Original article

Aspirin esterase activity Evidence for skewed distribution in

healthy volunteers
G.I. Adebayo
a,

, J. Williams
b
, S. Healy
a
a
Department of Medicine, Sligo General Hospital, The Mall, Sligo, Ireland
b
Department of Biochemistry, Sligo General Hospital, The Mall, Sligo, Ireland
Received 13 February 2006; received in revised form 26 June 2006; accepted 14 December 2006
Abstract
Background: Aspirin, with its analgesic, anti-inflammatory, antipyretic, and anti-platelet actions, is one of the most frequently used drugs.
Although its use as prophylaxis against thromboembolism is well established, an optimal dose, conferring maximal anti-platelet action
without increased risk of bleeding, remains elusive.
Method: We assessed the possible pharmacokinetic contribution to this problem in 107 healthy, non-medicated volunteers. Serum aspirin
esterase activity was evaluated at 37 C with 1 mM aspirin as substrate. On the basis of the report that most of aspirin esterase activity is
accounted for by pseudocholinesterase, we additionally quantified the activity of this enzyme, with and without dibucaine as an inhibitor,
using Ellman's reaction, in 41 of our volunteers.
Results: Aspirin esterase activities in all of our volunteers (33.90 nmol/ml/min to 222.65 nmol/ml/min, median 103.45 nmol/ml/min) showed
a continuous and skewed distribution with eight outliers. In the 41 subjects so studied, aspirin esterase activities correlated positively with
both pseudocholinesterase activities (Spearman's rho=0.593, pb0.001) and dibucaine numbers (Spearman's rho=0.422, pb0.01).
Conclusions: Our results support previous observations that the rate of aspirin hydrolysis is not determined by aspirin esterase alone and that
other factors are probably involved. Additionally, the skewed distribution of aspirin esterase activities makes a case for its possible
contribution to the phenomenon of aspirin resistance.
2007 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
Keywords: Aspirin; Aspirin esterase; Aspirin resistance; Pseudocholinesterase
1. Introduction
Since its synthesis in 1887 by Felix Hoffmann, acetylsa-
licylic acid, otherwise known as aspirin, has been used as an
analgesic, anti-inflammatory, and antipyretic drug. It also has
anti-platelet action, and its use as prophylaxis against
thromboembolism is well established [1].
Despite its universal usage, aspirin is not an innocuous
drug. Of the recognised adverse reactions to aspirin [2],
haemorrhagic complication is the antithesis to its use against
thromboembolism. Used as such, the ideal dose would be the
one which, while providing maximal anti-platelet action,
confers no risk of haemorrhage. Such an optimal dose,
however, remains unknown.
There was about seven-fold variation in doses employed
in the five large studies that have assessed the efficacy of
aspirin in the primary prevention of cardiovascular events. A
dose of 500 mg/day was employed in the British Male
Doctors' Trial [3], and while 325 mg on alternate days and
100 mg daily were used, respectively, in the Physicians'
Health Study [4] and the Primary Prevention Project trial [5],
a 75-mg daily was the chosen dose in both the Hypertension
Optimal Trial [6] and the Thrombosis Prevention Trial [7].
More recently, there has been a suggestion that a dose as low
as 30 mg daily may be effective [8].
Given the fact that the anti-platelet effect of aspirin is due
to the intact molecule, it is possible that its metabolism could
be contributory to the range of effective doses employed in
European Journal of Internal Medicine 18 (2007) 299303
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Corresponding author.
E-mail address: gani@eircom.net (G.I. Adebayo).
0953-6205/$ - see front matter 2007 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.ejim.2006.12.004
different studies, as well as to the difficulty in identifying an
optimal dose. To assess this possibility, we studied the break-
down of aspirin in a sizeable number of normal volunteers.
2. Materials and methods
One hundred and seven non-medicated volunteers (39
males) participated after informed consent. Their average
age was 30.6 years (range 1480 years) and their average
weight was 67.8 kg (44114.6 kg). The study had the
approval of our hospital ethics committee. The volunteers
were recruited as previously reported [9].
Serum aspirin esterase activity at 37 C was quantified as
described by Williams et al. [10] on the same day of blood
sampling; otherwise, the sample was frozen and the activity
quantified within a week. Then, 1 mM of acetylsalicylic acid,
freshly prepared, was incubated with 200 l of serum in 3 ml
of tris calcium buffer, pH 7.4, for 20 min. The salicylate
produced was quantified by absorbance reading at 300 nm
on a model U-2000 Hitachi spectrophotometer.
Given the report that plasma cholinesterase catalyses
aspirin hydrolysis [11], we deemed it appropriate to addi-
tionally quantify the activity of the enzyme. This was done in
41 of our volunteers using the method of Dietz et al. [12].
Data analysis was carried out using the SPSS statistical
package.
3. Results
The intra-assay coefficient of variation with the sample of
mean activity of 73.40 nmol/ml serum/min (n=4) was 4.8%
and the inter-assay value (n=4 over a week) was 5.1%. The
corresponding values for pseudocholinesterase were as
previously reported [9].
Aspirin esterase activity in all of our volunteers ranged from
33.90 to 222.65 nmol/ml/min (median 103.45 nmol/ml/min).
A histogram plot of these values suggests some degree of
positive skewness (Fig. 1a), and the deviation from normality
is supported by the corresponding normal plot (Fig. 1b).
Aspirin esterase activities in females (33.90199.00 nmol/
ml/min) were lower than those in males (40.20222.65 nmol/
ml/min, pb0.005).
Fig. 1. Aspirin esterase activity (nmol/ml/min at 37 C) in 107 healthy
volunteers expressed as (a) frequency histogram and (b) normal plot.
Fig. 2. Relationship between age and aspirin esterase activity in 107 non-
medicated volunteers.
300 G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299303
In the 41 volunteers so studied, pseudocholinesterase
activities were 3.0910.53 U/ml (meanSD, 6.531.64 U/
ml). Aspirin esterase activities in this subgroup ranged
from 40.20 to 222.65 nmol/ml/min (median=105.65 nmol/
ml/min).
4. Discussion
Our finding of significantly lower aspirin esterase activity
in females is at variance with that of Williams et al. [10], who
noted no gender difference in the activities of the enzyme. It
is, however, in accord with the report by Menguy et al. [13],
who suggested it as a possible reason why women seem
more prone to develop one of the adverse reactions to the use
of aspirin, namely gastric ulceration.
Data from our volunteers suggest a possible lack of
normal distribution of aspirin esterase activity. Eight values
were outside the range 34.05 nmol/ml/min to 175.05 nmol/
ml/min, representing mean2 SD of aspirin esterase
activities in our 107 volunteers. Of these outliers, the one
less than 34.05 nmol/ml/min was in a female. Six of the
Fig. 3. Frequency histogram (a) and normal plot (b) of pseudocholinesterase
activity (mmol/ml/min at 37 C) in 41 healthy volunteers.
Fig. 4. Frequency histogram (a) and normal plot (b) of aspirin esterase
activity (nmol/ml/min at 37 C) in 41 healthy volunteers.
301 G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299303
seven greater than 175.05 nmol/ml/min were in males,
possibly reflecting the generally higher level of activity in
males in this study. The distribution is unlikely to be age-
dependent, given the lack of a significant relationship bet-
ween this parameter and aspirin esterase activity (Fig. 2).
This is in consonance with the reports by other workers
[10,14]. The pattern of distribution rules out a genetic poly-
morphism but is supportive of the fact that differences in
aspirin metabolism are probably multi-factorial (Fig. 1a).
Both albumin and pseudocholinesterase are capable of
catalysing hydrolysis of aspirin [11]. We did not measure
plasma albumin in any of our volunteers. However, while, as
expected, pseudocholinesterase activities were normally
distributed in 41 of our volunteers so studied (Fig. 3a and b),
the same cannot be said of aspirin esterase activities in the
same group of people (Fig. 4a and b). Despite this discordance
in the patterns of distribution, assessment of a possible asso-
ciation between pseudocholinesterase and aspirin esterase
activities revealed a modest but significant correlation (Spear-
man's rho=0.593; pb0.001). Because percentage inhibition
by dibucaine, dibucaine number, reveals variants of pseudo-
cholinesterase that are otherwise unidentifiable on the basis of
catalytic activities alone, we evaluated the association between
this parameter and aspirin esterase activity. The result
Spearman's rho=0.422; pb0.01 is not superior to that
between pseudocholinesterase and aspirin esterase activities.
Surprising though this may be, it could be due, at least in part,
to the fact that although inhibition by dibucaine is routinely
employed to identify pseudocholinesterase variants, it is not
very specific, R02-0683 having been used to differentiate
variants of the enzyme indistinguishable by dibucaine number
values [15,16]. Given the fact that the catalytic activity of
albumin is minor compared to that of pseudocholinesterase
[11], these observations could well mean, as previously
suggested [17], that other factors are probably operative in
determining the rate of aspirin breakdown in human blood.
Whatever the cause(s), the continuous, skewed distribu-
tion of aspirin esterase activities brings in its trail a consid-
eration of its possible contribution to aspirin resistance,
which is probably a multi-factorial phenomenon. A study in
five healthy volunteers showed a 39% reduction in throm-
boxane B
2
formation in serum ex vivo before aspirin was
detected in the systemic circulation. This was ascribed to the
pre-systemic acetylation of platelet prostaglandin synthetase
[18]. It has been suggested that because of this pre-systemic
acetylation, the esterolytic cleavage of the acetyl group by
plasma aspirin esterase, resulting in the formation of salicylic
acid metabolite, is of no relevance in aspirin inhibition of
platelet aggregation [19].
Attributing significant inhibition of platelet aggregation,
before detection of aspirin in the systemic circulation, to pre-
systemic inhibition of platelet prostaglandin synthetase is
logical. However, concluding that pre-systemic hydrolysis of
aspirin is of no relevance in its effect on platelet aggregation
may not be entirely correct. In the portal circulation, aspirin
hydrolysis takes place parri-passu with the interaction of the
drug with platelet prostaglandin synthetase. Intuitive logic
would suggest that in a situation where aspirin hydrolysis is
such as to be relatively dominant, the parallel inhibition of
platelet prostaglandin synthetase, and hence platelet aggre-
gation, could be significantly reduced and probably contrib-
utes to aspirin resistance.
In this regard, an observation by Weber et al. [20] is
relevant. A 100-mg daily dose of aspirin for 5 days had no
effect on collagen-induced platelet aggregation or thrombox-
ane formation. However, the addition of aspirin in vitro
resulted in a greater than 95% inhibition of thromboxane
formation and a complete inhibition of collagen-induced
platelet aggregation. The investigators ruled out non-compli-
ance on the basis of the fact that participants in the study were
hospitalised and that aspirin intake was supervised by the
hospital staff. Unfortunately, however, plasma aspirin levels
were not quantified to ascertain the (extent of) absorption or
otherwise of ingested aspirin. Nor was aspirin esterase activity
assayed. To suggest a lack of absorption of aspirin by the
volunteers in this study would be too cynical a view; that
aspirin was absorbed but somehow rendered ineffective in
inhibiting platelet aggregation would appear more within the
bounds of probability. If it is accepted that aspirin absorption
did take place, the observation by Weber et al. [20] would be
supportive of a possible role of aspirin esterase activity in
determining the efficacy or otherwise of a given dose of orally
administered aspirin in inhibiting thromboxane formation and
platelet aggregation.
An implication of our finding, namely, considerable and
significant variation in the pharmacokinetics of aspirin, has
indeed been confirmed by other workers [21]. With such
variability, the 75-mg daily dose of aspirin commonly
employed for inhibition of platelet aggregation in patients
may not be enough for everyone. Although the lack of a parallel
pharmacodynamic study precludes us from demonstrating this
possibility, there is evidence that, far from being conjectural, it
could translate to a clinical problem. Mueller et al. [22] showed
that among 100 claudicant patients, re-occlusion at the sites of
angioplasty occurred exclusively in those whose platelet
aggregation by adenosine 5 diphosphate and collagen was
not inhibited despite being on a 100-mg daily dose of aspirin.
The similarity between the reports by Weber et al. [20] and
Mueller et al. [22] is striking, and both studies are supportive of
the possibility that a difference in aspirin breakdown
contributes to the phenomenon of aspirin resistance.
5. Learning points
Aspirin, as prophylaxis against thromboembolism, is
usually prescribed at a dose that is expected to be effective
in every individual, namely, 75 mg daily.
Because aspirin undergoes rapid hydrolysis but the
product thus formed, salicylic acid, is not an effective
inhibitor of platelet prostaglandin synthetase, the rate of
its breakdown could be expected to determine, at least in
part, the efficacy or otherwise of a given dose.
302 G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299303
In this study, we observed an approximately seven-fold
difference in the rate of aspirin hydrolysis in 107 non-
medicated volunteers, and with a distribution that devi-
ates from normality.
In seven of our volunteers, a 75-mg daily dose of aspirin
could be ineffective as prophylaxis against thromboem-
bolism because of the rapidity of its breakdown.
It is suggested that such a high rate of aspirin hydrolysis
could be a contributory factor to the already recognised
phenomenon of aspirin resistance.
Acknowledgement
This project was supported by a grant from the Sligo
Research and Education Foundation.
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