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CLIN. CHEM. 20/8. 1086-1096 (1974) Acetaminophen Metabolism in Man, as Determined by High-Resolution Liquid Chromatography1 J. E. Mrochek, S. Katz, W. H. Christie, and S. R. Dinsmore Acetaminophen is a commonly used analgesic, avail- able without prescription. Several of its metabolites have heretofore been isolated from physiologic fluids and analytically characterized. In general, the separation methods are complicated, usually requiring extensive sample pretreatment, and do not measure the individual conjugated metabolites. High-resolution anionexchange separation of urinary samples from subjects receiving acetaminophen reveals eight chromatograph- ic peaks, representing seven metabolites and the free drug itself. Metabolites separated include 2-methoxyacetaminophen, its glucuronide and sulfate conjugates, the sulfate conjugate of 2-hydroxyacetaminophen, the glucuronide and sulfate conjugates of acetaminophen, S-(5-acetamido-2-hydroxyphenyl)cysteine, and S-(5- acetamido-2-glucuronosidophenyl)cysteine. Urinary and serum concentrations of the drug and its seven metabolites were determined by high-resolution liquid chromatography as a function of time after two clinically normal men ingested 1950 mg of the drug. Concentrations in urine and serum are compared, and estimated urinary excretion rates are reported for all metabolites except S-(5-acetamido-2-hydroxyphenyl)cysteine. Serum concentrations of the glucuronide were higher than concentrations of the free drug 2 h after the drug was ingested, indicating that solvent-extraction procedures for serum will yield low estimates of total drug unless hydrolysis precedes the extraction step. AdditIonal Keyphrases: and metabolites chromatography in serum detoxication and urine #{149}mass spectrometry cysteine amido-2-glucuronosidophenyl) #{149}drug mechanisms #{149}gas #{149} toxicology #{149}S-( 5-acet- concentrations A number bolic products of drugs and the kinetand elimination coupled with clinical observations of pharmacologic activity can be invaluable aids to the clinical pharmacologist. Complete studies including identificationand monitoring of metabolites can be highly informative regarding drug activity, toxicity, and mechanism of ac- tion. From these data, new and more effective drug structuresmay be formulated. High-resolution liquid chromatography isan idealanalyticaltool for studies such as these because both unconjugated and conjugated metabolites can be separated and individual ‘Research cal Sciences Corporation’s 2Operated sponsored by the National Institute of General Medi. and National Cancer Institute under Union Carbide can be for Metabolites of investigators have studied the meta(AAP) in humans. included the sulfate and glu- of acetaminophen3 identified ters were computed. Prescott et al. (7) reported plas- ma concentrations of AAP as a function of time and total percent excreted (free + conjugated AAP) in 24-h urine samples. Each of these studies plete in one or more ways; sample treatment is incom- and separation difficulties precluded analyses of both serum and urine, and few data are presented on additional metabolites that are now known to be produced in humans (2). Metabolism of the homologous drug acetophenetidin (phenacetin), which is enzymatically de-ethylated to acetaminophen in man, has been more thoroughly studied. Metabolic products identified include the glucuronide and sulfate conjugates of AAP, 2-hydroxyphenetidine, 2-hydroxyphe- nacetin, S- (1-acetamido-4-hydroxyphenyl)cysteine, and 3-methoxy-4-hydroxyacetanilide and itsglucuronide (2, 7-11). Considering the similarityin the two drugs and the metabolic conversion of acetophenetidin to AAP, it isnot unreasonable to anticipatethat the metabolites of AAP would be similar to those found foracetophenetidin. Using high-resolution anion-exchange chromatography in the study presented here, we report urinary excretion rates for free AAP and seven metabolites, and serum concentrations of free AAP and three metabolites. Urinary excretion was determined periodically over the 24-h period after ingestion of a total of 1950 mg of AAP by each of two clinically normal contract with the U. S. Atomic Energy Commission. for the USAEC by the Union Carbide Corp. Oak Ridge National Laboratory,2 Received April 12, 1974; accepted 1058 metabolites curonide conjugates of AAP (1) and S-(1-acetamido4-hydroxyphenyl)-cysteine (2). Analytical studies of acetaminophen metabolism have generally focused on the 24-h urinary excretion of free AAP and total conjugated AAP, the latter determined after enzymatic or chemical hydrolysis (3-6). In one of the few studies reporting analyticalresultsfor severalmetabolites,Cummings et al. (1) listedurinary excretion rates forthe freedrug and itsglucuronide and sulfate conjugates; from these data, certain kinetic parame- 4-phenetidine, Studies of the metabolism ics of metabolite formation of all detectable determined in a single sample without the need hydrolysis or extensive sample pretreatment. CLINICAL CHEMISTRY Oak Ridge, Tenn. June 3, 1974. VoL 2O No. 5 1974 37830. ‘ Trade name, Tylenol; also known aminophenol, and 4-hydroxyacetanilide. as paracetamol, N.acetyl-p- men. Concentrations in serum were also determined periodically during a 3-h period after drug ingestion. Several new metabolites of AAP in humans are reported. Metabolites were identified by gas chromatography, mass spectrometry, and the two techniques combined. Details of the identification of one important new metabolite of acetaminophen are presented here; complete details of studies on other new metabolites,including gas-chromatographic and mass-spectrometric data, will be presented in a subsequent paper. Materials and Methods Chromatographic System The chromatographic system used for these studies Comparative ultraviolet spectrophotometric data were used to determine positional substitution on the aromatic ring where model compounds were available. Ultraviolet spectral data (maxima) were reported for synthetic S-(5-acetamido-2-hydroxyphenyl)cysteine in 0.1 mol/liter HC1 and 0.1 mol/liter KOH by Focella et a!. (compound 18, ref. 18). Drug-Ingestion Two Studies clinically normal men (both nonsmokers; Subject 1 age 41 and Subject 2 age 58 y) each ingest- ed six tablets of AAP (Tylenol, McNeil, 325 mg/tablet) after overnight fasting (12 h). The total of 1950 mg ingested was equivalent to 23 mg/kg of body weight for each subject (Subject 1, 185 lb; Subject 2, 187 ib). The amount of drug ingested is three times isgenerally similarto that described previously (12). A more complete descriptionof the system used here can be found in a laterpublication by Katz et al.(13). Certain modifications were introduced into the sys- the recommended tem, which enabled the chromatographic analysis of acetaminophen and related metabolites to be completed in 21 h. Thus, including system regeneration, the total cycle time was 24 h/sample. These modifications included the following: (a) reduction in column length from 150 to 100 cm, (b) increasingthe column temperature from 60 to 70 #{176}C, and (c) decreasing the total amount of eluting buffer from 350 g to 225 g while at the same time sharpening the gradient. The concentration gradient of buffer eluent was formed by using a nine-chamber gradient box (Phoenix Precision Instrument Co., Philadelphia, Pa.) with 25 g of pH 4.4 ammonium acetate-acetic acid buffer per chamber (15 mmol/liter in the first two chambers, 4 at 0.5,1, 2,and 3 h afterwards. The subjects concluded the fast after the 3-h blood and urine samples mol/literin the second two chambers, and 6 mol/liter in the finalfivechambers). This relativelysteep gra- dient and higher temperature enabled elution of three highly anionic sulfate conjugates in 21 h, allowing one sample per day to be analyzed. With use of the previous gradient (12) and a 150-cm column at 60 #{176}C, these strongly retained compounds required 42 h to elute. Extensive band-broadening resulted from such long retention on the column, making quantitation difficult. The measured molar absorptivity of AAP at 254 nm (7950 litersmole’ cm’ was used in allquantifi- cations, and the metabolite excretion was computed in terms of equivalent AAP. Ultraviolet spectra of the individual metabolites were similar to that of AAP, at leastwith respect to the main transitionat 244 nm. Thus, our use of a singlemolar absorptivity in the calculationsisexpected to yield reasonably accurate results. Identification of Metabolites Metabolites of acetaminophen fied by multiple analytical chromatography or mass urinary products isolated have been identi- techniques (including gas spectrometry or both) on by means of preparative- scale high-resolutionliquid chromatography (14-17). single dosage but within the rec- ommended 24-h dosage, 1950 to 2600 mg (two tablets taken three or four times daily). Blood samples were collected immediately before they took the drug and were collected, consuming a light lunch. Total urinary output was collected during each of the following time periods after ingesting the drug: 0-0.5, 0.5-1, 1-2, 2-3, 3-5, 5-7, 7-12, and 12-24 h. Urine samples were filteredthrough a 0.2-zm (average pore diameter) filter (Nalge, Sybron Corp., Rochester, N. V. 14602) to remove particulatematter and then frozen at -60 #{176}C untilchromatographic analysis.Blood samples were allowed to clotat ambient temperature. The clotwas centrifuged and the serum was ultrafiltered overnight (0 #{176}C) through dialysistubing (Union Carbide Corp., Food Products Division) with an air overpressure of 104 kPa (15 psig).The ultrafiltered serum samples were stored at -60 #{176}C until analyzed by liquidchromatography. Results Metabolite Identification Figure 1 depicts a typical urinary chromatogram obtained for a patient receivingthe analgesicmedication, AAP. The anion-exchange system used to obtain this chromatogram employed a more gentle gradient (12) and did not incorporate the changes men- tioned above (Materials and Methods); therefore, the time required for complete development of this illustrativechromatogram was 42 h instead of the 21 h required for the data of our study. We have identified seven metabolites of acetaminophen in urine samples from human subjects receivingthe drug as shown in Figure 1: (I)S-(5-acetamido-2-hydroxyphenyl)cysteine (IV) 2-methoxy-4-glucuronosidoacetanilide (V) 4-glucuronosidoacetanilide (VI) S- (5-acetamido-2-glucuronosidophenyl)cys- teine (VII) 2-methoxyacetaminophen (VIII)acetaminophen (IX) 2-hydroxyacetaminophen CLINICAL sulfate sulfate CHEMISTRY. sulfate Vol. 20. No. 8. 1974 1087 0 2 I 3 4 5 6 7 6 9 10 II 12 3 4 IS C IT C 19 20 SI 22 23 24 25 25 27 26 29 30 rIME It.,) ANION 02 ce 31 32 33 34 35 31 37 31 39 40 41 42 43 44 s CNCIIANGE CIOMATOONAPW 11101 CONIOTIONS .150cm STAINLESS STEEL WITH 11-12,. DISK AMINEX 0-27 11(519. TEMPENATIIWE. AMSIENT TO t.OC AT 3 ELIlEIT GNAOIENT, 0015 E TO NOW AWNONIUN ACETATE. 111144. ELUENT TLOW NATE. 7ml9c. COLUMIUPU(SSUNE Fig. 1. Anion exchange chromatogram obtained with use of the two-detector who had received acetaminophen Shaded peaks are acetaminophen 100 r 60 N#p H11C-II Lc.I > I) C-S.) 209 101 I- _______ 50 70 00 urine sample from a subject 110 i II’ io#{149}io tho Fig. 2. Mass spectrum for a probe-inserted acetamido-2-hvdroxvohenvl)cvsteine 210 230 sample of S-(5- Note that the conjugated metabolites are easily 20, No.8, elsewhere. Ultraviolet spectra for compounds I and VI methanol were virtually identical, with maxima the former observed at 244 and 299.5 nm and for latter at 244 and 300.5 nm. Figure 2 illustrates in for the the mass spectrum obtained for a probe-inserted sample separated from the unconjugated metabolites, thus illustrating a major advantage for liquid-chromatographic analysis of physiologic body fluids. The conjugate S-(5-acetamido-2-glucuronosidophenyl)cysteine is an important new metabolite of acetaminophen, and identification studies for it will be discussed with reference to our data for S-(5-acetamido-2-hydroxypheny!)cysteine, partially characterized by Jagenburg and Toczko (2) and completely identified by means of the total synthesis of I by FoCLINICAL CHEMISTRY, Vol. celia et a!. (18). Ultraviolet absorption maxima obtained for I in 0.1 mol/liter HC1 and 0.1 mol/liter NaOH agreed with those reported for the synthetic compound in Table 2 of this reference. Identification data on other metabo!ites, including mass spectral and gas-chromatographic retention data, will be presented U/E 1088 system for a typical and its metaboiltes >- 30 .27001111.5 1974 of I. Empirical formulas for all ions were confirmed by high-resolution mass spectrometry except for m/e 226 (not observed in the high-resolution spectrum). However, a metastable transition observed in the mass spectrum at m/e 171.7 (m*)4 related the ions m/e 226 and 197 and suggested a loss of methylene imine (CH2=NH). Metabolite I had very low volatility in the mass spectrometer, requiring high temperature (>200 #{176}C) for a useful spectrum. No masses were observed above those indicated in Figure 2. The identity of I was confirmed by the mass spectrum of the trimethylsi!yl derivative, as shown in Figure 3. EmParent m4 = d2/p. (p) and daughter (d) ions are related by the expression CTLI1iO&l 33 110 13) 17) 170 133210 23) 27)270217 310 1077370 A4- 311 a) 4)0 43) Fig. 3. Mass spectrum for the trimethylsilyl acetamido-2-hydroxyphenyl)cysteine 47 470 o F l7)5)0S33’77570 derivative 2O pirical formulas for all ions pictured in Figure 3 were confirmed by high-resolution mass spectrometry (see Table 1). The molecular weight of 270 for I [558-288 (four trimethylsilyl groups)] coupled with the presumed identity of the m/e 226 ion in Figure 2 (based on the metastable transition in the low-resolution mass spectrum) suggests partial thermal degradation and loss of CO2 before volatilization of the probe sample. This isa reasonable assumption, considering the probe temperature required to volatilize the compound and its chemical structure. Conjugated compound VI was most intractable and difficult to study. A probe sample required even higher temperatures than were required for I, to obtain a mass spectrum of reasonable intensity. The spectrum was identical to that observed for I, except that no m/e 226 ion was observed. High-resolution spectrometry verified that i,ick 17 170 I 1 170 1 210 77) 2T 270 27) 310 37) 7) 370 110 033 07) 070 410 47 QT 011) the previously 7) ii 110 iLL rIL 13) 210 I Ih 27) 07) ALl 270 47 1 1 1 310 77 L 77 370 77 411 Fig. 4. (a) Mass spectrum of S-(5-acetamldo-2-glucuronoskloafter silylation Underlined masses are characterIstic of the trimethylsilyl-derlvatlzed giuclsophenyl)cysteine nidemoiety (b) Mass spectrum of S-(5-acetamido-2-glucuronosidoph#{232}nyl)cysteineafter derivatiz#{226}tionwith diazomethane and bis(trimethylsilyl)trifluoroacetamide Interpretation of the data suggested the ions Illustrated above and then silylated with bis(trimethylsilyl)trifluoroa- cetamide, that in hopes esterification of the carboxy!- ic acid moieties with methyl groups would result in a compound that would volatilize in the mass spectrometer without thermal fragmentation. A comparifor m/e <500 is shown in Figure 4. Previously published data have established characteristic masses for the glucuronic acid moiety of conjugated urinary constituents (17). These masses are present in the spectrum of the trimethylsilyl son of the two mass spectra iden- inlet)was quite complex and impossible to interpret. was methylated I , 13D (b) Li 70 tified ions (Figure 2) were present. A mass spectrum of the trimethylsilyl derivative (gas chromatographic The compound 110 of S.(50:117 mass JI 70 with diazomethane Table 1. High-Resolution Mass Spectral Data for the Trimethylsilyl Derivative of S-(5-Acetamido-2-hyd roxyphenyl)-cysteine Differences from theoretical, Measured Empirical mass 558.2254 543.1998 515.2085 486.1890 441.1919 341.1337 218.1050 formula Suggested C23H4N204SSi4#{176} C22H43N2O4SSi4 C21H43N2O3SSi44 CH38N204SSi3 C19H27N2O2SSi3 C15H27N02SS12 + + CH,=Si(CH11), COOTMSS fragment losses millimass 1 +0.1 -2.3 I +1.4 +3.0 +3.5 +3.6 I TMSN=CHCOOTM5 C8H25N02S12 units +1.7 CH o= N-TMS “S-CH-Cl-I-COOTMS OTMS b Metastable HNTMS transition observed. CLINICAL CHEMISTRY, Vol. 20, No.8. 1974 1089 derivative, as shown in Figure 4a. Methylation of the carboxylic acid groups by means of diazomethane would decrease those masses characteristic of glucuronic acid by 58 mass units yielding m/e 407 and m/e 317 in place of the characteristic glucuronide ions m/e 465 and 375, respectively (19). As illustrated in Figure 4b, these were observed and the compound was verified as a glucuronide. Highest mass observed for the trimethylsilyl-derivatized conjugate was 691 and for the methylated trimethylsilyl derivative 681, neither of which is nearly high enough for a fully derivatized glucuronide of compound I (mol wt, 950; mol wt of comp. VI, 446 + 7 trimethylsilyl groups). However, we did observe masses that we interpreted as verifying the aglycon as I (see Figure 4b). Unfortunately, the conjugate was resistant to hydrolysis by fi-glucuronidase (72-h treatment at 37 #{176}C), and acid hydrolysis probably would have cleaved the sulfurcontaining side chain we were attempting to identify. Both I and VI showed a positive reaction with ninhydrin, indicating the presence of a free primary amine functional group. From the nearly identical ultraviolet spectra for I and the glucuronide, the mass spectra! data obtained for the methylated and tn- methylsilylated conjugate, and its positive reaction with ninhydnin, we believe the metabolite is S-(5acetamido-2-glucuronosidophenyl)cysteine. The ultraviolet spectral data indicate that the position of aromatic substitution for the cysteine moiety is identical for compounds land VI. Metabolite inUrine Excretion The urinary excretion of the previously listed seven metabolites of AAP was followed as a function of time after two clinically normal subjects ingested 23 mg of AAP per kg of body weight (‘1950 mg). This was the first time either subject had ingested this drug. Figure 5 graphically illustrates the chromatographic changes in urinary period. Note that excretion rates and cumulative excretion of AAP and its glucuronide, were initially much higher for Subject 2 than for Subject 1. Simi- TINIE. 11 CLINICAL CHEMISTRY. Vol. 20, No.8, 1974 concentra- metabolites for both subjects during each collection Fig. 5. Liquid-chromatographic analyses of urine samples from Subject 1, illustrating concentration changes undergone by acetaminophen and its metabolites 1090 metabolite tions for Subject 1 as a function of time. Figures 6 and 7 compare the urinary excretion rates (in milligrams per hour) for AAP and six of its metabolites (all computed as equivalent AAP) for Subjects 1 and 2, respectively; Table 2 lists the total excretion of all SUBJECT I o ACETAMINOPHEN (AAP) () #{149} 4.GLUIJB0NOSI55ETANILIDE ACETAMINOPHEN SULFATE() #{149} 2-METHOXY 4-GLUCURONOSIDO* tNt A o 2-METHOXV ACETAMINOPHEN SULFATE O 2-HTOROXY ACETANINOPHEN SULFATE #{149} S(IACET CYSTEINE I ‘C 4.. E 10 E 01 LI I- I- 4 4 11 z z 0 0 I- Iii LII C) 5) 5< III LI x 5. z 4 z 8 10 TIME, Is TIME,), Fig. 6. Rates of urinary excretion metabolites after its oral ingestion for acetaminophen by Subject 1 and six larly, peak excretion by Subject 2 occurred at 2.5 h (collection period 1 h, the 2nd to 3rd hour), while it was delayed until 4 h (collection period 2 h, the 3rd to 5th hour) for Subject 1. An examination of the excretion rates of AAP during the first half-hour sampling period indicated Subject 1 excreted the free drug at a nate of 1.4 mg/h, whereas the rate for Subject 2 averaged 8 mg/h. Similar differences were noted in a comparison of the excretion rates for the glucuronide during this initial time period, with 2.4 mg/h observed for Subject 1 and 16.3 mg/h for Subject 2. However, during this same period, excretion rates for the sulfate conjugate (VIII) were approximately the same for both subjects-12.4 mg/h for Subject 1 and 12.9 mg/h for Subject 2. Complicating the interpretation effects of differences data were possible of the rate in the rate of Fig. 7. Rates of urinary excretion for acetamiriophen metabolites after Its oral ingestion by Subject 2 drug absorption and average hourly urine and six flow for each subject. Urinary flow for Subject 1 averaged only 35 ml/h, as compared with 100 ml/h for Subject 2. Peak overlap in the chromatographic S-(5-acetamido-2-hydroxyphenyl)cysteine area where eluted (see Figure 5, 2.75 h) prevented accurate estimations of its excretion measurements rate. Crude estimations based on of the fluorescence of CeS+ suggested that its excretion tended to follow that of compound VI; however, this measurement was complicated by the co-elution of tryptophan with I, resulting in a non-gaussian peak shape and additional fluorescence caused by Ce4 oxidation of this amino acid. Collecting this chromatographic peak and rechromatograph- ing it on a high-resolution (‘A INI(’AI CHIMISTRY cation-exchange VnI 2fl system Nn R 1974 ini Table 2. Comparison of Total Urinary Excretion Data for Acetaminophen and Its Metabolites in Two Clinically Normal Male Adults Amount 0.9 5.0 Vc 1.5 VIII 7.8 10.3 2 1.2 3.7 1 2.0 2 4.3 1 2.8 81.9 55.2 2 5.2 157.8 66.0 3.0-5.0 1 2 7.9 4.6 257.0 182.2 110.8 78.6 5.0-7.0 1 2 6.3 3.5 222.8 131.1 1 2.9 116.4 7.4 322. 1.4 3.5 428.8 157.6 ingestion (h)b Subject 0-0.5 1 2 0.5-1.0 1.0-2.0 2.0-3.0 7.0-12.0 1 2 12.0-24.0 1 2 AAP excreted, mg11 IV VII N.D. 0.2 12.5 56.7 8.1 16.6 26.2 0.9 0.8 0.8 1.5 3.4 50.8 36.8 2.6 7.0 108.8 55.0 83.7 68.8 IX VI (mg) 0.02 0.05 10.7 25.9 0.5 34.4 1.3 1.8 2.0 1.4 1.5 0.4 94.0 4.8 3.9 3.4 3.5 4.2 1.1 100.8 5.2 1.8 184.5 9.5 0.6 5.1 7.9 10.8 7.8 6.3 3.7 12.3 4.0 5.3 4.4 9.3 5.5 7.4 3.7 19.7 5.3 164.0 248.7 427.8 288.2 8.4 12.8 21.9 14.8 11.2 11.5 7.4 17.5 360.5 219.0 18.5 11.2 196.8 531.2 756.9 280.2 10.1 27.2 38.8 14.4 4.8 3.2 3.6 4.2 53.8 167.1 208.1 102.4 7.0 7.9 29.0 4.6 2.9 6.3 31.5 1.7 3.9 10.8 17.4 4.5 9.9 8.9 40.6 6.1 Total, mg 1 2 25.6 37.1 1171.8 1127.4 572.8 572.2 66.6 44.3 70.0 25.1 48.5 35.3 96.8 30.4 2052.1 1871.8 Percent” 1 1.3 1.9 60.1 57.8 29.4 29.3 3.4 2.3 3.6 1.3 2.5 1.8 5.0 1.6 105.2 96.0 2 Percent” 0.3 0.7 All metabolites computed as equivalent acetaminophen. Each subject ingested 1950 mg of acetaminophen, equivalent to 23 mg/kg. AAP = acetaminophen; V = 4-glucuronosidoacetanilide; VIII = acetaminophen sulfate; IV = 2-methoxy-4-glucuronosidoacetanilide; VII = 2-methoxyacetaminophen sulfate; IX = 2-hydroxyacetaminophen sulfate; VI S.(5-acetamido.2.glucuronosidophenyl) cysteine. d Based on 1950mg ofacetaminopheningested. #{149} Not detected. (17) would enable accurate quantitation of this compound to be accomplished; however, rechromatography of the fraction containing these compounds was not performed for this study. It is interesting to note that excretion of the conjugate VI by Subject 1 was threefold higher than was observed for Subject 2 (see Table 2). This difference in excretion by the two subjects was also qualitatively observed for I by following the Ce3+ detector response in the two sample series. Concentrations of AAP and Its Metabolites in Serum Figure 8 shows that neither I nor VI was detected (absence of detector response at 2.75 h and 6.75 h) in any of the serum samples from Subject 1, and this was alsotrue for Subject 2. With the cerium oxidimetric detector the lower limit of detectabilityfor the two cysteine-containing metabolites was estimated to be about 50 nanograms per ml of serum. Thus, although we did detect traces of VII in the serum of both subjects (see, for example, 1- and 2-h samples in Figure 8 at about 16 h), neither I nor VI was detected even though the urinary concentration of the latter generally exceeded that of VII (see Table 2). It is possible that these two metabolites were bound to Blood samples (about 5 ml each) were collected before ingesting AAP and 0.5, 1.0, 2.0, and 3.0 h afterwards. Figure 8 illustrates the concentration changes we observed for AAP and its metabolites in serum macromolecules in the serum. Compounds with molecular weights greter than about 1000 would have been removed from the samples by the ultrafiltration step. samples from Subject 1, by use of the cerate oxidimetric detector. Generally, this detector was more sensi- Figure 9 illustratescomparisons of urine and serum concentrations of AAP and its two major metabolites,V and VIII, for Subjects 1 and 2. There is a striking similarity, for both subjects,in the concen- tive than ultraviolet detection for the two cysteinecontaining metabolites by a factor of two to three under our instrumental conditions. It was also more sensitive to the methoxy-substituted metabolites IV and VII; however, it exhibited about the same sensitivity as ultraviolet detection for the other compounds. 102 CLINICAL CHEMISTRY. Vol. 20. No. 8. 1974 tration changes undergone by AAP in urine and serum. As reported in the discussionof urinary excre- tion, a relatively high urinary excretion of AAP was observed for Subject 2 (compared to Subject 1) in the firstsample taken at 0.5 h. As illustratedin Figure 9, URIC ACID NO SO 70 NUVIC ACID NO DO ON 40 SO DO 10 0 Do 40 I/I H 30 00 lO Fig. 8. Cerium oxidimetric detector response for serum samples from Subject 1 0 SO The identity of the compound eluting at 12 h In the 2-h sample is not known 40 30 20 0 10 0 SO 40 30 20 I0 0 SO 40 So 20 0 U U I 2 3 4 5 3 7 3 3 0 TIME. II 2 3 4 IX IN Il IS 20 II N a twofold-higher concentration of AAP in the urine of Subject 2 was accompanied by an equivalent difference in the concentration in serum (see also Table 3). Concentrations of AAP in serum remained fairly constant over the 3-h period for Subject 1, which does not appear to fit known kinetics of drug elimination at normal dose levels. Concentrations in serum for Subject 2 decreased substantially from the initial highest concentration of 12.4 ig/ml (Table 3). However, the concentrations in the urine of both subjects seem to more or lessmirror these relativechanges in concentrations in serum (Figure 9). The later peaking of metabolite excretion rates in the urine (3 to 5 h period) for Subject 1 compared to that for Subject 2 (2 to 3 h period) seems to correlate with the difference in the rates of elimination for AAP from the sera of the two subjects. Note that appreciable quantities of conjugate V. even exceeding those of the free drug, were present in the serum. Thus, it should be recognized that gas-liqi II.JIrAI (‘WIAISTRV \inI ‘fl Jn R 1974 1002 Table 3. Comparisons of Serum Concentrations of Acetaminophen and Its Metabolites in Two Clinically Normal Men 5000 2000 Serum Time bOO after Ingestion” 0.5 drug (h) 1.0 200 2 2 100 2.0 50 C) 2 3.0 0 U ,,, 2 20 II H. -----. 10.0 >... Each subject ingested lent to 23 mg/kg. 5AAP = acetaminophen; - 0 5.0 VIII acetaminophen phen sulfate. #{149} Not detectable. / IA 2.0 = VS VIII), 0.5 N.D.c 2 1.0 12.4 4.3 6.6 8.6 7.0 5.4 7.0 5.5 3.2 9.1 0.9 1.0 0.2 1 2 1 2 1 2 AAPS 1 E 0 ,g/mI 6.2 Subject 500 concentration, 7.2 13.4 9.4 16.6 V = 0.1 1.2 0.04 2.4 2.1 1.2 1.9 1.6 0.2 1950 mg of acetaminophen, sulfate; VIII N.D.’ equiva- 4-glucuronosidoacetanilide; VII = 2.methoxyacetamino- 1.0 0.5 A-- IUJECT I 2 DUBJECT URINARY CONCENTRATION SERUM CONCENTRATION 02 #{149} ----.--- 0.1 4 0 0.5 1.0 1.5 2.0 2.5 3.0 4.0 TIME, Fig. 9. Comparisons for urinary and serum concentrations of acetaminophen (AAP) and its glucuronide (t) and sulfate (VIII) conjugates uid chromatography in which extraction methods are used (20, 21) for determination of AAP in blood are probably measuring only about half of the drug actu- ally present. Sample hydrolysis performed before the extraction step may overcome this problem if additional complications are not introduced by such harsh treatment of the serum. Discussion Significance of Compounds /and VI Studies aimed at defining the mechanism by which overdoses of AAP cause hepatic necrosis in rats and mice, and the possible role that glutathione may play in preventing this liver damage, have been performed by Mitchell et al. (22-25). These workers found markedly enhanced hepatic necrosis in AAP-loading experiments on rats and mice pretreated with pheno- barbital, which stimulated the disappearance of AAP from tissue. In contrast, pretreatment with piperonyl butoxide, tabolizing a known inhibitor of microsomal drug-meenzymes, decreased the metabolism of AAP, delayed its disappearance from tissues, and dramatically protected against hepatic damage (22). Using [3H]acetaminophen, Jollow et a!. (23) reported that amounts of covalently bound, radiolabeled hepatic material paralleled the severity of the histologically recognizable necrosis, with maximal binding preceding liver damage by at least 1 to 2 h. Based on the data, these workers concluded that hepatic damIOQA (I IMIfAI (I.1IAIQTQV I/,,I Oil 0 107A age is mediated by a toxic metabolite of AAP rather than by the drug itself. Probably influenced by numerous reports of glutathione conjugation with foreign aromatic or unsaturated compounds and subsequent excretion of these detoxified metabolites as mercapturic acids in animals (26-29), Mitchell et al. (25) examined the possibility that glutathione may prevent AAP-induced hepatic necrosis. They found that either glutathione or cysteine completely inhibited the binding of radiolabeled AAP to microsomal protein in vitro. Pretreating mice so as to artificially deplete hepatic gluta- thione enhanced AAP-induced hepatic necrosis, whereas pretreatment with cysteine, a glutathione precursor, prevented the hepatic necrosis. Finally, administration of AAP resulted in a dose-dependent depletion of hepatic glutathione, and significant covalent bonding of radiolabeled AAP to hepatic macromolecules did not occur until at least 70% of the hepatic glutathion&was depleted. These authors, on the basis of their experimental findings in rats and mice, suggested that a toxic metabolite of AAP ed from covalent binding to hepatic isprevent- macromolecules by preferentialreaction and subsequent detoxication with glutathione. In man, the importance of glutathione in preventing hepatic damage induced by AAP and other aryl and unsaturated drugs is unknown. Conjugation of drugs with glutathione occurs, but enzyme activities in the liver are reported to be lower in man than in rats and mice (30). Warner and Lorincz were unable to isolatea glutathione conjugate afteradministering bromobenzene to human subjects (31). Jagenburg and Toczko (2) reported the identificationof S-(1acetamido-4-hydroxyphenyl)cysteine as a metabolite of both AAP and phenacetin in man; however, there was no evidence cited that indicated glutathione participated in the formation of this metabolite. We crudely estimated excretion of I by the two subjects of this report to be no more than 1% of the ingested dose, while that of conjugate VI was 5% and 1.6%, respectively, for Subjects 1 and 2. Jagenburg et al. (32) reported urinary excretions for I of 0.4, 3.0, and 5.9% by three healthy subjects after oral doses of 1.5 or 2 g of AAP. These same authors reported the isolation of another cysteine-containing metabolite, excreted in amounts ranging from 4.5 to 6.1% of the ingested dose of AAP. The compound which they isolated yielded alanine after desulphurization and subsequent acid hydrolysis. Based on the absence of reaction with ninhydrin (I did react) and also on “the well-known fact that monohalogenated benzenes form N-acetylcysteine derivatives (mercapturic acids) in the animal body,” Jagenburg et al. assumed the metabolite to be S-(1-acetamido-4-hydroxyphenyl)-mercapturic acid. It is possible that the metabolite isolated by these tin with nucleophiles in acid solution. They reported formation of 4-hydroxy-3-methylthioacetanilide when methionine was the nucleophile used and postulated that the intermediate involved was N-acetylp-benzoquinoneimine. They postulated that S-(5- authors is the same metabolite that we have identi- cleophilic sulfur group. Further studies and other questions should add much edge on metabolism and detoxication fied as S-(5-acetamido-2-glucuronosidophenyl)-cysteine; however, we found that both it and I show a positive reaction with ninhydrin. This compound, shown by us to be a glucuronide by mass spectrometry, was completely resistant to hydrolysis with figlucuronidase after exposure for 72 h at 37 #{176}C at a pH of 5.04. This suggests the possibility of steric hindrance to attachment of the enzyme or an intramolecular complex between the aglycon and the carboxylic acid group of glucuronic acid. Our evidence for the identification of cysteine as the group attached to the aromatic ring is conclusive only with regard to the attachment of sulfur to the ring (see discussion regarding the mass-spectrometric studies on probe samples of VI). Based on our interpretation of mass spectral data obtained for VI after treatment with diazomethane and bis(trimethylsilyl)trifluoroacetamide (see Figure 4b) and its positive response to ninhydrin, we believe only an alanine moiety is attached to the sulfur (giving However, we did not observe a molecular cysteine). ion (M) [and the characteristicM -15(CH3) ion fortrimethylsilyl derivatives] for the high-molecular-weight conjugate, so that some possibilityfor error stillexists.Considering the high molecular weight, the numpresent, and the possibility of an bond, it is not surprising that the data failed to give a good structural representation of the total molecule. Important new questions can be answered about the nature of a possible toxic metabolite of AAP by using high-resolution liquid-chromatography coupled ber of polar groups intramolecular mass spectral with additional studies of animals. Focella et a!.(18) identified 4-hydroxy-3-methylthioacetanilide as a new urinary metabolite of phenacetin in the dog. To attempt to clarifythe mechanism by which the 3substituted metabolites might be formed, Calder et al. (33) studied the reactions of N-hydroxyphenace- acetamido-2-hydroxyphenyl)cysteine might be formed via a reaction of cysteine with this same intermediate. These results, coupled with our failure to identify any mercapturic acid metabolite, may argue against reaction of a presumed toxic metabolite of AAP with glutathione as suggested by Mitchell et al. (25). The protective role suggested by the studies of these authors on rats and mice may actually be played by either cysteine or methionine in human metabolism. Identifying the precursor of the new conjugated metabolite VI isvitalto further in vivo studies on the nature of the presumed toxic metabolite. The precursor could be either I or V, the glucuronide of AAP. If it is the latter, this probably would rule out epoxide formation (23) as preceding conjugation with the nuto clarify this to our knowl- of aromatic compounds in the human body, facilitating the ftrmulation of safer and, perhaps, more effective drugs. It is important to note that recovery of the ingested drug dose averaged 100.6% for the two subjects in this study. This again illustrates the utilityof liquid- chromatographic a wide spectrum techniques in drug studies to enable of metabolic changes to be observed. In this way, unexpected metabolic changes induced by a drug can be observed, enabling mechanistic in- ferences regarding its mode of action to be further tested. We gratefully acknowledge Jr., of the Analytical Rainey, the technical Chemistry assistance of Dr. W. T. Division at ORNL. References 1. Cummings, study A. J., King, of drug elimination: metabolites (1967). in man. M. L., and The excretion Brit. J. Phormacol. Martin, B. 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