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Effects of hypoxia and dietary aspirin on plasma and urinary amino acids in the rat
Effects of Hypoxia and Dietary Aspirin on Plasma and Urinary Amino Acids in the Rat By
DAVID A. VAUGHAN,PATRICIAR. KORTY AND JOHN L. STEELE
Plasma and urinary free amino acids were studied in rats receiving 0.5 percent acetylsalicylic acid (aspirin) while exposed to simulated high altitude. Rats were placed in a chamber at simulated 17,500 feet for 2 weeks. They were then fed a casein-based diet containing 0.5 percent aspirin as meals of 2 hour duration for a period of 2 weeks. Altitude and diet controls were run simultaneously. Plasma concentrations of 15 amino acids were measured at intervals of 4, 8, 15, and 22 hours after the last meal. Both aspirin and altitude modified the post prandial pattern of these amino acids. Glutamic acid levels rose sharply in the
aspirin fed rats at both ground level and altitude. Dietary controls showed no such rise. Both aspirin feeding and altitude exposure depressed peak concentrations of the other amino acids independently and there were no interactions between the two treatments. Aminoaciduria did not occur in either the aspirin-fed or altitude exposed rats. Although changes in renal function cannot be entirely ruled out, it would appear that both aspirin and altitude may affect amino acid absorption and metabolism and that these effects are often additive. (Metabolism 18: No. 12, December, 1055-1061, 1969)
A
CUTE SALICYLATE INTOXICATION has been reported to cause severe disturbances in amino acid metabolism. Aside from its apparent specific effect on glutamate metabolism,l.” salicylate may inhibit transport of several other amino acids into the cell or block renal reabsorption,3*4 leading to a generalized aminoaciduria.5zF Recently Levy et al .? have reported inhibition of transport of amino acids across the small intestine in the presence of salicylate and correlate this with uncoupling of oxidative phosphorylation. We are not aware, however, of any studies of protein or amino acid metabolism during prolonged chronic intake of salicylates at levels, for example, similar to those used for treatment of rheumatic disorders. Salicylates at these ~~~~. ___ __From the Pharmacology-Biochemistry Branch, Biosciences Division, USAF School of Aerospace Medicine, Brooks Air Force Base, Texas. Recei\,ed for publication May 26, 1969. The research reported irz this paper was conducted by personnel of the USAF School of Aerospace Medicine, Aerospace Medical Division, AFSC, United States Air Force, Brooks AFB. Texas. Further reproduction is authorized to satisfy the needs of the United States Co~~crnment. The arzimals involved in this study were maintained in accordance with the Guide for Laboratory Animal Facilities and Care as published by the National Academy of SciencesNational Research Council. DAVID A. VAUGHAN, PH.D.: Biochemist, Pharmacology-Biochemistry Branch, Biosciences Di~Ysiorl. USAF School of Aerospace Medicine, Brooks Air Force Base, Texas. PATRICIA R. KORTY, B.S.: Biologist, Pharmacology-Biochemistry Branch, Biosciences Division, USAF School of Aerospace Medicine, Brooks Air Force Base, Texas. JOHN L. STEELE, MASTER SERGEANT, USAF: Administrative Assistant, Physiology Division, USAF School of Aerospace Medicine, Brooks Air Force Base, Texas. METAROLISM. VOL. 18, No.
12 (DECEMBER),
1969
1055
1056
VAUGHAN, KORTY AND STEELE
levels stimulate metabolisms without severe toxic side effects. On the other hand, animals exposed to altitude are thought to have depressed nitrogen metabolism9 based on observations of lower urea excretion, although plasma levels of specific amino acids varied in both directions. We have already reported salicylate-altitude interactions on some parameters associated with carbohydrate metabolism. lo Controlled diets and measured daily food intakes were used in that experiment and a restricted time was allowed for eating. These feeding techniques are, in our opinion, the procedure of choice for the study of the effect of any agent or environment upon tissue metabolites. We therefore decided to measure plasma amino acid levels at intervals following rigidly defined meals and the effects thereupon of chronic acetylsalicylic acid feeding concomitant with hypoxic altitude exposure, in order to further define this one aspect of altitude-drug interactions. METHODS Young adult male Sprague-Dawley rats weighing 200-300 Gm. were trained to eat their daily ration as 2-hour meals. The control diet consisted of casein, 20 per cent; sucrose, 71 per cent; U.S.P. XIV salts, 4 per cent; corn oil, 5 per cent; and all required vitamins (Nutritional Biochemicals Corporation), 22 Gm./Kg. diet. After this, those rats to be kept at altitude were placed in a walk-in chamber maintained at the equivalent of 17,500 feet. They continued to eat the control diet until food intakes and growth rates reached prechamber levels (2 weeks). At this time, half of the rats at altitude and half of those at ground level began eating a diet containing 0.5 per cent aspirin (acetylsalicylic acid) mixed with the control diet. Aspirin and control feeding continued for 2 weeks. The animals were then sacrificed by decapitation at 4, 8, 15, and 22 hours after they had finished their last meal. Blood was collected and immediately processed for the amino acid analyses. Protein was precipitated with 3 per cent sulfosalicylic acid and amino acids were assayed Reon the filtrate by the method of Moore and Stein,rt using a Technicon AutoAnalyzer. sults are expressed as pmoles/lOO ml. of plasma. Arithmetic means were compared by Fisher’s t test and differences referred to as significant have a probability level of 0.05 or less. RESULTS
AND DISCUSSION
The plasma amino acid concentrations for ground level and altitude exposed rats are shown in Fig. 1 and 2, respectively. Twenty-four-hour excretions of amino acids in the urine are shown in Table 1. In Table 2, a summary of the standard error data for each amino acid is presented. In the ground level rats receiving the control diet, the highest levels were generally observed at either 8 or 15 hours after eating. Glycine and glutamic acid concentrations, however, did for not peak at all following the daily meal. lo The most probable explanation glycine is that its content in casein is extremely low and consequently plasma concentrations remained at fasting levels or dropped even lower in response to requirements for cellular synthesis, which, in turn, were stimulated by the large influx of dietary amino acids. On the other hand, nearly 25 per cent of casein consists of glutamic acid and its homeostasis in the plasma of animals fed the control diet is no doubt a result of very efficient removal from the plasma, via a variety of conversions such as transamination, decarboxylation, and dehydrogenation. The effect of aspirin in the post-prandial glutamic acid levels was unique when compared with its effect on the other amino acids measured. Levels rose rapidly to maximum values at 8 hours but returned to concentrations not sig-
HYPOXIA
AND DIETARY
1057
ASPIRIN
60
Fig. amino
l.-Plasma acid levels.
Black triangles indicate ground control; b I a c k dots, ground aspirin; white altitude triangles, control; white dots, altitude aspirin. Six rats per data
10
UTAMIC
ACID
LYSINE
0
60
point.
THREONINE
10
0 04
0
15
22048 HOURS
15 AFTER
22
0
4
6
15
22
EATING
nificantly different from controls by the end of 22 hours. While this may suggest inhibition of some pathway(s) by which glutamic acid is transformed, this inhibition is probably a transitory and recurring phenomenon, since the animal is able to eventually dispose of glutamic acid, although it takes it all day to reduce these concentrations to levels found in the control rats. Glutamic acid levels did not rise in this way when the aspirin treated animals were fed a single meal without aspirin. Excretion of free glutamic acid in the urine is apparently not a factor in this disposal, for the 24-hour values shown in Table 1 are infinitesimal fractions of the amount ingested in casein during the 2-hour meal. Gould and SmithlJ have shown that several reactions associated with glutamic acid are inhibited by large in vivo doses or by large in vitro concentrations of salicylate. Transitory reversible inhibition of glutamic acid transformations may therefore coincide with the daily peak levels of plasma salicylate observed in rats receiving
1058
VAUGHAN,
KORTY
AND STEELE
Fig. 2.-Plasma amino acid levels. Black triangles indicate ground control; black dots, ground aspirin; white triancongles, altitude trol; white dots, altitude aspirin. Six rats per data point.
% 8 14% 12 10 8 6 ISOLEUCJNE
4
+ ALANINE
FOOD h
048
METWJNINE 000
FOOO
15
22
0
HOURS
4
8
AFTER
15
22048
1s
22
EATING
daily 2-hour meals containing aspirinlo Transaminase reactions involving glutamic acid are not affected adversely at this level of aspirin feeding, however, for we found increased activity in the livers of all aspirin fed rats both at ground level and at altitude.lO Other possibilities are being investigated. The effects of aspirin feeding on the post-meal patterns of the other amino acids will now be considered. With a few exceptions, the effect of aspirin seems to be to displace the plasma concentration patterns downward (Figs. 1 and 2). Leucine, isoleucine, tyrosine, phenylalanine, histidine, serine, lysine, and threonine were all affected in this manner in the ground level rats. The fasting levels of alanine, valine, methionine, leucine, tyrosine, phenylalanine, lysine, and arginine were depressed in the ground level aspirin fed rats. The downward displacement of the post prandial patterns was also evident in the altitude ex-
HYPOXIA
AND DIETARY
1059
ASPIRIN
Table 1 .-Twenty-four-hour Ground
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
Control Level
Urinary Excretion of Amino Acids
(pmoles) Altitude
0.112.02* 1.80 k .24 1.08 2 .15 1.51 k .19 2.67 k .18 1.55 rt .lO 0.68 * .12 0.39 i .06 0.39 f .07 0.65 t .12 0.44 2 .06 0.40 f .07 1.41 f .I5 0.84 -c .20 0.68 -c .I6
0.80 1.47 1.05 2.30 2.43 2.00 0.56 0.47 0.65 0.63 0.41 0.30 1.17 0.75 0.59
rfr .18 $ * .09 2 .06 k .43 f. .26 k .45 2 .I8 k .05 2 .17 k .18 r .07 & .06 _’ .21 2 .06 2 .lO
Ground
0.12 0.90 0.80 1.02 3.40 1.38 0.38 0.22 0.24 0.40 0.38 0.22
0.5% Aspirin Level
f k k ? k k ? k e -c 2 * 1.04 h 0.50 * 0.34 2
(~moles) ~~ Altitude
0.99 * 1.41 & 1.29 ?I 3.76 2 4.08 * 2.96 e 0.90 k 0.45 i0.59 rt 0.80 k 0.48 f 0.48 f 1.25 k 0.63 k 0.65 -c
.05 .29 t .21 .31 .38 .I1 .16 .08 .09 .18 .13 .lO .26 .09 .20
.21 : .lO .lO .63 9 .61 : .33 0 .13 : .I0 .I4 .15 .lO .09 .09 .lO .09 _
* SE of mean: 6 animals per mean. i p < .05: control vs. aspirin. $ p < .05: ground vs. altitude. Table Z.-Standard
Errors of Points on Amino Acid Curves Range
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
0.3-1.1 1.4-8.4 0.8-4.5 1.8-6.2 1.3-5.3 2.2-7.3 0.7-2.3 0.3-1.0 0.4-l .5 0.5-2.0 0.2-1.6 0.1-0.7 1.3-7.5 0.3-1.0 0.7-2.7
Meall
0.4* 3.9 3.0 3.4 2.7 4.0 1.6 0.6 0.8 1.2 0.9 0.5 3.7 0.6 1.4
-
-
* I6 standard errors per mean.
posed aspirin fed rats, although in some instances, notably with tyrosine, phenylalanine, valine, serine, and lysine, the depression was not as marked. This may be due to a compressing effect on the curves as a result of generally lower levels in the altitude exposed rats. Especially is this noticeable at the fasting (22hour) interval, when only threonine, tyrosine, and lysine concentrations were significantly depressed. In no case, however, was the effect of aspirin reversed at altitude. Twenty-four-hour excretions of free amino acids were measured and are recorded in Table 1. No aminoaciduria attributable to aspirin feeding was found. In fact, the excretion of any amino acid was a very small fraction (less than 0.1% ) of the amount eaten in the daily meal. This absence of an amino-
1060
VAUGHAN,
KORTY
AND STEELE
aciduria is different than the observations of other investigators”,” and could be a result of a temporary adaptation to chronic dosage or to a lower level of intake of salicylate. In any case, the lowered plasma concentration of most of the amino acids measured can hardly be a result of depressed reabsorption or any other kidney dysfunction in this experiment. In our opinion, a more probable hypothesis is that amino acid transport across the small intestine is inhibited’ which might in turn cause a delay in absorption and thereby flatten the curves of plasma concentration, of several amino acids, as can be seen in the figures. The other contributing factor to plasma concentrations is removal for catabolic processes or for protein synthesis. Salicylates are metabolic stimulants, due to their uncoupling effect on oxidative phosphorylation,13 and the fact that transaminatiorP and oxygen consumption* are increased would support the idea that, where specific abnormalities such as glutamic acid responses are not involved, removal of amino acids from plasma might proceed at higher rates in aspirin fed rats for the purpose of supporting increased catabolism. If it is true, as Van Liere and Stickney state, 14 that gastric emptying is delayed by chronic exposure to altitude ( 18,000 feet), then the time at which plasma amino acids are measured would become critical in comparing altitude animals with ground level animals. We have measured concentrations at 4 intervals following a 2 hour meal, and the results indicate that plasma amino acids did not begin to rise in the altitude exposed rats until after 8 hours post prandium, while at ground level this rise occurred between 4 and 8 hours, the exception being glycine which did not rise at all because of its extremely low content in casein. These observations suggest that absorption is delayed during hypoxic altitude of most of the exposure and confirm the work cited above. l1 Concentrations amino acids measured (except serine and arginine), even when at maximum recorded values, were depressed when compared to respective ground level concentrations. It is quite possible that the maximum plasma concentrations in the altitude exposed rats may have occurred at a time immediately before or after 15 hours post prandium, at an interval not measured in this experiment, and so a definite conclusion regarding the effect of altitude on absolute maximum values is probably not warranted. In spite of these reservations, it is abundantly clear that either the release of amino acids into the plasma or the removal of amino acids from the plasma, or both, may be modified by continuous exposure to altitude for 4 weeks. Whether the animal is, at this stage, on an adaptive course, and would eventually be indistinguishable in this regard from its ground control, is not known. One might speculate that an increase in certain synthetic processes, for example erythropoiesis, as a result of prolonged hypoxic altitude exposure, might “permanently” alter the way in which amino acids are utilized following protein intake and might also change the distribution or composition of amino acid pools. The urinary excretion of some of the amino acids (aspartic acid on both diets, and glutamic acid, alanine, and valine on the aspirin supplemented diet) increased during altitude exposure. The significance of these effects is not clear, and the direction of change is the reverse of that reported by others.” We have
demonstrated
that
aspirin
fed in 2-hour
meals
modifies
the post
HYPOXIA
AND DIETARY
1061
ASPIRIN
prandial pattern of plasma amino acid concentrations. Glutamic acid levels rise to peaks not seen in control rats. In general, other amino acids peak later and levels are not as high as in controls. Altitude exposure further modifies these effects in that it depresses plasma amino acid concentrations and also delays the appearance of maximum levels. Interactions between aspirin feeding and altitude exposure were not generally observed. ACKNOWLEDGMENT The authors express their thanks to Sgt. William T. Knowles
for his technical
assistance.
REFERENCES 1. Gould. B. J., and Smith, M. J. H.: Inhibition of rat brain glutamate decarboxylase activity by salicylate in vitro. J. Pharm. Pharmacol. 17:15-18, 1965. 2. -, and Smith, M. J. H.: Salicylate and aminotransfernses. J. Pharm. Pharmacol. 17: X3-88, 1965. 3. Segal. S.. and Blair, A.: In vitro effect of salicylate on amino acid accumulation by kidney cortex slices. Nature 200: 139-141. 1963. 4. Elliot. H. C., Jr., and Murdaugh, H. V., Jr.: Effects of acetylsalicylic acid on excretion of endogenous metabolites by man. Proc. Sot. Exp. Biol. Med. 109:333-335, 1962. 5. Andrews, B. F.. Bruton, D. C., and Knoblock. E. C.: Aminoacidurin in salicylate intoxication. Amer. J. Med. Sci. 242: 411-414, 1961. 6. Berry, H. K.. and Guest, G. M.: The cfJect of salicylate intoxication on amino acid excretions in rats. Metabolism 12:760770. 1963. 7. T.evy. G., Angelino, N. J., and Matsuznwa. T.: Effect of certain nonsteroid antirheumatic drugs on active amino acid transport across the small intestine. J. Pharm. Sci. 56:681-683, 1967. 8. Becker. E. .I.: Effect of sodium sali-
cylate on the food and oxygen consumption of rats. J. Nutrition 66:237-244, 1958. 9. Mefferd, R. B., Jr., and Hale, H. B.: Altitude-induced changes in plasma and urinary nitrogen and electrolyte constituents of rats. Zrl Weihe, W. H. (Ed.): The Physiological Effects of High Altitude. New York. Macmillan, 1962. 10. Vaughan. D. A.. Steele, J. I_.. and Korty, P. R.: Metabolic effects of feeding aspirin to rats at altitude. Fed. Proc. 28: 1110-1114, 1969. 11. Moore, S., and Stein, W. H.: Procedures in the chromatographic determination of amino acids on 4% cross-linked sulfonated polystyrene resins. J. Biol. Chem. 211:893-906, 1954. 12. Rogers, Q. R.. Spolter, P. D.. and Harper, A. E.: Effect of leucine-isoleucine antagonism on plasma amino acid pattern of rats. Arch. Biochem. Biophys. 97:497-504. 1962. 13. Brody. T. M.: Action of sodium salicylate and related compounds on tissue metabolism in vitro. J. Pharmacol. Exp. Ther. 117:39-51, 1956. 14. Van Liere, E. J., and Stickney, J. C.: Hypoxia. Chicago, University of Chicago Press, 1963, p. 66.
DAVID A. VAUGHAN,PATRICIAR. KORTY AND JOHN L. STEELE
Plasma and urinary free amino acids were studied in rats receiving 0.5 percent acetylsalicylic acid (aspirin) while exposed to simulated high altitude. Rats were placed in a chamber at simulated 17,500 feet for 2 weeks. They were then fed a casein-based diet containing 0.5 percent aspirin as meals of 2 hour duration for a period of 2 weeks. Altitude and diet controls were run simultaneously. Plasma concentrations of 15 amino acids were measured at intervals of 4, 8, 15, and 22 hours after the last meal. Both aspirin and altitude modified the post prandial pattern of these amino acids. Glutamic acid levels rose sharply in the
aspirin fed rats at both ground level and altitude. Dietary controls showed no such rise. Both aspirin feeding and altitude exposure depressed peak concentrations of the other amino acids independently and there were no interactions between the two treatments. Aminoaciduria did not occur in either the aspirin-fed or altitude exposed rats. Although changes in renal function cannot be entirely ruled out, it would appear that both aspirin and altitude may affect amino acid absorption and metabolism and that these effects are often additive. (Metabolism 18: No. 12, December, 1055-1061, 1969)
A
CUTE SALICYLATE INTOXICATION has been reported to cause severe disturbances in amino acid metabolism. Aside from its apparent specific effect on glutamate metabolism,l.” salicylate may inhibit transport of several other amino acids into the cell or block renal reabsorption,3*4 leading to a generalized aminoaciduria.5zF Recently Levy et al .? have reported inhibition of transport of amino acids across the small intestine in the presence of salicylate and correlate this with uncoupling of oxidative phosphorylation. We are not aware, however, of any studies of protein or amino acid metabolism during prolonged chronic intake of salicylates at levels, for example, similar to those used for treatment of rheumatic disorders. Salicylates at these ~~~~. ___ __From the Pharmacology-Biochemistry Branch, Biosciences Division, USAF School of Aerospace Medicine, Brooks Air Force Base, Texas. Recei\,ed for publication May 26, 1969. The research reported irz this paper was conducted by personnel of the USAF School of Aerospace Medicine, Aerospace Medical Division, AFSC, United States Air Force, Brooks AFB. Texas. Further reproduction is authorized to satisfy the needs of the United States Co~~crnment. The arzimals involved in this study were maintained in accordance with the Guide for Laboratory Animal Facilities and Care as published by the National Academy of SciencesNational Research Council. DAVID A. VAUGHAN, PH.D.: Biochemist, Pharmacology-Biochemistry Branch, Biosciences Di~Ysiorl. USAF School of Aerospace Medicine, Brooks Air Force Base, Texas. PATRICIA R. KORTY, B.S.: Biologist, Pharmacology-Biochemistry Branch, Biosciences Division, USAF School of Aerospace Medicine, Brooks Air Force Base, Texas. JOHN L. STEELE, MASTER SERGEANT, USAF: Administrative Assistant, Physiology Division, USAF School of Aerospace Medicine, Brooks Air Force Base, Texas. METAROLISM. VOL. 18, No.
12 (DECEMBER),
1969
1055
1056
VAUGHAN, KORTY AND STEELE
levels stimulate metabolisms without severe toxic side effects. On the other hand, animals exposed to altitude are thought to have depressed nitrogen metabolism9 based on observations of lower urea excretion, although plasma levels of specific amino acids varied in both directions. We have already reported salicylate-altitude interactions on some parameters associated with carbohydrate metabolism. lo Controlled diets and measured daily food intakes were used in that experiment and a restricted time was allowed for eating. These feeding techniques are, in our opinion, the procedure of choice for the study of the effect of any agent or environment upon tissue metabolites. We therefore decided to measure plasma amino acid levels at intervals following rigidly defined meals and the effects thereupon of chronic acetylsalicylic acid feeding concomitant with hypoxic altitude exposure, in order to further define this one aspect of altitude-drug interactions. METHODS Young adult male Sprague-Dawley rats weighing 200-300 Gm. were trained to eat their daily ration as 2-hour meals. The control diet consisted of casein, 20 per cent; sucrose, 71 per cent; U.S.P. XIV salts, 4 per cent; corn oil, 5 per cent; and all required vitamins (Nutritional Biochemicals Corporation), 22 Gm./Kg. diet. After this, those rats to be kept at altitude were placed in a walk-in chamber maintained at the equivalent of 17,500 feet. They continued to eat the control diet until food intakes and growth rates reached prechamber levels (2 weeks). At this time, half of the rats at altitude and half of those at ground level began eating a diet containing 0.5 per cent aspirin (acetylsalicylic acid) mixed with the control diet. Aspirin and control feeding continued for 2 weeks. The animals were then sacrificed by decapitation at 4, 8, 15, and 22 hours after they had finished their last meal. Blood was collected and immediately processed for the amino acid analyses. Protein was precipitated with 3 per cent sulfosalicylic acid and amino acids were assayed Reon the filtrate by the method of Moore and Stein,rt using a Technicon AutoAnalyzer. sults are expressed as pmoles/lOO ml. of plasma. Arithmetic means were compared by Fisher’s t test and differences referred to as significant have a probability level of 0.05 or less. RESULTS
AND DISCUSSION
The plasma amino acid concentrations for ground level and altitude exposed rats are shown in Fig. 1 and 2, respectively. Twenty-four-hour excretions of amino acids in the urine are shown in Table 1. In Table 2, a summary of the standard error data for each amino acid is presented. In the ground level rats receiving the control diet, the highest levels were generally observed at either 8 or 15 hours after eating. Glycine and glutamic acid concentrations, however, did for not peak at all following the daily meal. lo The most probable explanation glycine is that its content in casein is extremely low and consequently plasma concentrations remained at fasting levels or dropped even lower in response to requirements for cellular synthesis, which, in turn, were stimulated by the large influx of dietary amino acids. On the other hand, nearly 25 per cent of casein consists of glutamic acid and its homeostasis in the plasma of animals fed the control diet is no doubt a result of very efficient removal from the plasma, via a variety of conversions such as transamination, decarboxylation, and dehydrogenation. The effect of aspirin in the post-prandial glutamic acid levels was unique when compared with its effect on the other amino acids measured. Levels rose rapidly to maximum values at 8 hours but returned to concentrations not sig-
HYPOXIA
AND DIETARY
1057
ASPIRIN
60
Fig. amino
l.-Plasma acid levels.
Black triangles indicate ground control; b I a c k dots, ground aspirin; white altitude triangles, control; white dots, altitude aspirin. Six rats per data
10
UTAMIC
ACID
LYSINE
0
60
point.
THREONINE
10
0 04
0
15
22048 HOURS
15 AFTER
22
0
4
6
15
22
EATING
nificantly different from controls by the end of 22 hours. While this may suggest inhibition of some pathway(s) by which glutamic acid is transformed, this inhibition is probably a transitory and recurring phenomenon, since the animal is able to eventually dispose of glutamic acid, although it takes it all day to reduce these concentrations to levels found in the control rats. Glutamic acid levels did not rise in this way when the aspirin treated animals were fed a single meal without aspirin. Excretion of free glutamic acid in the urine is apparently not a factor in this disposal, for the 24-hour values shown in Table 1 are infinitesimal fractions of the amount ingested in casein during the 2-hour meal. Gould and SmithlJ have shown that several reactions associated with glutamic acid are inhibited by large in vivo doses or by large in vitro concentrations of salicylate. Transitory reversible inhibition of glutamic acid transformations may therefore coincide with the daily peak levels of plasma salicylate observed in rats receiving
1058
VAUGHAN,
KORTY
AND STEELE
Fig. 2.-Plasma amino acid levels. Black triangles indicate ground control; black dots, ground aspirin; white triancongles, altitude trol; white dots, altitude aspirin. Six rats per data point.
% 8 14% 12 10 8 6 ISOLEUCJNE
4
+ ALANINE
FOOD h
048
METWJNINE 000
FOOO
15
22
0
HOURS
4
8
AFTER
15
22048
1s
22
EATING
daily 2-hour meals containing aspirinlo Transaminase reactions involving glutamic acid are not affected adversely at this level of aspirin feeding, however, for we found increased activity in the livers of all aspirin fed rats both at ground level and at altitude.lO Other possibilities are being investigated. The effects of aspirin feeding on the post-meal patterns of the other amino acids will now be considered. With a few exceptions, the effect of aspirin seems to be to displace the plasma concentration patterns downward (Figs. 1 and 2). Leucine, isoleucine, tyrosine, phenylalanine, histidine, serine, lysine, and threonine were all affected in this manner in the ground level rats. The fasting levels of alanine, valine, methionine, leucine, tyrosine, phenylalanine, lysine, and arginine were depressed in the ground level aspirin fed rats. The downward displacement of the post prandial patterns was also evident in the altitude ex-
HYPOXIA
AND DIETARY
1059
ASPIRIN
Table 1 .-Twenty-four-hour Ground
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
Control Level
Urinary Excretion of Amino Acids
(pmoles) Altitude
0.112.02* 1.80 k .24 1.08 2 .15 1.51 k .19 2.67 k .18 1.55 rt .lO 0.68 * .12 0.39 i .06 0.39 f .07 0.65 t .12 0.44 2 .06 0.40 f .07 1.41 f .I5 0.84 -c .20 0.68 -c .I6
0.80 1.47 1.05 2.30 2.43 2.00 0.56 0.47 0.65 0.63 0.41 0.30 1.17 0.75 0.59
rfr .18 $ * .09 2 .06 k .43 f. .26 k .45 2 .I8 k .05 2 .17 k .18 r .07 & .06 _’ .21 2 .06 2 .lO
Ground
0.12 0.90 0.80 1.02 3.40 1.38 0.38 0.22 0.24 0.40 0.38 0.22
0.5% Aspirin Level
f k k ? k k ? k e -c 2 * 1.04 h 0.50 * 0.34 2
(~moles) ~~ Altitude
0.99 * 1.41 & 1.29 ?I 3.76 2 4.08 * 2.96 e 0.90 k 0.45 i0.59 rt 0.80 k 0.48 f 0.48 f 1.25 k 0.63 k 0.65 -c
.05 .29 t .21 .31 .38 .I1 .16 .08 .09 .18 .13 .lO .26 .09 .20
.21 : .lO .lO .63 9 .61 : .33 0 .13 : .I0 .I4 .15 .lO .09 .09 .lO .09 _
* SE of mean: 6 animals per mean. i p < .05: control vs. aspirin. $ p < .05: ground vs. altitude. Table Z.-Standard
Errors of Points on Amino Acid Curves Range
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
0.3-1.1 1.4-8.4 0.8-4.5 1.8-6.2 1.3-5.3 2.2-7.3 0.7-2.3 0.3-1.0 0.4-l .5 0.5-2.0 0.2-1.6 0.1-0.7 1.3-7.5 0.3-1.0 0.7-2.7
Meall
0.4* 3.9 3.0 3.4 2.7 4.0 1.6 0.6 0.8 1.2 0.9 0.5 3.7 0.6 1.4
-
-
* I6 standard errors per mean.
posed aspirin fed rats, although in some instances, notably with tyrosine, phenylalanine, valine, serine, and lysine, the depression was not as marked. This may be due to a compressing effect on the curves as a result of generally lower levels in the altitude exposed rats. Especially is this noticeable at the fasting (22hour) interval, when only threonine, tyrosine, and lysine concentrations were significantly depressed. In no case, however, was the effect of aspirin reversed at altitude. Twenty-four-hour excretions of free amino acids were measured and are recorded in Table 1. No aminoaciduria attributable to aspirin feeding was found. In fact, the excretion of any amino acid was a very small fraction (less than 0.1% ) of the amount eaten in the daily meal. This absence of an amino-
1060
VAUGHAN,
KORTY
AND STEELE
aciduria is different than the observations of other investigators”,” and could be a result of a temporary adaptation to chronic dosage or to a lower level of intake of salicylate. In any case, the lowered plasma concentration of most of the amino acids measured can hardly be a result of depressed reabsorption or any other kidney dysfunction in this experiment. In our opinion, a more probable hypothesis is that amino acid transport across the small intestine is inhibited’ which might in turn cause a delay in absorption and thereby flatten the curves of plasma concentration, of several amino acids, as can be seen in the figures. The other contributing factor to plasma concentrations is removal for catabolic processes or for protein synthesis. Salicylates are metabolic stimulants, due to their uncoupling effect on oxidative phosphorylation,13 and the fact that transaminatiorP and oxygen consumption* are increased would support the idea that, where specific abnormalities such as glutamic acid responses are not involved, removal of amino acids from plasma might proceed at higher rates in aspirin fed rats for the purpose of supporting increased catabolism. If it is true, as Van Liere and Stickney state, 14 that gastric emptying is delayed by chronic exposure to altitude ( 18,000 feet), then the time at which plasma amino acids are measured would become critical in comparing altitude animals with ground level animals. We have measured concentrations at 4 intervals following a 2 hour meal, and the results indicate that plasma amino acids did not begin to rise in the altitude exposed rats until after 8 hours post prandium, while at ground level this rise occurred between 4 and 8 hours, the exception being glycine which did not rise at all because of its extremely low content in casein. These observations suggest that absorption is delayed during hypoxic altitude of most of the exposure and confirm the work cited above. l1 Concentrations amino acids measured (except serine and arginine), even when at maximum recorded values, were depressed when compared to respective ground level concentrations. It is quite possible that the maximum plasma concentrations in the altitude exposed rats may have occurred at a time immediately before or after 15 hours post prandium, at an interval not measured in this experiment, and so a definite conclusion regarding the effect of altitude on absolute maximum values is probably not warranted. In spite of these reservations, it is abundantly clear that either the release of amino acids into the plasma or the removal of amino acids from the plasma, or both, may be modified by continuous exposure to altitude for 4 weeks. Whether the animal is, at this stage, on an adaptive course, and would eventually be indistinguishable in this regard from its ground control, is not known. One might speculate that an increase in certain synthetic processes, for example erythropoiesis, as a result of prolonged hypoxic altitude exposure, might “permanently” alter the way in which amino acids are utilized following protein intake and might also change the distribution or composition of amino acid pools. The urinary excretion of some of the amino acids (aspartic acid on both diets, and glutamic acid, alanine, and valine on the aspirin supplemented diet) increased during altitude exposure. The significance of these effects is not clear, and the direction of change is the reverse of that reported by others.” We have
demonstrated
that
aspirin
fed in 2-hour
meals
modifies
the post
HYPOXIA
AND DIETARY
1061
ASPIRIN
prandial pattern of plasma amino acid concentrations. Glutamic acid levels rise to peaks not seen in control rats. In general, other amino acids peak later and levels are not as high as in controls. Altitude exposure further modifies these effects in that it depresses plasma amino acid concentrations and also delays the appearance of maximum levels. Interactions between aspirin feeding and altitude exposure were not generally observed. ACKNOWLEDGMENT The authors express their thanks to Sgt. William T. Knowles
for his technical
assistance.
REFERENCES 1. Gould. B. J., and Smith, M. J. H.: Inhibition of rat brain glutamate decarboxylase activity by salicylate in vitro. J. Pharm. Pharmacol. 17:15-18, 1965. 2. -, and Smith, M. J. H.: Salicylate and aminotransfernses. J. Pharm. Pharmacol. 17: X3-88, 1965. 3. Segal. S.. and Blair, A.: In vitro effect of salicylate on amino acid accumulation by kidney cortex slices. Nature 200: 139-141. 1963. 4. Elliot. H. C., Jr., and Murdaugh, H. V., Jr.: Effects of acetylsalicylic acid on excretion of endogenous metabolites by man. Proc. Sot. Exp. Biol. Med. 109:333-335, 1962. 5. Andrews, B. F.. Bruton, D. C., and Knoblock. E. C.: Aminoacidurin in salicylate intoxication. Amer. J. Med. Sci. 242: 411-414, 1961. 6. Berry, H. K.. and Guest, G. M.: The cfJect of salicylate intoxication on amino acid excretions in rats. Metabolism 12:760770. 1963. 7. T.evy. G., Angelino, N. J., and Matsuznwa. T.: Effect of certain nonsteroid antirheumatic drugs on active amino acid transport across the small intestine. J. Pharm. Sci. 56:681-683, 1967. 8. Becker. E. .I.: Effect of sodium sali-
cylate on the food and oxygen consumption of rats. J. Nutrition 66:237-244, 1958. 9. Mefferd, R. B., Jr., and Hale, H. B.: Altitude-induced changes in plasma and urinary nitrogen and electrolyte constituents of rats. Zrl Weihe, W. H. (Ed.): The Physiological Effects of High Altitude. New York. Macmillan, 1962. 10. Vaughan. D. A.. Steele, J. I_.. and Korty, P. R.: Metabolic effects of feeding aspirin to rats at altitude. Fed. Proc. 28: 1110-1114, 1969. 11. Moore, S., and Stein, W. H.: Procedures in the chromatographic determination of amino acids on 4% cross-linked sulfonated polystyrene resins. J. Biol. Chem. 211:893-906, 1954. 12. Rogers, Q. R.. Spolter, P. D.. and Harper, A. E.: Effect of leucine-isoleucine antagonism on plasma amino acid pattern of rats. Arch. Biochem. Biophys. 97:497-504. 1962. 13. Brody. T. M.: Action of sodium salicylate and related compounds on tissue metabolism in vitro. J. Pharmacol. Exp. Ther. 117:39-51, 1956. 14. Van Liere, E. J., and Stickney, J. C.: Hypoxia. Chicago, University of Chicago Press, 1963, p. 66.