January 1977
130
The Journal of P E D I A T R I C S
Age-related differences in salicylamide and acetaminophen conjugation in man Following a concomitant oral dose of salicylamide and acetaminophen (5 mg/kg of each) the urinary excretion of glucuronide and sulfate conjugates of the drugs were followed in children (ages seven to ten years) and adults. No significant difference was observed between the two age groups in the half-lives for appearance of salicylamide conjugates in urine. Age-related changes in the metabolic pathways, however. were observed. The mean percentage of dose excreted as salicylamide sulfate was significant!v higher in children (78%) than in adults (36%). In contrast, salicylamide glucuronide was the major excretory product in adults. Similar age-related differences were observed for acetaminophen conjugation. Pharmaeokinetic ana(vsis indicated that the deficiency in glucuronide conjugation of these drugs in children is accompanied bv a higher rate of sulfate/brmation.
S. N. Alam, Ph.D., R. J. Roberts, Ph.D., M.D., and L. J. Fischer, Ph.D.,* I o w a City, I o w a
CONJUGATION of drugs with glucuronic acid and sulfate is usually necessary for their rapid removal from the body through excretion. From limited studies it is thought that by the age of three years children can conjugate drugs and endogenous substances with glucuronic acid at rates similar to adults. Gladtke and Rind ' and Weiss and associates-' reported that the rates of glucuronide conjugation of bilirubin and chloramphenicol, although low at birth, reached the adult level in children by three years of age. Vest :~ reported that the conjugation of p-aminophenol with glucuronic acid in children reached an adult level within the first year. Recent studies by Miller and associates,' however, in which the elimination kinetics of acetaminophen was examined in human subjects of various ages, showed that the rates of glucuronide and sulfate conjugation continued to change through early childhood. The From the Departments of Pharmacology and Pediatrics, The University of Iowa. Supported by United States Public Health Service Grant No. GM 12675 from The National Institutes of Health. *Reprint address: The Toxicologr" Center, Department of Pharmacology, Basic Sciences Building, The University of Iowa, Iowa City. IA 52242.
VoL 90, No. 1, pp. 130-135
percentage of the dose excreted in the urine as acetaminophen sulfate was found to be significantly higher in newborn infants (0 to two days old) and children (three to nine years old) when compared to 12-year-old children and adults. Acetaminophen glucuronide, in contrast, was the major metabolite in the urine of adults and 12-yearold children but accounted for a minor fraction of the dose in younger children and neonates. In an attempt to determine whether age-related differences in sulfate and glucuronide conjugation exist for drugs other than acetaminophen we have investigated the elimination kinetics of salicylamide in children (age seven to ten years) and adults. Salicylamide, a mild analgesic and antipyretic agent, is metabolized in man mainly to glucuronide and sulfate conjugates which are excreted rapidly in urine? -~' Simultaneous administration of a subtherapeutic dose of acetaminophen with salicylamide was employed in this study to assure that the previously observed age-related differences in conjugation rates for acetaminophen were also present in the subjects who participated in this investigation. METHODS Three healthy male volunteers and four normal children (ages seven to ten years) participated in this investi-
Volume 90 Number 1
SalicFlamide and acetaminophen conjugation
13 1
100 Acetaminophen
80 60
40 r ~ l UnchongedDrug
20
[~
GlucuronideConjugote
mR SulfoteConjugate
Age Group Glucuronide to Sulfate Ratio (Mean_+S.E.)
7 -10 Yrs. N=4 022 +0.04
Adults N=3
7-10 Y r s . N=4
1.14+0.02
OB1 +-0.20
Adults N=3 2.3 s
Fig. 1. Excretion of salicylamide, acetaminophen and their glucuronide and sulfate conjugates in the urine (0 to 36 hours) following a concomitant oral dose of 5 mg/kg of each drug in children (seven to ten years) and adults. The ratio of glucuronide to sulfate conjugates of each drug in urine is also presented. An asterisk (*) denotes significant differences (p < 0.05) between age groups.
gation. Consent from the children as well as full informed consent from the parents of the children was obtained. Each subject received a single, subtherapeutic, oral dose ofsalicylamide (5 m s / k s ) as a suspension in Tylenol elixir (acetaminophen 5 m s / k s ) in the morning before breakfast. The dose was followed by 4 to 8 ounces of water to assure complete ingestion of the drugs. Subjects were requested to abstain from food for one hour after the dose. Urine was collected for 36 hours and frozen until the time of analysis. Analytical procedure. Salicylamide and acetaminophen in urine were determined by the gas chromatographic method for acetaminophen described by Miller and associates? The glucuronide and sulfate conjugates of the drugs in urine were determined using selective hydrolysis with bovine fl-glucuronidase (Sigma Chemical, St. Louis, Mo.) and Glusulase (Endo Laboratories, Garden City, N.Y.) followed by gas chromatographic analysis for salicylamide and acetaminophen. 4 Gas liquid chromatographic analysis was performed using a Packard Model 7700 series gas chromatograph equipped with a flame ionization detector. A 6-foot glass column (ID 6 mm) packed with 3% OV-I, 80-100 mesh Supelcoport (Supelco, Inc., Bellefonte, Pa.) was used. Operating conditions were: injector temperature, 250 ~ C; detector temperature, 250 ~ C; column temperature, 170 ~ C; nitrogen carrier gas flow, 45 ml/minute: the air and hydrogen flows were optimized. The retention times of the trimethylsilyl derivatives of acetaminophen, salicylamide, and the internal standard (3-acetaminophenol), under these conditions, were 2.8, 3.6, and 2.3 minutes,
respectively. Gentisamide, a minor metabolite of salicylamide found under certain conditions,; .... did not interfere with the assay because GC-MS analysis of the gas chromatographic peaks representing salicylamide, acetaminophen, and internal standard indicated that they were homogeneous. Pharmacokinetic and statistical analyses were carried out as previously described for acetaminophen by Miller and associates.' The elimination of the drugs and the excretion of the metabolites were assumed to follow the one compartment model proposed by Cummings and associates? ~ In that model, the overall elimination rate constant for salicylamide or acetaminophen is equal to the sum of the individual formation rate constants for glucuronide, sulfate, and any minor metabolites and the excretion rate constant for unchanged drug. The rates of appearance of salicylamide and acetaminophen conjugates in the urine are limited by their rates of formation rather than their relatively rapid rates of renal excretion.'....... According to the model, the half-life of the drug in the body is reflected by the half-lives for appearance of conjugates and unchanged drug in the urine. The pharmacokinetic constants for elimination of the drugs were determined solely from the urinary excretion data according to the methods described by Cummings and associates.' ..... Semilogarithmic ptots of the percent of the dose remaining to be excreted versus time were constructed using data from each subject. The overall elimination rate constant for each subject was determined by linear regression analysis. Some apparently deviant
13 2
A lain, Roberts, and Fischer
The Journal of Pediatrics January 1977
A. ChiLdren
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Hours Afler Dose Fig. 2. Pharmacokinetic plots depicting the urinary excretion of salicylamide (A) and acetaminophen ([2) in children (panel A) and adults (panel B). The percent of the dose remaining to be excreted as glucuronide conjugate, sulfate conjugate and unchanged drug are shown for all subjects. Excretion of unchanged salicylamide was too low to be included in the analysis. The lines drawn through the data points represent the mean of the regression lines calculated for the individuals in each group, Closed data points indicate values not included in the regression analysis (see Methods).
experimental points were excluded from the regression analysis and these are clearly indicated in Fig. 2. The criteria used to exclude points were that they represent less than 1% of the dose and deviate from the other points to an extent that caused a marked change (i.e., > 20%) in the slope of the line. The rationale for elimination of these experimental data points from the regression analysis was that they would have a disproportionate weight in the determination of the slope relative to the very small fraction (i.e,, < 1%) of the dose that they represented. RESULTS Administration of an oral dose of salicylamide and acetaminophen (5 mg/kg of each) resulted in the urinary excretion of the major portion of the dose as the glucu-
ronide and sulfate conjugates with small amounts appearing as the unchanged drugs. Total recovery of salicylamide (mean _ SE) was 95.2 +_ 8,7 for children and 79.0 ___ 7.9 for adults. Although mean recovery of this drug was somewhat higher in children, this was not a statistically significant difference from the recovery in adults. Acetaminophen recoveries (mean _+ SE) were 75.7 +__3.6 and 81.3 +_ 7.1 in the children and adults, respectively, values not significantly different from each other. Fig. 1 shows the mean percentages of the dose of salicylamide and acetaminophen excreted as the unchanged drugs and their glucuronide and sulfate conjugates in children and adults, A very small fraction of the dose was excreted as salicylamide and acetaminophen
Volume 90 Number 1
Salicylamide and acetaminophen conjugation
13 3
Table L Pharmacokinetic constants for the elimination of a 5 m g / k g oral dose of salicylamide and acetaminophen in children and adults
Formation rate constants: (hr ~)for
Half-life (hr)* for appearance in the urine of
Children (7-10 yr) N = 4 Adults N = 3
Salicylamide
Salicvlamide glucuronide
-t -t
1.4 • 0.1 1.3 • 0.1
[ A cetaminophen Children (7-10 yr) N = 4 Adults N = 3
3.5 _+ 0.4 3.4 _+ 0.9
Salicvlamide glucuronide
Salicylamide sulfale
1.3 • 0.2 1.9 _+ 0.1
0.083 • 0.015w 0.185 _+ 0.006
0.379 • 0.047w 0.162 • 0.007
A cetaminophen
A cetaminophen
glucuronide
sulfate
A cetaminophen glucuronide
A cetaminophen sulfate
4.4 _+ 0,1 3.8 • 0+8
5.0 _+ 0.4 4.5 • 0.4
0.048 • 0.007w 0.085 _+ 0.010
0.067 • 0.010 0.038 • 0.007
]
Salicylamide sulfate
*Calculated from the terminal log-linearportions of the individual urinary excretioncurves. Values are mean _+ SE. tNot calculated because of insufficientdata points. :~Calculatedfrom urinary excretiondata by the method of Cummingsand associates"and expressed as the mean _+ SE of the kineticconstantsfor subjectsin each age group. w differentat P < 0.05 from the correspondingadult formation rate constant.
and no significant differences between the two age groups occurred in excretion of the unchanged drugs. There were significant age-related differences in the excretion of the conjugates of both drugs. The percentages of the dose excreted as salicylamide glucuronide and acetaminophen glucuronide were significantly higher in adults than in children. In contrast, the mean percentages of the dose excreted as sulfate conjugates were significantly higher in the children when compared to adults. The smaller fraction of the dose of each drug excreted as a glucuronide conjugate in the children was accompanied by excretion of a correspondingly larger fraction as the sulfate conjugate. Consequently+ the total recoveries of salicylamide and acetaminophen were not significantly different between the two age groups. Fig. 1 also presents the glucuronide/sulfate ratios for both salicylamide and acetaminophen. This ratio indicates the relative importance of the two metabolic pathways in the age-groups studied. The mean glucuronide/ sulfate ratios for salicylamide and acetaminophen in children were significantly lower than the corresponding values for adults. Kinetic analysis of the excretion data from all subjects in each age group is presented in Fig. 2. Examination of these plots reveals a similar pharmacokinetic picture for the drugs in both age groups. Apparent first order kinetics were observed since the semilogarithrnic plots of percent remaining to be excreted versus time exhibited linearity. Excretion of the glucuronide and sulfate conjugates became linear and parallel within one hour after the administration of salicylamide and four hours after acetaminophen. Regardless of age+ over 90% of the
a m o u n t of salicylamide eventually excreted by the subjects had appeared within six hours after administration of the dose. The somewhat slower excretion of acetaminophen was greater than 90% complete by 16 hours. Half-lives for the appearance of the drugs and their conjugates in the urine were calculated using data from each subject. Table I shows the mean half-lives for appearance of the drugs and their glucuronide and sulfate conjugates in the urine of children and adults. Comparison of the values between age groups indicates that no age-related differences in the half-life for each drug were found. This indicates that the overall rate of elimination of salicylamide and acetaminophen from the body was not different in children when compared to adults. The half-lives for the appearance of the glucuronide and sulfate conjugates of salicylamide were the same within each age group. Excretion of acetaminophen and its conjugates showed a similar pattern in that half-lives of the drug and its conjugates were not significantly different within the age groups. This similarity in the half-life for appearance of drug and the two major metabolites in the urine allowed the calculation of formation rate constants for each metabolite (Table I). The formation rate constant for salicylamide glucuronide and acetaminophen glucuronide are significantly lower in children when compared to adults. Formation ofsalicylamide sulfate, in contrast+ is significantly faster in children as reflected by the higher rate constant. This would also seem to be the case for acetaminophen sulfate in children but the higher mean formation rate constant was not significantly different from the adult value. These data show that the rate of
13 4
A lain, Roberts, and Fischer
formation of glucuronide conjugates of salicylamide and acetaminophen was slower in seven- to ten-year-old children than in adults. For salicylamide there was a faster rate of sulfate formation in children. DISCUSSION Differences between children and adults were found in their ability to conjugate these drugs with sulfate and glucuronic acid. In children (ages seven to ten). salicylamide and acetaminophen were excreted predominantly as a sulfate conjugate; whereas in adults, the major metabolite was the glucuronide conjugate. It appears that a deficiency in glucuronide conjugation exists in children but increased sulfate conjugation adequately compensates for this. No differences between age groups, therefore. were observed in the overall elimination rate constants (i.e., apparent half-lives) for these drugs. Thus, there were age-related changes in the metabolic pathways but not in the rate of elimination of the drugs from the body. The time course of the urinary excretion of salicylamide and its major metabolites followed apparent first-order kinetics. Similar apparent first-order kinetics for elimination of salicylamide metabolites have been reported by Song and associates ...... in normal human adults and in patients with pyrogen-induced fever or acute intermittent porphyria. Even though the elimination kinetics of salicylamide appear to be first-order. Levy and Matsuzawa ~ have shown that, even at low doses, there is a dose dependence in the relative rates of sulfate and glucuronic acid conjugation in adults. Rate constants for conjugate formation were calculated in this study to provide an estimate of the quantitative changes occurring with age in the conjugation of a 5 mg/kg oral dose of salicylamide. The values of the formation rate constants so calculated are not likely to be the same as those calculated using other doses of the drug. In contrast to salicylamide, dosedependent conjugation of acetaminophen in adults is not apparent until higher doses of the drug (e.g.. > 2 gin) are given/Thus, the formation rate constants for acetaminophen glucuronide and sulfate calculated for children and adults in this study are not different from those reported by Miller and associates ~using similar age groups and a 10 mg/kg dose of acetaminophen. Observation of similar patterns of age-related development in the conjugation of various substrates have been used as evidence for homogeneity of the enzymatic systems involved?:' On this basis, it would appear that similar, if not identical, sulfotransferase and glucuronyl transferase enzymes are involved in the conjugation of salicylamide and acetaminophen. The observation that the relative changes with age in the formation rate constants for sulfate and glucuronide were the same for
The Journal of Pediatrics Januao~ 1977
each drug provides some support for this speculation. The similar chemical structure of the drugs may allow them to be conjugated in human beings via the same transferase enzymes. It is apparent from studies in laboratory animals, however, that there are several substrate-specific transferases utilized in conjugation to sulfate or glucuronic acid.' ..... The changes with age in the activity of some of these transferase enzymes may be different than the pattern of development seen here for salicylamide and acetaminophen conjugation. Only through studies using other drugs can the rates of development of conjugation pathways after birth in the human being be completely elucidated. The biochemical basis for opposing age-related changes in glucuronide and sulfate conjugation of salicylamide and acetaminophen is not known and remains an intriguing question. Studies with acetaminophen ~showed these changes occurred slowly throughout childhood and. therefore, may not be related to sexual maturation. Although the liver is known to be the most important site for glucuronidation and sulfation.' ..... these processes are also known to take place in gastrointestinal mucosa. -'''-~ The possibility exists that the relative importance of these organs in conjugation processes may be different in children and adults. It is apparent from the data presented here for salicylamide and acetaminophen, and previously for acetaminophen given alone' that an adult pattern for conjugation of these drugs is not reached until late childhood. Miller and associates 4 showed that 12-year-old children had an adultlike pattern of acetaminophen conjugation. ' Although 12-year-old children were not included in the present study, it is reasonable to suggest that they would conjugate salicylamide like adults. Knowledge of the age at which children conjugate various drugs at adult rates has important clinical implications. Conjugation is a primary mechanism for detoxification and excretion of many drugs.-'..... An underdeveloped conjugation process, in the absence of effective alternate metabolic pathways, could lead to enhanced pharmacologic effects or toxic manifestations. Since it has been shown that salicylamide and acetaminophen are not conjugated with glucuronic acid and sulfate at adult rates until late childhood, it is important to determine whether other drugs show a similar developmental pattern for conjugation in human beings. REFERENCES
1. Gladtke E, and Rind H: Bilirubinstoffwechsel beim Neugeborenen, Mschr Kinderheilk 115:231, 1967. 2. Weiss CV, Blaska AJ, and Weston JK: Chloramphenicol in the newborn infant. A physiologic explanation of its toxicity
Volume 90 Number 1
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
when given in excessive doses, N Engl J Med 262:787, 1960. Vest, M: Insufficient glucuronide formation in the newborn and its relationship to the pathogenesis of icterus neonatorium, Arch Dis Child 33:473, 1958. Miller RP, Roberts RJ, and Fischer LJ: Kinetics of acetaminophen elimination in newborns, children and adults, Clin Pharmacol Ther 19:284, 1976. Mandel GH, Rodwell VW, and Smith PK: A study of the metabolism of C"-salicylamide in the human, J Pharmacol Exp Ther 106:433, 1952. Crampton JM, and Voss E: Salicylamide 1. Absorption, excretion and blood levels, J Am Pharm Assoc 43:470. 1954. Levy G, and Matsuzawa T: Pharmacokinetics of salicylamide elimination in man, J Pharmacol Exp Ther 156:285, 1967. Levy G, and Yamada H: Drug biotransformation interactions in man III. Acetaminophen and salicylamide, J Pharm Sci 60:215, 1971. Becher A, Miksch J, Rambacher P, and Sch~ifer A: Uber das verhalten des salicylamid in stoffwechsel des menschen, Klin Wochenschr 30:913, 1952. Song CS, Gelb NA, and Wolff SA: The influence of pyrogen-induced fever on salicylamide metabolism in man, J Clin Invest 52:2959, 1972. Cummings AJ, King ML, and Martin BK: A kinetic study of drug elimination: The excretion of paracetamol and its metabolites in man, Br J Pharmacol 29:150. 1967. Hartiala K, Hasanen A, and Raussi M: The absorption of salicylamide in pernicious anemia, gastric achylia and peptic ulcer, Ann Med Exp Fennial (Helsinki) 41:549, 1963. Cummings AJ, Martin BK, and Park GS: Kinetic considerations relating to the accrual and elimination of drug metabolites, Br J Pharmacol 29:136, 1967. Song CS, Bonkowsky HL, and Tschudy DP: Salicylamide metabolism in acute intermittent porphyria, Clin Pharmacol Ther 15:431. 1974,
Salicylarnide and acetaminophen
conjugation
13 5
15. Dutton D J: The biosynthesis ofglucuronides, in Dutton GJ, editor: Glucuronic acid free and combined, New York, 1966. Academic Press, Inc., p 234. 16. Villar ED, Sanchez E, Autor AP, and Tephly TR: Morphine metabolism IlL Solubilization and separation of morphine and p-nitrophenol uridine diphosphoglucuronyl transferases. Mol Pharmacol 11:236, 1975. 17. Roy AB: Sulphate conjugation enzymes, in Brodie BB, and Gillette JR, editor: Handbook of experimental pharmacology, Vol XXVII/2, New York, 1971, Springer-Verlag, Publisher, p 542. 18. Mettinen TA, and Leskinen E: Glucuronic acid paihway, in Fishman WH, editor: Metabolic conjugation and metabolic hydrolysis, Vol 1, New York, 1970, Academic Press, Inc., p 157. 19. Dodgson KS. and Rose FA: Sulfoconjugation and sulfohydrolysis, in Fishman WH, editor: Metabolic conjugation and metabolic hydrolysis. Vol 1, New York, 1970, Academic Press, Inc., p 239. 20. Barr WH, and Riegelman S: Intestinal drug absorption and metabolism II. Kinetic aspects of intestinal glucuronide conjugation, J Pharm Sci 59:164, 1970. 21. Barr WH and Riegelman S: Intestinal drug absorption and metabolism t. Comparison of methods and models to study physiological factors of in vitro and in vivo intestinal absorption, J Pharm Sci 59:154, 1970. 22. Conway WD, Minatoya H, Lands M. and Shekowski J: Absorption and elimination profile ofisoproterenol Ill. The metabolic fate of dl-isoproterenol-7-:'H in the dog, J Pharm Sci 57:1134, 1968. 23. Josting D, Winne D, and Bock KW: Glucuronidation of paracetamol, morphine and l-naphthol, Biochem Pharmacol 25:613, 1976. 24. Williams RT: Detoxification mechanisms, ed 2, New York, 1959. John Wiley & Sons, Inc., p 279. 25. Williams RT: The biogenesis of conjugation and detoxification products, in Peter Bernfeld. editor: Biogenesis of natural products, New York, 1967, Pergamon Press. Inc, p 589.
130
The Journal of P E D I A T R I C S
Age-related differences in salicylamide and acetaminophen conjugation in man Following a concomitant oral dose of salicylamide and acetaminophen (5 mg/kg of each) the urinary excretion of glucuronide and sulfate conjugates of the drugs were followed in children (ages seven to ten years) and adults. No significant difference was observed between the two age groups in the half-lives for appearance of salicylamide conjugates in urine. Age-related changes in the metabolic pathways, however. were observed. The mean percentage of dose excreted as salicylamide sulfate was significant!v higher in children (78%) than in adults (36%). In contrast, salicylamide glucuronide was the major excretory product in adults. Similar age-related differences were observed for acetaminophen conjugation. Pharmaeokinetic ana(vsis indicated that the deficiency in glucuronide conjugation of these drugs in children is accompanied bv a higher rate of sulfate/brmation.
S. N. Alam, Ph.D., R. J. Roberts, Ph.D., M.D., and L. J. Fischer, Ph.D.,* I o w a City, I o w a
CONJUGATION of drugs with glucuronic acid and sulfate is usually necessary for their rapid removal from the body through excretion. From limited studies it is thought that by the age of three years children can conjugate drugs and endogenous substances with glucuronic acid at rates similar to adults. Gladtke and Rind ' and Weiss and associates-' reported that the rates of glucuronide conjugation of bilirubin and chloramphenicol, although low at birth, reached the adult level in children by three years of age. Vest :~ reported that the conjugation of p-aminophenol with glucuronic acid in children reached an adult level within the first year. Recent studies by Miller and associates,' however, in which the elimination kinetics of acetaminophen was examined in human subjects of various ages, showed that the rates of glucuronide and sulfate conjugation continued to change through early childhood. The From the Departments of Pharmacology and Pediatrics, The University of Iowa. Supported by United States Public Health Service Grant No. GM 12675 from The National Institutes of Health. *Reprint address: The Toxicologr" Center, Department of Pharmacology, Basic Sciences Building, The University of Iowa, Iowa City. IA 52242.
VoL 90, No. 1, pp. 130-135
percentage of the dose excreted in the urine as acetaminophen sulfate was found to be significantly higher in newborn infants (0 to two days old) and children (three to nine years old) when compared to 12-year-old children and adults. Acetaminophen glucuronide, in contrast, was the major metabolite in the urine of adults and 12-yearold children but accounted for a minor fraction of the dose in younger children and neonates. In an attempt to determine whether age-related differences in sulfate and glucuronide conjugation exist for drugs other than acetaminophen we have investigated the elimination kinetics of salicylamide in children (age seven to ten years) and adults. Salicylamide, a mild analgesic and antipyretic agent, is metabolized in man mainly to glucuronide and sulfate conjugates which are excreted rapidly in urine? -~' Simultaneous administration of a subtherapeutic dose of acetaminophen with salicylamide was employed in this study to assure that the previously observed age-related differences in conjugation rates for acetaminophen were also present in the subjects who participated in this investigation. METHODS Three healthy male volunteers and four normal children (ages seven to ten years) participated in this investi-
Volume 90 Number 1
SalicFlamide and acetaminophen conjugation
13 1
100 Acetaminophen
80 60
40 r ~ l UnchongedDrug
20
[~
GlucuronideConjugote
mR SulfoteConjugate
Age Group Glucuronide to Sulfate Ratio (Mean_+S.E.)
7 -10 Yrs. N=4 022 +0.04
Adults N=3
7-10 Y r s . N=4
1.14+0.02
OB1 +-0.20
Adults N=3 2.3 s
Fig. 1. Excretion of salicylamide, acetaminophen and their glucuronide and sulfate conjugates in the urine (0 to 36 hours) following a concomitant oral dose of 5 mg/kg of each drug in children (seven to ten years) and adults. The ratio of glucuronide to sulfate conjugates of each drug in urine is also presented. An asterisk (*) denotes significant differences (p < 0.05) between age groups.
gation. Consent from the children as well as full informed consent from the parents of the children was obtained. Each subject received a single, subtherapeutic, oral dose ofsalicylamide (5 m s / k s ) as a suspension in Tylenol elixir (acetaminophen 5 m s / k s ) in the morning before breakfast. The dose was followed by 4 to 8 ounces of water to assure complete ingestion of the drugs. Subjects were requested to abstain from food for one hour after the dose. Urine was collected for 36 hours and frozen until the time of analysis. Analytical procedure. Salicylamide and acetaminophen in urine were determined by the gas chromatographic method for acetaminophen described by Miller and associates? The glucuronide and sulfate conjugates of the drugs in urine were determined using selective hydrolysis with bovine fl-glucuronidase (Sigma Chemical, St. Louis, Mo.) and Glusulase (Endo Laboratories, Garden City, N.Y.) followed by gas chromatographic analysis for salicylamide and acetaminophen. 4 Gas liquid chromatographic analysis was performed using a Packard Model 7700 series gas chromatograph equipped with a flame ionization detector. A 6-foot glass column (ID 6 mm) packed with 3% OV-I, 80-100 mesh Supelcoport (Supelco, Inc., Bellefonte, Pa.) was used. Operating conditions were: injector temperature, 250 ~ C; detector temperature, 250 ~ C; column temperature, 170 ~ C; nitrogen carrier gas flow, 45 ml/minute: the air and hydrogen flows were optimized. The retention times of the trimethylsilyl derivatives of acetaminophen, salicylamide, and the internal standard (3-acetaminophenol), under these conditions, were 2.8, 3.6, and 2.3 minutes,
respectively. Gentisamide, a minor metabolite of salicylamide found under certain conditions,; .... did not interfere with the assay because GC-MS analysis of the gas chromatographic peaks representing salicylamide, acetaminophen, and internal standard indicated that they were homogeneous. Pharmacokinetic and statistical analyses were carried out as previously described for acetaminophen by Miller and associates.' The elimination of the drugs and the excretion of the metabolites were assumed to follow the one compartment model proposed by Cummings and associates? ~ In that model, the overall elimination rate constant for salicylamide or acetaminophen is equal to the sum of the individual formation rate constants for glucuronide, sulfate, and any minor metabolites and the excretion rate constant for unchanged drug. The rates of appearance of salicylamide and acetaminophen conjugates in the urine are limited by their rates of formation rather than their relatively rapid rates of renal excretion.'....... According to the model, the half-life of the drug in the body is reflected by the half-lives for appearance of conjugates and unchanged drug in the urine. The pharmacokinetic constants for elimination of the drugs were determined solely from the urinary excretion data according to the methods described by Cummings and associates.' ..... Semilogarithmic ptots of the percent of the dose remaining to be excreted versus time were constructed using data from each subject. The overall elimination rate constant for each subject was determined by linear regression analysis. Some apparently deviant
13 2
A lain, Roberts, and Fischer
The Journal of Pediatrics January 1977
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Hours Afler Dose Fig. 2. Pharmacokinetic plots depicting the urinary excretion of salicylamide (A) and acetaminophen ([2) in children (panel A) and adults (panel B). The percent of the dose remaining to be excreted as glucuronide conjugate, sulfate conjugate and unchanged drug are shown for all subjects. Excretion of unchanged salicylamide was too low to be included in the analysis. The lines drawn through the data points represent the mean of the regression lines calculated for the individuals in each group, Closed data points indicate values not included in the regression analysis (see Methods).
experimental points were excluded from the regression analysis and these are clearly indicated in Fig. 2. The criteria used to exclude points were that they represent less than 1% of the dose and deviate from the other points to an extent that caused a marked change (i.e., > 20%) in the slope of the line. The rationale for elimination of these experimental data points from the regression analysis was that they would have a disproportionate weight in the determination of the slope relative to the very small fraction (i.e,, < 1%) of the dose that they represented. RESULTS Administration of an oral dose of salicylamide and acetaminophen (5 mg/kg of each) resulted in the urinary excretion of the major portion of the dose as the glucu-
ronide and sulfate conjugates with small amounts appearing as the unchanged drugs. Total recovery of salicylamide (mean _ SE) was 95.2 +_ 8,7 for children and 79.0 ___ 7.9 for adults. Although mean recovery of this drug was somewhat higher in children, this was not a statistically significant difference from the recovery in adults. Acetaminophen recoveries (mean _+ SE) were 75.7 +__3.6 and 81.3 +_ 7.1 in the children and adults, respectively, values not significantly different from each other. Fig. 1 shows the mean percentages of the dose of salicylamide and acetaminophen excreted as the unchanged drugs and their glucuronide and sulfate conjugates in children and adults, A very small fraction of the dose was excreted as salicylamide and acetaminophen
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Salicylamide and acetaminophen conjugation
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Table L Pharmacokinetic constants for the elimination of a 5 m g / k g oral dose of salicylamide and acetaminophen in children and adults
Formation rate constants: (hr ~)for
Half-life (hr)* for appearance in the urine of
Children (7-10 yr) N = 4 Adults N = 3
Salicylamide
Salicvlamide glucuronide
-t -t
1.4 • 0.1 1.3 • 0.1
[ A cetaminophen Children (7-10 yr) N = 4 Adults N = 3
3.5 _+ 0.4 3.4 _+ 0.9
Salicvlamide glucuronide
Salicylamide sulfale
1.3 • 0.2 1.9 _+ 0.1
0.083 • 0.015w 0.185 _+ 0.006
0.379 • 0.047w 0.162 • 0.007
A cetaminophen
A cetaminophen
glucuronide
sulfate
A cetaminophen glucuronide
A cetaminophen sulfate
4.4 _+ 0,1 3.8 • 0+8
5.0 _+ 0.4 4.5 • 0.4
0.048 • 0.007w 0.085 _+ 0.010
0.067 • 0.010 0.038 • 0.007
]
Salicylamide sulfate
*Calculated from the terminal log-linearportions of the individual urinary excretioncurves. Values are mean _+ SE. tNot calculated because of insufficientdata points. :~Calculatedfrom urinary excretiondata by the method of Cummingsand associates"and expressed as the mean _+ SE of the kineticconstantsfor subjectsin each age group. w differentat P < 0.05 from the correspondingadult formation rate constant.
and no significant differences between the two age groups occurred in excretion of the unchanged drugs. There were significant age-related differences in the excretion of the conjugates of both drugs. The percentages of the dose excreted as salicylamide glucuronide and acetaminophen glucuronide were significantly higher in adults than in children. In contrast, the mean percentages of the dose excreted as sulfate conjugates were significantly higher in the children when compared to adults. The smaller fraction of the dose of each drug excreted as a glucuronide conjugate in the children was accompanied by excretion of a correspondingly larger fraction as the sulfate conjugate. Consequently+ the total recoveries of salicylamide and acetaminophen were not significantly different between the two age groups. Fig. 1 also presents the glucuronide/sulfate ratios for both salicylamide and acetaminophen. This ratio indicates the relative importance of the two metabolic pathways in the age-groups studied. The mean glucuronide/ sulfate ratios for salicylamide and acetaminophen in children were significantly lower than the corresponding values for adults. Kinetic analysis of the excretion data from all subjects in each age group is presented in Fig. 2. Examination of these plots reveals a similar pharmacokinetic picture for the drugs in both age groups. Apparent first order kinetics were observed since the semilogarithrnic plots of percent remaining to be excreted versus time exhibited linearity. Excretion of the glucuronide and sulfate conjugates became linear and parallel within one hour after the administration of salicylamide and four hours after acetaminophen. Regardless of age+ over 90% of the
a m o u n t of salicylamide eventually excreted by the subjects had appeared within six hours after administration of the dose. The somewhat slower excretion of acetaminophen was greater than 90% complete by 16 hours. Half-lives for the appearance of the drugs and their conjugates in the urine were calculated using data from each subject. Table I shows the mean half-lives for appearance of the drugs and their glucuronide and sulfate conjugates in the urine of children and adults. Comparison of the values between age groups indicates that no age-related differences in the half-life for each drug were found. This indicates that the overall rate of elimination of salicylamide and acetaminophen from the body was not different in children when compared to adults. The half-lives for the appearance of the glucuronide and sulfate conjugates of salicylamide were the same within each age group. Excretion of acetaminophen and its conjugates showed a similar pattern in that half-lives of the drug and its conjugates were not significantly different within the age groups. This similarity in the half-life for appearance of drug and the two major metabolites in the urine allowed the calculation of formation rate constants for each metabolite (Table I). The formation rate constant for salicylamide glucuronide and acetaminophen glucuronide are significantly lower in children when compared to adults. Formation ofsalicylamide sulfate, in contrast+ is significantly faster in children as reflected by the higher rate constant. This would also seem to be the case for acetaminophen sulfate in children but the higher mean formation rate constant was not significantly different from the adult value. These data show that the rate of
13 4
A lain, Roberts, and Fischer
formation of glucuronide conjugates of salicylamide and acetaminophen was slower in seven- to ten-year-old children than in adults. For salicylamide there was a faster rate of sulfate formation in children. DISCUSSION Differences between children and adults were found in their ability to conjugate these drugs with sulfate and glucuronic acid. In children (ages seven to ten). salicylamide and acetaminophen were excreted predominantly as a sulfate conjugate; whereas in adults, the major metabolite was the glucuronide conjugate. It appears that a deficiency in glucuronide conjugation exists in children but increased sulfate conjugation adequately compensates for this. No differences between age groups, therefore. were observed in the overall elimination rate constants (i.e., apparent half-lives) for these drugs. Thus, there were age-related changes in the metabolic pathways but not in the rate of elimination of the drugs from the body. The time course of the urinary excretion of salicylamide and its major metabolites followed apparent first-order kinetics. Similar apparent first-order kinetics for elimination of salicylamide metabolites have been reported by Song and associates ...... in normal human adults and in patients with pyrogen-induced fever or acute intermittent porphyria. Even though the elimination kinetics of salicylamide appear to be first-order. Levy and Matsuzawa ~ have shown that, even at low doses, there is a dose dependence in the relative rates of sulfate and glucuronic acid conjugation in adults. Rate constants for conjugate formation were calculated in this study to provide an estimate of the quantitative changes occurring with age in the conjugation of a 5 mg/kg oral dose of salicylamide. The values of the formation rate constants so calculated are not likely to be the same as those calculated using other doses of the drug. In contrast to salicylamide, dosedependent conjugation of acetaminophen in adults is not apparent until higher doses of the drug (e.g.. > 2 gin) are given/Thus, the formation rate constants for acetaminophen glucuronide and sulfate calculated for children and adults in this study are not different from those reported by Miller and associates ~using similar age groups and a 10 mg/kg dose of acetaminophen. Observation of similar patterns of age-related development in the conjugation of various substrates have been used as evidence for homogeneity of the enzymatic systems involved?:' On this basis, it would appear that similar, if not identical, sulfotransferase and glucuronyl transferase enzymes are involved in the conjugation of salicylamide and acetaminophen. The observation that the relative changes with age in the formation rate constants for sulfate and glucuronide were the same for
The Journal of Pediatrics Januao~ 1977
each drug provides some support for this speculation. The similar chemical structure of the drugs may allow them to be conjugated in human beings via the same transferase enzymes. It is apparent from studies in laboratory animals, however, that there are several substrate-specific transferases utilized in conjugation to sulfate or glucuronic acid.' ..... The changes with age in the activity of some of these transferase enzymes may be different than the pattern of development seen here for salicylamide and acetaminophen conjugation. Only through studies using other drugs can the rates of development of conjugation pathways after birth in the human being be completely elucidated. The biochemical basis for opposing age-related changes in glucuronide and sulfate conjugation of salicylamide and acetaminophen is not known and remains an intriguing question. Studies with acetaminophen ~showed these changes occurred slowly throughout childhood and. therefore, may not be related to sexual maturation. Although the liver is known to be the most important site for glucuronidation and sulfation.' ..... these processes are also known to take place in gastrointestinal mucosa. -'''-~ The possibility exists that the relative importance of these organs in conjugation processes may be different in children and adults. It is apparent from the data presented here for salicylamide and acetaminophen, and previously for acetaminophen given alone' that an adult pattern for conjugation of these drugs is not reached until late childhood. Miller and associates 4 showed that 12-year-old children had an adultlike pattern of acetaminophen conjugation. ' Although 12-year-old children were not included in the present study, it is reasonable to suggest that they would conjugate salicylamide like adults. Knowledge of the age at which children conjugate various drugs at adult rates has important clinical implications. Conjugation is a primary mechanism for detoxification and excretion of many drugs.-'..... An underdeveloped conjugation process, in the absence of effective alternate metabolic pathways, could lead to enhanced pharmacologic effects or toxic manifestations. Since it has been shown that salicylamide and acetaminophen are not conjugated with glucuronic acid and sulfate at adult rates until late childhood, it is important to determine whether other drugs show a similar developmental pattern for conjugation in human beings. REFERENCES
1. Gladtke E, and Rind H: Bilirubinstoffwechsel beim Neugeborenen, Mschr Kinderheilk 115:231, 1967. 2. Weiss CV, Blaska AJ, and Weston JK: Chloramphenicol in the newborn infant. A physiologic explanation of its toxicity
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15. Dutton D J: The biosynthesis ofglucuronides, in Dutton GJ, editor: Glucuronic acid free and combined, New York, 1966. Academic Press, Inc., p 234. 16. Villar ED, Sanchez E, Autor AP, and Tephly TR: Morphine metabolism IlL Solubilization and separation of morphine and p-nitrophenol uridine diphosphoglucuronyl transferases. Mol Pharmacol 11:236, 1975. 17. Roy AB: Sulphate conjugation enzymes, in Brodie BB, and Gillette JR, editor: Handbook of experimental pharmacology, Vol XXVII/2, New York, 1971, Springer-Verlag, Publisher, p 542. 18. Mettinen TA, and Leskinen E: Glucuronic acid paihway, in Fishman WH, editor: Metabolic conjugation and metabolic hydrolysis, Vol 1, New York, 1970, Academic Press, Inc., p 157. 19. Dodgson KS. and Rose FA: Sulfoconjugation and sulfohydrolysis, in Fishman WH, editor: Metabolic conjugation and metabolic hydrolysis. Vol 1, New York, 1970, Academic Press, Inc., p 239. 20. Barr WH, and Riegelman S: Intestinal drug absorption and metabolism II. Kinetic aspects of intestinal glucuronide conjugation, J Pharm Sci 59:164, 1970. 21. Barr WH and Riegelman S: Intestinal drug absorption and metabolism t. Comparison of methods and models to study physiological factors of in vitro and in vivo intestinal absorption, J Pharm Sci 59:154, 1970. 22. Conway WD, Minatoya H, Lands M. and Shekowski J: Absorption and elimination profile ofisoproterenol Ill. The metabolic fate of dl-isoproterenol-7-:'H in the dog, J Pharm Sci 57:1134, 1968. 23. Josting D, Winne D, and Bock KW: Glucuronidation of paracetamol, morphine and l-naphthol, Biochem Pharmacol 25:613, 1976. 24. Williams RT: Detoxification mechanisms, ed 2, New York, 1959. John Wiley & Sons, Inc., p 279. 25. Williams RT: The biogenesis of conjugation and detoxification products, in Peter Bernfeld. editor: Biogenesis of natural products, New York, 1967, Pergamon Press. Inc, p 589.
