Blood ammonia in rats with diabetic acidosis

Blood ammonia in rats with diabetic acidosis Experimental

study

in the pregnant

ALFRED Madison, With

L.

to explain

diabetic

reproductive

wastage

woman

KENNAN,

M.D.

Wisconsin the technical

GEORGE

assistance

of

PROPER

1 N A study of nitrogen metabolism in fetal tissues, blood ammonia determinations were made on patients with severe complications of pregnancy. In one patient with unexpected intrauterine fetal death, we were surprised to find markedly elevated blood ammonia levels. Further investigation led to a diagnosis of uncontrolled diabetes mellitus with acidosis. It occurred to us that the elevated blood ammonia level might have been an important factor in this fetal loss. It is widely recognized that the perinatal loss in patients with diabetes mellitus is increased if clinical acidosis is present.l Even “subclinical acidosis” or “silent acidosis” has been suggested as a cause of perinatal mortality.2 There is good evidence that meticulous management of diabetes to prevent acidosis is significant in preventing this fetal wastage.3 In considering diabetic acidosis, one usually thinks in terms of the ketogenic intermediates (ketone bodies) from fat metabolism. When these substances were given to rabbits, in amounts found in diabetic acidosis and coma, no significant toxicity was observed. 4 It appears from this that

From the Department of Gynecology and Obstetrics, University of Wisconsin School of Medicine. Presented at a meeting Gynecological Society.

of the Chicago

Supported in part by Grants from the United States Public Health Service A-3313 and the Wisconsin Alumni Research Foundation.

the ketone bodies are not the proximate cause for the toxicity in severe diabetic acidosis. There are only a few studies dealing with the nitrogen metabolism in the uncontrolled diabetic patient. Diabetic patients in acidosis excrete more ammonia5 and urea than normaL6 Fuld7 has described a patient in diabetic coma with a high blood ammonia level. The liver from a diabetic rat synthesized urea at twice the rate of normal liver when the two were perfused.* If the blood ammonia level is regularly elevated when the blood sugar Ieveh are high and ketosis is present, even in the absence of coma, this factor should be seriously considered as one of the possible causes of the increased perinatal mortality seen in the offspring of diabetic pregnant women. Patients of this type are rare in the Madison hospitals; therefore, we investigated the relationship between acidosis and blood ammonia levels in rats made diabetic with alloxan. In a previous experiment, coma was produced in rats with a large dose of alloxan administered intraperitoneally. The blood ammonia level was elevated, but these results were complicated by the effects of this dose upon the liver and kidney tubule. This experiment has been modified to take this into account. Materials Female (Holtzman)

and

methods

rats weighing 190 to 210 grams were made diabetic with

516

Eebluary

Kennan

.\m.

alloxan given intravenously in a dose of 50 to 65 mg. per kilogram. They were maintained on insulin to allow sufficient time for healing of any liver or kidney lesions. This time interval averaged 10 days. The insulin was then stopped and they were permitted to go into acidosis. Blood was obtained b> insertion of a 20 gauge needle into the aorta at the iliac bifurcation and withdrawal of 5 ml. of blood into a heparinized syringe. The blood ammonia level was determined b) a modification of the Seligson method and the blood sugar level by the method of Somogyi and Nelson. Rats with a blood sugar level above 300 11%~. per cent have excessive ketonuria. We have arbitrarily considered those with a higher blood sugar level than this to be in acidosis. Results

The data are presented in Fig. 1. The blood ammonia level for each animal is arranged in order rather than grouped frequencies. The average and a standard deviation are indicated for each group. This shows that rats given alloxan have on the average a higher blood ammonia level. In 58 per cent of these it was higher than the highest value obtained in animals with a normal blood sugar level. Several rats with blood sugar levels over 500 mg. per cent

15, 1962

.J. Obst.

& Gynec.

+TPN+

(CR CfN’)

ENZYME SCTEM3 FlXlffi AMhON!A I. GLUTAMlc DWYWDGENASE

coo$0

cm+ NH;

+ TPNH tOR DPNHI c==tdNH;

Y2 5-t? coo--KETccLul~TE

23 &GLurAbHrE MUSCLT, BRAIN, HURT)-

OLIVER, nlCtW,

3. GLUTAMiNE

_-_ “c$% COO-

+ NH;

isi GLlrrAM4TE (LIVER. KIDNEI.

SYNTSTASE UXJ+ATP+N$ Ek2 GLUTAMINE BRAIN, HEART)

Mu3cLE.

--.

Fig. 2. The enzyme mechanism and the tissue in which it occurs are given for the 3 reactions which fix ammonia in mammalian tissues.

did not have an elevated blood ammonia level and in a few instances a high ammonia level occurred with a blood sugar level of less than 400 mg. per cent. We had the livers and kidneys of 10 animals examined in the pathology department* to be sure that we were observing only the effects of diabetic acidosis and not the effects of a coincident liver or kidney lesion. The liver was normal in all cases. There was vacuolar degeneration of tubular epithelium in one animal and slight tubular dilatation in 4 others. These are minimal nonspecific changes. Comment

LOXANTREATED

i

0123456

I

I

pq

I

I

I

I

NHS-N/ml.

Fig. 1. The diabetic and control animals are arranged in order of increasing blood ammonia level with the standard deviation.

The adverse effect of diabetes upon the infant in pregnancy is far too complicated to be explained by a simple cause and effect relationship.g In general, there are three changes which should be considered: (1) the rffects of an increase in activity of the anterior pituitary, adrenal, and thyroid glands upon the diabetes; (2) depletion of available essential nutritional substances in the diahetic mother by the use of protein and fat rather than of carbohydrate; (3) the metabolism of nitrogen and excretion of the ketone bodies. The untoward effect of the endocrine hor-

of

*Acknowledgment the Pathology

is herewith Department

for

made to Robert D. Coyr examining these tissues.

Volurlle Number

83 4

mones upon diabetes has been studied in some detail. Their general effects are to make the diabetes worse. A detailed discussion of this aspect of the disease is outside the scope of this paper. The diabetic patient must use fat and protein for energy and by this the supplies available for the infant are reduced. It is unlikely that fetal synthetic mechanisms would be affected for the placenta picks up amino acids from the blood more efficiently than the liverlo and the placenta is bathed with fat precursors from fat oxidation. Assimilation of the products from fat and protein oxidation can be a serious problem when large excesses are formed. The “ketone bodies” are moderately strong acids and the kidney cannot excrete them as such.5 The kidney would ordinarily neutralize these acids with sodium but in acidosis this cation must be conserved and hydrogen, ammonium, and, in severe cases, calcium are exchanged for it. These mechanisms work reasonably well with large volumes of fluid, but in persistent acidosis ammonia formation is quantitatively the most significant.5 With excretion of these salts and glucose in the urine, large volumes of fluid are lost. The kidney is unable to control the pH with insufficient water and in trying to conserve water reabsorbs the ammonia formed by the tubule. This sequence of events increases the ammonia arriving in the liver for detoxication. The organic acids (ketone bodies) also accumulate in the blood and tissues of the mother because of the oliguria and produce a severe acid-base disturbance. In mammals, ammonia is usually detoxified by the formation of urea or glutamine. The urea is excreted by the kidney without modification. Ammonia may be released in the kidney from glutamine by the action of kidney glutaminase and be excreted as a base to conserve sodium or be taken up by the venous blood and returned to the circulation (described above). The role of glutamate synthesis in ammonia detoxication has not been clearly defined. These three enzymatic mechanisms are given in Fig. 2. An elevated blood ammonia level has been

Blood

ammonia

in rats

with

diabetic

acidosis

517

found in various types of liver disease and is thought to be the toxic factor in hepatic coma. The correlation of blood ammonia level and toxicity is good for the most part, but there has been no correlation in several cases. This may have been due to the measurement of venous rather than arterial ammoniall or to the presence in blood of less ammonia (NH,) * when compared to ammonium (NH,+). I3 Both of these factors have been considered important in the production of coma. Nevertheless, it is clear that this substance is toxic in humans, and the dose necessary to produce toxicity is much lower than for other species.l”l I5 There are a limited number of studies of ammonia metabolism in the fetus and newborn infant. In infants of normal women, the ammonia level in the umbilical vein was significantly lower than the maternal venous ammonia level but became elevated when NH&I was given to the mother.16 In the newborn infant, the blood ammonia level reaches the adult level in 3 days, corresponding to the time necessary for evolution of the bacterial flora in the intestinal tract.16 Ammonium chloride was toxic when given to premature infants for they could not exI7 KaiseP and Carrington, Reardon, crete it. and Shumanlg have found a reduced blood pH (more acid) in infants of diabetic and prediabetic mothers. An associated increase in carbon dioxide was also noted. Kaiser was able to produce these same changes by giving large doses of NH&I to normal mothers. Acidosis produced in premature infants by calcium chloride did not result in toxicity but only increased urine formation.“O These studies suggest that acidosis alone is not the single cause for fetal loss in diabetes and it may be conjectured that the abnormalities which Kaiser discovered in the blood after giving NH&l are due to ammonia. The human infant at term can synthesize 3.19 Gm. of urea in a 24 hour period but the synthesis of glutamine could not be de-

*The pH 01 this buffer system is 9.5; hence, at the usual pH quantity of free ammonia (NHz)

in 0.15M saline at 38’ of mammalian blood, would be ~mall.‘~

C. the

518

Kennon

tected in the same liver.21 This suggests that an elevation of blood ammonia in the mother is of vital importance to the infant, for only one of the enzyme systems that detoxifies ammonia is developed. If the blood ammonia level is elevated in diabetic acidosis, this could account for the fetal loss.

Summary

REFERENCES

1. 2. 3.

4. 5.

6. 7. 8. 9. 10.

il.

Jones, W. S.: AM. J. OBST. & GYNEC. 66: 322, 1953. Kramer, D. W.: AM. J. OBST. & GYNEC. 30: 68, 1935. Eastman, N. J.: Williams Obstetrics, ed. 11, New York, 1956, Appleton-Century-Crofts, Inc., p. 783. Fisher, P.: Diabetes 1: 108, 1952. White, A., Handler, P., Smith, E. L., and Stetten, D., Jr.: Principles of Biochemistry, New York, 1954, McGraw-Hill Book Company, Inc.. Geiger, E., and Pinsky, J. J.: Metabolism 4: 166. 1955. Ful& H.: Klin. Wchnschr. 12: 1364, 1933. Haft, D. E., and Miller, L. L.: Am. J. Physiol. 193: 469, 1958. Hoet, J. P.: Diabetes 3: 1, 1954. Whipple, G. H., Hill, R. B., Jr., Terry, R., Lucas, F. V., and Yuile, C. L.: J. Exper. Med. 101: 617, 1955. Bessman, S. P.: In McElroy, W. D., and Glass, B., editors: Inorganic Nitrogen Metabo-

and

conclusions

The blood ammonia level is elevated in rats with diabetic acidosis. It is suggested that an elevation of the blood ammonia level may account for the fetal loss with acidosis in the pregnancies of diabetic women.

12.

13.

14. 15. 16. 17. 18. 19. 20.

21.

lism, Baltimore, 1956, Johns Hopkins Press, p. 408. Robin, E. D., Travis, D. M., Bromberg, P. A., Forkner, C. E., Jr., and Tyler, J. M.: Science 129: 270, 1959. Jacquez, J. A., Poppcll, J. W., Lawrence, W., Jr., and Roberts, K. E.: Clin. Res. Proc. 5: 20, 1957. Bollman, J. L., and Mann, F. C.: Am. J. Physiol. 92: 92, 1930. Hugounenq, L., and Florence, G.: Bull. Sot. chim. biol. 3: 174, 1921. Anderson, J. A., and Miller, E.: A. M. A. J. Dis. Child. 94: 444, 1957. Gorden, H. H., McNamara, H., and Benjamin, H. R.: Pediatrics 2: 290, 1948. Kaisor, I. H.: AM. J. OBST. & GYNEC. 77: 573, 1959. Carrington, E. R., Reardon, H. S., and Shuman, C. R.: J. A. M. A. 166: 245, 1958. Ruben, B. L., Calcagno, P. L., Rubin, M. I., and Weintraub, D. H.: A. M. A. J. Dis. Ohild. 92: 513, 1956. Kennan, A. L., and Cohen, P. P.: Proc. Sot. Exper. Biol. & Med. 106: 170, 1961.

Discussion DR. JOSEPH blood ammonium tween ammonium tract, ammonium

uptake

H.

SKOM, Chicago, Illinois. The level represents a balance beproduction from the intestinal production from the kidney,

and release from peripheral

tissues and

brain, and removal by the liver. Dr. Kennan’s paper, as he has pointed out, deals with rats in which there is not only the problem of energy production but which are also dehydrated and in electrolyte imbalance. Although acidosis itself, and not the sodium depIetion that occurs, is responsible for increased ammonium production by the kidney, there are obviously many other hazardous variables involved in alloxan-treated acidotic rats. .4 more important problem arises, however, and that is-“what actually is being measured?” The rate of release of ammonium differs in oxalated and in heparinized blood from the same source. The quantities involved are minute, rang-

ing from 50 to 100 mg. per 100 ml. There is no absolute proof that the substance (“volatile base”) is actually ammonium, or at least that all of it is. Since the quantity present increases progressively after blood is shed, there is reason to believe that there are no detectable amounts free in the circulating blood. This “volatile base” increases relatively rapidly during the first 3 minutes, presumably as it is liberated from tbc amino acids and other breakdown products, and then much more sIowly. Therefore, the amount present after 3 minutes has arbitrarily been accepted by many as the best measure of the blood “ammonium*’ level. What does a blood ammonium level actually mean? It has long been recognized that hepatic coma is quite distinct from uremic and diabetic coma. Most of our knowledge in this field derives from work done on patients and experimental animals