. 15 (6)
I
mg/100 ~~sa liva
Protein ... ......
433 . 3 ± 32 . o1 58. 5" ± 14. 61133. 6" ± 50 .5 I 173.9" ± 32. 8 m!f/ /00 mg protein
Protein-bound carhydrate Sialic acid ... . . Fucose . . . . . . . . . Hexose . Hexosamine
12.1 14. 5 25 .1 38 .1
± ± ± ±
1.9 1. 2 2.0 4.9
18 .6' 7 .8" 25.9'' 29.3'
± ± ± ±
4 .3 1. 8 5.1 5 .6
21 .2" 7. 1" 22. 1' 32 . 21
± ± ± ±
1.9 1.1 4.6 3.2
0 .4 (6)
0.2 (6)
18 . 1" 9 .3 1 24 .4' 32 . 7h
± 0. 9 ± 1.7 ± 2 .1 ± 3. 3
I 451. 4' 12 . ge 16. 3h 28 .8• 38 . 4•
± 76 .5
± ± ± ±
1. 9 1.8 1.7 4.1
Nerve stimulation : represents data received for maximum ra te of secretion. v, volts; c, cycles per sec. ' Numbers in parentheses indicate number of dogs used. <1 p < 0.001. ' Not significant. I p < 0.03. • P < 0.005. h p < 0.2. a h
the saliva resulting from the pilocarpine stimulation is significantly less than with nerve stimulation with dosages up to 0.4 mg per kg of body weight. At the dosage of 0.4 mg per kg of body weight, the protein content of saliva is not significantly different from that observed with nerve stimulation . The differences in carbohydrate content in the saliva compared with that found in nerve-stimulated saliva were significant in some areas, but not in others. The fucose concentrations were
less in most cases than any other carbohydrate fraction . As will be noted, the carbohydrate fractions were quite similar to those observed with nerve stimulation when the dosage was at the 0.4 mg per kg level. The consistency with which the differences in chemical concentration of saliva appear with nerve and pilocarpine stimulation, as observed by the small standard error, definitely supports the concept of the presence of a basic response difference between nerve and pilocarpine stimulation at the cellular level.
July 1971
Effects of aspirin administration on salivary secretion and its composition. The 5 dogs used in these experiments were given aspirin orally in doses of 4 grains per kg per day for a period ranging from 29 to 33 days. In all the tests made, the stimulation rate for nerve and pilocarpine effects was made at the previously determined maximum rate of secretion: 7 vat 20 cycles per sec for nerve stimulation and 0.2 mg per kg of body weight for pilocarpine. The average results obtained are presented in table 4. It will be noted that the maximum rate of secretion of the submaxillary gland was not significantly different from that reported in tables 2 and 3. Also, there was no difference in rate of secretion over that observed before aspirin was given. The data also show that the longtime administration of high doses of aspirin had no significant effect on the protein content of the saliva with either nerve or pilocarpine stimulation. In the case of the effects of aspirin on the protein-bound carbohydrates present in the saliva, however, there was a significant reduction found in all four fractions studied. The amounts of each carbohydrate studied are expressed as milligrams of carbohydrate bound per 100 mg of protein present in the collected sample. The actual distribution of the protein-bound carboTABLE 4.
hydrates shows that the sialic acid and fucose were the lowest in concentration with hexosamine the highest. The difference in carbohydrate content in the saliva collected via pilocarpine stimulation was greater than that observed with nerve stimulation. The calculated difference resulting from aspirin administration in terms of percentage gave a decrease for each of the four carbohydrates tested. The decrease with the nervestimulated group ranged from 25 to 44% while the pilocarpine-stimulated group gave a range of 56 to 73%. Of the four proteinbound carbohydrates studied, it appears that the sialic acid fraction is the most sensitive to aspirin administration and hexosamine the least. There seems to be little doubt that aspirin can have a marked effect on the concentration of the protein-bound carbohydrates. In experiments where the free nonprotein carbohydrates were determined, the amounts present were small and not significant. Effects of cortisone administration on salivary secretion and its composition. In a similar experiment, 5 dogs were tested with orally administered cortisone in daily dosages of 100 mg per dog. As indicated in table 5, there was no difference in the maximum secretion rate of saliva before and after 30 consecutive days of cortisone administration either with nerve stimulation
Effects of aspirin on salivary secretion and its composition a
Nerve stimulation P Value
Condition
Change from control
ml/a rvet wt /min
0 . 49 ± 0 . 03 j 0 . 46 ± 0 . 06
Pilocarpine stimulation P Value
Before aspirin
After aspirin
Before aspirin
Secretion rate .
NS•
413 ± 68
I
14.7 15.6 24.6 39.5
a Average of 5 dogs. • NS, not significant.
± ± ± ±
1.3 2.1 3.8 6.3
ml/g tt•et wt/min
0.47 ± 0.09 10.41 ± 0.13
NS
-12
NS
-27
m g/100 ml saliua
411 ± 48
8.2 9 .5 16 . 0 29.8
'/,
Yr
NS
-1
226 ± 116 1164 ± 115 mR/100 mg protein
mp,l /()(} m g protem
Protein-bound carbohydrate Sialic acid .. Fucose Hexose . Hexosa mine
Change from control
After aspirin
- 6
mg/ 100 ml saliua
Protein concentration .
73
EFFECTS OF ASPIRIN AND CORTISONE ON SALIVA
± ± ± ±
1.7 2.3 3.3 7.0
< 0 . 001 < 0.005 < 0.01 < 0 .1
-44 -39 -35 -25
18. 3 11.5 28.1 36 .8
± ± ± ±
8.0 3.1 5.5 4.5
4.9 4.6 10.1 16.3
± ± ± ±
2.2 3.9 3.5 6.7
< 0.001 < 0.05 < 0.01 < 0.001
-73 -60 - 64 -56
Vol. 61 , No . I
TAKEUCHI ET AL.
74 TABLE
5. Effects of cortisone on salivary secretion and its composition" Nerve stim ulation
Condition Before cort isone
ml/g
Secretion rate .
Change from control
P Valu e
Pilocarpine stimulation P Value
wel wt/m in
c·
0 .45 ± 0.1110.44 ± 0.09
' -2
NS '
417 ± 79 m~/100
0 .33 ± 0.091 0.45 ± 0 . 09 mg/ 100 ml
Protein-bound carbohydrate Sialic acid . Fucose Hexose . Hexosamine
14.7 17.6 24.1 42 . 1
± ± ± ±
2.4 2.5 1.7 3 .0
1 377 ± 93
- 10
NS
222 ± 114 mg/ 100
mp protein
15.9 12.2 18 . 2 33.3
± 2.5 ± 3.1 ± 2.0 ± 3.4
NS <0.05 < 0.001 < 0.005
+8 -31 - 24 -21
',
ml/g wet wt/min
mg/ 100 ml saliua
Protein concentration .
After cortisone
Before cortisone
After cortisone
22.1 ± 16.8 ± 28.3 ± 36.4 ±
5 .0 7.5 7.5 4.9
Change from control
I 180 ±50
~ ;:
NS
+36
NS
-19
NS < 0 .2 < 0 .01 < 0.01
+5 - 46 -47 - 26
~aliua
protein
23.2 ·9.1 14.9 27.0
± 5.2 ±7.5 ± 7.5 ± 0 .9
Average of 5 dogs. • NS, not significant.
a
or injection of pilocarpine. As in the case of the aspirin experiments, the protein content showed no significant difference. A depressant effect of cortisone on the composition of the protein-bound carbohydrate was noted in the fucose, hexoses, and hexosamines, but not so great as that observed in the aspirin experiment (see table 4). The decrease in concentration of these fractions was more statistically significant with nerve-produced saliva than in the pilocarpine-stimulated saliva samples, although in percentages, the pilocarpine samples gave greater differences. The one notable difference observed was that cortisone did not depress the sialic acid carbohydrate fraction, while with aspirin administration the sialic acid fraction was inhibited more than any of the other fractions studied. In general, there was less change in the production of carbohydrate than was observed with the aspirin treatment. As indicated in table 5, the percentage differences in concentration of the four carbohydrate fractions gave a range of + 8 to -31 % with the nerve stimulation and +5 to -47% when pilocarpine was used. Discussion In making comparative studies of the effects of longtime administration of drugs on the secretory function of an organ, such
as a salivary gland, it is important to eliminate such factors as the nature of the response to the stimulus, compensatory hypertrophy of the exposed gland, as well as the presence of any dietary differences that might occur during the time of drug administration. The establishment of a fixed response to nerve and pilocarpine stimulation, such as maximum rate of secretion, is important in making these studies so that the gland under these conditions is not overstimulated to the point where structural damage might be induced and the secreting power of the gland cells exceeded. The fact that the remaining salivary gland studied in these experiments, after the longtime administration of aspirin or cortisone, responded in their rate of secretion to either nerve or pilocarpine stimulation with the same degree of efficiency as previously observed supports the fact that the gland was not damaged as far as its responses to nerve and pilocarpine are concerned. Furthermore, to test whether the chemical changes observed in the saliva after the 30 days of drug administration were actually due to the drug and not to some type of compensatory reaction that might have taken place to alter the chemical composition of the salivary secretion, the following experiment was performed. The submaxillary
July 1971
EFFECTS OF ASPIRIN AND CORTISONE ON SALIVA
salivary glands of 5 dogs were exposed and their ducts cannulated. Saliva was then collected at the maximum rate of secretion by nerve stimulation as well as with an established pilocarpine rate of secretion. The samples in each case were tested for protein and carbohydrate composition as outlined above. The dogs were then kept for 60 days (30 days recuperation and 30 days to represent the drug administration period), after which . the salivary samples were similarly collected and tested for protein and carbohydrate composition. Theresults of all 5 dogs showed no significant change between the first and second salivary collection periods. The dogs ate all their provided food every day, and there was no change in total body weight. These experiments support the concept that the operative procedures.used did not alter the normal functioning of the salivary gland. As already mentioned, with the 5 dogs used in the aspirin experiment there was an occasional day when the dogs did not eat all their food, and on one or two occasions 1 of the dogs vomited. Although no significant weight loss occurred in these experimental animals, there still remained the possibility that the dietary upset could be responsible for the changes produced in the protein-bound carbohydrates observed in table 2. To rule out this possibility, the following experimental procedure was used. Saliva was collected from 3 dogs and a chemical analysis was made of the samples. The dogs were then kept on a daily routine of commercial dog biscuit for 30 days, except that the total dietary allotment was reduced by 50%. In this case the dog lost weight, but when the 30-day experimental period was over and the salivary secretion was chemically analyzed, it was noted that all the parameters studied showed no decrease as was observed in the aspirin and cortisone experiment. These findings are supported by the observations of Sreebny et al. 10 They also lend support to the fact that the experimental results observed and reported were not closely associated with any occasional small dietary upset resulting from aspirin administration. Although our observations on nerve and
75
pilocarpine stimulation of the salivary gland and its composition of different proteins and certain protein-bound carbohydrates are somewhat similar to those reported by Dische et al 9 they differ markedly in the strength and frequency of nerve stimulation used; Dische et al. 9 varied the voltage and kept the frequency constant (40 cycles per sec). The voltage variation is less a factor in obtaining quantitative results in physiological stimulations of live tissues than frequency changes. Furthermore, Dische et al. 9 used pilocarpine as a stimulant for salivary secretion by varying the dose from 0.4 mg to 4.0 mg at a continuous rate of infusion. This dosage and manner of administration is far different from that used in the above experiments and does not lend itself to the establishment of a threshold for maximum rate of secretion. Diamant et al., 11 from their experiments on submaxillary secretion in man, reported that the maximum rate of secretion occurred at a frequency of 10 cycles per sec with a voltage variation between 2 and 3 v. This low rate of frequency and voltage may be associated with species difference or the depth of anesthesia used. We believe that our method of calibration of nerve and pilocarpine administration was necessary to establish a standard for comparison of saliva composition before and after the longtime administration of such drugs as aspirin and cortisone. To explain the cause for the differences in salivary secretion and salivary composition that were observed between the aspirin- and·cortisone-treated dogs is not easy. Menguy and Masters, 2 • 3 in their experiments on gastric mucin production in dogs receiving aspirin and cortisone, discuss the possible theoretical cause for the decreases in mucin secretions that they observed. Our observations may be interpreted in a similar manner. An examination of their experimental procedure showed that they administered a total of 80 g of aspirin orally every 3rd day for about 40 days, and administered cortisone intravenously in doses of 150 mg every day for 15 days. In our experiments, much greater daily dosages of aspirin (4 grains per kg of
TAKEUCHI ET AL.
76
body weight) for 30 days were tolerated without any consistent ill effects except for slight losses of body weight. The cortisone dosage of 100 mg per day per dog was an estimate based on the effectiveness of this drug in clinical use and, therefore, it is admitted that any real comparison between the two drugs must be treated with caution.
5. 6.
7.
8. REFERENCES 1. Winkelman EI, Summerskill WHJ: Gastric secretion in relation to gastrointestinal bleeding from salicylate compounds. Gastroenterology 40:5663, 1961 2. Menguy R, Masters YF: Effect of cortisone on mucoprotein secretion by gastric antrum of dogs: pathogenesis of steroid ulcer. Surgery 54:19-28, 1963 3. Men guy R, Masters YF: Effects of aspirin on gastric mucous secretion. Surg Gynec Obstet 120:9298, 1965 4. Lowry OH, Rosenbrough NJ, Farr AL, et al:
9.
10.
11.
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Protein measurement with the Falin phenol reagent. J Bioi Chern 193:265- 275, 1951 Warren L: The thiobarbituric acid assay of sialic acids. J Bioi Chern 234:1971-1975, 1959 Dische Z, Shettles LB: A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J Bioi Chern 175: 595-603, 1948 Dubois M, Gills KA, Hamilton JD, et al : Colorimetric method for determination of sugars and related substances. Anal Chern 28:350-356, 1956 Remington C: Seromucoid and the bound carbohydrate of the serum proteins. Biochem J 34: 931-940, 1940 Dische Z, Pallavicini C, Kanasaki H, et al: Influence of the nature of the secretory stimulus on the composition of the carbohydrate moiety of glycoproteins of the submaxillary saliva. Arch Biochem 97:459-469, 1962 Sreebny LM, Johnson DA: Effect of food consistency and decreased food intake on rat parotid and pancreas. Amer J Physiol 215:455-460, 1968 Diamant H, Enfors B, Holmstedt B: Salivary secretion in man elicited by means of stimulation of the chorda tympani. Acta Physiol Scand 45: 293-299, 1959