Alma Hiller

THE EXCRETION OF AMMONIA AND TITRATABLE ACID IN NEPHRITIS

As the result of past metabolic studies, especially those of Hender-son and Palmer (12) it is known that the non-volatile acids produced in excess of fixed base by human metabolism are excreted in the urine in two forms, viz., as free acids, and as ammonium salts. Since the kidney is unable to form urine with a pH much lower than 5.0, it can excrete, in significant amounts, free acids of only the weak buffer type. In this class fall acid phosphates and the various organic adds. It appears, however, that free acid excretion may assist also in elimination of strong acids, which can react with buffer salts (e.g., HC + Na2HPO4 = H(NaHPO4) + NaCl), the free buffer acid being excreted in place of the strong acid. Thus Marriott and Howland (19) found that HCl ingestion increased the output of free buffer acids. Ammonia serves to neutralize either weak or strong acids. Consequently both ingestion of HC1, (either as such, or as NH4Cl or CaC12) and diabetic ketosis (producing weak beta-hydroxybutyric acid) cause increase in ammonia excretion. In fact both the above conditions increase both ammonia and free buffer acid output, without greatly altering the NHF/acid ratio (Marriott and Howland (19), Fitz and Van Slyke (4)). Peculiarly, however, ingestion of acid phosphate was found bv Marriott and Howland to increase only the output of titratable acid, without any effect whatever on the ammonia output. Since the nature of the acid eliminated, and presumably other unknown factors, can influence the proportions excreted as ammonium salts and titratable free buffer acids respectively, it is not surprising 255 Downloaded from

STUDIES OF UREA EXCRETION. IX. COMPARISON OF UREA CLEARANCES CALCULATED FROM THE EXCRETION OF UREA, OF UREA PLUS AMMONIA, AND OF NITROGEN DETERMINABLE BY HYPOBROMITE

According to the usually accepted concept, the urea removed from the blood by the kidneys is excreted in two forms, chiefly as urea itself, but partly as ammonia. That the ammonia of the urine is formed in the kidneys was demonstrated by Nash and Benedict (1921), in work which has been confirmed by other authors (cf. Peters and Van Slyke, I, p. 373). Evidence from animal experiments in v4vo has indicated urea as the probable chief source of the ammonia formed by the kidney (cf. Peters and Van Slyke (1931) p. 373). Krebs recent work (1933) with kidney tissue in vitro has indicated the possibility that blood amino acids may, also be a direct source of the ammonia formed by the kidney, but whether an important part of the ammonia actually excreted comes from them has not been demonstrated. If urinary ammonia is entirely or chiefly formed from the blood urea, the urea cleared from the blood per minute by the kidneys is represented more accurately by the excretion of urea + ammonia rather than of urea alone. It appears possible, therefore, that urea clearances calculated from the excretion of urea + ammonia may measure the work of the kidneys in excreting urea from the blood more accurately than clearances calculated in the usual manner from the output of urea alone. It therefore appears to be a problem of physiological interest to ascertain whether, when the ammonia: urea ratio in urine varies markedly, more consistent values for the urea clearance are calculated from the excretion rate of urea alone, or of urea + ammonia. For clinical interpretation, as already pointed out by Bell, Gilmour and Cameron (1934), it would seldom make a serious difference if, in the formula for calculating the urea clearance (Moller, McIntosh and Van Slyke (1928, a)), urine urea nitrogen were replaced by urea + ammonia nitrogen. Since the ammonia nitrogen ordinarily equals 1 to 10 per cent of the urea nitrogen, the clearance calculated from urea + ammonia would be from 1 to 10 per cent higher than that calculated from the urea excretion alone. In nephritis the proportion of nitrogen excreted as ammonia is likely to be less than in normal subjects, because the damaged kidney turns a smaller proportion than normal of the urinary nitrogen into ammonia (see literature quoted by Peters and Van Slyke (1931), p. 378; also data in Table III, present paper). Conditions do occur, however, in which the ammonia: urea ratio may be unusually high. Such are the acidoses of diabetes and starvation, and the depression of urea output caused by very low protein, high calory, diets. If the urea clearance is measured in such a condition, it is of interest to know whether more consistent values may be expected when the calculation is based on the output of urea, or of urea + ammonia. Aside from this, and the physiological interest of the problem, the question has some practical bearing on the convenience of procedures for determining the urea clearance. If a method for determining urinary urea is used which depends upon estimation of the ammonia formed when the urea is hydrolyzed by heat or urease, it is simpler to determine urea + ammonia than urea alone, since thereby one avoids an extra operation to remove or determine the preformed ammonia. Likewise, if the hypobromite gasometric method is used, it is simpler to determine urea + ammonia, since the reagent gives practically the same yields of N2 from both. Furthermore, if, by bladder infection or delay in analyzing urine, part of the urea is decomposed into ammonia before analysis, no error is introduced into the figure representing the sum of urea + ammonia.

Determination of Albumin and Globulin in Urine.

Albumin and globulin are separated by precipitating the latter with sodium sulfate, as in Howes 1 technique for plasma protein separation. The separated proteins are determined by the colorimetric method of Autenrieth, 2 which can be made practical for general colorimetry by introducing pure biuret as a standard. One mg. of biuret gives a color equal to that of 0.924 mg. of urinary proteins treated with alkali and copper sulfate, as described by Autenrieth. 2 The standard solution is made by dissolving 0.4 gm. of biuret in water and diluting to 150 cc. Five cc, containing 13.33 mg. of biuret, is colorimetrically equivalent to 12.3 mg. of urinary proteins. For total protein, enough urine to contain 8 to 20 mg. is precipitated with an equal volume of 10 per cent trichloroacetic acid. The precipitate is redissolved in 3 per cent NaOH, treated at 10 cc. volume with 0.25 cc. of 20 per cent CuSO4 · 5H2O, and compared with 1 cc. of the biuret standard similarly treated. Globulins are precipitated by treating the urine at 38° with an equal volume of 44 per cent Na2SO4. In the filtrate, the albumin is precipitated with trichloroacetic acid, and determined as described for total proteins. Globulins are calculated as (total protein)—(albumin).

Studies of Renal Metabolism.

A kidney is drawn out of the body cavity of a dog and sutured under the skin of the back, with the renal vein located in such a position that it can be punctured through the skin with a needle. The metabolism of the kidney has been studied by simultaneous analyses of arterial and renal bloods and of urine secreted during the observation periods. The rate of blood flow through the kidney has been estimated by comparing the amount of urea removed from a unit volume of blood in passing through the kidney with the amount excreted per minute. In most of our experiments only one kidney at a time has been studied; one is brought out under the skin, and the other is removed, in order to simplify the experiments. In a few controls in which one kidney was brought out and the other left in situ excretion was similar in both. Through a single kidney in clogs of 15 to 20 kilos weight the blood flow is in the neighborhood of 200-250 cc. per minute. Increasing the urea content of the blood as much as 10-fold does not significantly accelerate the blood flow. The urea excretion increases 10-fold, but the increase is due to the fact that the proportion of blood urea removed during perfusion of the kidney remains constant, so that the amount removed per liter of blood passing through the kidneys rises in proportion to the amount present in the blood. This explains the manner in which the blood urea clearance is kept constant during wide fluctuations in blood urea content. The proportion of oxygen removed from the blood by the kidney is rather low, 10 to 20% of the arterial oxygen content as a rule.

The non-protein nitrogenous constituents of normal human muscle

The material for these analyses was obtained at operations for severe lacerations, carcinoma of the breast, or gangrene of the extremities. Only such specimens were utilized as were not involved by the pathological process. This seemed to be the only available source from which muscle tissue that might be regarded as normal could be procured. In every case the blood of these subjects was analyzed for its non-protein nitrogen as well as urea content. Any patients in whom these were above the normal would not have been utilized in this series. As a matter of fact, no abnormal bloods were encountered among these cases. The maximal, minimal and average figures, as well as the number of determinations for each substance, are given in the appended table. The results for the non-protein nitrogen are much lower than they should be. These data were obtained by extracting the muscle tissue by alcohol. Alcohol does not dissolve the kreatin. Since this substance forms such a large portion of the non-protein nitrogen of muscle, a considerable error is introduced. It is our aim in completing this series of determinations to employ trichloracetic acid or some other fluid for extraction which will approximate the true values more closely. It is hoped that these studies may form a basis for comparison with pathological muscle tissue.