STUDIES OF UREA, CREATININE, AND AMMONIA EXCRETION IN DOGS IN ACIDOSIS*

BY ALF S. ALVING AND WAYNE GORDON

(From the Department of Medicine and the Lasker Foundation for Medical Research of the University of Chicago, Chicabo)

(Received for publication, May 6, 1937)

Van Slyke, Page, Hiller, and Kirk (1) have reported, in recent studies on man, that when the proportion of urea in the urea and ammonia mixture of the urine was markedly decreased by induced acidosis and a low protein diet, the urea clearance calculated from the excretion rate of urea alone suffered a parallel reduction. If, however, values for excretion of urea and ammonia were sub- stituted for urea, the clearance calculated remained at the usual level. In two subjects the simultaneously performed creatinine clearances were found to have normal values. They considered these results “favor the hypothesis that the ammonia excreted in the urine of man is formed in the kidney chiefly from urea removed from the blood.” Pitts (2) found that, in the dog, the urea clearance relative to the creatinine clearance was essentially the same in acidosis and alkalosis as in the normal. At low plasma urea values in acidosis the urea + ammonia clearance was con- siderably higher than the urea clearance, and in some instances exceeded the creatinine clearance. He concluded that if urea is the precursor of urinary ammonia, it is not the urea that has passed into the glomerular filtrate. The possibility that urea might be removed directly from the postglomerular blood and transformed to ammonia in the tubules could not be excluded.

In the present work, various physiological functions of the kid- ney have been studied during acidosis in dogs with only one kidney in which the kidney had previously been explanted by a modifica- tion of Rhoads’ technique (3). Because explantation permits the

* This work was aided by a grant from the Douglas Smith Foundation for Medical Research.

103

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104 Urea, Creatinine, and Ammonia Excretion

simultaneous withdrawal of renal venous and femoral arterial blood, investigation can be made of the possibility of formation of urinary ammonia from postglomerular blood urea, as well as the formation of ammonia from filtered urea.

Methods

Female dogs were used in all experiments, and were prepared by the explantation of the left kidney and a subsequent right nephrectomy., They were maintained on the low protein diet of Jolliffe and Smith (4). All experiments were carried out after a fast of 18 hours or more. Acidosis was produced by feeding the dogs calcium chloride in water for several days before, and again an hour before, the experiment was begun. The total C!OZ con- tent of the serum was found to be as low as 8.2 rnM per liter in some experiments. A saline solution containing creatinine was injected intravenously throughout the experiment. The tech- nique for explantation of the kidney, and details employed in determining clearances and in the withdrawal of blood, were the same as described by Gordon, Alving, Kretzschmar; and Alpert

(5). Urea nitrogen in urine and in whole blood was determined by

the method of Van Slyke (6). Creatinine was determined in urine, in plasma, and in blood by the method of Folin and Wu (7). Urinary ammonia nitrogen was determined by the method of Van Slyke and Cullen (8). Cell and plasma volumes were determined by the Wintrobe (9) hematocrit. All analyses were carried out in duplicate, and in triplicate when blood urea determinations and creatinine determinations did not agree within 0.1 mg. per 100 cc. and within 0.5 per cent, respectively.

Calculations

Experimental and theoretical support for the validity of for- muhe expressing various physiological activities of the kidneys has been presented by Moller, McIntosh, and Van Slyke (lo), Van Slyke, Rhoads, Hiller, and Alving (ll), and Van Slyke, Hiller, and Miller (12), and, therefore, will not be discussed. The formulae and definitions of symbols are as follows:

Clearance (C or C&-The volume of whole blood or of plasma containing the amount of excretory substance that is eliminated per minute in the urine.

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A. S. Alving and W. Gordon 105

Creatinine, C, = UV/A,

s

Urea + ammonia, C = ((U + NHdV)/A

s

A or A, = mg. excretory substance contained in 1 cc. arterial whole blood or arterial plasma

Ii = mg. excretory substance in 1 cc. urine

NK = mg. ammonia nitrogen in 1 cc. urine

V = cc. urine excreted per minute AS = surface area expressed in sq.m.

Estraction Percentage (E or &-The percentage of urea or creatinine removed from the blood or plasma as it perfuses through the kidney.

Urea (observed), E = E, = Li2 x 100

A, - R, Plasma creatinine (observed), E, = -7 x 100

P

Calculated by assuming that all the extracted urea comes from plasma,

R or R, = mg. excretory substance contained in 1 cc. renal venous blood or renal venous plasma

0.75 = ratio of cell water to plasma water V, or V, = volume of cells or plasma in 1 volume of blood

CJrea fleubsorption-The fraction of filtered urea reabsorbed in the tubules during the 30 to 60 minute period of urea clearance determination or during the 1 to 2 minute period of blood withdrawal.

Urea reabsorption over 30 to 60 minute period, 1 - es&; ‘Y

Urea reabsorption over 1 to 2 minute period, 1 - calculated urea E,

100a

a = fraction of arterial water filtered in glomeruli (lOOa = creatinine E,)

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Ext

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108 Urea, Creatinine, and Ammonia Excretion

Renal Blood Flow (F)-The number of cc. of blood flowing through the kidney per minute.

Creatinine or urea, F =i UV/(A - R)

S

Urea + ammonia, F = (U + NHd/(A - R)

S

Because A - R is much less variable in the case of creatinine than in the case of urea (5), the blood flow calculated from creat- inine values is less subject to error than blood flow calculated from urea values, and is used as the standard of reference in judging the validity of blood flow determinations in which other values are employed.

Results

The mean of individual experiments on each dog and the average for three dogs are given in Table I. Results on individual dogs were in good agreement.

Clearances-During acidosis the average plasma creatinine clearance fell 36 per cent, and the blood urea clearance, 34 per cent. In individual experiments the fall varied greatly according to the degree and duration of acidosis, but the ratio of the two clearances remained fairly constant. The average blood urea + ammonia clearance, in contrast to the urea clearance, fell only 22 per cent. The urea clearance to creatinine clearance ratio remained constant (+6 per cent) during acidosis, while the urea + ammonia clear- ance to creatinine clearance ratio increased 25 per cent, exceeding unity in one experiment. These results agree with those of Fitts

(2). Reabsorption of Urea-Acidosis did not affect significantly the

reabsorption of urea, either as estimated from simultaneous urea and creatinine clearances or from the simultaneous extraction percentages of urea and creatinine.

Extraction Percentages-The average plasma creatinine extrac- tion fell slightly during acidosis. The average observed whole blood urea extraction and the plasma urea extraction, estimated after allowing for diffusion of urea from cells, were similarly slightly decreased. The fall in extractions was of questionable significance.

Renal Blood Flow-During control periods when ammonia

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A. S. Alving and W. Gordon 109

excretion was negligible, average renal blood flow estimations based on urea and urea + ammonia values were 6 per cent and 12 per cent higher, respectively, than the renal blood flow estimated from creatinine. In individual experiments the blood flow esti- mated from creatinine and urea values were frequently at variance because occasionally great momentary fluctuations in the reab- sorption of urea make its extraction less constant than the creat- inine extraction, which changes only with variations in filtration (5). The inconstancy of urea extraction invalidates comparison of renal blood flow figures in single experiments, but does not vitiate the significance of comparisons based on the averages of several experiments. During acidosis average diminution in renal blood flow, estimated either from creatinine or urea values, was of the same order of magnitude as the diminution in urea and creatinine clearances. Both blood flow estimations were 37 per cent below the control (creatinine) blood flow measured during periods with- out acidosis. The average blood flow during acidosis estimated by calculating urinary ammonia as urea was only 19 per cent lower than the (creatinine) control, and was 28 per cent higher than the blood flow calculated either from creatinine or urea values during acidosis.

DISCUSSION

As premises for the following discussion it will be assumed, as appears probable (11, 12, 5), that normally approximately 20 per cent of the water and filtrable solutes of the plasma, including creatinine and urea, is filtered in the glomeruli, and that, of the filtered urea, on the average about 40 to 50 per cent is reabsorbed. No creatinine appears to be reabsorbed. (A summary of values expected on the basis of the theoretical considerations discussed below, compared to average values obtained experimentally, is given in Table II.)

In the dog, the urea clearance calculated from the excretion rate of urea alone has the same ratio to the creatinine clearance during acidosis that it has under normal conditions. Therefore, if the creatinine clearance represents the glomerular filtrate, the urea clearance is proportional to that filtrate. When, on the other hand, the value for the excretion of urea + ammonia is substituted for urea, the ratio of the clearance thus calculated to the creatinine

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TAB

LE

II

Val

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Exp

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d fro

m

Theo

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Col

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ions

C

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with

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und

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tally

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expl

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of

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a..

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omen

- ta

ry)..

......

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al

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a.

. ._

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a.

. . .

.

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(U

+ N

Hr)V

A

A-R

-

x 10

0 A

l- ca

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E,

crea

tinin

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A-R

(U

+ N

&)V

A-R

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s to

be

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osis

_-

-

If ur

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H2

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m

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Cor

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Cor

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(+

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ar

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as s

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of

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nce

in

dete

rmin

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“cor

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” ur

ea

5

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A. S. Alving and W. Gordon 111

clearance increases with increased ammonia output. These results, in agreement with those found by Pitts (2) in experiments on the dog, render improbable the formation of urinary ammonia in significant amounts from urea that has passed into the glomer- ular filtrate. The possibility of formation of urinary ammonia from reabsorbed or postglomerular urea,l however, cannot be excluded. These results with dogs are opposite to those observed in man by Van Slyke, Page, Hiller, and Kirk (l), who found that the ratio, ammonia + urea clearance to creatinine clearance, rather than the ratio, urea clearance to creatinine clearance, re- mained unaffected by acidosis and high ammonia formation in human subjects.

The percentage of urea extracted from the arterial blood during passage through the kidney is, like the extraction of creatinine, only slightly diminished, or normal during acidosis.

Urea reabsorption, calculated from either the clearances or the extraction ratios of urea and creatinine, is normal during acidosis. The approximate constancy of the ratio, creatinine extraction to urea extraction, would be expected during increased ammonia production only if (a) the ammonia were formed from some pre- cursor other than urea, or (b) the ammonia were formed from the fraction of filtered urea that is not reabsorbed, or (c) the ammonia were formed from reabsorbed or postglomerular urea, and the reabsorption of urea were increased exactly enough to balance the formed and excreted ammonia. Of these possibilities (a) appears compatible with all the observed facts, (6) does not appear prob- able for reasons advanced previously in the discussion of clearance ratios, and (c) would appear improbable because increased urea reabsorption was not observed. The normal extraction percent- ages and normal estimated urea reabsorption, therefore, add to the weight of evidence against urea as the source of ammonia in the kidney of the dog.

During both control periods and periods of acidosis, estimations of renal blood flow based on the excretion rate of urea alone are the same as those based on creatinine, but those based on the excre- tion of urea + ammonia are higher (significantly so during acido- sis). This fact, for reasons similar to those given above, also weighs against ammonia formation from urea.

1 By postglomerular urea is meant the urea that has passed through the glomerulus without being filtered.

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112 Urea, Creatinine, and Ammonia Excretion

When a fall in the urea or creatinine clearance occurred during acidosis, it was accompanied in our experiments by a diminution in renal blood flow, and only to an insignificant extent, if at all, by a diminution of the percentages of these substances extracted from the arterial blood during passage through the kidney. The paral- lelism between the urea and creatinine clearance changes and renal blood flow changes, observed in dogs by previous authors (11, 12) holds equally well when ammonia output is increased by CaCh acidosis.

SUMMARY

The ammonia to urea ratio in the urine of dogs with kidneys explanted according to Rhoads’ procedure was increased by a combination of low protein diet and CaCh acidosis, and the results were compared with those observed in the same animals on low protein diets, with relatively slight ammonia outputs.

The creatinine to urea ratio, with regard to both the percentages extracted from the blood plasma during passage through the kid- ney, and with regard to the clearances of the two substances, remained the same during acidosis as before. Also the blood flows, calculated from the extraction and excretion rates of the two substances, were alike.

On the other hand, if urea + ammonia was substituted for urinary urea in calculating the urea clearance, the ratio of the clearance thus calculated to the creatinine clearance was increased by the acidosis; the blood flow calculated by similar substitution also became greater than that estimated from creatinine.

These results appear contrary to the probability that in dogs urea is the source of the urinary ammonia formed in the kidneys.

BIBLIOGRAPHY

1. Van Slyke, D. D., Page, I. H., Hiller, A., and Kirk, E., J. Clin. Inv., 14, 901 (1935).

2. Pitts, R. F., J. Clin. Inv., 16,671 (1936). 3. Rhoads, C. P., Am. J. Physiol., 109, 324 (1934). 4. Jolliffe, N., and Smith, H. W., Am. J. Physiol., 99, 101 (1931). 5. Gordon, W., Alving, A. S., Kretzschmar, N. R., and Alpert, L., Am. J.

PhysioZ., 119,483 (1937). 6. Van Slyke, D. D., J. BioZ. Chem., 73, 695 (1927). 7. Folin, O., and Wu, H., J. BioZ. Chem., 38,81 (1919).

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A. S. Alving and W. Gordon 113

8. Van Slyke, D. D., and Cullen, G. E., J. Biol. Chem., 19, 211 (1914); 24, 117 (1916).

9. Wintrobe, M. M., J. Lab. and Cl&. Med., 16,287 (1929-30). 10. Mtiller, E., McIntosh, J. F., and Van Slyke, D. D., J. Cl&. Inv., 6,427

(1928). 11. Van Slyke, D. D., Rhoads, C. P., Hiller, A., and Alving, A. S., Am. J.

Physiol., 109, 336 (1934). 12. Van Slyke, D. D., Hiller, A., and Miller, B. F., Am. J. Physiol., 113,

611 (1935); llS, 629 (1935).

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Alf S. Alving and Wayne GordonACIDOSIS

AMMONIA EXCRETION IN DOGS IN STUDIES OF UREA, CREATININE, AND

1937, 120:103-113.J. Biol. Chem. 

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