THE METABOLISM OF p-AMINOSALICYLIC ACID IN THE ORGANISM OF THE RABBIT

BY ALAMELA VENKATARAMAN,* POONAMALLE R. VENKATARAMAN,t AND HOWARD B. LEWIS

(From the Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor)

(Received for publication, December 27, 1947)

The importance of p-aminosalicylic acid in the treatment of tuberculosis has been demonstrated both clinically and experimentally (l-6). The close structural relationships between benzoic, salicylic, p-aminobenzoic (PABA), and p-aminosalicylic (PASA) acid warrant a comparative study of the metabolism of these substances in the animal body.* Recent workers, interested in the behavior of PABA in man, the dog, and the rabbit, have concerned themselves chiefly with the acetylation of this compound, with little consideration for other possible metabolic reactions such as conjugation with glycine to form the conjugate, p-aminohippuric acid. It is of interest to note that in vitro synthesis of this latter compound from PABA by homogenates and tissue liver slices has been demonstrated recently (8-10). In a comprehensive study of the fate of salicylic acid in man (ll), 80 per cent of a single dose was excreted by the kidneys; of this 20 per cent was unchanged, 55 and 25 per cent in conjugation with glycine (as salicyluric acid) and glucuronic acid respectively, and a small amount (4 to 8 per cent) as gentisic acid and other products of oxidation. In children and in febrile patients, oxidation of salicylic acid was increased, while, in fever, the conjugation to form salicyluric acid was diminished. Lutn-ak-Mann (12) isolated gentisic acid from the urine of rats to which salicylic and acetylsalicylic acids had been fed, and observed that when the animals were poisoned with carbon tetrachloride or yellow phosphorus the formation of gentisic acid was decreased. Detailed studies of the fate of salicylic acid in the rabbit are not available. In general, however, as pointed out by Quick (7), the conjugation of glycine with an aromatic carboxyl group is markedly inhibited by a substituent group in the ortho position as in salicylic acid. Systematic studies of the metabolism of salicylic acid and related compounds with more specific methods for the determination of products of metabolism are needed. It has seemed,

* Government of India Scholar. t Scholar of the Watumull Foundation of Los Angeles. 1 For a review of older work on the metabolism of substituted benzoic acids, the

studies of Quick (7) should be consult,ed. 641

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642 P-AMINOSALICYLIC ACID

therefore, desirable to have information as to the biological behavior of PASA in as many animals as possible in view of the marked species varia- tions known with other aromatic acids, e.g., benzoic acid in man, the dog, or the rabbit. As an initial contribution to this problem, we present here the results of an investigation of the metabolism of PASA in the rabbit.

EXPERIMENTAL

Collection of Specimefas-Male rabbits, 2 to 4 kilos in weight, were main- tained on a diet of cabbage and oats. The procedures of caging and the collection of the urine were those usually employed in this laboratory (13). Decomposition was prevented by the addition of 10 ml. of 1 per cent nitric acid to the collection bottle. The PASA was administered orally through a stomach tube either as a solution of the sodium salt or as the acid in fine suspension in water, or was injected intramuscularly as a freshly prepared solution of the sodium salt.

Compounds Fed; Stability-The PASA was crystallized from absolute alcohol and dried over phosphorus pentoxide. The crystalline white material melted at 150” (decomposition) and contained 9.09 per cent of nitrogen (theoretical, 9.15 per cent) on analysis (semimicro-Kjeldahl). A 10 mg. per cent solution of the acid had an initial pH of 3.2, which changed to 4.2 on standing at room temperature for 48 hours and to 4.7 in a week; after being heated on the steam bath for 1 hour, the pH of the solution was 5.4. This change in reaction is believed to be due to a slow decarboxyla- tion of the compound in aqueous solution with the formation of m-amino- phenol. As shown by the iron reaction (to be discussed subsequently), the decarboxylations in 24 and 48.hours were 41 and 54 per cent respec- tively. When the material was dissolved in 4 per cent toluenesulfonic acid (pH l.O), only 18 per cent decarboxylation occurred at room temperature in 24 hours, although this reaction was complete after heating the solution on the steam bath for 1 hour.

Crystallized PASA was suspended in 95 per cent alcohol and the mixture was carefully neutralized with alcoholic sodium hydroxide until a definite pink color with phenolphthalein was obtained. This solution of sodium salt was poured slowly into cold ether, with shaking. The precipitated sodium salt was filtered, washed with cold ether, and dried. The salt ap- peared to form a loose molecular compound wit,h ether but the solvent could be removed by continuous evacua,tion at 0.5 mm. pressure over phosphorus pentoxide until the weight became constant. A sample thus dried gave a loss of 14.31 per cent. The dried sample contained 7.48 per cent of nitrogen (theoretical 7.11 per cent). In all cases in which the sodium salt was administered orally to the rabbits, the amount adminis- tered is calculated as the dried salt. The sodium salt was very soluble

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VENHATARAbUN, VENKATARAbUN, AND LEWIS 643

in water. A 100 mg. per cent aqueous solution of the sodium salt (pH approximately 6.8) appeared to be stable even at a pH of 9.0 and no de- carboxylation (as was evidenced by the iron reaction) occurred when the solution was heated on the steam bath for 1 hour. There was slight dis- coloration in concentrated solutions on exposure to light, but samples of the dry sodium salt have been kept for several months without deteriora- tion.

Methods of Analysis-Two color reactions have been employed to follow the metabolism of PASA. Compounds containing the orthohydroxy- benzoyl group give an amethyst color with ferric salts in an acid medium, due to the formation of an iron complex. The quantitative aspect of this test has been the subject of a careful study by Nicholls (14) and Brodie and coworkers (15). In I’UA, the color formation should not take place if the OH group has been blocked by substitution or if decarboxylation of the COOH group has occurred. 2-Hydroxy-4-aminohippuric acid (the PASA analogue of salicyluric acid) also gives a similar color, but of different intensity.

The free arylamino group is characterized by the lemon yellow color formed with p-dimethylaminobenzaldehyde (Ehrlich’s reagent). This re- action has been applied to the determination of sulfanilamide derivatives in plasma and urine (16-19). Morris (20) has shown that, for the success- ful application of the p-dimethylaminobenzaldehyde reaction to the estima- tion of sulfanilamides, the test must be carried out in acid media (pH 1.5 to 1.7) and in the presence of an excess of the aldehyde reagent. Both of these conditions are met in our procedures by carrying out the reaction at a pH of 2.0 to 2.2 and adding a constant amount of Ehrlich’s reagent buffered with a mixture of sodium acetate and acetic acid. N-Acetylated PASA did not give this reaction and could be estimated after hydrolysis with acid as m-aminophenol.

Reagents- 1. 20 per cent p-toluenesulfonic acid (TSA). 200 gm. of TSA mono-

hydrate (21, 22) were dissolved in 600 ml. of water, stirred with a small amount of norit K for 10 minutes, filtered, and the fihrate made up to 1 liter. 2 volumes of this stock solution were diluted with 3 volumes of water to give 8 per cent TSA.

2. Iron reagent. A 1.68 per cent solution of Fe(N0&.9Hz0 (Merck) in 0.07 N nitric acid.

3. Ehrlich’s reagent. 2 gm. of p-dimethylaminobenzaldehyde (East- man) were dissolved in 100 ml. of glacial acetic acid and added to an equal volume of a 4 M solution of anhydrous sodium acetate. When 1 ml. of this reagent was added to 10 ml. of 4 per cent TSA, the pH was 2.0 to 2.2.

Measurements of Color-All measurements of color intensity were made

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644 ~AhU.NOfULICYLIC ACID

with a Coleman Universal spectrophotometer (model 11). The blank was set at 100 per cent transmission and matched tubes were used for containers.

Standard K Values-A sample of PASA, twice crystallized from absolute alcohol and dried over phosphorus pentoxide, was used to prepare a stand- ard stock solution containing 1 mg. per ml. Since PASA is not freely soluble in water and is stable in aqueous solution only as the sodium salt, the acid (100 mg.) was neutralized with 1 per cent sodium carbonate solu- tion and made up to 100 ml. For the iron reaction, a lo-fold dilution of the stock solution was made. 1 to 8 ml. samples were added to tubes containing 2 ml. of 20 per cent TSA and the volume was made up to 10 ml., so that the final concentration of TSA was 4 per cent. 2 ml. of the iron reagent were added to each tube and the color was read at 500 rnp with a water blank similarly treated. There seemed to be no delay in the develop- ment of the color, nor any fading within a reasonable length of time. How- ever, the readings were taken between 5 and 20 minutes. The average K value for the concentrations of from 0.1 to 0.8 mg. was used for the calcula- tions of the unknowns.

For the Ehrlich reaction, the stock solution was so diluted that 1 ml. of the diluted solution contained 0.005 mg. of PASA. The procedure as outlined above was followed, except that 1 ml. of Ehrlich’s reagent was used to develop the color, which was read at 450 mp. Here also the color developed immediately and was quite stable; readings were usually taken between 5 and 20 minutes. The average K value obtained over the con- centration range of 0.005 to 0.040 mg. was used for calculations.

Similarly the average K value (Ehrlich) for m-aminophenol was obtained for a range of 0.005 to 0.040 mg. to calculate the m-aminophenol (MAP) produced after hydrolysis. As would be anticipated, this value agrees very well with that obtained directly from known amounts of PASA after hydrolysis in 4 per cent TSA.

Iron Reaction-To 5 ml. of urine so diluted as to contain 0.2 to 1.2 mg. of PASA, 5 ml. of 8 per cent TSA were added, followed by 2 ml. of the iron reagent. The pink color was read at 500 rnp with a similarly treated nor- mal urine blank for 100 per cent transmission.

Ehrlich’s Z&action-(a) The free arylamino group n-as estimated by adding 5 ml. of 8 per cent TSA to 5 ml. of a suitably diluted urine sample, followed by 1 ml. of Ehrlich’s reagent. The color was read at 450 rnp with a similarly treated normal urine blank for 100 per cent transmission. (b) For the determination of total arylamino groups, 5 ml. of the suitably diluted urine were hydrolyzed with 5 ml. of 8 per cent TSA in a loosely stoppered graduated tube in a boiling water bath for 1 hour. The tube was then cooled, the level of the fluid again brought to the mark as neces- sary, and the color developed with 1 ml. of Ehrlich’s reagent. A similarly

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VENKATARAMAN, VENKATARAMAN, AND LEWIS 645

treated normal urine was used as the blank and the color was read at 450 rnp. Since PASA, under the above conditions, is completely decarboxy- lated to MAP, the results calculated as MAP were converted into PASA by multiplying by the factor 153/109. In the above estimations, the 24 hour sample of urine previous to the administration of PASA was suitably diluted to give suitable blanks to correspond to 6 and 18 hour specimens of normal urine.

Glucuronic acid was estimated by the modification of the Maughan, Evelyn, and Browne method (13) commonly used in this laboratory, and creatinine and urinary sulfur partition by the methods of Folin. PASA did not interfere with the estimations of glucuronic acid or creatinine.

DISCUSSION

150 mg. daily of PASA (as the sodium salt) per kilo of body weight could be administ,ered orally to male rats for 2 weeks without signs of toxicity or impairment of growth. To determine whether any significant fraction of the dosage escaped absorption, the feces were analyzed for PASA in two experiments with rabbits. The amount found was so small, less than 5 mg. in a 24 hour feces, that it was thought that the routine analysis of the feces was not necessary.

In Table I, the results of urinary excretion of PASA and derivatives based on the Ehrlich reaction are summarized. 53 to 80 per cent of the compound ingested orally could be accounted for in the urine. On the other hand, when the compound was administered by intramuscular injec- tion, there was a greater recovery, about 90 per cent, as indicated by the Ehrlich reaction. In either case, 70 to 90 per cent of the total amount of PASA recovered was excreted in the first 6 hours, and during this period 55 to 80 per cent of the amount excreted was in the acetylated form. The lower levels of acetylation, about 40 per cent in the 6 hour period, when the compound was administered parenterally, may be due to the rapidity of excretion. The rapid elimination of PASA makes it improbable that the fraction not recovered could be stored in the tissues. It has been believed that a certain amount of salicylic acid may be destroyed in the organism (23). This is in contrast to the unsubstituted compound, benzoic acid, which may be almost completely accounted for in the urine after administration.

Urine was collected from four rabbits, each fed 1 gm. of PASA on 2 successive days. The sample was treated with neutral lead acetate. The precipitate was centrifuged and the centrifugate was made just alkaline with ammonium hydroxide and treated with basic lead acetate. The precipitate was centrifuged and was decomposed by treatment with hydro- gen sulfide. The lead sulfide was filtered, washed, and the filtrate and the

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646 P-AMINOSALICYLIC ACID

washings, after aeration, were kept in the refrigerator for 72 hours. The crystalline white solid (400 mg.) was filtered, washed with a small quantity of ice-cold water, and dried in the desiccator. The acetyl PASA thus ob- tained melted at 212” with decomposition. The mother liquor was ex- tracted four times with 200 ml. portions of ether and from the ether extract 1 gm. of an impure white solid was obtained. On washing this with small quantities of ether, 150 mg. of the sparingly soluble acetyl compound re- mained on the filter. The ether-soluble fraction (800 mg.) after crystalli-

TABLE I Urinary Excretion of PASA and Derivatives by Rabbits (Ehrlich Reaction)

The sodium salt used for oral administration was prepared as described in the text. For injection, the sodium salt was prepared freshly by the neutralization of the PASA with the theoretical amount of sodium bicarbonate.

PASA adminie

tered

%.

397 397 502 362 330 571 796 750 580 740

Method of administration

Salt, oral “ “ “ I‘ ,‘ “ ‘I “

Acid, “ “ ‘I ‘I “

Salt, intramuscular “ “

PASAexcreted

6 hrs. 24 hrs.

-

Jnchanged 1 Lcetylated T Jnchanged

“v;;;,” Py$$ Py$yf

21 42 22 25 34 26 16 45 18 11 27 13 9 39 10

20 25 25 14 27 17 12 22 17 46 28 48 47 32 49

A - 1

icetylated

ICI cent inlake

of

58 48 62 40 53 47 36 42 39 42

_

PASA acety- Wed, of total

excreted

6 hrs. !4 hrs.

- m

cent

67 58 74 70 82 56 65 65 38 40

Per cm1

72 65 77 75 84 65 68 71 45 46

zation from absolute alcohol was identified as PASA. The acetyl com- pound was purified by solution in dilute sodium bicarbonate solution (charcoal) and precipitation with dilute hydrochloric acid. The sample thus obtained was filtered, washed with cold water, dried first in a desic- cator, and finally in an oven at 60”. The isolated compound and a pure sample of acetyl PASA were heated in a bath whose temperature was rising slowly. They melted at 212” and 214“ respectively with decomposition.2

2 According to a private communication, Mr. Leonard Doub has observed a decom- position point of 233-234’ (uncorrected) in a bath when the temperature was increased rapidly. The decomposition point was not precise and depended on the rate of heat- ing.

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VENKATARAMAN, VENKATARAMAN, AND LEWIS 647

A mixed sample of the two showed no depression in the melting point. Found, nitrogen 7.23 per cent; theory, nitrogen 7.19 per cent. The iso- lated acetyl derivative was also analyzed by the color reaction with the iron reagent to give 98.5 per cent purity and after hydrolysis with 4 per cent TSA for 1 hour under the usual conditions, as m-aminophenol, 98 per cent, by the Ehrlich reaction.

Since in most experiments the PASA was administered orally, an at- tempt was made to demonstrate whether the contents of the stomach or the intestines would decompose the compound in vitro. The stomach and the small intestines of rats and rabbits were removed separately and the

TABLE II Incubation of PASA with Homogenized Gastric and Intestinal

Contents of Rabbit and Rat In all experiments except Experiment 19-9, the contents were incubated with

finely divided PASA; in Experiment 19-9 the acid was dissolved in 4 ml. of 5 per cent sodium bicarbonate.

Experiment No. OrgaIl

19-9 (rabbit). . . Stomach Intestine

20-10 (rabbit). Stomach

21-11 (rat). . . ~ Intestine Stomach

! Intestine I

Time 31 incu bation

win. w. 90 104

210 101 90 108

210 101 90 32

210 50

PASA

PASA recovered

mat- tion Free

(0) -- per cen1 per mni

97 84 103 80

96 70 97 79 94 81 92 86

---L

Con- Tota1 jugated

@I (b - a) -- per cen1 fier cm;

98 14 98 18 92 22 96 17 91 10 94 8

contents were homogenized with water in a Waring blendor. PASA or its sodium salt was incubated with the emulsion for a definite period, and the mixture was then treated with a sufficient quantity of 20 per cent TSA to make a final concentration of 4 per cent TSA and filtered. The filtrate, centrifuged as necessary, was analyzed by the iron and the Ehrlich reac- tions. The results were also checked by estimating by the Ehrlich reaction the m-aminophenol formed after heating the filtrate for 1 hour on the steam bath. All necessary dilutions were made in 4 per cent TSA. A control experiment was run in each case without PASA, which served as a blank in the estimations. The results are summarized in Table II.

One might have expected the decarboxylation of PAM, associated with the acidity available in the stomach and the activity of the intestinal flora.

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648 P-AMINOSALICTLIC lCID

However, the results of the incubation of PASA with gastric and intestinal contents of the rabbit and the rat definitely indicated that there was no decarboxylation in z&o, since essentially all the material originally added was recovered by the iron reaction. The only change detected was a small amount of acetylation, 14 to 23 per cent in the case of the rabbit and 6 to 10 per cent with the rat. This needs further investigation.

The earlier experiments indicated conjugation with acetic acid to give the N-acetyl derivative. However, PASA might be conjugated in the animal body in more than one way, as there are three groups in the molecule which might be used in such reactions. Owing to the ease with which the compound is decarboxylated in the test-tube, it might have been expected that this reaction would take place in the animal body. If this were the case to any considerable extent, the resulting m-aminophenol should be acetylated or possibly conjugated with glucuronic acid or sulfuric acid (24). One might also observe the int.roduction of a hydroxyl group in the mole- cule of PASA in viva, either by direct oxidation of the compound and its acetyl derivative or by a formation and rearrangement of the hydroxy- amino derivative,3 to form 2,5-dihydroxy-4-amino- or acetylaminobenzoic acid. These compounds might be conjugated with glucuronic or sulfuric acid, particularly at the hydroxyl group in the 5 position.

Studies of urinary sulfur after feeding PASA indicated practically no increase in ethereal sulfate. No uniformity is displayed in the excretion of extra glucuronic acid after the administration of PASA. From experi- ence in this laboratory, we consider that, with rabbits, any value for extra glucuronic acid less than 70 mg. either in the 6 hour or 18 hour period is of little significance. In only two of ten experiments was the extra glu- curonic acid in the 6 hour period, the period of rapid excretion of PASA, greater than 70 mg., values of 75 and 103 mg. being obtained. On this basis we are inclined to believe that the amounts of extra glucuronic acid after the administration of PASA orally or parenterally are not indicative of any significant conjugation.

It may, therefore, be assumed that there is neither considerable decar- boxylation of PASA nor introduction of a new hydroxyl group in viva, and the amounts of free and combined derivatives in the urine as estimated by the Ehrlich reaction represent primarily free and acetylated PASA.

The question of the conjugation of glycine with the carboxyl group of PAS-4 may be discussed. 2-Hydroxy-4-aminohippuric acid gives a color with the iron reagent similar to those with PASA and acetyl PASA; how- ever, it is not decomposed completely to m-aminophenol under the condi- tions used for hydrolysis, but still gives considerable color with the iron

3 Williams (25) has discussed the possible formation of p-hydroxylaminobenxene- sulfonamide in metabolism. A number of hydroxy derivatives of the sulfonamides have been isolated and characterized in studies both in vivo and in vitro (25-27).

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VENRATARAMAN, VEPiXATARAMAX, AND LETVIS 649

reagent after such a procedure. The experimental urine did not give any color with the iron reagent after hydrolysis, indicating the absence of the glycine conjugate. This is in accordance with the belief that there is little or no conjugation of glycine with ortho substituted benzoic acids with the exception of o-chlorobenzoic acid (7). It may be noted, however, that a considerable proportion of salicylic acid, an orthohydroxyl derivative of benzoic acid, when fed to man, is reported to be excreted in conjugation with glycine (11).

The color observed in the iron reaction with the experimental urine should be due to the presence of both the free and acetylated PASA. It may be pointed out that, since the two compounds give different intensities of color with the iron reagent, the two K values being 0.524 and 0.264 re- spectively under our experimental conditions, the evaluation of the ob- served optical activity in terms of PASA alone might suggest considerable decarboxylation of the compound in viva. Therefore, the values for PASA and acetylated PASA obtained by the Ehrlich reaction with the experi- mental urines have been used to calculate the expected individual optical densities in the iron reaction. The difference between the observed optical density and the sum of the calculated optical densities of the two forms of PASA present might well be due to the presence of small amounts of other metabolites.

The presence of m-aminophenol and its N-acetyl derivative, which do not react with the iron reagent, would give negative values in such a cal- culation, while the presence of fl-resorcylic acid, which reacts with the iron reagent, would give positive values. The difference (“unknown”) has been somewhat arbitrarily expressed as PASA for the calculation of the net results of the iron reaction (Column 3). The values calculated in this manner are presented in Table III.

It may be noted that the amounts of unknown metabolite thus calcu- lated as differences represent a relatively small proportion of the total PASA (free and acetylated) excreted and that the values are both positive and negative. The data fail to offer any significant evidence of decarboxy- lation of PASA in the organism of the rabbit. This would be shown by consistent negative values. On the contrary, the differences, thus calcu- lated for the 6 hour period, the period in which maximal excretion of the PASA is occurring, are only slightly positive in six of the ten experiments recorded and none of the negative values represents any large proportion of the total amount excreted.

The algebraic sum of the amounts in Columns 1, 2, and 3 represents the total amount of PASA or derivatives excreted, as indicated by the iron reaction. The per cent of the total PASA administered which is repre- sented by this value is calculated in Column 4. This figure may be com- pared with the total PASA, excreted in the 6 hour period as obtained by

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650 p-AMINOSALICYLIC ACID

the Ehrlich reaction, which is shown in Column 5. The average total recoveries for the eight feeding experiments as calculated by the two methods are 45.3 and 48.6 per cent respectively. The differences between these average values are not significant.

We are indebted to Mr. Leonard Doub of Parke, Davis and Company for generous gifts of p-aminosalicylic acid and some of its derivatives and for the analysis of the sodium salt of PASA, and to Dr. A. C. Bratton, Jr., for suggestions relating to the calorimetric methods of analysis used in

TABLE III Interpretation of Iron Reaction in Terms of Excretions of PASA and

Derivatives As Determined bg Ehrlich Reaction

The calculations are for the 6 hour period immediately after the administration of PASA. The “unknown” metabolites (Column 3) are expressed in terms of PASA. Column 5 is included for comparison. For explanation of the calculation, the text should be consulted. The PASA administered can be obtained from the correspond- ing data of Table I.

Acetyl

(2)

m&T. mt.

82 165 98 136 82 228 41 98 29 129

112 145 113 214 88 162

268 165 351 239

-

_

!-

UIlkllOWIl

(3) -

m.

-42 -27 -34 +9

-29 +13

+5 +4

+17 +87

Sum of intake

(4)

Ehrlich reaction of intake

(5)

per ccst )C? cent

52 63 52 59 55 61 41 38 39 48 47 45 42 41 34 34 78 74 91 79

this study. In particular the interest and helpful criticism of Dr. Bratton are gratefully acknowledged.

SUMMARY

1. An aqueous solution of p-aminosalicylic acid (PASA) undergoes a gradual decarboxylation at room temperature with the formation of m- aminophenol. This is evidenced by the change in pH toward the alkaline side as well as by the decrease in the intensity of the reaction with ferric salts. The sodium salt, however, forms a stable solution when dissolved in water.

2. The metabolic fate of PASA in the rabbit has been studied. PASA

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VENKATARAMAN, VENKATARAMAN, AND LEWIS G51

is rapidly excreted in the urine in the unchanged and acetylated forms. From 40 to 60 per cent of the amount administered orally could be ac- counted for in 24 hours as the acetyl derivative. Both free and acetylated PASA have been isolated from the experimental urines.

3. PASA is absorbed almost completely from the gastrointestinal tract and there is no indication of decarboxylation in vitro in the presence of gastric or intestinal contents. No clear-cut evidence of any appreciable decarboxylation in viva has been obtained.

4. Data obtained for extra glucuronic acid do not suggest any significant conjugation as a glucuronide, while the possibility of conjugation with glycine appears remote. No increase in ethereal sulfate formation is indi- cated.

5. The chief mechanism of detoxication of PASA in the rabbit appears to be through acetylation.

BIBLIOGRAPHY

1. Lehmann, J., Lancet, 260, 15 (1946). 2. Lehmann, J., Svenska Liikarlidn., 43, 2029 (1946); Chem. Abstr., 41, 1334 (1947). 3. Sievers, O., Svenska Ltikartidn., 43, 2041 (1946); Chem. Abstr., 41, 1277 (1947). 4. Vallentin, G., Svenska Liikartidn., 43,2047 (1946); cited by Tobie, W. G., private

communication. 5. Youmans, G. P., Raleigh, G. W., and Youmans, A. S., J. Bact., 64, 409 (1947). 6. Alin, K., and Difs, H., Nord. med. Ark., 33, 151 (1947); cited by Tobie, W. G.,

private communication. 7. Quick, A. J., J. Biol. Chem., 66,83 (1932); 97,403 (1932). 8. Cohen, P. P., and McGilvery, R. W., J. Biol. &em., 166, 261 (1946). 9. Cohen, P. P., and McGilvery, R. W., J. Biol. Chem., 169, 119 (1947).

10. Cohen, P. P., and McGilvery, R. W., J. Biot. Chem., 171, 121 (1947). 11. Kapp, E. M., and Coburn, A. F., J. Biol. Chem., 146,549 (1942). 12. Lutwak-Mann, C., Biochem. J., 37,246 (1943). 13. Dziewiatkowski, D. D., and Lewis, H. B., J. BioZ. Chem., 168, 77 (1945). 14. Nicholls, J. R., Analyst, 63, 19 (1928). 15. Brodie, B. B., Udenfriend, A., and Coburn, A. F., J. Pharmacol. and Exp. Therap.,

80, 114 (1944). 16. Kimmig, Arch. Dermat. u. Syph., Vienna, 6, 176 (1938); quoted by Morris (20). 17. Ktihnau, W. W., Klin. Wochschr., 17, 116 (1938). 18. Werner, A. E. A., Lancet, 236, 18 (1939). 19. Andrews, M. C., and Strauss, A. F., J. Lab. and CZin. Med., 26,887 (1940-41). 20. Morris, C. J. O., Biochem. J., 36,952 (1941). 21. Marshall, E. K., Jr., J. BioZ. Chem., 122, 263 (1937-38). 22. Schmidt, E. G., J. Lab. and CZin. Med., 31, 694 (1946). 23. Hanzlik, P. J., Actions and uses of the salicylates and cinchophen in medicine,

Baltimore, 45 (1927). 24. Williams, R. T., Biochem. J., 32, 878 (1938). 25. Williams, R. T., Biochem. J., 40. 219 (1946). 26. Bray, H. G., Ryman, B. E., andThorpe, W. V., Biochem. J., 41,212 (1947). 27. Stubbs, A. L., and Williams, R. T., Biochem. J., 41, p. xlix (1947).

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Venkataraman and Howard B. LewisAlamela Venkataraman, Poonamalle R.

ORGANISM OF THE RABBIT-AMINOSALICYLIC ACID IN THE

pTHE METABOLISM OF

1948, 173:641-651.J. Biol. Chem. 

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