- Email: [email protected]
A glucuronide metabolite of sulfamethomidine in human urine
TOXICOLOGY
AND
APPLIED
A Glucuronide
FREDERICK
J.
DICARLO,
Biochemistry
5, 392-400
PHARMACOLOGY
Metabolite
of
in
Human
SYLVIA
G.
Department, Morris
(1963)
Sulfamethomidine
Urine
MALAMENT,
AND
GEORGE
Warner-Lambert Research Plains, New Jersey
Received
July
E.
PHILLIPS
Institute,
27, 1962
Previous reports on sulfamethomidine [Nl- (2-methyl-6-methoxy-4pyrimidinyl) sulfanilamide] dealt with its metabolism and binding by three animal species (DiCarlo et al., 1962) and by man (DiCarlo et al., 1963). The present investigation concerns the characterization of the sulfamethomidine metabolites in human urine. In this work, the BrattonMarshall sulfonamide assay procedure (1939) was employed to prepare pigmented derivatives of sulfamethomidine and related compounds. These colored compounds are easily detected on paper chromatograms and adsorption columns. Abbreviated terms such as “azo sulfamethomidine” refer to the purple compounds produced by diazotization of the indicated amine and subsequent coupling with N-( 1-napthyl)ethylenediamine. The abbreviation “SM urine” is employed to designate human urine collected after the ingestion of sulfamethomidine. METHODS
“Free” and Total Sulfamethomidine Assay Using a Coleman Junior spectrophotometer and selected round cuvettes (6-310A) in the analytical procedure of Bratton and Marshall (1939)) the azo pigment of sulfamethomidine gives an optical density of 0.084 for a I pg/ml solution. The expression “free sulfonamide” refers to metabolites which can be diazotized directly and coupled. “Total sulfonamide” covers N4-acetylated sulfonamide as well as “free sulfonamide,” and the assay method includes a preliminary hydrolysis to free the N4amino group for diazotization. Glucuronide Assay “Free” and conjugated glucuronic acid levels were determined by the method of Fishman and Green (1955). Using a Coleman Junior spec392
GLUCURONIDE
FROM
SULFAMETHOMIDINE
393
trophotometer at 565 rnp and selected round cuvettes (6-310A), standard curves were developed from glucuronic acid and 6-bromo-2-naphthyl-pglucuronide (Mann). A linear response was obtained up to an optical density of 0.600 from each reference compound. The optical density value corresponding to 1 ug of glucuronic acid per milliliter was 0.032. Paper Chromatography Three solvent systems were employed. Solvent A was prepared by mixing n-butyl alcohol, glacial acetic acid, and water (5: 1:4 v/v), collecting the upper phase and adding 50 ml of n-butyl alcohol per liter. Solvent B was prepared by adding 50 ml of n-butyl alcohol to 1 liter of the upper phase obtained from a mixture of n-butyl alcohol, concentrated NHIQH, and water (4: 1:3 v/v). Solvent C consisted of n-butyl alcohol saturated with 0.1 M phosphate buffer at pH 7.4. The ascending technique was employed for both one- and two-dimensional chromatography. The development periods were 18-24 hours for one-dimensional chromatography and 6-8 hours for two-dimensional work. Chromatograms of urine and of compounds which form colorless solutions were inspected under a Mineralite lamp in a dark room to locate compounds by their absorption of ultraviolet light (Carter, 1950). Chromatograms of azo compounds developed in solvents B and C were exposed to HCl vapors in order to convert the compounds to their purple form for ready detection. Collection and Chromatography of Human Urine Control human urine (50 ml) in dialysis tubing was concentrated to onefifth of its original volume at room temperature with the aid of a stream of air. The concentrate was filtered, and the filtrate was submitted to paper chromatography with solvent B. Inspection of the chromatograms under a Mineralite lamp showed the presence of several spots which absorbed ultraviolet light and of one spot which fluoresced in ultraviolet light. Eluates of these spots failed to produce color in the BrattonMarshall test, either with or without prior hydrolysis. Each of three subjects swallowed a gelatin capsule containing 1.0 g of sulfamethomidine and collected all of his urine for 24 hours thereafter. The pooled urine from the three subjects measured 3.5 1 and contained 780 mg of total sulfamethomidine; 19% was acetylated. The total glucuronic acid content of the urine was about 700 ug/ml, and more than 90% of the glucuronic acid was conjugated. After concentration as above, SM urine was submitted to paper chromatography in solvents A and B.
394
FREDERICK
J.
DICARLO
ET AL.
Three new ultraviolet-absorbing spots were found. Two of the spots yielded eluates which responded directly to the Bratton-Marshall test and the other compound required hydrolysis to permit diazotization and coupling to form a pink solution. By comparative paper chromatography, one of the directly reacting compounds was shown to be sulfamethomidine and the compound requiring preliminary hydrolysis was identified as N4acetylsulfamethomidine. Glucuronic acid was found in eluates of the spot containing the other compound which responded directly to the BrattonMarshall assay. The glucuronic acid present in the eluates was found to be bound by applying the differential assay method of Fishman and Green ( 1955). Paper chromatography in different developing solvents yielded eluates with approximately the same molar proportion of glucuronic acid to sulfonamide (calculated as sulfamethomidine) . This molar proportion was 1.4-1.6. After using solvents A and B in two-dimensional paper chromatography, the glucuronide eluate showed a molar proportion of glucuronic acid to sulfamethomidine of 1.25. Conzpakon of Urinary Glucwonide with Synthetic SuljamethomidineI?+‘-glucuronide A few milligrams of crude urinary sulfamethomidine glucuronide was prepared by lyophilizing the eluates obtained from two-dimensional chromatograms. The metabolite was compared with synthetic sulfamethomidine-N’-glucuronide and the compounds were found to be different. The comparisons were: (1) paper electrophoresis in Tris buffer at pH 10 whereupon the N4-glucuronide migrated 2.3 times farther than the natural product; (2) paper chromatography in solvent B whereupon the N”-glucuronide remained at Rf 0.0 and the urinary glucuronide moved to RI 0.22 ; (3) acidification followed by paper chromatography in solvent B whereupon a small portion of the N4-glucuronide remained at Rf 0.0 while a large new spot appeared at Rj 0.38 (corresponding to sulfamethomidine) whereas the urinary conjugate remained intact and traveled its usual distance; (4) paper chromatography in solvent A resulting in extensive decomposition of sulfamethomidine-N4-glucuronide into sulfamethomidine (Rf 0.88) whereas the urinary metabolite migrated to R, 0.22. Preparation of Azo Derivatives One milliliter of SM urine (adjusted to pH l-2) and 0.2 ml of 0.1% sodium nitrite solution were mixed with a Vortex Jr. Mixer. After 3
GLUCURONIDE
FROM
SULFAMETHOMIDINE
395
minutes, 0.2 ml of 0.5% ammonium sulfamate solution was added and mixed. Two minutes later, a deep purple solution was produced by adding 0.2 ml of 0.1% N-( 1-naphthyl)ethylenediamine solution. The azo derivatives of sulfamethomidine, sulfanilic acid, 2-hydroxysulfanilic acid, 3-hydroxysulfanilic acid, and sulfanilamide were prepared similarly from saturated aqueous solutions. All the compounds except 3hydroxysulfanilic acid responded by forming purple solutions as soon as the last reagent had been added. 3-Hydroxysulfanilic acid became brown and then red after 5 minutes. The R, values obtained from paper chromatograms of the reaction products are listed in Table 1. After exposure to HCl vapors, azo 3-hydroxysulfanilic acid became blue whereas all of the other compounds became violet. Tests for Ester Glucuronide SM urine was tested for the presence of ester with several modifications of the sensitive ferric hydroxamate method (Cheronis and Entrikin, 1957). The tests were negative. Schachter’s scheme (1957a,b) was also employed to check SM urine for ester glucuronide. The method involved treating the azo derivative with hydroxylamine and identifying the new pigment chromatographically. SM urine (5 ml) was mixed with 1 ml of hydroxylamine solution (20 mg/ml) at pH 7 and allowed to stand at room temperature for 1 hour. The reaction mixture was diazotized and coupled to form the azo derivative as described above. Identical chromatograms were obtained in solvents A and B from the azo derivatives of the SM urine and the hydroxylamine-treated SM urine. It was concluded that sulfamethomidine was not excreted in human urine as an ester-type glucuronide. Partial Purification
of Azo Sulfamethomidine
Glucuronide
Concentrated HCl was added dropwise to 20 ml of SM urine to produce a pH of about 1.5. The acidified urine was treated with 15 ml of each Bratton-Marshall test reagent on the usual time schedule. The entire reaction mixture was poured onto a 15mm (inside diameter) adsorption column packed to a height of 16 cm with 12 g of Florisil (60/100 mesh, Floridin Co.). The column was then washed with 55 ml of 0.1 N HCl followed by 300 ml of distilled water. The azo fraction was retained in the uppermost 2.5 cm of the column. The column was washed with 35 ml of 95% ethyl alcohol and the yellow
396
FREDERICK
J.
DI CARLO
ET
AL.
eluate was discarded. In order to elute the azo compound, a mixture of 10ml of water, 10 ml of concentrated NH40H and 1Oml of 95% ethyl alcohol was passedthrough the column. The colored eluate was collected and reduced to a small volume at room temperature to remove ammonia and alcohol. The concentrate was diluted with water to 18 ml and found to contain 270 ug of sulfonamide (calculated as sulfamethomidine) per milliliter. Enzymatic
of Azo Sulfamethomidine Glucuronide
Hydrolysis
Following the hydrolytic procedure of Bernfeld and Fishman (1950)) 5 ml of the above solution of partially purified azo sulfamethomidine glucuronide was mixed with 10 ml of a 37~ solution of deoxyribonucleic acid (sperm origin, Nutritional Biochemicals Corp.) at pH 5.0 and 200 ml of 0.1 M acetate buffer at pH 4.5. After the mixture had been kept at 37” in a constant temperature water bath for 15 minutes, 50 ml of l!l-glucuronidase (Ketodase, Warner-Chilcott) was added. Three more SO-mlaliquots of the same enzyme solution were added to the digestion mixture at 30-minute intervals and the incubation was continued at 37°C for a total period of 67 hours. The hydrolyzate was clarified by centrifugation and concentrated to 70 ml in vacua. The Rf values obtained by paper chromatography of the concentrate in solvents A, B, and C are given in Table 1. ,4&d Hydrolysis
of SM
Urine
SM urine (5.0 ml) was rendered 0.5 N in HCl and heated in a boiling water bath for 15 minutes. The cooled hydrolyzate was diazotized and coupled in the usual manner. Table 1 shows the results of paper chromatography. Acid Hydrolysis
of Azo Suljamethomidine
Glucuronide
SM urine (5.0 ml) was diazotized and coupled as described above. The reaction mixture was made 0.5 N with respect to HCl and heated for 15 minutes in a boiling water bath. Chromatographic data obtained on the hydrolyzate are shown in Table 1. Alkaline
Hydrolysis
of SM Urine
SM urine (5.0 ml) was made 0.2 N in NaOH and heated in a boiling water bath for I5 minutes. The reaction mixture was cooled, acidified to pH l-2 with concentrated HCl and converted into the azo form. Chromatographic findings are presented in Table 1.
GLUCURONIDE
RESULTS
FROM
AND
SULFAMETHOMIDINE
397
DISCUSSION
Paper chromatograms from concentrated SM urine showed the presence of sulfamethomidine and two metabolites. One of the metabolites was concluded to be N”-acetylsulfamethomidine on the basis of comparative paper chromatography, its requirement for hydrolysis before responding to the Bratton-Marshall test method, and the fact that the R, of the hydrolysis product corresponds to sulfamethomidine in different solvent systems. The second metabolite contained bound glucuronic acid after paper chromatography in two directions. The fact that the molar proportion of glucuronic acid to sulfonamide (calculated as sulfamethomidine) was 1.25 rather than 1.0 suggested that the metabolite base has a higher molecular extinction coefficient than sulfamethomidine. Researchers in Japan (Ogiya and Kataoka, 1959; Ogiya, 1959, 1960: Uno and Ueda, 1960; Uno and Kono, 1961) have reported that several sulfonamides are excreted in human urine as N4-glucuronides. In the present study, the urinary sulfamethomidine glucuronide was distinguished from synthetic sulfamethomidine-N”-glucuronide by differences in stability toward acid, paper electrophoresis, and paper chromatography. The failure of the urinary glucuronide to form a hydroxamic acid derivative demonstrated that the metabolite is not an ester glucuronide. It was concluded, therefore, that urinary sulfamethomidine glucuronide belongs to the class known as ether glucuronides (Williams, 1959). A study of the azo products formed from sulfanilic acid, sulfanilamide! and sulfamethomidine by the Bratton-Marshall assay method showed that these compounds were separable by paper chromatography (cf. Table 1). Since the azo compounds are intensely colored (purple in acid, red in base), very small quantities can be seen on paper chromatograms. Another investigation revealed that the azo derivative of urinary sulfamethomidine glucuronide is adsorbed from an acid medium by Florisil and is eluted from Florisil by base. With this technique, azo sulfamethomidine glucuronide was purified for enzyme treatment. (Purification was necessary because SM urine contained material which inhibited the action of l3-glucuronidase.) The enzymatic hydrolysis of the azo form of the metabolite gave a product which traveled much faster (R, 0.46) than the original material (0.19) in the strongly basic solvent B. In the strongly acidic solvent A, the hydrolyzed product migrated at about the same speed as the starting material. The chromatographic pattern obtained from solvent C (pH 7.4) was R, 0.37 for azo sulfamethomidine glucuronide,
39%
FREDERICK
J. DI CARLO
ET
AL.
R, 0.29 for the product of enzymatic hydrolysis and RI 0.61 for azo sulfamethomidine. These findings confirmed the glucuronide nature of the metabolite, but did not elucidate the structure of the azo hydroxysulfamethomidine formed by treating the azo glucuronide with glucuronidase. TABLE PAPER
Test
CHROMAT~CRAPHY
1
OF SULFAMETHOMIDINE
AND RELATED
Solvent
material
A
Solvent
COMPOUNDS
B
Solvent
0.22a
0.20a
Sulfamethomidine
0.86
0.38
Sulfanilamide
0.67
0.53
0.90 -
0.11
Concentrated
Sulfanilic
SM
urine
acid
0.07
0.15
2-Hydroxysulfanilic
acid
0.13
0.08
0.17
3-Hydroxysulfanilic
acid
0.12
0.05
0.88
0.45
0.13 -
0.86
0.00
-
0.35
0.19
0.37
NJ-Acetylsulfamethomidine Sulfamethomidine-N4-glucuronide Azo sulfamethomidine (in urine)
glucuronide
Azo
sulfamethomidine
0.51
0.47
Azo
sulfanilamide
0.39
0.65
0.61 -
i\zo
sulfanilic
0.25
0.27
0.32
Azo
2-hydroxysulfanilic
acid
0.30
0.27
0.28
Azo
3-hydroxysulfanilic
acid
0.38
0.24
0.35
Enzymatic hydrolyzate of azo sulfamethomidine glucuronide
0.35
0.46
0.29
Azo derivatives formed after hydrolysis of SM urine
0.25,a
Acid hydrolyzate methomidine
acid
of azo sulfaglucuronide
Azo derivative formed after alkaline hydrolysis of SM urine (L Principal
acid
0.51
0.46
0.27
0.25 0.39.a
0.27,a
C
0.53
0.66
-
spot.
Having established that the urinary metabolite is an ether glucuronide, the next problem was to ascertain whether the hydroxy group is attached to the benzene or to the pyrimidine ring. This point was clarified by utilizing the observation of Koechlin et al. (1959) that the sulfonamide link of sulfadimethoxine glucuronide is easily hydrolyzed by acid and alkali.
GLUCURONIDE
FROM
399
SULFAMETHOMIDINE
If the glucuronic acid were joined to the phenyl group of sulfamethomidine, acid cleavage would yield either 2-hydroxysulfanilic acid or 3hydroxysulfanilic acid. If the point of attachment were to the pyrimidine ring, the cleavage product would be sulfanilic acid. The azo derivatives of the three possible acids were found to be separated chromatographically (Table 1). In addition, azo 3-hydroxysulfanilic acid gives a distinctive blue color on chromatograms rather than the usual violet. After SM urine was hydrolyzed with acid, diazotized, and coupled with N-( l-naphthyl)ethylenediamine, two colored spots appeared in the chromatograms. In each solvent, the R, values corresponded to azo sulfamethomidine and azo sulfanilic acid. When sulfamethomidine glucuronide was diazotized and coupled prior to acid hydrolysis, only one colored spot was formed and its Rf values corresponded to azo sulfanilic acid. In another experiment, SM urine was hydrolyzed with dilute alkali and coupled for identification. The chromatograms showed the presence of one pigment which corresponded to azo sulfanilamide. These results indicated that the hydroxy group of the ether glucuronide is not located on the phenyl nucleus, but is attached to the pyrimidine ring. SUMMARY Human urine collected after the oral administration of sulfamethomidine contains a glucuronide metabolite in addition to the N4-acetyl conjugate. Urinary sulfamethomidine glucuronide is an ether glucuronide having the glucuronic acid moiety linked to the pyrimidine nucleus. The Bratton-Marshall test procedure was employed to form azo derivatives of sulfanilamide, sulfanilic acid, hydroxysulfanilic acids, sulfamethomidine, and of sulfamethomidine glucuronide and its aglycon. These pigmented products were shown to be separable by paper chromatography and are considered to have general application to the metabolic study of sulfonamides and other aromatic amines. The azo product of sulfamethomidine glucuronide was partially purified by column chromatography with Florisil as the adsorbent. ACKNOWLEDGMENTS The authors are grateful to Dr. Eric Goldschmidt and Mr. Edward Merrill of this Institute for synthesizing sulfamethomidine NJ-glucuronide and to Dr. Thomas M. Goecke (Pollack Hospital, Jersey City, New Jersey) for cooperation in the excretion study. REFERENCES BERNFELD, P., and FISHMAN, W. H. (1950). A coenzyme of spleen Science 112, 653-654. BRATTON, A. C., and MARSHALL, E. K., JR. (1939). A new coupling sulfanilamide determination.‘J. Biol. Chem. P28, 537-550.
B-glucuronidase. component
for
400
FREDERICK
J.
DI CARLO
ET
AL.
CARTER, C. E. (1950). Paper chromatography of purine and pyrimidine derivatives of yeast ribonucleic acid. J. ‘4%~. Chem. Sot. 72, 14661471. CHERONIS, N. D., and ENTRIKIN, J. B. (1957). Semimicro Qualitative Organic Analysis, pp. 229-230. Interscience, New York. DICARLO, F. J., MALAMENT, S. G., HAYNES, L. J., and PHILLIPS, G. E. (1962). Metabolism of Nt-(2-methyl-6-methoxy-4-pyrimidinyl) sulfanilamide (Sulfamethomidine) in the rat, the rabbit, and the dog. Toxicol. Appl. Pharmacol. 4, 475-488. DIC~LO, F. J., MALAMENT, S. G., HAYNES, L. J., and PHILLIPS, G. E. (1963). Metabolism and binding of N1-(Z-methyl-6-methoxy-4-pyrimidinyl) sulfanilamide (Sulfamethomidine) in man. Toxicol. Appl. Pharmacol. 5, 61-70. FISHMAN, W. H., and GREEN, S. (1955). Microanalysis of glucuronide glucuronic acid as applied to fi-glucuronidase and glucuronic acid studies. J. Biol. Chem. 215, 527-537. KOECHLIN, B. A., KERN, W., and ENGELBERG, R. (1959). A comparative study of the metabolism and the urinary excretion of 2,4-dimethoxy-6-sulfanilamido-1,3 diazine (Madribon). Antibiotic Med. Clin. Therup. 6, Suppl. 1, 22-31. OGIYA, S. (1959). On the N-glucuronides of sulfonamides. II. N-glucuronide as a metabolite of sulfapyridine. J. Pharm. Sot. Japan 79. 953-9.57. OGIYA, S. (1960). On the N-glucuronides of sulfonamides. III. N-glucuronide as a metabolite of sulfathiazole, sulfanilamide, and sulfadiazine. J. Phavm. Sot. Japan 66, 15’38-1542. OGIYA, S., and KATAOKA, H. (1959). On the N-glucuronides of sulfonamides. I. Separatory estimation of mixture of N-glucuronide of sulfonamide and other glucuronic acid conjugates. J. Pharm. Sot. Japan 79, 949-953. SCHACRTER, D. (1957a). The chemical estimation of acyl glucuronides and its application to studies on the metabolism of benzoate and salicylate in man. J. Clin. Invest. 36, 297-302. SCHACHTER, D. (1957b). Nature of the glucuronide in direct-reacting bilirubin. Science 126, 507-508. UNO, T., and UEDA, M. (1960). Studies on the metabolism of sulfathiazole. I. On the excreted substance in the human urine after administration of sulfathiazole. J. Pharm. Sot. Japan 60, 1785-1788. UNO, T., and KONO, M. (1961). Studies on the metabolism of sulfisoxazole. II. On the suhisoxazole N-glucuronide. J. Pharm. Sot. Japan 81, 72-76. WILLIAMS, R. T. (1959). Detosijication Mechanisms, 2nd ed., p. 5. Wiley. New York.
AND
APPLIED
A Glucuronide
FREDERICK
J.
DICARLO,
Biochemistry
5, 392-400
PHARMACOLOGY
Metabolite
of
in
Human
SYLVIA
G.
Department, Morris
(1963)
Sulfamethomidine
Urine
MALAMENT,
AND
GEORGE
Warner-Lambert Research Plains, New Jersey
Received
July
E.
PHILLIPS
Institute,
27, 1962
Previous reports on sulfamethomidine [Nl- (2-methyl-6-methoxy-4pyrimidinyl) sulfanilamide] dealt with its metabolism and binding by three animal species (DiCarlo et al., 1962) and by man (DiCarlo et al., 1963). The present investigation concerns the characterization of the sulfamethomidine metabolites in human urine. In this work, the BrattonMarshall sulfonamide assay procedure (1939) was employed to prepare pigmented derivatives of sulfamethomidine and related compounds. These colored compounds are easily detected on paper chromatograms and adsorption columns. Abbreviated terms such as “azo sulfamethomidine” refer to the purple compounds produced by diazotization of the indicated amine and subsequent coupling with N-( 1-napthyl)ethylenediamine. The abbreviation “SM urine” is employed to designate human urine collected after the ingestion of sulfamethomidine. METHODS
“Free” and Total Sulfamethomidine Assay Using a Coleman Junior spectrophotometer and selected round cuvettes (6-310A) in the analytical procedure of Bratton and Marshall (1939)) the azo pigment of sulfamethomidine gives an optical density of 0.084 for a I pg/ml solution. The expression “free sulfonamide” refers to metabolites which can be diazotized directly and coupled. “Total sulfonamide” covers N4-acetylated sulfonamide as well as “free sulfonamide,” and the assay method includes a preliminary hydrolysis to free the N4amino group for diazotization. Glucuronide Assay “Free” and conjugated glucuronic acid levels were determined by the method of Fishman and Green (1955). Using a Coleman Junior spec392
GLUCURONIDE
FROM
SULFAMETHOMIDINE
393
trophotometer at 565 rnp and selected round cuvettes (6-310A), standard curves were developed from glucuronic acid and 6-bromo-2-naphthyl-pglucuronide (Mann). A linear response was obtained up to an optical density of 0.600 from each reference compound. The optical density value corresponding to 1 ug of glucuronic acid per milliliter was 0.032. Paper Chromatography Three solvent systems were employed. Solvent A was prepared by mixing n-butyl alcohol, glacial acetic acid, and water (5: 1:4 v/v), collecting the upper phase and adding 50 ml of n-butyl alcohol per liter. Solvent B was prepared by adding 50 ml of n-butyl alcohol to 1 liter of the upper phase obtained from a mixture of n-butyl alcohol, concentrated NHIQH, and water (4: 1:3 v/v). Solvent C consisted of n-butyl alcohol saturated with 0.1 M phosphate buffer at pH 7.4. The ascending technique was employed for both one- and two-dimensional chromatography. The development periods were 18-24 hours for one-dimensional chromatography and 6-8 hours for two-dimensional work. Chromatograms of urine and of compounds which form colorless solutions were inspected under a Mineralite lamp in a dark room to locate compounds by their absorption of ultraviolet light (Carter, 1950). Chromatograms of azo compounds developed in solvents B and C were exposed to HCl vapors in order to convert the compounds to their purple form for ready detection. Collection and Chromatography of Human Urine Control human urine (50 ml) in dialysis tubing was concentrated to onefifth of its original volume at room temperature with the aid of a stream of air. The concentrate was filtered, and the filtrate was submitted to paper chromatography with solvent B. Inspection of the chromatograms under a Mineralite lamp showed the presence of several spots which absorbed ultraviolet light and of one spot which fluoresced in ultraviolet light. Eluates of these spots failed to produce color in the BrattonMarshall test, either with or without prior hydrolysis. Each of three subjects swallowed a gelatin capsule containing 1.0 g of sulfamethomidine and collected all of his urine for 24 hours thereafter. The pooled urine from the three subjects measured 3.5 1 and contained 780 mg of total sulfamethomidine; 19% was acetylated. The total glucuronic acid content of the urine was about 700 ug/ml, and more than 90% of the glucuronic acid was conjugated. After concentration as above, SM urine was submitted to paper chromatography in solvents A and B.
394
FREDERICK
J.
DICARLO
ET AL.
Three new ultraviolet-absorbing spots were found. Two of the spots yielded eluates which responded directly to the Bratton-Marshall test and the other compound required hydrolysis to permit diazotization and coupling to form a pink solution. By comparative paper chromatography, one of the directly reacting compounds was shown to be sulfamethomidine and the compound requiring preliminary hydrolysis was identified as N4acetylsulfamethomidine. Glucuronic acid was found in eluates of the spot containing the other compound which responded directly to the BrattonMarshall assay. The glucuronic acid present in the eluates was found to be bound by applying the differential assay method of Fishman and Green ( 1955). Paper chromatography in different developing solvents yielded eluates with approximately the same molar proportion of glucuronic acid to sulfonamide (calculated as sulfamethomidine) . This molar proportion was 1.4-1.6. After using solvents A and B in two-dimensional paper chromatography, the glucuronide eluate showed a molar proportion of glucuronic acid to sulfamethomidine of 1.25. Conzpakon of Urinary Glucwonide with Synthetic SuljamethomidineI?+‘-glucuronide A few milligrams of crude urinary sulfamethomidine glucuronide was prepared by lyophilizing the eluates obtained from two-dimensional chromatograms. The metabolite was compared with synthetic sulfamethomidine-N’-glucuronide and the compounds were found to be different. The comparisons were: (1) paper electrophoresis in Tris buffer at pH 10 whereupon the N4-glucuronide migrated 2.3 times farther than the natural product; (2) paper chromatography in solvent B whereupon the N”-glucuronide remained at Rf 0.0 and the urinary glucuronide moved to RI 0.22 ; (3) acidification followed by paper chromatography in solvent B whereupon a small portion of the N4-glucuronide remained at Rf 0.0 while a large new spot appeared at Rj 0.38 (corresponding to sulfamethomidine) whereas the urinary conjugate remained intact and traveled its usual distance; (4) paper chromatography in solvent A resulting in extensive decomposition of sulfamethomidine-N4-glucuronide into sulfamethomidine (Rf 0.88) whereas the urinary metabolite migrated to R, 0.22. Preparation of Azo Derivatives One milliliter of SM urine (adjusted to pH l-2) and 0.2 ml of 0.1% sodium nitrite solution were mixed with a Vortex Jr. Mixer. After 3
GLUCURONIDE
FROM
SULFAMETHOMIDINE
395
minutes, 0.2 ml of 0.5% ammonium sulfamate solution was added and mixed. Two minutes later, a deep purple solution was produced by adding 0.2 ml of 0.1% N-( 1-naphthyl)ethylenediamine solution. The azo derivatives of sulfamethomidine, sulfanilic acid, 2-hydroxysulfanilic acid, 3-hydroxysulfanilic acid, and sulfanilamide were prepared similarly from saturated aqueous solutions. All the compounds except 3hydroxysulfanilic acid responded by forming purple solutions as soon as the last reagent had been added. 3-Hydroxysulfanilic acid became brown and then red after 5 minutes. The R, values obtained from paper chromatograms of the reaction products are listed in Table 1. After exposure to HCl vapors, azo 3-hydroxysulfanilic acid became blue whereas all of the other compounds became violet. Tests for Ester Glucuronide SM urine was tested for the presence of ester with several modifications of the sensitive ferric hydroxamate method (Cheronis and Entrikin, 1957). The tests were negative. Schachter’s scheme (1957a,b) was also employed to check SM urine for ester glucuronide. The method involved treating the azo derivative with hydroxylamine and identifying the new pigment chromatographically. SM urine (5 ml) was mixed with 1 ml of hydroxylamine solution (20 mg/ml) at pH 7 and allowed to stand at room temperature for 1 hour. The reaction mixture was diazotized and coupled to form the azo derivative as described above. Identical chromatograms were obtained in solvents A and B from the azo derivatives of the SM urine and the hydroxylamine-treated SM urine. It was concluded that sulfamethomidine was not excreted in human urine as an ester-type glucuronide. Partial Purification
of Azo Sulfamethomidine
Glucuronide
Concentrated HCl was added dropwise to 20 ml of SM urine to produce a pH of about 1.5. The acidified urine was treated with 15 ml of each Bratton-Marshall test reagent on the usual time schedule. The entire reaction mixture was poured onto a 15mm (inside diameter) adsorption column packed to a height of 16 cm with 12 g of Florisil (60/100 mesh, Floridin Co.). The column was then washed with 55 ml of 0.1 N HCl followed by 300 ml of distilled water. The azo fraction was retained in the uppermost 2.5 cm of the column. The column was washed with 35 ml of 95% ethyl alcohol and the yellow
396
FREDERICK
J.
DI CARLO
ET
AL.
eluate was discarded. In order to elute the azo compound, a mixture of 10ml of water, 10 ml of concentrated NH40H and 1Oml of 95% ethyl alcohol was passedthrough the column. The colored eluate was collected and reduced to a small volume at room temperature to remove ammonia and alcohol. The concentrate was diluted with water to 18 ml and found to contain 270 ug of sulfonamide (calculated as sulfamethomidine) per milliliter. Enzymatic
of Azo Sulfamethomidine Glucuronide
Hydrolysis
Following the hydrolytic procedure of Bernfeld and Fishman (1950)) 5 ml of the above solution of partially purified azo sulfamethomidine glucuronide was mixed with 10 ml of a 37~ solution of deoxyribonucleic acid (sperm origin, Nutritional Biochemicals Corp.) at pH 5.0 and 200 ml of 0.1 M acetate buffer at pH 4.5. After the mixture had been kept at 37” in a constant temperature water bath for 15 minutes, 50 ml of l!l-glucuronidase (Ketodase, Warner-Chilcott) was added. Three more SO-mlaliquots of the same enzyme solution were added to the digestion mixture at 30-minute intervals and the incubation was continued at 37°C for a total period of 67 hours. The hydrolyzate was clarified by centrifugation and concentrated to 70 ml in vacua. The Rf values obtained by paper chromatography of the concentrate in solvents A, B, and C are given in Table 1. ,4&d Hydrolysis
of SM
Urine
SM urine (5.0 ml) was rendered 0.5 N in HCl and heated in a boiling water bath for 15 minutes. The cooled hydrolyzate was diazotized and coupled in the usual manner. Table 1 shows the results of paper chromatography. Acid Hydrolysis
of Azo Suljamethomidine
Glucuronide
SM urine (5.0 ml) was diazotized and coupled as described above. The reaction mixture was made 0.5 N with respect to HCl and heated for 15 minutes in a boiling water bath. Chromatographic data obtained on the hydrolyzate are shown in Table 1. Alkaline
Hydrolysis
of SM Urine
SM urine (5.0 ml) was made 0.2 N in NaOH and heated in a boiling water bath for I5 minutes. The reaction mixture was cooled, acidified to pH l-2 with concentrated HCl and converted into the azo form. Chromatographic findings are presented in Table 1.
GLUCURONIDE
RESULTS
FROM
AND
SULFAMETHOMIDINE
397
DISCUSSION
Paper chromatograms from concentrated SM urine showed the presence of sulfamethomidine and two metabolites. One of the metabolites was concluded to be N”-acetylsulfamethomidine on the basis of comparative paper chromatography, its requirement for hydrolysis before responding to the Bratton-Marshall test method, and the fact that the R, of the hydrolysis product corresponds to sulfamethomidine in different solvent systems. The second metabolite contained bound glucuronic acid after paper chromatography in two directions. The fact that the molar proportion of glucuronic acid to sulfonamide (calculated as sulfamethomidine) was 1.25 rather than 1.0 suggested that the metabolite base has a higher molecular extinction coefficient than sulfamethomidine. Researchers in Japan (Ogiya and Kataoka, 1959; Ogiya, 1959, 1960: Uno and Ueda, 1960; Uno and Kono, 1961) have reported that several sulfonamides are excreted in human urine as N4-glucuronides. In the present study, the urinary sulfamethomidine glucuronide was distinguished from synthetic sulfamethomidine-N”-glucuronide by differences in stability toward acid, paper electrophoresis, and paper chromatography. The failure of the urinary glucuronide to form a hydroxamic acid derivative demonstrated that the metabolite is not an ester glucuronide. It was concluded, therefore, that urinary sulfamethomidine glucuronide belongs to the class known as ether glucuronides (Williams, 1959). A study of the azo products formed from sulfanilic acid, sulfanilamide! and sulfamethomidine by the Bratton-Marshall assay method showed that these compounds were separable by paper chromatography (cf. Table 1). Since the azo compounds are intensely colored (purple in acid, red in base), very small quantities can be seen on paper chromatograms. Another investigation revealed that the azo derivative of urinary sulfamethomidine glucuronide is adsorbed from an acid medium by Florisil and is eluted from Florisil by base. With this technique, azo sulfamethomidine glucuronide was purified for enzyme treatment. (Purification was necessary because SM urine contained material which inhibited the action of l3-glucuronidase.) The enzymatic hydrolysis of the azo form of the metabolite gave a product which traveled much faster (R, 0.46) than the original material (0.19) in the strongly basic solvent B. In the strongly acidic solvent A, the hydrolyzed product migrated at about the same speed as the starting material. The chromatographic pattern obtained from solvent C (pH 7.4) was R, 0.37 for azo sulfamethomidine glucuronide,
39%
FREDERICK
J. DI CARLO
ET
AL.
R, 0.29 for the product of enzymatic hydrolysis and RI 0.61 for azo sulfamethomidine. These findings confirmed the glucuronide nature of the metabolite, but did not elucidate the structure of the azo hydroxysulfamethomidine formed by treating the azo glucuronide with glucuronidase. TABLE PAPER
Test
CHROMAT~CRAPHY
1
OF SULFAMETHOMIDINE
AND RELATED
Solvent
material
A
Solvent
COMPOUNDS
B
Solvent
0.22a
0.20a
Sulfamethomidine
0.86
0.38
Sulfanilamide
0.67
0.53
0.90 -
0.11
Concentrated
Sulfanilic
SM
urine
acid
0.07
0.15
2-Hydroxysulfanilic
acid
0.13
0.08
0.17
3-Hydroxysulfanilic
acid
0.12
0.05
0.88
0.45
0.13 -
0.86
0.00
-
0.35
0.19
0.37
NJ-Acetylsulfamethomidine Sulfamethomidine-N4-glucuronide Azo sulfamethomidine (in urine)
glucuronide
Azo
sulfamethomidine
0.51
0.47
Azo
sulfanilamide
0.39
0.65
0.61 -
i\zo
sulfanilic
0.25
0.27
0.32
Azo
2-hydroxysulfanilic
acid
0.30
0.27
0.28
Azo
3-hydroxysulfanilic
acid
0.38
0.24
0.35
Enzymatic hydrolyzate of azo sulfamethomidine glucuronide
0.35
0.46
0.29
Azo derivatives formed after hydrolysis of SM urine
0.25,a
Acid hydrolyzate methomidine
acid
of azo sulfaglucuronide
Azo derivative formed after alkaline hydrolysis of SM urine (L Principal
acid
0.51
0.46
0.27
0.25 0.39.a
0.27,a
C
0.53
0.66
-
spot.
Having established that the urinary metabolite is an ether glucuronide, the next problem was to ascertain whether the hydroxy group is attached to the benzene or to the pyrimidine ring. This point was clarified by utilizing the observation of Koechlin et al. (1959) that the sulfonamide link of sulfadimethoxine glucuronide is easily hydrolyzed by acid and alkali.
GLUCURONIDE
FROM
399
SULFAMETHOMIDINE
If the glucuronic acid were joined to the phenyl group of sulfamethomidine, acid cleavage would yield either 2-hydroxysulfanilic acid or 3hydroxysulfanilic acid. If the point of attachment were to the pyrimidine ring, the cleavage product would be sulfanilic acid. The azo derivatives of the three possible acids were found to be separated chromatographically (Table 1). In addition, azo 3-hydroxysulfanilic acid gives a distinctive blue color on chromatograms rather than the usual violet. After SM urine was hydrolyzed with acid, diazotized, and coupled with N-( l-naphthyl)ethylenediamine, two colored spots appeared in the chromatograms. In each solvent, the R, values corresponded to azo sulfamethomidine and azo sulfanilic acid. When sulfamethomidine glucuronide was diazotized and coupled prior to acid hydrolysis, only one colored spot was formed and its Rf values corresponded to azo sulfanilic acid. In another experiment, SM urine was hydrolyzed with dilute alkali and coupled for identification. The chromatograms showed the presence of one pigment which corresponded to azo sulfanilamide. These results indicated that the hydroxy group of the ether glucuronide is not located on the phenyl nucleus, but is attached to the pyrimidine ring. SUMMARY Human urine collected after the oral administration of sulfamethomidine contains a glucuronide metabolite in addition to the N4-acetyl conjugate. Urinary sulfamethomidine glucuronide is an ether glucuronide having the glucuronic acid moiety linked to the pyrimidine nucleus. The Bratton-Marshall test procedure was employed to form azo derivatives of sulfanilamide, sulfanilic acid, hydroxysulfanilic acids, sulfamethomidine, and of sulfamethomidine glucuronide and its aglycon. These pigmented products were shown to be separable by paper chromatography and are considered to have general application to the metabolic study of sulfonamides and other aromatic amines. The azo product of sulfamethomidine glucuronide was partially purified by column chromatography with Florisil as the adsorbent. ACKNOWLEDGMENTS The authors are grateful to Dr. Eric Goldschmidt and Mr. Edward Merrill of this Institute for synthesizing sulfamethomidine NJ-glucuronide and to Dr. Thomas M. Goecke (Pollack Hospital, Jersey City, New Jersey) for cooperation in the excretion study. REFERENCES BERNFELD, P., and FISHMAN, W. H. (1950). A coenzyme of spleen Science 112, 653-654. BRATTON, A. C., and MARSHALL, E. K., JR. (1939). A new coupling sulfanilamide determination.‘J. Biol. Chem. P28, 537-550.
B-glucuronidase. component
for
400
FREDERICK
J.
DI CARLO
ET
AL.
CARTER, C. E. (1950). Paper chromatography of purine and pyrimidine derivatives of yeast ribonucleic acid. J. ‘4%~. Chem. Sot. 72, 14661471. CHERONIS, N. D., and ENTRIKIN, J. B. (1957). Semimicro Qualitative Organic Analysis, pp. 229-230. Interscience, New York. DICARLO, F. J., MALAMENT, S. G., HAYNES, L. J., and PHILLIPS, G. E. (1962). Metabolism of Nt-(2-methyl-6-methoxy-4-pyrimidinyl) sulfanilamide (Sulfamethomidine) in the rat, the rabbit, and the dog. Toxicol. Appl. Pharmacol. 4, 475-488. DIC~LO, F. J., MALAMENT, S. G., HAYNES, L. J., and PHILLIPS, G. E. (1963). Metabolism and binding of N1-(Z-methyl-6-methoxy-4-pyrimidinyl) sulfanilamide (Sulfamethomidine) in man. Toxicol. Appl. Pharmacol. 5, 61-70. FISHMAN, W. H., and GREEN, S. (1955). Microanalysis of glucuronide glucuronic acid as applied to fi-glucuronidase and glucuronic acid studies. J. Biol. Chem. 215, 527-537. KOECHLIN, B. A., KERN, W., and ENGELBERG, R. (1959). A comparative study of the metabolism and the urinary excretion of 2,4-dimethoxy-6-sulfanilamido-1,3 diazine (Madribon). Antibiotic Med. Clin. Therup. 6, Suppl. 1, 22-31. OGIYA, S. (1959). On the N-glucuronides of sulfonamides. II. N-glucuronide as a metabolite of sulfapyridine. J. Pharm. Sot. Japan 79. 953-9.57. OGIYA, S. (1960). On the N-glucuronides of sulfonamides. III. N-glucuronide as a metabolite of sulfathiazole, sulfanilamide, and sulfadiazine. J. Phavm. Sot. Japan 66, 15’38-1542. OGIYA, S., and KATAOKA, H. (1959). On the N-glucuronides of sulfonamides. I. Separatory estimation of mixture of N-glucuronide of sulfonamide and other glucuronic acid conjugates. J. Pharm. Sot. Japan 79, 949-953. SCHACRTER, D. (1957a). The chemical estimation of acyl glucuronides and its application to studies on the metabolism of benzoate and salicylate in man. J. Clin. Invest. 36, 297-302. SCHACHTER, D. (1957b). Nature of the glucuronide in direct-reacting bilirubin. Science 126, 507-508. UNO, T., and UEDA, M. (1960). Studies on the metabolism of sulfathiazole. I. On the excreted substance in the human urine after administration of sulfathiazole. J. Pharm. Sot. Japan 60, 1785-1788. UNO, T., and KONO, M. (1961). Studies on the metabolism of sulfisoxazole. II. On the suhisoxazole N-glucuronide. J. Pharm. Sot. Japan 81, 72-76. WILLIAMS, R. T. (1959). Detosijication Mechanisms, 2nd ed., p. 5. Wiley. New York.