Acta ledica Scandinavica. Yol. CXXVII, fasc. V, 1947.

From the Third Medical Department, Eommunehospital, Copenhagen. (Physician-in-Chief: Poul Iversen, M. D.)

On the Excretion of p-Aminohippuric Acid through the Kidneys.1

BY ULAUS BRUN, TAGE HILDEN and FLEMMINQ RAASCHOU,

Copenhagen.

(Suhmitted for publication November 5, 1946.)

In 1938 Smith, Goldring & Chasis (11) described the excretion of diodrast (Per-Abrodil) through the kidneys by tubular secre- tion. They assumed that with low plasma concentrations diodrast is removed almost completely from plasma in a single passage through the kidneys, and therefore the diodrast clearance at low plasma concentrations becomes a measure of the renal plasma flow.

In 1940 Finkelstein, Aliminosa & Smith (6) showed that other compounds of pyridon and of hippuric acid, including p-amino- hippuric acid, have the same high clearance and that they mutual depressed the clearance of each other. They assumed that all these substances were excreted by the same tubular mechanism of secretion and that for all of them the extraction from the plasma was almost complete.

This was confirmed by Warren, Brannon & Merrill (12) as far as p-aminohippuric acid was concerned, for they made direct determinations of the plasma extraction on man; they found that between 85 and 100 per cent. of the plasma content in arterial blood was removed in the course of a single kidney passage.

In many recent American works on kidney physiology, p-amino- hippuric acid has now supplanted diodrast (5 , 8, 9, 10). However,

1 The investigation carried out with support from King Christian X’S Pund.

472 CLAUE BRUN, TAGE HILDEN AND FLEMMINO RAASCHOU.

in the available literature we have been unable t o find any descrip- tion of the excretion of the substance with higher plasma concen- trations, including the determination of the Tm-value and the position of the self-depression limit and of the saturation 1imit.l Accordingly we considered we must have these questions cleared up before proceeding to utilize the substance in our research work on kidney physiology.8

Analytical Method. Determination of p-aminohippuric acid is carried out according

t o the principle described by Bratton & Marshall jr. (2). The pro- cedure is as follows:

Plasma. The plasma is diluted with water to 1 : 20 or more according to

the concentration so that the dilution contains between 1 and 6 y per ml. Of the dilution take 4 ml and add 1 ml30 per cent. trichloracetic acid; let the mixture stand at least 30 minutes, then filter; the filtrate must be quite clear; if the long precipitation time is not observed, the fil- trate will be cloudy and there will be too high a blank value by the colorimetry.

Take 2 ml. of the filtrate and add 0.1 ml of a 0.9 per cent. freshly prepared sodium-nitrite solution; shake, and allow to stand for 3 min- utes, then add 0.1 ml of an 0.5 per cent. amidosulphonic acid solution. Shake well, and allow the mixture to stand for 3 minutes more. Then add 1 ml of an 0.15 per cent. solution of N-ethyl-a-naphthylaminhydro- bromide in absolute alcohol. After thorough shaking allow it to stand a t east 30 minutes before colorimetry.

Urine or aqueous solutions. Prepare a suitable dilution so that i t contains between 1 and 6 y per

ml. Of the dilution take 4 ml, add 1 ml 2 n hydrochloric acid; of this mixture take 2 ml, treat it with sodium nitrite etc. as described above.

Colorimetry. The instrument used is a Pulfrich Stufen photometer, Filter 8. 53.

The colorimetry curve is determined on pure, aqueous solutions; within the concentrations indicated it follows Beer’s law. Blank tests must always be made on both blood plasma and urine. After subtracting the blank tests extinction from the actual test extinction, multiply the extinction found by a factor found by testing pure, aqueous solutions.

The drug was placed at our disposal by Lsvens kemiske Fabrik, Copenhagen, for which we tender o w best thanks.

Since the preparation of this work publications have appeared on this subject: Chasis, H.. J. Redish, W. Goldring, H. A. Ranges & H. W. Smith: J. Clin. Invest. 1946: 24: 683, and H. W. Smith, N. Finkelstein, L. Aliminosa, B. Crawford & M. Ctraber: J. Clin. Invest. 1946: 24: 388.

ESCRHTION OF P-AMINOHIPPURIC ACID. 473 We found the factor 0.7 I. By this multiplication we get the concentra- tion in the dilution, and from there we arrive at the final concentration in plasma or urine by multiplying by the degree of dilution.

By this method, however, it is possible only to determine free p- aminohippuric acid; any combined (acetylated?) quantity can be de- termined after boiling acidified solutions.

As a result of the protein precipitation described under the plasma analyses there is a certain loss of p-aminohippuric acid; with the pro- cedure indicated, however, in which the plasma is diluted at least 20 times before the protein precipitation, the loss is only between 1 and 2 per cent.; we have therefore disregarded this low.

Experimental Technique. Regarding experimental persons and the technique employed,

we refer to another work (3). It may be added here that the p- aminohippuric acid was used in a 20 per cent. solution of the sodium salt. Of this 20 per cent. solution one can employ about the same number of ml in the test as of a 35 per cent. diodrast solution (3). Thus the calculations of priming injections and in- fusions given by Goldring, Chasis, Ranges & Smith (7) are applic- able.

Results of Experiments. Fig. 1 shows a curve for the course of the clearance and tubular

secretion of p-aminohippuric acid with increasing plasma concen- trations. As to the more detailed explanation of these curves we shall refer to our work on Diodrast-Tm (3) in order to avoid unnecessary repetition. As might have been anticipated, the re- sults for p-aminohippuric acid were found to be quite in analogy with the excretion of diodrast.

We have followed the excretion of p-aminohippuric acid with increasing plasma concentrations in altogether five male experi- mental persons. Regarding the plasma concentrations a t which self-depression sets in (the self-depression limit) and the maximum tubular secretion is reached (the saturation limit), we refer to a separate work (4); we need merely add here that in our experi- ments these limits lay a t the following:

1) Self-depression limit, between 7 and 13 mg%. 2) Saturation limit, between 13 and 20 mg%. Thus in every case the clearance and Tm for p-aminohippuric

acid must be determined with plasma concentrations lying below 7 and above 20 mg% respectively.

31-470729. Acta med. scandinav. Vol. C X X V I I .

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Table I contains the values for p-aminohippuric acid clearance and Tm arrived a t by experiments on 8 healthy young men. In every case a correction is inserted for a body surface of 1.73 8q.m.

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70 $ 60

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40

30

20

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Table I. P-aminohippuric acid cleararcs and p-aminohippuric acid 2'm in h

normal subjects.

$ y .a rr

i t

Mean ........ l o ...........

1.92 1.96 1.85 1.96 1.78 1.89 1.52 1.98

=

!3 f h

B Y

- 1.80 1.81 1.73 1.85 1.76 1.83 1.62 1.80

149 160 122 137 121 116 109 136

- 74 77 73 74 66 70 61 80

............. ' 130

............. 116.4 lorn ....................... 6.43

~ ~

- 698 666 647 668 613 668 647

628 44.8 16.9

600

-

-

=

;J.!y 2 I p

- 69.0 67.7 61.8 72.0 63.2 79.0 64.3 57.8

66.6

3.07

60.6

8.7 _ _ ~

- - 24.9 22.9 22.3 20.5 19.8 17.2 16.9

20.6 2.96

-

1.ia 21.4

- 2.16 2.22 2.36 1.90 1.92 1.45 1.70 2.33

2.01

0.11 2.51

0.3a - __ -

- 0.356 O.34D 0.268 0.371 0.326 0.407 0.332 0.298

0.338 I 0.045 1 0.016 1

0.109 ~

EXCRETION OF P-AMINOH[PPURIC ACID. 475

As previously mentioned, there was no account of the p-amino- hippuric acid Tm in the literature available to 119.1 I n a monograph just published by Goldring & Chasis (8), however, the authors say in a footnote that they have found the p-aminohippuric acid Tm to be 76.1 mg/min. on making tests with 22 normal people; they do not refer to any publication on the subject.

The lowest line in Table I represents the values which we have found for diodrast for 10 healthy young male experimental persons (3). Of these, there are 4 on whom tests were made with both dio- drast and p-aminohippuric acid on different days; thus these results are more suitable for comparison than the averages of the rather small normal materials for diodrast and p-aminohippuric acid. Table I1 contains the results from the double tests with these 4 subjects.

Tnble 11. P-arninohippuric acid clearance and p-aminohippuric acid Tm in 4 normal subjects compared with the corresponding diodrast values in

the same persons.

1 - 1 I - B

3 251 1.52 Diodrast ' 132 p. a. h. ac. 109

8 22 I 1.SO 1 Diodrast 126 I j p. a. h. W. 115 I 1.98 Diodrast 1 132

1.92 Diodrast 1 126 p. a. h. ac. 1 135

p. a. h. ac. I 149

I

Fa Y

&. I . P '

I v

484 43.6

684 44.6 668 79.0 750 56.2

502 56.0 598 69.0

50.1 67.5

647 64.3

- 57.8

2 $-

B- @ -9

- 0.172 0.306 0.175 0.318 0.221 0.276 0.220 0.328 0.197 D.348 '

g Y g. gr

g 38

- 27.3

18.5 . 17.2 17.6

25.1 24.9

16.9

-

=

9

f? r B

- 3.03 1.70 2.84 1.45 3.35 3.33 3.25 3.16

11.1 j 10.0 15.4 I 8.5 13.3 1

! 8.96 ! 8.68 I -

It will be seen that the diodrast clearance and the p-amino- hippuric acid clearance are equal, which agrees with the American reports (6, 8). On the other hand, the maximum tubular secretion, Tm, is higher for p-aminohippuric acid than for diodrast. Among the form subjects referred to the proportions between them were

1 See prevom footnote 2 p. 472.

3

Fig. 2. The plasma concentrations of p-aminohippuric acid after subcutaneous injection of 0.04 and 0.07 g/kg of sodium paminohippurate.

&otr;umpura - om;m d,>pura t sutcut int . .

1.0/1.35; Goldring C Chasis (8) give the proportions of l .O/l .S for diodrast iodine Tmlp-aminohippuric acid-Tm.

The difference between the two Tm values becomes more pro- minent when the Tm is expressed in milli-equivalents; indeed this is the more correct, as it tells us something about the secreted number of molecules of the substance. It will be seen from Table I1 that the proportion between diodrast iodine Tm and p-amino- hippuric acid Tm expressed in milli-equivalents is as l.O/l. 77; this means that the capacity of the tubulus cells to secrete p-amino- hippuric acid is almost twice as great as that of diodrast. It looks as if p-aminohippuric acid ))fitted)) better in with the enzymatic secre- tion mechanism of the tubules; the reason of this may be that from a chemical point of view p-aminohippuric acid to a greater extent than diodrast is related to substances which nature has endowed tubular secretion with the ability t o handle. It is not impossible that pre-formed p-aminohippuric acid occurs in the organism. Just as benzoic acid is excreted through the kidneys as hippuric acid, it is perhaps presumable that p-aminobeneoic acid, well known as an important factor in bacterial metabolism, is excreted as p-ami- nohippuric acid.

Like diodrast, p-aminohippuric acid can be administered sub- cutaneously as well as intravenously; we have employed a 20 per cent. solution of sodium-para-amino-hippurate diluted with equal parts of physiological salt solution without observing any discom-

EXCRETION OF 1’-AMINOHIPl’URIC ACID. 477 Table 111.

P-aminohippuric ocid clearances after subcutaneous injections.

I

h

I 1

fort afterwards. I n fig. 2 is a curve showing the plasma concentra- tion after the subcutaneous injection of 0.04 and 0.07 g/kg respectively in two normal persons.

Four persons were injected subcutaneously; of these four curves it will be seen that after about 90 minutes the curves reach their peak and begin to fall. This falling, straight curve is suitable for the determination of the p-aminohippuric acid clearance for example in two hours.

The results of the p-arninohippuric acid clearance in these four persons after subcutaneous injection are shown in Table 111.

Furthermore, we have tried giving p-aminohippuric acid by mouth; the dose was 4 g p-aminohippuric acid, but we did not suc- ceed in recovering the free drug in either plasma ur urine.

Finally, p-aminohippuric acid in blood and urine was not found in the combined (acetylated) state.

The question now remaining is whether p-aminohippuric acid has advantages over diodrast. In principle the two substances must be said to be equal, as the physiological information obtained when testing their excretion is the same. Nevertheless, p-amino- hippuric acid seems to have certain practical advantages, where- fore it will undoubtedly be used in the future.

In the first place, the analytical method is more realiable and easier to carry out than the diodrast analysis, and presumably it will be more suitable for an ordinary hospital laboratory, be- cause the method is already in use for the determination of sul- phonamides.

In the second place, unlike diodrast p-aminohippuric acid can

478 CLAUS BHUN, TAGE HILDEN AND FLEMMINQ I1AASCHOU.

be determined quantitatively in plasma. When analysing for diodrast iodine in plasma the preliminary precipitation of plasma protein will cause a certain loss of iodine (about 10 per cent.), depending on the precipitating agent and on the plasma dilution during precipitation. It has not been possible to eliminate this loss (1). In the p-aminohippuric acid determination there is a loss of only 1 or 2 per cent. during the precipitation of the protein.

Finally, the fact that the drug does not contain iodine may be taken as an advantage, in so far as, in contrast to diodrast, it does not involve a risk to patients hypersensitive to iodine.

All in all, p-aminohippuric acid will undoubtedly prove to be a more handy substance than diodrast.

Summary.

The excretion of p-aminohippuric acid through the kidneys with various plasma concentrations has been examined; conditions corresponded to those in the case of diodrast.

In 8 healthy young male persons p-aminohippuric acid clear- ance was found to be 628 f 44.8 ml/min. and the maximum tubu- lar secretion of p-aminohippuric acid ( TMPJ 66.6 f 8.7 mglmin.

The p-aminohippuric acid clearance is of the same value as the diodrast clearance, whereas the maximum tubular secretion (Tm) is higher for p-aminohippuric acid than for diodrast; expressed in milli-equivalents the proportion of Tm, to Tmpah is 1.0/1.77.

P-aminohippuric acid can be injected subcutaneously; this mode of supply will be very suitable for isolated clearance tests.

Of 4 g p-aminohippuric acid administered by mouth we were unable to recover any in blood or urine.

P-aminohippuric acid is determined in plasma and urine accord- ing to Bratton & Marshall's method; this method being more simple and rel'able, we must regard p-aminohippuric acid as being more suitable for clinical tests than diodrast.

Literature,

1) Bak, B., C. Brun & F. Raaschou: Acta med. scand. 1943: 114: 271. - 2) Bratton, A. C., & E. K. Marshall jr.: J. Biol. Chem. 1939: 128: 537. - 3) Brm, C., T. Hilden & F. Raaschou: Acta med. scand. 1947. - 4) Brun, C., T. Hilden & F. Raaschou: Acta physiol. scand.

EXCRETION OF P-AMINOHIPPURIC ACID. 479

in preparation. - 5) Craig, F. N., F. Visscher & R. Houok: Amer. J. Physiol. 1945: 143: 108. - 6) Finkelstein, N., L. M. Aliminosa & H. W. Smith: Amer. J. Physiol. 1941: 133: 276. -7) Goldring, W., H. Chasis, H. A. Ranges & H. W. Smith: J. Clin. Invest. 1940: 19: 739. - 8) Gold- ring, W., & H. Chasis: Hypertension and hypertensive disease. NewYork. The Commonwealth Fund 1944. - 9) Selkurt, E., & R. Heuch Amer. J. Phyaiol. 1944: 141: 423. - 10) Selkurt, E.: Amer. J. Phyaiol. 1944: 142: 182. - 11) Smith, H. W., W. Goldring & H. Chasis: J. Clin. In- vest. 1938: 17: 263. - 12) Warren, J. V., E. s. Brannon & A. 5. Merrill: Science 1944: 100: 10s.