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1. T h e metabolism of a 900 mg oral dose of aspirin has been investigated in 129 healthy
volunteers. For this purpose, the 0-12 h urine was collected and analysed for the following
excretion products: salicylic acid, its acyl and phenolic glucuronides, salicyluric acid, its
phenolic glucuronide and gentisic acid.
2. T h e total excretion of salicylate and metabolites was normally distributed within the
population group studied, showing a 2.5-fold variation: a mean of 68.1 yo of the dose was
recovered in 12 h .
3. T h e excretion of salicylic acid was found to be highly variable within the study panel
(1.3-31’), of dose in 12 h), and was related to both urine volume and pH.
4. Salicyluric acid was the major metabolite in the majority of the volunteers and its
excretion was normally distributed amongst the study panel. T h e elimination of this
metabolite ranged from 19.8 to 65O;, of the dose and was related to the total recovery of
For personal use only.
salicylate.
5. T h e excretion of the two salicyl glucuronides was highly variable, ranging from 0.8 to
42YA of the dose. The elimination of the glucuronides was inversely related to that of
salicyluric acid.
6. Gentisic acid and salicyluric acid phenolic glucuronide were minor metabolites of
salicylate, accounting for 1 and 3?, of the dose, respectively.
7. T h e recovery of gentisic acid was statistically significantly greater in female subjects
than in males, whilst the opposite was found for salicyluric acid and total salicylate.
However, these differences were small in magnitude.
Introduction
Variability in metabolism is known to be an important determinant of an
individual’s response to a drug. There is thus considerable interest in identifying,
and where possible quantifying, the significance of discrete factors as sources of
human variation in drug metabolism and response. It is accepted that inter-
individual variations in biotransformation have their origins in differences between
subjects in the nature and activities of the various drug-metabolizing enzymes,
which are subject to regulation by a plethora of genetic, environmental, physiological
and pathological factors.
Inter-individual variability in drug metabolism has been almost exclusively
studied from the viewpoint of the oxidative reactions, and very little attention has
been paid to the metabolic conjugation reactions. Variability in the activity of these
latter pathways may be more complicated, as factors governing the supply of the
endogenous conjugating agent will be involved in addition to those governing
enzymic activity.
factors have been associated with this variability, including gender (Menguy et al.
1972, Rainsford et al. 1980, Miaskiewicz, Shively and Vesell 1982), age (Montgom-
ery and Sitar 1981), genetic factors (Furst, Gupta and Paulas 1977), disease
processes (Kapp and Coburn 1942, Graham et al. 1977), auto-induction (Furst et al.
1977, Day, Shen and Azarnoff 1983) and glycine availability (Notarianni et al. 1983).
Of particular note is the study of Furst et al. (1977), who showed, using pairs of
identical twins, that genetic factors were significant in determining inter-individual
differences in salicylate disposition. However, an earlier study showed that the
plasma level of salicylate following administration of sodium salicylate to tubercu-
lous patients was found to be normally distributed amongst the study population
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(Evans 1960, quoted in Evans and Clarke 1961), suggesting that the existence of a
major polymorphism in the metabolism of salicylate is unlikely. However, taken
alone, this conclusion must be treated with some caution, as there do occur instances
of apparently normal distributions concealing polymorphically distributed variables
among the population.
Recently, in a preliminary study, Caldwell et al. (1980) have found the
Hydrolysis
Salicylic (2-hydroxybenzoic) acid was obtained from the Pharmacy of S t Mary’s Hospital (London
W2, UK). The following compounds were purchased: aspirin (acetylsalicylic acid), gentisic (2,s-
dihydroxybenzoic) acid (Sigma Chemical Co., Poole, Dorset, UK); salicyluric (2-hydroxyhippuric) acid,
2-methoxybenzoic acid, hydroxylamine hydrochloride (Aldrich Chemical Co., Gillingham, Dorset,
UK); salicylhydroxamic acid (Koch-Light Laboratories, Colnbrook, Middlesex, UK). Soluble aspirin
tablets B.P., containing 300mg aspirin (Disprin, Reckitt and Colman, Hull, UK) were supplied by the
Pharmacy of St Mary’s Hospital.
Human investigation
The investigation used a panel of healthy volunteers, drawn from the students and staff of St Mary’s
Hospital and Medical School, made up of 67 males and 62 females, ages 19-35 years (mean 21.1), body wt
41-92 kg (mean 65.4). The subjects had taken no drugs for at least seven days before their participation in
the study; no other exclusion criteria were applied.
Each subject took a single oral dose of 900 mg aspirin dissolved in water (3 x 300 mg soluble aspirin
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tablets B.P., equivalent to 690mg salicyclic acid) at 9 a.m. following a light breakfast. They collected their
urine for 12 h after dosing. The volume and pH of the sample was recorded at the time of collection, and
samples stored at -20°C until analysed.
The study was approved by the local Ethics Committee, and all volunteers gave their informed consent
to the experimental procedure.
Results
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% TOTFlL RECOVERY
Figure 2. Frequency distribution of the total recovery of salicylate and metabolites in the 0-12 h urine
following a 900mg oral dose of aspirin to 129 subjects.
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10 20 30 40 50
% DOSE EXCRETED FlS SRLICYLIC RCIO
Figure 3. Frequency distribution of the excretion of salicylic acid in the 0-12h urine of 129 subjects
given an oral dose of 900 mg aspirin.
Table 1 presents quantitative details of the urinary excretion of salicylate and its
metabolites in this panel of subjects.
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0 20 40 60 80 100
% DOSE EXCRETED f l S S A L I C Y L U R I C A C I D
Figure 4. Frequency distribution of the excretion of salicyluric acid in the 0-12 h urine of 129 subjects
given an oral dose of 900 mg aspirin.
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0 10 20 30 40 50
% DOSE EXCRETED RS SRLICYL PHENOLIC GLUCURONIDE
Figure 6. Frequency distribution of the recovery of salicyl phenolic glucuronide in the 0-12 h urine of
129 volunteers given an oral dose of 900 mg aspirin.
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Table 1. Urinary excretion of salicylate and metabolites in the 0-12 h urine of 129 healthy volunteers
each given 900mg aspirin orally.
yo Dose excreted
Number of subjects in
Metabolite which metabolite
detected Mean fS.D. Range
?Values for salicyl phenolic glucuronide were calculated as outlined in the text.
salicyl acyl glucuronide (R(s)-0.338, P<O.OOl), which may be due to the more
facile hydrolysis of ester glucuronides under mild alkaline conditions (Caldwell et al.
1983). NQother statistically significant relationships were revealed by this analysis of
the data.
Gender. T h e excretion of salicylate and its metabolites in the two sexes was
compared by the unpaired Student t test, and the data are presented in table 2.
Females excreted significantly more gentisic acid than males, while the reverse was
the case for salicyluric acid. T h e total recovery of salicylate and metabolites was
greater in males, presumably due to the increased excretion of salicylurate. No
246 A. J . Hutt et al.
Table 2. Sex differences in the urinary excretion of salicylate and metabolites in the G12 h urine of 129
healthy volunteers given 900 mg aspirin orally.
% Dose excreted in
(mean+S.D.)
Salicyl phenolic
glucuronide 11 +
8.0 8.2 6.3k 6.8 ns.
Total salicylate 70.2k11.6 65.9+12.0 0.05 > P> 0.02
+
t N = 6 7 ; mean age, 21.3 3.4 years; mean weight, 73.0f9.0kg; mean urine val., 843 +458ml; mean
pH value, 6.5 f0.7.
$ N = 6 2 ; mean age, 21.0f3.6 years; mean weight, 57.1 k 6 . 1 kg; mean urine vol., 813k376ml; mean
pH value, 6.3 kO.5.
§Unpaired t test, level of significance P=O.O5.
I/Salicyl phenolic glucuronide values were calculated as outlined in the text.
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statistically significant differences at the P= 0.05 level were observed between the
sexes in the excretion of salicylic acid or its glucuronides.
Competing metabolic options. The major routes of salicylic acid metabolism are
conjugation with glycine at the -COOH group, and glucuronidation at either the
-COOH or -OH groups. The formation of the two glucuronides appears to
represent the principal metabolic alternative to glycine conjugation, which nearly
always predominates. In only five of the 129 subjects in this study did the sum of the
two glucuronides exceed the percentage dose eliminated as salicyluric acid. T h e sum
of both the salicyl glucuronides ranges from 0.8 to 41.6% (mean 12.3%) of the dose,
and in only one individual was neither detected. It must be stressed that this subject
did excrete 5.5yoof the administered dose as the glucuronide of salicyluric acid.
Spearman rank correlation revealed an inverse relationship between salicyluric acid
elimination and the excretion of the two glucuronides (R(s)-0.389, P<O.OOl). T h e
excretion of salicyluric acid was related to the total recovery of salicylate and
metabolites (R(s)0.675, P<O.OOl),which is to be expected since salicyluric acid is
nearly always the major excretion product. However, the sum of the two glu-
curonides was unrelated to the total elimination of salicylate.
Discussion
The findings presented in this paper show that the excretion of salicylate and its
metabolites following the administration of aspirin to a panel of 129 healthy
volunteers is highly variable. This confirms both expectations from the literature
based on small groups of subjects (for references, see Introduction) and a previous
report from this laboratory based on a separate group of 85 volunteers (Caldwell,
O’Gorman and Smith 1980). The variability within the population, like that in our
previous preliminary report (Caldwell et al. 1980), was normally distributed.
As would be expected from the literature, the major metabolite was the glycine
conjugate, salicyluric acid, which was excreted principally free but also to a small
Population study of aspirin metabolism 247
showed a skewed normal distribution. However, the distribution of the sum of these
two glucuronides did not follow a normal distribution by probit analysis (data not
shown). T h e significance of this is unclear. T h e total formation of both these
glucuronides (acyl plus phenolic) was inversely related to the elimination of
salicyluric acid, indicating that these two reactions are alternative metabolic options
for the salicylic acid molecule. T h e formation of the acyl glucuronide was always less
than that of the glycine conjugate, both metabolites involving conjugation of the
-COOH group. T h e glucuronides are formed by the UDP-glucuronyl transfer-
ases located in the endoplasmic reticulum (Kasper and Henton 1980) whereas the
glycine-conjugating mechanisms are found in the mitochondria (Killenberg and
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Webster 1980), and the choice between these two alternative options may involve
aspects of the subcellular distribution of salicylate (see Dixon, Caldwell and Smith
1977).
T h e two glucuronides of salicylate are also noteworthy from the analytical
viewpoint. Authentic standards of these compounds are not available, and the
majority of the work reported in the literature has not attempted to differentiate
between these metabolites of salicylate. T h e present report would appear to be the
first to do so since the work of Levy some 1 2 years ago (Levy, Tsuchiya and Amsel
1972).
Acyl glucuronides are known to be unstable, undergoing both hydrolysis to the
aglycone and glucuronic acid and the interesting phenomenon of acyl migration, in
which the biosynthetic 1-0-acyl glucopyranosiduronates undergo intramolecular
rearrangement in mild (pH 7 and above) alkaline conditions to yield the correspond-
ing 8-glucuronidase-resistant2-, 3- and 4-glucuronic acid esters (see Heirwegh and
Compernolle 1979, Sinclair and Caldwell1982, Caldwell et al. 1983). T h e use here of
hydroxamic acid formation for the analysis of the ester glucuronide, rather than
reliance upon enzymic hydrolysis, obviates analytical problems which might be
caused by the migration of the salicyl moiety around the sugar ring. Care was also
taken to prevent errors due to the spontaneous hydrolysis of the acyl glucuronide by
either analysing the sample immediately upon collection, or by freezing at - 20°C.
The phenolic glucuronide was estimated by difference, following the hydrolysis of
metabolites with concentrated acid, an approach validated by our previous study of
the fate of I4C-aspirin in man (Hutt et al. 1982).
The oxidation product gentisic acid was found to be a minor metabolite of
aspirin, in agreement with the results of Levy et al. (1972) and Bochner et al. (1981).
T h e excretion of salicylic acid by these volunteers was highly variable, and in this
case the variability was related to the urinary p H and volume. It is important to
appreciate that the metabolic fate of that fraction of the salicylate dose which does
A. J. Hutt et al.
The influence of gender upon the fate of aspirin is the subject of controversy in
the literature (see literature cited in the Introduction). This study shows the existence
in this panel of small but statistically significant differences between the sexes in the
elimination of gentisic acid, salicyluric acid and its phenolic glucuronide, and total
salicylate (see table 2). These differences just may be relevant to the higher incidence
of aspirin intolerance in females (Reynolds 1982).
It is likely that the explanation for the observed inter-individual differences in
aspirin metabolism lies in differences in the relative activities of the major metabolic
pathways open to the salicylic acid molecule, which in turn presumably arise from
differences in the activities of the enzymes catalysing the reactions. This is hard to
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assess on the basis of the present data, as the various routes of metabolism exhibit
different kinetic characteristics: thus, at the present dose level (a reasonable one in
terms of the intermittent use of aspirin as an analgesic/anti-inflammatory), studies in
very small numbers of subjects have shown that the glycine conjugation and phenolic
glucuronidation follow zero-order (i.e., capacity-limited) kinetics, while the other
pathways follow first-order kinetics (see Levy 1979, Bochner et al. 1981). It appears
from the present data that the activities of the individual metabolic pathways vary
independently, and that the extensive elimination of one product does not necessari-
ly result in a low excretion of others. It is apparent that the percentage dose
eliminated as salicylic acid has little impact on the pattern of disposition of that
fraction of the dose undergoing metabolism. However, there was a poor
(R(s)- 0.39) but significant ( P <0.001) inverse relationship between the elimin-
ation of salicyluric acid and that of salicyl glucuronides. No other rela-
tionships emerged upon rank correlation of the excretion of salicylic acid with
that of the various metabolites. T h e present paper has defined the nature and extent
of the inter-individual differences in the handling of aspirin by a group of 129 young
healthy volunteers, and the kinetic aspects of this are addressed in a subsequent
paper (Hutt et al. to be submitted).
Acknowledgements
This work was supported by a grant from the Sir Halley Stewart Trust. We are
grateful to D r N. S. Oates for help with statistical analysis.
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