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A colorimetric micromethod for determination of ammonia; the ammonia content of rat tissues and human plasma
& R C H I V E S OF B I O C H E M I S T R Y AND B I O P H Y S I C S
66, ~01-309 (1957)
A Colorimetric Micromethod for Determination of Ammonia; the Ammonia Content of Rat Tissues and H u m a n Plasma I Rose H. Brown, George D. Duda, Seymour Korkes2 and Philip Handler From the Department of Biochemistry, Duke University ~chool of Medicine, Durham, North Carolina Received May 7, 1956 INTRODUCTION
The determination of ammonia in the presence of labile amides, such as glutamine, is a frequently encountered problem which has been particularly difficult for those biological systems wherein the ammonia concentration is small relative to that of the amide. This problem has been approached most successfully by rapid removal of the ammonia under conditions such that amide hydrolysis is negligible (1, 2). On the assumption that amide decomposition is first order with time, it follows that such interference is best minimized if the sample can be diluted before microdiffusion. Since this would also dilute the ammonia, the ideal method also requires a sensitive method for estimation of the diffused ammonia. The present report describes a procedure which combines microdiffusioa with a sensitive colorimetric procedure. The reaction between NH3 and phenol in the presence of hypochlorite to yield a blue dye, described by Berthelot in 1859, has served as the basis for several procedures for ammonia determination (3-5). The addition of small amounts of nitroprusside markedly accelerates color production, and Zalta and Lubochinsky (6) have so employed it in a method for NH3 determination in Kjeldahl digests. Seligson and Seligson (2) have described a simple, inexpensive modification of the microdiffusion These studies were supported by grants from the Committee on Growth, National Research Council (MET-47) ; National Institutes of Health [B-906(C9) ]; and Contract AT-(40-1)-289 between Duke University and the U. S. Atomic Energy Commission. 2 Deceased: December 10, 1955. 301
302
BROWN~ DUDA~ KORKES AND HANDLER
p r o c e d u r e of T o m p k i n s a n d Kirk: (7) w h e r e i n r a p i d diffusion is a s s u r e d b y r o t a t i n g t h e a m m o n i a s a m p l e as a film on t h e surface of a s m a l l vial. B y c o m b i n i n g t h i s diffusion t e c h n i q u e w i t h t h e colorir~etric m e t h o d of Z a l t a a n d L u b o c h i n s k y , a m m o n i a m a y r e a d i l y be d ~ e r m i n e d in t h e presence of a 30-fold ~excess of g l u t a m i n e . E x P F ~IMENTAL
Reagents (a) Sodium hypochlorite, 0.06 N, is prepared by dilution of "Clorox" 1:20. I t is standardized by addition of solid KI, acidification with H2SO4, and titration with standard thiosulfate. The absolute concentration is not critical. No detectable differences in color have been observed from 0.05 to 0,07 N. (b) Sodium nitroprusside, stock 0.5%. Fresh 1:100 dilutions are prepared for each analytical series. (c) Sodium carbonate, 0.05 M. (d) Sodium carbonate, 50%. (e) Sodium phenolate. Dissolve 2.5 g. of freShly distilled phenol and 1.25 g. of NaOtt in 100 mI. of water and allow to stand 2 days before using. (f) Sulfuric acid, 1 N.
Procedure The sample, not to exceed 1.0 ml. and containing not more than 0.2 ~mole of NH3, is pipetted into the bottom of a bottle (Kimble, Neutraglass N-51A, 35 ml., 3~% cm. outside diam., 7 ~ cm. long) which has previously been prepared by making a small annulus around the bottle, approximately 3 ~ era. from the base. The bottle is placed on its side, and 0.5 ml. of 50% Na~CO~ is carefully added on the distal side of the annulus so that it does not mix with the sample. A previously prepared paddle-shaped strip of filter paper, 1 cm. at the base and about 1.5 cm. long, is inserted into the open end of the glass tubing which passes through the No. 1 one-hole rubber stopper used to seal the vessel. Then 0.01 ml. of 1 N H2SO4 is placed on the paper at its base; this should not saturate the paper. After the vessel is thus sealed, the contents are carefully mixed and the bottle is then mechanically rotated on its axis until diffusion is complete. Our device operates at 78 r.p.m. The diffusion bottle, before mixing, is shown in Fig. 1. The paper is then transferred, with forceps, to a euvette containing 1.0 ml. of water. In the follow~
FIG. 1. The diffusion bottle with absorption paddle, sample, and alkali in place before mixing.
DETERMINATIO N OF AMMONIA
303
ing order are added 1.0 ml. each of 2.5% sodium phenolate, 0.005~ sodium nitroprusside, 0.05 M sodium carbonate, and 0.06 N sodium hypoehlorite. After mixing, the tube is allowed to stand, in the dark, for 30 rain. The volume is then brought to 10 ml. with water, and the optical density at 625 m~ is measured in a spectrophotometer. Studies of the rate of diffusion as a function of time in the mechanical rotater have shown that, with NH4+ concentration§ of 0.05-0.20 ~mole of ammonium salts in water per vessel, diffusion was, in all~cases, virtually complete after 20 rain. Using whole blood, plasma, or protein-free filtrates, diffusion was invariably complete in 20-30 min. The dye formed has the properties of an indophenol and is red in acid media, but blue under the conditions described. Its absorption spectrum, in alkali, is shown in Fig. 2. As showli in Fig. 3, the color obeys Beer's law and the complete procedure yields a linear relationshi p between the ammonia of the initial sample before diffusion and the color obtained. Because of the sensitivity of the method the blanks are appreciably colored, but no difficulty has been occasioned on this account• When glutamine, in amounts up to 3 ~mole, was added to diffusion vessels containing 0.1 ~mole of ammonia, there was no increase in apparent ammonia• However, 4, 7, and 10 ~moles of glutamine yielded increments in color equivalent to 0.016, 0.04, and 0.06 ~mole of ammonia, respectively. Thus it would appear that, since the liberation of ammonia is probably first order with time, the ability of the present procedure to (a) separate ammonia from glutamine rapidly and (b) to determine relatively small concentrations of ammonia and, thus, permit appreciable dilution of biological samples, essentially obviates serious interference by glutamine. 0.4
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F~o. 2. Absorption spectrum of the dye formed in the NHrphenol-hypochlorite reaction in the presence of nitroprusside, pH 12.
304
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FIG. 3. Relationship between ammonia content of diffusion flask and optical density of the final colored solution. Each point is the mean of not less than six samples. As a check on the method, varying amounts of ammonia were added to aliquots of a single specimen of human plasma which was deproteinized by adiusting to pH 5 and heating for 3 min. The filtrates were then used as samples in the diffusion bottle. The apparent ammonia content of plasma, treated in this manner, was 0.05 pmole/ml. When 0.05, 0.1, and 0.15 #mole of ammonia were added, the recoveries were 112,100, and 103%, respectively. AMMONIA CONCENTRATION IN NORMAL HUMAN PLASMA
T h e simplicity of the analytical procedure suggested its application to the determination of ammonia in h u m a n plasma, where the presence of relatively large amounts of glutamine and glutamine-containing proteins has long presented serious analytical difficulties. The simplest procedure proved to be the most reliable. I n t o the diffusion bottles are placed 0.5 nil. of freshly drawn, oxalated plasma, 0.5 ml. of water and 0.5 ml. of 50 % sodium carbonate. T h e samples are rotated for 20 rain. and color is then developed in the usual manner. T e n specimens of plasma drawn from healthy young males yielded values varying from 30 to 55 gg. hTH~ N / 1 0 0 ml. with a mean of 39/~g./100 ml. Experiments in which
DETERMINATION OF AMMONIA
305
0.1 ~mole NHa was added to the plasma in the diffusion bottles yielded recoveries of 94-105%. Several procedures were attempted for deproteinization of the plasma prior to alkalinization and ammonia diffusion including isoelectrie, tungstic acid, and trichloroacetic acid precipitations. Each of these resuited in values for the ammonia content of plasma which were from 25 to 175 % higher than those obtained without deproteinization. Since the recovery experiments indicated that ammonia diffusion was essentially complete in 20 rain., it was concluded that the deproteinization procedures also entail a varying degree of glutamine or other amide hydrolysis. In consequence, the simple procedure, described above, is recommended. Relatively few values for the ammonia content of plasma, determined by reasonable procedures, have been reported. Seegmiller, Schwartz, and Davidson found a mean of 130 ~g. NHa N/100 ml. with a range of 50-220 ug./100 ml. (8). The ammonia content of whole blood has been reported as 10.6 ~ 3.1 pg./100 ml. (9), 120 ~g./100 ml, with a range of 20-180 (10), and 55.6 ~ 10 ~g./100 ml. (11). In general, therefore, the values obtained here are appreciably lower than those reported hitherto. This is readily understood from the fact that plasma glutamine-N has been stated to be of the order of 5.8-8.0 rag./100 ml. (8, 12, 13) or about 200 times the ammonia content observed in the present study. AMMONIA CONTENT OF RAT TISSUES
Male rats weighing about 300 g. were killed by decapitation, and the appropriate tissues were removed quickly and frozen in a dry ice-acetone mixture. The frozen tissue was then weighed, pulverized, and placed in a chilled Waring blendor. For each milliliter of tissue, 1 ml. of each of the following reagents was rapidly added: water, 10 % sodium tungstate, and 2~ N sulfuric acid. The mixture was homogenized for 2-3 rain. and then centrifuged at 25,000 X g for 10 min. The protein-free supernatant fluid was neutralized and frozen until used. Aliquots of 0.5 ml. were used for ammonia determination as described above. As a check on the method, recovery experiments were performed in which 2 ~moles of ammonia was added to homogenates of one-half a rat liver. The variation in recovery was of the same order noted previously with plasma. Brain
The brains of only three rats were examined for ammonia. Approximately 1 min. elapsed between the death of each animal and the freezing
306
BROWN~ DUDA, KORKES AND HANDLER
of the brain. The values obtained were 48, 40, and 36 ~moles/100 g., respectively, equivalent to 0.67, 0.56, and 0.50 mg. NHa N/100 g. tissue. These values are of the same order as reported by Richter and Dawson (14) but, as explained below, little reliance is placed on these data as an indication of the ammonia concentration of brain in situ. Muscle
A set of four rats was killed by decapitation, and approximately 1 re_in, after death was required before samples of thigh muscle could be frozen. These yielded values of 20, 24, 40, and 63 umoles/100 g., respectively. However, the muscles of animals killed in this manner twitch violently. In consequence, a second group of six rats was first anesthetized with Nembutal and then given intraperitoneM MgS04 (15, 16). No twitching was observed, and it was possible to reduce the time necessary to dissect and freeze the samples to about 30 sec. This series yielded ammonia values of about 1.1 ~moles (18 ~g. NH~ 1N)/100 g. tissue. Thus, resting muscle contains a trivial amount of ammonia and, indeed, since 30 sec. was still required to obtain the samples, it appears likely that the true value for resting muscle approaches zero. Violent activity by the anaerobic muscles of dying rats results in the appearance of as much as 60 times the ammonia found in resting muscle. This does not necessarily indicate that ammonia appears in aerobic muscle contracting in situ under normal circumstances. Liver In an initial study, a group of seven rats was sacrificed by decapitation and their livers were frozen at times which varied from 5 to 90 see. after death. These yielded values for ammonia which varied from 4 to 28 ~moles/100 g. liver, increasing with increasing time in obtaining the sample. This was then carefully repeated with two series of rats. The livers of group A were allowed to remain in situ but were loosened and placed over the intestine until removed and frozen. The livers of group B were removed as rapidly as possible (5-10 sec. after death) and kept at ambient temperature o n weighing paper until frozen. The data are shown in Fig. 4. Again, it was apparent that ammonia is generated rapidly post mortem. The data obtained after 10, 20, and 30 sec. in both series were pooled, and the best straight line among them was plotted by the method of least squares, as shown in Fig. 5. The value for the intercept is - 1.3 ~ 1.5 gmoles/100 g. liver.
DETERMINATION
OF
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Fzo. 4. Apparent ammonia content of rat liver. O . . . . O group B; @ : (D group A (see text). Unquestionably, the data for liver are more reliable than are those for muscle or brain insofar as they purport to represent the ammonia concentration of these tissues in the living steady state. If credence is placed in the data of Fig. 5, the ammonia concentration of liver, in situ, is trivial and m a y truly be zero. Since the rats were not fasted and the livers were obtained early in the morning, these livers should exhibit an active postprandial metabolism. To be sure, it is entirely possible that liver m a y possess a minute pool of ammonia which turns over at a great rate, but the possibility should be entertained that ammonia, qua ammonia, is not an important metabolite in normal liver despite the existence of such systems as glutaminase, glutamine synthetase, glutamic dehydrogenase, glycine oxidase, i)- and L-amino acid oxidases, and the
308
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FIG. 5. Apparent ammonia content of rat liver at 10, 20, and 30 sec. after death. The line was calculated by the method of least squares. Each point is the mean of a group of three rats. system for making carbamyl phosphate from ammonia. This suggestion recalls the observation that, when dogs were infused with massive amounts of a mixture of L-amino acids, sufficient to saturate their ureasynthesizing capacity, no ammonia appeared in plasma, whereas lesser quantities of a mixture of D-amino acids rapidly resulted in the appearance of large amounts of ammonia in plasma (17) and the death of the animals. To be sure, the plasma ammonia in this situation may arise outside the liver since kidney has an extremely active D-amino acid oxidase. Administration, intraperitoneally, of 0.25 g. of D- or L-leueine, or 0.4 g. of either isomer orally to groups of five rats each, 30 rain. before sacrificing, was without effect on the NH3 content of livers sampled in such fashion that 20 sec. elapsed between the death of the animal and the freezing of its liver.
DETERMINATION OF AMMONIA
309
~UMMARY
A method is described for the determination of ammonia in the presence of labile amides. The method combines rapid mierodiffusion with an extremely sensitive colorimetric procedure. When applied to normal human plasma, concentrations of the order of 40 ~g. NH3 N/100 ml. were obtained. The method was also applied to rat brain, skeletal muscle, and liver. Resting muscle was found to contain less than 1 #mole/100 g. tissue, and liver less than 4 ~moles/100 g. Data are presented which indicate that, in the living steady state, these values are markedly reduced and approach zero. ]:~EFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14. 15. 16. 17.
CONWAr, E. J., Bioehem. J. 29, 2755 (1935). SELI~SON,D., AND SELIGSON,H., J. Lab. Clin. Med. 38,324 (1951). BORSOOK,H. J., J. Biol. Chem., 110, 481 (1935). RUSSELL,J. A., J. Biol. Chem. 156,457 (1944). VAN SLYKE, ~). D., AND HELLER, A., J. Biol. Chem. 102,499 (1933). ZALTA,J. R., AND LUBOCHINSKr,B., Bull. sOC. chim. biol. 36, 1363 (1954). TOMPKINS,E. R., AND KIRK, P. L., J. Biol. Chem. 142, 477 (1942). SEEGMILLER,J. E., SCtlWARTZ,R., AND DAVIDSON,C. S., J. Clin. Invest. 33, 984 (1954). SINGH, ~'. D., BARCLAY,J. A., AND COOKE, W. T., Lancet 1954, 1004. TI~AEGER,H. S., GABUDZA,G. J., BALLOU,A. •., AND DAVIDSON,C. S., Metabolism 3, 99 (1954). MCDERMOTT, W. V., ADAMS, ]:t. D., AND RIDDELL, A: G., Proc. Soc. Exptl. Biol. Med. 88, 380 (1955). KaEBS, H. A., E6GLESTON, L. V., AND HEMS, R., Biochem. J. 44, 159 (1949). NIUNKVAD,I., AND VESTERDAL,J., Acta Pediatrica 43, 320 (1954). RICHTER,D., AND DAWSON, I2~.M. C., J. Biol. Chem. 176, 1199 (1948). DuBols, K. P., ALnAVM,H. G., AND POTTER, V. R., J. Biol. Chem. 147, 699 (1943). FISHER, E. H., AND KREBS, E. G., J. Biol. Chem. 216, 246 (1948). KAMIN, H., AND HANDLER,P., J. Biol. Chem. 188, 193 (1951).
66, ~01-309 (1957)
A Colorimetric Micromethod for Determination of Ammonia; the Ammonia Content of Rat Tissues and H u m a n Plasma I Rose H. Brown, George D. Duda, Seymour Korkes2 and Philip Handler From the Department of Biochemistry, Duke University ~chool of Medicine, Durham, North Carolina Received May 7, 1956 INTRODUCTION
The determination of ammonia in the presence of labile amides, such as glutamine, is a frequently encountered problem which has been particularly difficult for those biological systems wherein the ammonia concentration is small relative to that of the amide. This problem has been approached most successfully by rapid removal of the ammonia under conditions such that amide hydrolysis is negligible (1, 2). On the assumption that amide decomposition is first order with time, it follows that such interference is best minimized if the sample can be diluted before microdiffusion. Since this would also dilute the ammonia, the ideal method also requires a sensitive method for estimation of the diffused ammonia. The present report describes a procedure which combines microdiffusioa with a sensitive colorimetric procedure. The reaction between NH3 and phenol in the presence of hypochlorite to yield a blue dye, described by Berthelot in 1859, has served as the basis for several procedures for ammonia determination (3-5). The addition of small amounts of nitroprusside markedly accelerates color production, and Zalta and Lubochinsky (6) have so employed it in a method for NH3 determination in Kjeldahl digests. Seligson and Seligson (2) have described a simple, inexpensive modification of the microdiffusion These studies were supported by grants from the Committee on Growth, National Research Council (MET-47) ; National Institutes of Health [B-906(C9) ]; and Contract AT-(40-1)-289 between Duke University and the U. S. Atomic Energy Commission. 2 Deceased: December 10, 1955. 301
302
BROWN~ DUDA~ KORKES AND HANDLER
p r o c e d u r e of T o m p k i n s a n d Kirk: (7) w h e r e i n r a p i d diffusion is a s s u r e d b y r o t a t i n g t h e a m m o n i a s a m p l e as a film on t h e surface of a s m a l l vial. B y c o m b i n i n g t h i s diffusion t e c h n i q u e w i t h t h e colorir~etric m e t h o d of Z a l t a a n d L u b o c h i n s k y , a m m o n i a m a y r e a d i l y be d ~ e r m i n e d in t h e presence of a 30-fold ~excess of g l u t a m i n e . E x P F ~IMENTAL
Reagents (a) Sodium hypochlorite, 0.06 N, is prepared by dilution of "Clorox" 1:20. I t is standardized by addition of solid KI, acidification with H2SO4, and titration with standard thiosulfate. The absolute concentration is not critical. No detectable differences in color have been observed from 0.05 to 0,07 N. (b) Sodium nitroprusside, stock 0.5%. Fresh 1:100 dilutions are prepared for each analytical series. (c) Sodium carbonate, 0.05 M. (d) Sodium carbonate, 50%. (e) Sodium phenolate. Dissolve 2.5 g. of freShly distilled phenol and 1.25 g. of NaOtt in 100 mI. of water and allow to stand 2 days before using. (f) Sulfuric acid, 1 N.
Procedure The sample, not to exceed 1.0 ml. and containing not more than 0.2 ~mole of NH3, is pipetted into the bottom of a bottle (Kimble, Neutraglass N-51A, 35 ml., 3~% cm. outside diam., 7 ~ cm. long) which has previously been prepared by making a small annulus around the bottle, approximately 3 ~ era. from the base. The bottle is placed on its side, and 0.5 ml. of 50% Na~CO~ is carefully added on the distal side of the annulus so that it does not mix with the sample. A previously prepared paddle-shaped strip of filter paper, 1 cm. at the base and about 1.5 cm. long, is inserted into the open end of the glass tubing which passes through the No. 1 one-hole rubber stopper used to seal the vessel. Then 0.01 ml. of 1 N H2SO4 is placed on the paper at its base; this should not saturate the paper. After the vessel is thus sealed, the contents are carefully mixed and the bottle is then mechanically rotated on its axis until diffusion is complete. Our device operates at 78 r.p.m. The diffusion bottle, before mixing, is shown in Fig. 1. The paper is then transferred, with forceps, to a euvette containing 1.0 ml. of water. In the follow~
FIG. 1. The diffusion bottle with absorption paddle, sample, and alkali in place before mixing.
DETERMINATIO N OF AMMONIA
303
ing order are added 1.0 ml. each of 2.5% sodium phenolate, 0.005~ sodium nitroprusside, 0.05 M sodium carbonate, and 0.06 N sodium hypoehlorite. After mixing, the tube is allowed to stand, in the dark, for 30 rain. The volume is then brought to 10 ml. with water, and the optical density at 625 m~ is measured in a spectrophotometer. Studies of the rate of diffusion as a function of time in the mechanical rotater have shown that, with NH4+ concentration§ of 0.05-0.20 ~mole of ammonium salts in water per vessel, diffusion was, in all~cases, virtually complete after 20 rain. Using whole blood, plasma, or protein-free filtrates, diffusion was invariably complete in 20-30 min. The dye formed has the properties of an indophenol and is red in acid media, but blue under the conditions described. Its absorption spectrum, in alkali, is shown in Fig. 2. As showli in Fig. 3, the color obeys Beer's law and the complete procedure yields a linear relationshi p between the ammonia of the initial sample before diffusion and the color obtained. Because of the sensitivity of the method the blanks are appreciably colored, but no difficulty has been occasioned on this account• When glutamine, in amounts up to 3 ~mole, was added to diffusion vessels containing 0.1 ~mole of ammonia, there was no increase in apparent ammonia• However, 4, 7, and 10 ~moles of glutamine yielded increments in color equivalent to 0.016, 0.04, and 0.06 ~mole of ammonia, respectively. Thus it would appear that, since the liberation of ammonia is probably first order with time, the ability of the present procedure to (a) separate ammonia from glutamine rapidly and (b) to determine relatively small concentrations of ammonia and, thus, permit appreciable dilution of biological samples, essentially obviates serious interference by glutamine. 0.4
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F~o. 2. Absorption spectrum of the dye formed in the NHrphenol-hypochlorite reaction in the presence of nitroprusside, pH 12.
304
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FIG. 3. Relationship between ammonia content of diffusion flask and optical density of the final colored solution. Each point is the mean of not less than six samples. As a check on the method, varying amounts of ammonia were added to aliquots of a single specimen of human plasma which was deproteinized by adiusting to pH 5 and heating for 3 min. The filtrates were then used as samples in the diffusion bottle. The apparent ammonia content of plasma, treated in this manner, was 0.05 pmole/ml. When 0.05, 0.1, and 0.15 #mole of ammonia were added, the recoveries were 112,100, and 103%, respectively. AMMONIA CONCENTRATION IN NORMAL HUMAN PLASMA
T h e simplicity of the analytical procedure suggested its application to the determination of ammonia in h u m a n plasma, where the presence of relatively large amounts of glutamine and glutamine-containing proteins has long presented serious analytical difficulties. The simplest procedure proved to be the most reliable. I n t o the diffusion bottles are placed 0.5 nil. of freshly drawn, oxalated plasma, 0.5 ml. of water and 0.5 ml. of 50 % sodium carbonate. T h e samples are rotated for 20 rain. and color is then developed in the usual manner. T e n specimens of plasma drawn from healthy young males yielded values varying from 30 to 55 gg. hTH~ N / 1 0 0 ml. with a mean of 39/~g./100 ml. Experiments in which
DETERMINATION OF AMMONIA
305
0.1 ~mole NHa was added to the plasma in the diffusion bottles yielded recoveries of 94-105%. Several procedures were attempted for deproteinization of the plasma prior to alkalinization and ammonia diffusion including isoelectrie, tungstic acid, and trichloroacetic acid precipitations. Each of these resuited in values for the ammonia content of plasma which were from 25 to 175 % higher than those obtained without deproteinization. Since the recovery experiments indicated that ammonia diffusion was essentially complete in 20 rain., it was concluded that the deproteinization procedures also entail a varying degree of glutamine or other amide hydrolysis. In consequence, the simple procedure, described above, is recommended. Relatively few values for the ammonia content of plasma, determined by reasonable procedures, have been reported. Seegmiller, Schwartz, and Davidson found a mean of 130 ~g. NHa N/100 ml. with a range of 50-220 ug./100 ml. (8). The ammonia content of whole blood has been reported as 10.6 ~ 3.1 pg./100 ml. (9), 120 ~g./100 ml, with a range of 20-180 (10), and 55.6 ~ 10 ~g./100 ml. (11). In general, therefore, the values obtained here are appreciably lower than those reported hitherto. This is readily understood from the fact that plasma glutamine-N has been stated to be of the order of 5.8-8.0 rag./100 ml. (8, 12, 13) or about 200 times the ammonia content observed in the present study. AMMONIA CONTENT OF RAT TISSUES
Male rats weighing about 300 g. were killed by decapitation, and the appropriate tissues were removed quickly and frozen in a dry ice-acetone mixture. The frozen tissue was then weighed, pulverized, and placed in a chilled Waring blendor. For each milliliter of tissue, 1 ml. of each of the following reagents was rapidly added: water, 10 % sodium tungstate, and 2~ N sulfuric acid. The mixture was homogenized for 2-3 rain. and then centrifuged at 25,000 X g for 10 min. The protein-free supernatant fluid was neutralized and frozen until used. Aliquots of 0.5 ml. were used for ammonia determination as described above. As a check on the method, recovery experiments were performed in which 2 ~moles of ammonia was added to homogenates of one-half a rat liver. The variation in recovery was of the same order noted previously with plasma. Brain
The brains of only three rats were examined for ammonia. Approximately 1 min. elapsed between the death of each animal and the freezing
306
BROWN~ DUDA, KORKES AND HANDLER
of the brain. The values obtained were 48, 40, and 36 ~moles/100 g., respectively, equivalent to 0.67, 0.56, and 0.50 mg. NHa N/100 g. tissue. These values are of the same order as reported by Richter and Dawson (14) but, as explained below, little reliance is placed on these data as an indication of the ammonia concentration of brain in situ. Muscle
A set of four rats was killed by decapitation, and approximately 1 re_in, after death was required before samples of thigh muscle could be frozen. These yielded values of 20, 24, 40, and 63 umoles/100 g., respectively. However, the muscles of animals killed in this manner twitch violently. In consequence, a second group of six rats was first anesthetized with Nembutal and then given intraperitoneM MgS04 (15, 16). No twitching was observed, and it was possible to reduce the time necessary to dissect and freeze the samples to about 30 sec. This series yielded ammonia values of about 1.1 ~moles (18 ~g. NH~ 1N)/100 g. tissue. Thus, resting muscle contains a trivial amount of ammonia and, indeed, since 30 sec. was still required to obtain the samples, it appears likely that the true value for resting muscle approaches zero. Violent activity by the anaerobic muscles of dying rats results in the appearance of as much as 60 times the ammonia found in resting muscle. This does not necessarily indicate that ammonia appears in aerobic muscle contracting in situ under normal circumstances. Liver In an initial study, a group of seven rats was sacrificed by decapitation and their livers were frozen at times which varied from 5 to 90 see. after death. These yielded values for ammonia which varied from 4 to 28 ~moles/100 g. liver, increasing with increasing time in obtaining the sample. This was then carefully repeated with two series of rats. The livers of group A were allowed to remain in situ but were loosened and placed over the intestine until removed and frozen. The livers of group B were removed as rapidly as possible (5-10 sec. after death) and kept at ambient temperature o n weighing paper until frozen. The data are shown in Fig. 4. Again, it was apparent that ammonia is generated rapidly post mortem. The data obtained after 10, 20, and 30 sec. in both series were pooled, and the best straight line among them was plotted by the method of least squares, as shown in Fig. 5. The value for the intercept is - 1.3 ~ 1.5 gmoles/100 g. liver.
DETERMINATION
OF
307
AMMONIA
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I 6
MINUTES
Fzo. 4. Apparent ammonia content of rat liver. O . . . . O group B; @ : (D group A (see text). Unquestionably, the data for liver are more reliable than are those for muscle or brain insofar as they purport to represent the ammonia concentration of these tissues in the living steady state. If credence is placed in the data of Fig. 5, the ammonia concentration of liver, in situ, is trivial and m a y truly be zero. Since the rats were not fasted and the livers were obtained early in the morning, these livers should exhibit an active postprandial metabolism. To be sure, it is entirely possible that liver m a y possess a minute pool of ammonia which turns over at a great rate, but the possibility should be entertained that ammonia, qua ammonia, is not an important metabolite in normal liver despite the existence of such systems as glutaminase, glutamine synthetase, glutamic dehydrogenase, glycine oxidase, i)- and L-amino acid oxidases, and the
308
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30
SECONDS
FIG. 5. Apparent ammonia content of rat liver at 10, 20, and 30 sec. after death. The line was calculated by the method of least squares. Each point is the mean of a group of three rats. system for making carbamyl phosphate from ammonia. This suggestion recalls the observation that, when dogs were infused with massive amounts of a mixture of L-amino acids, sufficient to saturate their ureasynthesizing capacity, no ammonia appeared in plasma, whereas lesser quantities of a mixture of D-amino acids rapidly resulted in the appearance of large amounts of ammonia in plasma (17) and the death of the animals. To be sure, the plasma ammonia in this situation may arise outside the liver since kidney has an extremely active D-amino acid oxidase. Administration, intraperitoneally, of 0.25 g. of D- or L-leueine, or 0.4 g. of either isomer orally to groups of five rats each, 30 rain. before sacrificing, was without effect on the NH3 content of livers sampled in such fashion that 20 sec. elapsed between the death of the animal and the freezing of its liver.
DETERMINATION OF AMMONIA
309
~UMMARY
A method is described for the determination of ammonia in the presence of labile amides. The method combines rapid mierodiffusion with an extremely sensitive colorimetric procedure. When applied to normal human plasma, concentrations of the order of 40 ~g. NH3 N/100 ml. were obtained. The method was also applied to rat brain, skeletal muscle, and liver. Resting muscle was found to contain less than 1 #mole/100 g. tissue, and liver less than 4 ~moles/100 g. Data are presented which indicate that, in the living steady state, these values are markedly reduced and approach zero. ]:~EFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14. 15. 16. 17.
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