Intracellular Potassium Concentration During Ammonia Intoxication

Vol. 61. No.6 Printed in U.S.A.

GASTROENTEROLOGY

Copyright © 1971 by The Williams & Wilkins Co.

INTRACELLULAR POTASSIUM CONCENTRATION DURING AMMONIA INTOXICATION 0.

ALBANO, AND

A.

FRANCAVILLA

Istituto di Patologia Speciale Medica e Metodologia Clinica, Facolta di Medicina e Chirurgia, Universita degli Studi di Bari, Bari, Italy

A study of the concentration of ammonia, K+, and Na + in the red cells of rats exposed to ammonia intoxication is presented. The results obtained show that ammonia, injected intraperitoneally, is rapidly taken up by red cells. Accumulation of ammonia is accompanied by a specific decrease of the cellular content of K+; there is, on the other hand, no significant change in the cellular content of N a+. The results are explained on the basis of the known effects of NH 4 + on the Na pump of red cells in vitro, and their possible relationship to the mechanism of ammonia toxicity is discussed. The toxicity of ammonia is well documented. l-a Accumulation of ammonia in the brain is generally considered to be one cause, if not the primary one, of hepatic encephalopathy. 4 • 5 Various mechanisms have been proposed to explain ammonia toxicity and hepatic encephalopathy. a- 11 Recently attention has been focused on the mutual relationship between ammonia metabolism and potassium distribution in red cells and body fluids. In 1957 Post and Jolly 12 demonstrated that in isolated human red cells ammonia competes with potassium to take Na + out of the cells, an adenosine triphosphatedependent process mediated by the "Na pump." In this investigation the effect in vivo of experimental ammonia intoxication on potassium concentration in red cells of rats has been studied. Ammonia intoxication was found to Received December 3, 1969. Accepted July 9, 1971. Address requests for reprints to: Dr. 0. Albano, Istituto di Patologia Speciale Medica, Facolta di Medicina e Chirurgia, Universita degli Studi di Bari, Bari, Italy. This study was supported by grants from Consiglio Nazionale delle Ricerche, Italy. The authors wish to thank Mr. Francesco Ardito and Mr. Angelo Palma for their expert technical assistance.

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cause significant decrease of potassium concentration in red cells. Materials and Methods In Vitro Experiments The effect of ammonium on the active transport of potassium. Red cells were obtained from albino Wistar rats, weighing 60 to 70 g. The animals were decapitated and the blood was collected from the neck in a heparin solution (final concentration, 5 mg per ml) . The red cells were prepared by allowing them to fill with sodium and to become empty of potassium during storage at 2 C in a sodium medium for 15 days. The composition of the sodium medium was : 110 mM NaCl, 25 mM Na 2 HPO 4 , 2 mM HCl, 2 mM MgCl 2, 3. 7 mM adenosine triphosphate (ATP), 10 mM glucose, and bovine serum albumin (1 g per liter) . Red cells [40 to 60 mg of hemoglobin (Hb)] were incubated for 2 hr in a medium containing 140 mM NaCI, 8 mM KCI, 2 mM MgCl 2, 3.7 mM ATP, 100 mM histidine imidazole-HCl buffer (pH 7.4) , and 1 mg per ml of bovine serum albumin ; final volume, 2 mi. The reaction temperature was 37 C. At the beginning of the incubation, sodium and potassium content were 118 and 13 mEq per 5 mg of Hb respectively. Where indicated NH.CI was added as the solid to the incubation medium at the final concentration specified in table 1. The final pH was 7.4. After incubation , the red cells were separated from the incubation medium (see below) . Na + and K + content were determined by flame photometry. Their concentration was

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LIVER PHYSIOLOGY AND DISEASE TABLE

Vol. 61, No . 6

1. Plasma and erythrocyte ammonia concentration in normal rats and in animals with NH, +

intoxication a Concentration (mM) of NH 4 + Rats

Normal . . . . . . . .. . . . Intoxicated .. . . . .

Plasma

Erythrocyte

Mean

SD

SE

Mean

SD

SE

0.1 2.5

±0.008 ± 0.23

± 0.001 ±0.41

0.1 6.8

± 0.001 ± 0.58

±0.0009 ± 0.12

a Values given in table are the means of 15 experiments. Experimental details are given in " Materials and Methods."

expressed as microequivalents per 100 mg of Hb. Hemoglobin was determined by the method ofKing. 13 Adenosine triphosphatase of erythrocy te ghosts from normal and NH •+-treated rats. Erythrocyte ghosts, prepared according to the method of Bonting and Caravaggio," were incubated for 1 hr at 30 C in a mixture containing histidine imidazole buffer, 100 mM (pH 7.1) ; MgC1 2, 5 mM; ATP-tris, 5 mM; KCN, 1 mM; and ethylenediaminetetraacetate, 0.1 mM. The final volume was 2 mi. The reaction was stopped by the addition of 0.2 ml of 35% perchloric acid , and inorganic phosphate was estimated according to the method of Martin and Doty. 1 5 The adenosine triphosphatase activity was expressed as millimicromoles of P liberated per hour per milligram of protein.

In Vivo Experiments Starved male albino Wistar rats were treated in one of the following ways: (1) One group of rats received intraperitoneally a single dose (7.9 mmoles per kg) of ammonium acetate. (2) The second group received intraperitoneally a single dose (50 mg per kg) of Nembutal. (3) The third group was treated with ethyl ether until comatose. The animals were killed by decapitation and the blood was immediately collected from the neck in a test tube containing heparin (5 mg per ml of blood) and ("C) carboxy-dextran (0.5 mg per ml of blood, 440.000 counts per min). The red cells were rapidly separated from the plasma by filtration centrifugation through a silicon layer as described by Pfaff. 16 The following analyses were carried out on the red cell pellets: (1) Hb determination 13 ; (2) determination of ( "C)carboxy-dextran radioactivity with a Packard liquid-scintillation counter. The radioactivity of ("C)carboxy-dextran in red cell pellets divided by the radioactivity per IL!iter of plasma gave an estimate of adhering plasma in the pellet. The red cell cation content was calculated by subtracting

the amount present in the adhering plasma from the total cation content of the pellet; (3) determination of K + and Na + by flame photometry; (4) determination of NH, + by the method of Schmidt and Schwarz 17 ; (5) H 20, determined gravimetrically, and corrected for adherent H 2 0 calculated from the radioactivity of ( "C)carboxy-dextran.

Determination of Blood Ammonia The ammonium of plasma was determined according to the method of Schmidt and Schwarz. 17

Results The effect on NH,Cl on K + transport in isolated red cells is shown in figure 1. It can be seen that increasing NH,Cl concentration caused a progressive decrease of the rate of K + influx into red cells. In table 1 the plasma and red cell concentration of ammonia in normal rats and in intoxicated rats is shown. It can be seen that, 10 min after the intraperitoneal injection of ammonium acetate, the concentration of ammonia in the plasma increased from 0.1 mM to 2.5 mM. Under these conditions ammonia was taken up by the red cells. The intracellular concentration of ammonia was, in fact, about three times higher than that in the plasma. The red cell concentration of ammonia in the intoxicated rats was 68 times higher than that in the control animals. In table 2 the intracellular K + and Na + content of red cells from control and from ammonia-intoxicated rats is given. The animals of both groups were killed 8 to 10 min after injection of sodium acetate or ammonium acetate. Mter this interval the intoxicated rats were in deep coma. In some animals convulsions also occurred,

December 1971

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FIG. 1. The influence of external ammonium concentration on rate of active K + transport in intact erythrocytes. Red cells were incubated in a medium containing NH.Cl at final concentrations of 2.5, 5, 7.5, 10, and 12.5 mM. Final pH, 7.4. Hb, hemoglobin.

but these animals were not used for cation determinations. No significant difference in the intracellular sodium content of red cells in the control and intoxicated rats was seen. On the other hand, the intracellular content of K + in the red cells of the intoxicated animals was significantly (P < 0.01) reduced in comparison with that of control animals. In another group of rats the intracellular K + content of red cells was measured during the various phases of ammonia intoxication (fig. 2). Three steps of the intoxication course were observed: (I) the induction phase lasted about 8 min from the time of injection of ammonium acetate; (2) onset of coma was between 8 and 10 min; and (3) a regression phase was seen from the 24th to the 60th min. After this interval the rats returned to their normal state. The experimental data of figure 2 show that the erythrocyte potassium content was unaffected during the first 6 min of the intoxication (precoma period). At the beginning of the coma, there was a 20Sf decrease of

the potassium content in the red cells. This decrease persisted during the entire coma phase, i.e., from the 8th to the 20th min after the injection of ammonia. After this a progressive restoration of K + content of red cells was observed. After 1 hr the K + content was completely restored. In table 3 red cell concentration of K + and Na+ during coma induced by Nem butal or ethyl ether are shown. These types of coma had no effect on the cellular content of K + and Na+ . In table 4 the activity of Na +- and K +dependent adenosine triphosphatase from erythrocyte ghosts of normal and intoxicated rats is reported. It can be seen that the adenosine triphosphatase activity, in the absence and in the presence of various concentrations of Na + and K +, was practically the same in normal and intoxicated rats.

Discussion Ammonia, injected intraperitoneally into rats, rapidly reaches a high level in the blood and accumulates in red cells. The accumulation of ammonia by red cells is accompanied by a decrease in their K + content. The Na + content, on the other hand, remains constant. These findings show that the competitive effect of NH 4 + on K + transport by the Na pump (Na-, Kadenosine triphosphatase) (see reference 12 and fig . I) has an actual effect in vivo on the K + concentration in erythrocytes. It is conceivable that NH 4 + is transported from TABLE

2. Effect of ammonia intoxication on the K + and Na + content of red celL~ " Norma l ra ts

I

Am moni aintox ica ted rats

Na·

K·

ILEq/100 m11 Hb'

Mean Standard deviation Standard error

P .

Na·

K·

" Eq / 100 mg Hb

13.86 86.50 13.52 67.42 ± 2.11 ± 8.34 ±2.44 ± 9.09 ± 0.44 ± 1.86 ± 0.51 ± 1.90 > 0.6 < 0.01

a Values given in table are the means of 23 experiments. Experimental details are given in "Materials and Methods. " • Hb, hemoglobin.

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LIVER PHYSIOLOGY AND DIS EASE

Vol. 61, No . 6

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FIG . 2. The effect of ammonia intoxication on intracellular K + content during the various phases of ammonia intoxication. Hb , hemoglobin . TABLE

3. Erythrocy te concentration of K +and Na + during coma induced by Nembutal or ethyl ether intoxication" K· Mean

SD

Na· SE

Mean

SD

SE

,Eq/100 mp Hb'

Normal rats Comatose rats Nembutal Ethyl-ether

..

.. ....

.

..

..

85.20

± 8.50

±1.83

13.86

±2. 10

± 0.44

86.20 84.50

± 7.45 ± 9.20

± 1.93 ± 2.01

13.51 13.98

±2.02 ± 1.96

± 0.36 ± 0.48

a Values given in table are the means of 15 experiments. Experimental details are given in " Materials and Methods." • Hb, hemoglobin .

TABLE

4. Na +- and K +-adenosine triphosphatase activity of erythrocyte membranes•

K'

Na •

mM

0 72 0 72

Rats

Normal

Intoxicated

m,.moles P/hr!mg protein

0 0 72 72

0 16.2 18.1 39.2

0 19 17.5 38.8

a Experimental details are given in " Materials and Methods ."

the plasma into the red cells in exchange with Na + and in competition with K +. Thus, at the concentrations of NH 4 + reached in the plasma under the present

experimental conditions, the influx of NH 4 + replaces a portion of the K + influx . The total activity of the system remains unaffected, however, as is revealed by the fact that the red cell content of Na + (see table 2) and adenosine triphosphatase activity (not shown) remained constant during ammonia intoxication. The data in figure 2 show that the decrease of the potassium content in red cells correlates chronologically with the neurological symptoms of the intoxication. Schenker et al. 18 have found that 15 min after injection of ammonium acetate the brain ammonia concentration was about 13-fold greater than control values. It is conceivable, on the basis of the present

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December 1971

observations, that the accumulation of ammonia in the brain is accompanied by a decrease in the intracellular content of K+. In fact, it is generally agreed that the sodium and potassium content of brain cells is controlled by a mechanism similar to that operative in red cells. REFERENCES 1. Van Caulaert C, Deviller C, Halff M: Troubles provoque par !'ingestion de sels ammoniacaux chez l'homme atteint de cirrhose de Laennec. C R Soc Bioi (Paris) 111:739-740, 1932 2. White LP, Phear EA, Summerskill WHJ, et a!: Ammonium intolerance in liver disease : observations based on catheterization of the hepatic veins. J Clin Invest 34:158-168, 1955 3. Stahl J: Studies of the blood ammonia in liver disease. Its diagnostic, prognostic and therapeutic significance. Ann Intern Med 58:1-24, 1963 4. Gabuzda GI: Hepatic coma: clinical considerations,. pathogenesis and management. Advances Intern Med 11:11-73, 1962 5. Chalmers TC: Pathogenesis and treatment of hepatic failure. New Eng J Med 263:23-30, 1960 6. Bessman SP, Bessman AN: The cerebral and peripheral uptake of ammonia in liver disease with an hypothesis for the mechanism of hepatic coma. J Clin Invest 34:622-628, 1955 7. McKhann GM, Tower DB: Ammonia toxicity and cerebral oxidative metabolism. Amer J Physiol200:420- 424, 1961

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8. Worcel A, Erecinska M: Mechanism of inhibitory action of ammonia on the respiration of rat-liver mitochondria. Biochim Biophys Acta 65:27-33, 1962 9. Weil-Malherbe H: Ammonia Metabolism in the brain in Neurochemistry. Second edition. Edited by KAC Elliot, IH Page, JH Quastel. Springfield, lll, Charles C Thomas Publisher, 1962, p 321 10. Zieve L: Pathogenesis of hepatic coma. Arch Intern Med (Chicago) 118:211, 1966 11. Shorey J, McCandless WD, Schenker S: Cerebral a-ketoglutarate in ammonia intoxication. Gastroenterology 53:706- 711, 1967 12. Post RL, Jolly PC: The linkage of sodium potassium and ammonium active transport across the human erythrocyte membrane. Biochim Biophys Acta 25:118- 128, 1957 13. King EJ: Microanalysis in Medical Biochemistry. London, J and A Churchill, 1931 14. Bonting SL, Caravaggio LL: Studies on sodium potassium activated adenosinetriphosphatase . Correlation of enzyme activity with cation flux in six tissues. Arch Biochem 101:37-46, 1963 15. Martin JB, Doty DM: Determination of inorganic phosphate. Anal Chern 21:965, 1949 16. Pfaff E: Ph.D thesis, Marbury, 1965 17. Schmidt FH, Schwarz H: Uber die enzymatische ammoniak bestimmung in blut. Klin Wschr 44 10:591- 592, 1966 18. Schenker S, McCandless DW, Brophy E, et a!: Studies on the intracerebral toxicity of ammonia. J Clin Invest 46:838- 848, 1967