THE RELATIONSHIP BETWEEN CHEWCAL STRUCTURE AND PHYSIOLOGICAL RESPONSE

IV. CONJUGATION OF SALICYLIC ACID WITH GLYCINE AND ITS ACTION ON URIC ACID EXCRETION

BY ARMAND J. QUICK

(From the Department of Surgery of the Fifth Avenue Hospital, New York)

(Received for publication, May 3, 1933)

The experimental studies on the fate of salicylic acid have yielded many conflicting results. In 1856, Bertagnini (1) re- ported that after ingesting salicylic acid he succeeded in isolating a compound consisting of glycine and salicylic acid which he named salicyluric acid because of its analogy to hippuric acid. Later investigators who also reported finding salicyluric acid are Nencki (2), Lesnik (3), Moss0 (4), Bass (5), Baldoni (6), and Stockman (7). Hanzlik (8), however, failed to find salicyluric acid even though he carefully followed the various procedures of the previous investigators. He therefore seriously questioned the occurrence of salicyluric acid in the urine. Several years later Holmes (9) reported the isolation of the conjugated product from human urine. He furthermore criticized the Thoburn-Hanzlik method (lo), which consists essentially in steam-distilling salicylic acid from urine strongly acidified with phosphoric acid, as unsatis- factory for determining salicyluric acid. From his quantitative studies Holmes concluded that salicylic acid is excreted in the ratio of 40 per cent free to 60 per cent combined with glycine. Holmes’ work has in turn been adversely criticized by Johnson (11). Recently the writer (12) in studying the conjugation of substituted benzoic acids discovered the rule that the union of glycine with a carboxyl group attached to a benzene ring is markedly inhibited by substitution in the ortho position. Since salicylic acid is o-hydroxybenzoic acid, its conjugation with glycine should be inhibited. It was found, as was anticipated, that the excretion of salicyluric acid was exceedingly small. On continuing

475

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476 Salicylic Acid

the study of salicylic acid, a simple method for isolating salicyluric acid was developed, which is described in this paper. Further quantitative studies on the conjugation of salicylic acid and on its influence on the excretion of uric acid are also reported.

EXPERIMENTAL

Both the successful isolation of salicyluric acid and the quanti- tative determination of free and combined salicylic acid employed in this work are based on the fact that these compounds can be completely removed from urine with ether by means of a con- tinuous extractor. The form01 titration method previously described by the author (13) for hippuric acid yields equally satis- factory results for salicyluric acid, and the writer’s adaptation of the Day and Taggard bromination method (14, 15) for total sali- cylic acid is simple and accurate. These methods have the ad- vantage that they can be applied to small volumes of urine and that they have a high degree of accuracy yet are simple and rapid enough to be suitable for the analysis of routine hourly specimens.

In studying the conjugation of salicylic acid and its influence on the excretion of uric acid, the drug was always given orally in the form of the sodium salt. A light breakfast consisting of coffee and a cruller or toast was eaten 1 hour before the test. During the experimental period the subject was kept on a low protein and low purine diet. With a fairly well maintained standard diet, a remarkable constancy not only in the conjugation of salicylic acid but also on its stimulation of uric acid excretion was observed. Even after an interval of 1 year, the response to a fixed dose of the drug showed no greater variation than could be accounted for by the experimental errors inherent in the methods of analysis. Uric acid in the urine was determined by the Benedict-Hitchcock method (16).

Isolation of Salicyluric Acid-Since hippuric acid and related compounds, which are always present in the urine after a mixed diet, cannot be separated from salicyluric acid, it is necessary for the isolation of pure salicyluric acid to put the subject on a low protein and a fruit- and vegetable-free diet for 24 hours prior to the test. 2.3 gm. of sodium salicylate are given orally and the urine collected for 24 hours, or if more convenient, for a shorter period. During the experiment only milk, bread, and sugar are

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A. J. Quick 477

allowed. If these dietary precautions are observed, pure salicyl- uric acid having the correct melting point is readily obtained, whereas on a mixed diet, the product although white and crystalline will not melt correctly even after repeated recrystallizations from water. The urine is made acid to litmus and concentrated on the water bath to about 100 cc. After the removal of the sediment, the urine is made acid to Congo red, and extracted with ether in a continuous extractor until the urine no longer gives a purple color with ferric chloride. After removing the ether by distillation, the gummy or crystalline residue is leached twice with hot toluene and once with chloroform. The crude product is dissolved in a small volume of boiling water, treated with decolorizing charcoal, filtered, and allowed to crystallize. Usually one recrystallization is sufficient to yield a colorless product but an additional simple extraction or washing with chloroform to remove traces of free salicylic acid may be necessary to obtain a pure compound. A yield of about 0.5 gm. was obtained. In one experiment, 0.61 gm. was isolated from a 24 hour specimen of urine which from analysis was known to contain 0.96 gm. of salicyluric acid.

The melting point was found to be 167”, corrected. The accuracy of the thermometer was tested by determining the melt- ing point of pure salicylic acid. The present finding agrees with the melting point of the synthetic salicyluric acid as recorded by both Hanzlik and Holmes. Sa.licyluric acid crystallizes in the form of needle crystals which tend to be grouped in rosettes. Microscopically, the crystals differ from those of salicylic acid in being more lanceolated, while those of the latter compound have square ends. Salicyluric acid is practically insoluble in chloroform and toluene. It is very soluble in hot water and in cold water it is about four times as soluble as hippuric acid. The dry compound easily becomes electrically charged, a property also noted by Bondi (17) for the synthetic product. The compound gives a purple color with ferric chloride, and after hydrolysis gives a positive ninhydrin reaction. Salicyluric acid, like p-hydroxyhippuric acid, unites with 2 atoms of bromine, and like the para isomer, it gives on titration with standard sodium hydroxide a higher titration than the expected based on the monobasic acid. This indicates that the hydrogen of the hydroxy group of salicyluric acid is more acidic, i.e. ionized, than it is in salicylic acid. Bertag-

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478 Salicylic Acid

nini stated that he suspected that salicyluric acid is dibasic and Bondi also noted this property in the synthetically prepared compound. This is another illustration of how a change in one substituted group attached to the benzene ring can affect a second group.

Analysis-Titration with 0.1 N sodium hydroxide: sample, 0.1 gm.; found, 6.4 cc.; calculated (for monobasic acid), 5.15 cc.

Bromination: sample, 0.1 gm.; found, 0.159 gm. bromine; calculated, 0.164 gm.

Glycine: sample, 0.1 gm.; found, 0.0352 gm.; calculated, 0.0384 gm.

Determination of Acetylsalicylic Acid-A simple procedure for determining acetylsalicylic acid has been developed on the basis that this compound will not combine with bromine, whereas free salicylic acid, which is liberated on hydrolysis, will take up 3 atoms of bromine. Therefore, by determining the amount of bromine consumed before and after hydrolysis, one can calculate the quantity of acetylsalicylic acid. If salicyluric acid is present, a correction must be made since this compound takes up 2 atoms of bromine, but is also hydrolyzed. For the determination of acetylsalicylic acid in urine, 10 cc. samples are extracted, after the urine has been acidified, with ether in a continuous extractor. One sample is analyzed by the bromination met)hod directly, care being taken to keep the solution cold and allowing only 15 minutes for the reaction. A second sample is hydrolyzed by refluxing with 5 per cent sodium hydroxide before applying the bromination method. From the difference in the two titrations, the amount of salicylic acid combined with acetic acid can be calculated. A small fraction of the acetylsalicylic acid will be hydrolyzed during the determination, but the amount is surprisingly small and can be adequately corrected by a blank obtained by making a deter- mination with pure acetylsalicylic acid.

DISCUSSION

The human organism can synthesize salicyluric acid but the amount excreted is very small and is not markedly influenced by exogenous glycine, as can be seen in Table I. In agreement with Holmes, a definite ratio of free salicylic acid to salicyluric acid seems to exist, but this ratio varies with the dose of sodium

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A. J. Quick

salicylate administered since the amount of uncombined salicylic acid increases more with increasing doses of the drug than the output of salicyluric acid. The excretion of salicylic acid is not rapid, contrary to the statement found in some text-books of pharmacology. Whereas the excretion of hippuric acid following

TABLE I

Conjugation of Xalicylic Acid in Man After Varying Doses of Sodium Salicylate

Subject &.; weight, 52 kilos.

Time

l.Ogm.

Excreted

Salicylic acid ingested

2.0 gm.

Excreted

3.5 gm.

Excreted

As sali- As sali- As sali- Free* cylurie Total Free &ic Total Free cylurio Total

acidt acid ____- ~____---

hrs. gm. gm. gm. gm. gm. gm. gm. om. om.

1 0.007 0.020 0.027 0.004 0.023 0.027 0.023 0.027 0.050 2 0.014 0.029 0.043 0.016 0.026 0.042 0.125 0.035 0.160 3 0.016 0.029 0.045 0.036 0.029 0.065 0.159 0.031 0.190 4 0.022 0.021 0.043 0.033 0.033 0.066 0.098 0.029 0.127

25 0.006 0.008 0.014 0.008 0.021 0.029 0.002 0.039 0.041 ______~.~~~~~

Total in 24 hrs. 0.260 0.280 0.540 0.420 0.680 1.100 1.262 0.658 1.920

With 15 gm. gelatin

1 0.025 0.009 0.034 0.021 0.011 0.032 0.033 0.027 0.060 2 0.033 0.021 0.054 0.026 0.049 0.075 0.091 0.032 0.123 3 0.026 0.033 0.032 0.065 0.134 0.042 0.176 4 0.031 0.022 0.053 0.027 0.038 0.065 0.148 0.039 0.187

* Probably includes a small amount of salicylic acid conjugated with sulfuric acid and glucuronic acid.

t Corrected for a blank which was found experimentally to be equivalent to 0.020 gm. of salicylic acid per hour.

the ingestion of 2 gm. of benzoic acid is complete in 4 hours, the elimination of 2 gm. of salicylic acid is only 50 per cent complete in 24 hours.

It is probable that the importance of salicyluric acid has been overemphasized. It is certainly erroneous to conclude that the

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480 Salicylic Acid

pharmacological differences between benzoic acid and salicylic acid are due to the fact that the former is detoxicated readily with glycine while the latter is only to a very limited degree. Other ortho-substituted benzoic acids exhibit the same limited conjugation with glycine as does salicylic arid, yet they are therapeutically inactive. Thus, o-chlorobenzoic acid not only resembles salicylic acid rather closely in its physical properties such as solubility and ionization constant, but it is also excreted partly uncombined and is conjugated only to a very small extent with glycine. Nevertheless, it possesses none of the physiological actions of salicylic acid. In fact, while the latter stimulates the excretion of uric acid, the chloro compound actually depresses it.

Since other ortho-substituted benzoic acids suppress the excre- tion of uric acid, the specific activity of salicylic acid must reside in the hydroxy group. That the activity of salicylic acid is not primarily dependent upon the inability of the organism to conjugate it readily with glycine is well illustrated by the fact that exogenous glycine, which tends to augment the formation of salicyluric acid, actually increases the physiological action of salicylic acid as measured by uric acid excretion. Although glycine alone in relatively large amounts will increase the output of uric acid, it seems rather certain that the synergistic action is not the result of a simple summation. Thus, while 5 gm. of glycine have practically no effect on the excretion of uric acid, 1.5 gm. when given with 2 gm. of salicylic acid will produce a striking augmentation. An answer for this somewhat paradoxical in- fluence of glycine can perhaps be found in the author’s previous studies (18, 19). It was found that while aromatic acids such as beneoic acid and substituted benzoic acids diminished the hourly output of uric acid, excess glycine significantly prevented, at least partially, this depressing effect. It seems, therefore, probable that salicylic acid in common with the other substituted beneoic acids may possess a masked inhibitory effect on uric acid excretion besides its predominant stimulatory action. When, therefore, the inhibitory factor is counteracted by glycine, the resulting stimulating effect becomes definitely more pronounced as is shown in Table II.

While glycine intensifies the action of salicylic acid, it also tends to shorten the period of stimulation. Thus, it was found that

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TABL

E II

Effe

ct

of Sa

licyli

c Ac

id

and

of Ac

etyls

alicy

lic

Acid

on

Ur

ic Ac

id

Excre

tion.

Syne

rgist

ic Ac

tion

of

Glyc

ine

Tim

e Ur

ic ac

id ex

cret

ed

hrs.

1 2 3 4 5

- 1 wl

.

23.0

23

.2

22.1

20

.5

w.

w.

29.3

21

.4

33.1

23

.4

29.2

21

.0

17.5

mu.

m

g.

mJ.

18.7

19

.5

34.3

26

.0

22.1

24

.5

23.0

22

.8

22.4

-- _-

WJ.

7ng.

m

l. w

. w

. m

g.

m&7

.

27.2

30

.0

25.5

30

.6

47.0

46

.0

43.3

36

.0

48.1

41

.0

41.0

49

.9

56.5

56

.0

38.3

35

.0

34.5

40

.0

39.6

50

.5

49.1

31

.8

18.0

33

.8

42.0

23

.0

23.1

48

.2

__-

~~_~

To

tal

in24

hrs..

...

k80.0

I

I lb

0 I

I I

ml.

35.2

58

.5

45.5

44

.0

__

.?+

mg.

57.7

5

85.6

a

77.0

fi.

68

.5

is

‘57.0

* 0.

008

gm.

of

salic

ylic

acid

as

ace

tylsa

licyli

c ac

id

excr

eted

du

ring

the

1st

hour

. t

0.01

4 gm

. of

sa

licyli

c ac

id

as a

cety

lsalic

ylic

acid

ex

cret

ed

durin

g th

e 1s

t ho

ur.

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Salicylic Acid

when 1 gm. of salicylic acid was given by mouth, the stimulation lasted approximately 4 hours, whereas, if glycine was supplied, the uric acid output returned to normal in 3 hours. It seems that glycine accelerates the excretion of salicylic acid sufficiently to bring about a quicker reduction of the concentration of the drug to the level at which it is no longer effective. No obvious explana- tion can be offered for the finding that salicylic acid stimulates the output of uric acid only after a certain concentration of the drug is present in the organism and that stimulation immediately ceases after the concentration falls below this crucial level. In repeated experiments the author has found that while 0.5 gm. of salicylic acid will produce no increase in the hourly out,put of uric acid, a 1.0 gm. dose will cause a very marked augmentation. A con- centration of about 0.8 gm. seems to be required in the subject studied to bring about an increase in the excretion of uric acid. On giving 0.6 gm. intravenously, however, a stimulation was observed during the 1st hour, but although only 0.02 gm. of salicylic acid was excreted during that period, the excretion of uric acid dropped to normal during the 2nd hour. In this case the loss of stimulation seems to have been brought about by a redis- tribution of the drug in the body. It should be observed that the level of salicylic acid needed to affect uric acid excretion is dis- tinctly higher than that required for mild analgesia, but much lower than the concentration necessary to bring about relief in acute rheumatic fever. Salicylic acid appears to act specifically on uric acid; it does not affect to any marked degree creatinine, urea, chlorides, or the other common constituents of urine. The excretion of uric acid is apparently independent of the volume of the urine, and frequently a diminished urine volume was observed when the elimination of uric acid was high. A con- centration of 250 mg. of uric acid per 100 cc. of urine was found on several occasions.

Although much work remains to be done before the mechanism involved in the excretion of uric acid can be explained, the fact remains that the synergistic action between glycine and salicylic acid should find definite therapeutic applications. It will be interesting to find whet#her glycine will aid salicylic acid in stim- ulating the excretion of uric acid in cases of pathological retention. Perhaps glycine may even augment the therapeutic action of salicylic acid in rheumatic fever.

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A. J. Quick

The results obtained on acetylsalicylic acid are interesting, for 1.5 gm., which is equivalent to 1.15 gm. of salicylic acid, produced practically the same stimulation on uric acid excretion as when 1 gm. of salicylic acid was fed, while 0.75 gm., equivalent to 0.57 gm. of salicylic acid, caused no stimulation. Since the author has previously shown that the replacement of the hydrogen of the hydroxy group in salicylic acid, as illustrated by o-methoxybenzoic acid, abolishes the physiological activity, one must assume that the st,imulatory action of acetylsalicylic acid is due rather to the liberated salicylic acid than to the intact compound. Acetyl- salicylic acid apparently undergoes rapid hydrolysis in the body; nevertheless, some of the compound enters the general circulation as proved by the fact that the compound can be detected in the urine during the 1st hour following its ingestion. This finding was also made by Hanzlik and Presho (20) who administered much larger doses than were employed in this study.

In attempting to explain the physiological action of salicylic acid on the basis of its chemical constitution, it should be noted that the compound possesses two active chemical radicals attached to a benzene ring. If it be permitted to borrow from immunology, one can look upon these as haptophore-like groups. It is probable that the carboxyl group, since it unites with glycine, can also unite with the amino group of a larger aggregate of amino acids, possibly with a protein. It has been noted by Vinci (21) that salicylic acid shows an elective action on the element of the blood with which it undergoes a relatively strong union. The hydroxy group of salicylic acid, on the other hand, can combine with glucu- ronic acid. Thus the possibility of a glucuronic acid-salicylic acid-protein compound must at least be considered in attempting to elucidate the physiological action of salicylic acid. Glucuronic acid combined with salicylic acid should act as a hapten very much like the glucoside and galactoside of p-aminophenol which Goebel and Avery (22) conjugated artificially with a protein and thereby produced specific antigens. It is well known that salicylic acid and, more commonly, acetylsalicylic acid can cause symptoms such as urticaria and edema which are typically allergic. Due probably to the small molecular weight of the theoretical salicylic acid hapten group, such actions are relatively infrequent. With cinchophen these reactions are more common, and from the recent work of Davis (23) it appears that an individual can become

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484 Salicylic Acid

sensitized to the drug and remain sensitive for over a year and probably longer. As the writer (19) has previously pointed out, experimental findings furnish strong indication t,hat cinchophen in the body is oxidized to a hydroxy derivative; moreover, the position of the carboxyl group which is on the a-carbon atom of the quinoline ring produces inhibition of the glycine conjugation similar to ortho substitution. Thus, cinchophen appears to have in common with salicylic acid, an acquired hydroxy group and a carboxyl group so situated that conjugation with glycine is in- hibited. Since the molecule of cinchophen is larger and more complex, it should give rise to a more active hapten. This seems t,o be borne out clinically since urticaria and other allergic reactions from cinchophen are relatively common.

SUMMARY

1. Pure salicylurie acid was isolated from human urine. 2. A method for the det,ermination of acetylsalicylic acid in

urine is described. 3. The rate of salicylic acid excretion is dependent upon the

concentration of the drug in the body. With increasing doses of salicylic acid, the excretion of free salicylic acid becomes definitely greater, while the output of salicyluric acid is only slightly affected. Exogenous glycine has little influence on the excretion of salicyluric acid.

4. The stimulatory effect of salicylic acid on uric acid excretion appears to be dependent upon a fixed concentration of the drug in the body. Below this crucial concentration, salicylic acid has no apparent influence on the output of uric acid.

5. The action of salicylic acid on uric acid elimination is strik- ingly augmented by glycine or foods rich in glycine. An explana- tion for this finding is offered, and the possible therapeutic applica- tions of this synergism between glycine and salicylic acid are pointed out.

6. A theory based on the possibility that salicylic acid con- jugated with glucuronic acid may act as a hapten is proposed as a possible explanation for some of the physiological properties of the salicylates.

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A. J. Quick 485

BIBLIOGRAPHY

1. Bertagnini, C., Ann. Chem., 97,248 (1856). 2. Nencki, M., Arch. Anat. u. Physiol., 399 (1870). 3. Lesnik, M., Arch. Path. u. Pharmakol., 24, 171 (1888). ezp. 4. MOSSO, U., Arch. Path. u. Pharmakol., 26, 267 (1889-90). ezp. 5. Bass, H. K., 2. physiol. Chem., 14, 416 (1890). 6. Baldoni, A., Arch.furmucoZ. 18, 151 (1915). sper., 7. Stockman, R., Edinburgh Med. J., 20,104 (1906). 8. Hanzlik, P. J., J. Phurmucol. and Exp. Therup., 10, 461 (1917-18). 9. Holmes, E. G., J. PhuwmacoZ. and Exp. Therap., 26,297 (1925).

10. Thoburn, T. W., and Hanzlik, P. J., J. BioZ. Chem., 23, 163 (1915). 11. Johnson, C., J. Pharnaacol. and Exp. Therup., 34, 437 (1928). 12. Quick, A. J., J. BioZ. Chem., 96, 83 (1932). 13. Quick, A. J., J. BioZ. Chem., 67,477 (1926). 14. Day, A. R., and Taggard, W. T., Ind. and Eng. Chem., 20,545 (1928). 15. Quick, A. J., J. BioZ. Chem., 97,403 (1932). 16. Benedict, S. R., and Hitchcock, E. H., J. Biol. Chem., 20, 619 (1915). 17. Bondi, S., 2. physiol. Chem., 62, 170 (1907). 18. Quick, A. J., J. BioZ. Chem., 92, 65 (1931). 19. Quick, A. J., J. BioZ. Chem., 98, 157 (1932). 20. Hanzlik, P. J., and Presho, E., J. Pharmacol. and Exp. Therap., 21,

247 (1923). 21. Vinci, G., Arch. furmacol. sper., 3,294 (1904). 22. Goebel, W. F., and Avery, 0. T., J. Exp. Med., 60, 533 (1929). 23. Davis, J. S., Am. J. Med. SC., 184,555 (1932).

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Armand J. QuickURIC ACID EXCRETION

WITH GLYCINE AND ITS ACTION ONCONJUGATION OF SALICYLIC ACID

PHYSIOLOGICAL RESPONSE: IV.CHEMICAL STRUCTURE AND

THE RELATIONSHIP BETWEEN

1933, 101:475-485.J. Biol. Chem. 

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