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Aspirin esterase activity — Evidence for skewed distribution in healthy volunteers
European Journal of Internal Medicine 18 (2007) 299 – 303 www.elsevier.com/locate/ejim
Original article
Aspirin esterase activity — Evidence for skewed distribution in healthy volunteers G.I. Adebayo a,⁎, J. Williams b , S. Healy a a
b
Department of Medicine, Sligo General Hospital, The Mall, Sligo, Ireland 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, p b 0.001) and dibucaine numbers (Spearman's rho = 0.422, p b 0.01). 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, acetylsalicylic 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,
⁎ Corresponding author. E-mail address: [email protected] (G.I. Adebayo).
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
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
300
G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299–303
weight was 67.8 kg (44–114.6 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 μl 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 additionally 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 (33.90–199.00 nmol/ ml/min) were lower than those in males (40.20–222.65 nmol/ ml/min, p b 0.005).
Fig. 1. Aspirin esterase activity (nmol/ml/min at 37 °C) in 107 healthy volunteers expressed as (a) frequency histogram and (b) normal plot.
different studies, as well as to the difficulty in identifying an optimal dose. To assess this possibility, we studied the breakdown 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 14–80 years) and their average
Fig. 2. Relationship between age and aspirin esterase activity in 107 nonmedicated volunteers.
G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299–303
301
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 mean ± 2 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.
In the 41 volunteers so studied, pseudocholinesterase activities were 3.09–10.53 U/ml (mean ± SD, 6.53 ± 1.64 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
Fig. 4. Frequency histogram (a) and normal plot (b) of aspirin esterase activity (nmol/ml/min at 37 °C) in 41 healthy volunteers.
302
G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299–303
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 agedependent, given the lack of a significant relationship between 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 polymorphism 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 association between pseudocholinesterase and aspirin esterase activities revealed a modest but significant correlation (Spearman's rho = 0.593; p b 0.001). Because percentage inhibition by dibucaine, dibucaine number, reveals variants of pseudocholinesterase 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; p b 0.01 – 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 distribution of aspirin esterase activities brings in its trail a consideration 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 thromboxane B2 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 presystemic 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 aggregation, could be significantly reduced and probably contributes 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 thromboxane 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-compliance 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.
G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299–303
• In this study, we observed an approximately seven-fold difference in the rate of aspirin hydrolysis in 107 nonmedicated volunteers, and with a distribution that deviates from normality. • In seven of our volunteers, a 75-mg daily dose of aspirin could be ineffective as prophylaxis against thromboembolism 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. References [1] Schror K. Aspirin and platelets: the antiplatelet action of aspirin and its role in thrombosis treatment and prophylaxis. Semin Thromb Hemost 1997;23:349–56. [2] Schror K. Antiplatelets drugs. A comparative review. Drugs 1995;50:7–28. [3] Peto R, Gray R, Collins R, Wheatley K, Hennekens C, Jamrozik K, et al. Randomised trial of prophylactic daily aspirin in British male doctors. Br Med J (Clin Res Ed) 1988;296:313–6. [4] Steering Committee of the Physicians' Health Study Research Group. Final report on the aspirin component of the ongoing Physicians' Health Study. N Engl J Med 1989;321:129–35. [5] Collaborative Group of the Primary Prevention Project. Low-dose aspirin and vitamin E in people at cardiovascular risk: a randomised trial in general practice. Lancet 2001;357:89–95. [6] Hansson L, Zanchetti A, Carruthers SG, et al. Effect of intensive blood pressure lowering and low-dose aspirin in patients with hypertension: principal results of the Hypertension Optimal Treatment (HOT) randomised trial. HOT Study Group. Lancet 1998;351:1755–62. [7] The Medical Research Council's General Practice Research Framework. Thrombosis prevention trial: randomised trial of low-intensity oral anticoagulation with warfarin and low-dose aspirin in the primary prevention of ischaemic heart disease in men at risk. Lancet 1998;351: 233–41.
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[8] Patrono C, Coller B, Dalen JE, et al. Platelet-active drugs: the relationships among dose, effectiveness, and side effects. Chest 2004;119: 39S–63S [Suppl]. [9] Adebayo GI, William J, Healy S. Pseudocholinesterase polymorphism in Irish population. Eur J Intern Med 2005;16:492–5. [10] Williams FM, Wynne H, Woodhouse KW, Rawlins MD. Plasma aspirin esterase: the influence of old age and frailty. Age Ageing 1989;18: 39–42. [11] Rainsford KD, Ford NLV, Brooks PM, Watson HM. Plasma aspirin esterase in normal individuals, patients with alcoholic liver disease and rheumatoid arthritis: characterisation and importance of the enzyme components. Eur J Clin Invest 1980;10:413–20. [12] Dietz AA, Rubinstein HM, Lubrano T. Colorimetric determination of serum cholinesterase and its genetic variants by propionylthiocholinedithiobis(nitrobenzoic acid) procedure. Sel Methods Clin Chem 1973;19: 1309–13. [13] Menguy R, Desbaillets L, Masters YF, Okabe S. Evidence for a sexlinked difference in aspirin metabolism. Nature 1972;239:102–3. [14] Khaled Abou-Hatab M, O'Mahony S, Patel S, Woodhouse K. Relationship between age and plasma esterases. Age Ageing 2001;30:41–5. [15] Rubinstein HM, Dietz AA, Lubrano T. E1k, another qualitative variant at cholinesterase locus 1. J Med Genet 1978;15:27–9. [16] Evans RT, Wardell J. On the identification and frequency of the J and K cholinesterase phenotypes in a Caucasian population. J Med Genet 1984;21:99–102. [17] Williams FM, Nicholson EN, Woolhouse NW, Adjepon-Yamoah KK, Rawlins MD. Activity of esterases in plasma from Ghanaian and British subjects. Eur J Clin Pharmacol 1986;31:485–9. [18] Pedersen AK, Fitzgerald GA. Dose-related kinetics of aspirin presystemic acetylation of platelet cyclooxygenase. N Engl J Med 1984;311:1206–11. [19] Gawaz M. Blood Platelets. Stuttgart: Georg Thieme Verlag; 2001. p. 57–84. [20] Weber A-A, Przytulski B, Schanz A, Hohlfeld T, Schror K. Towards a definition of aspirin resistance: a typological approach. Platelets 2002;13:37–40. [21] Benedek IH, Joshi AS, Pieniaszek HJ, King SY, Kornhauser DM. Variability in the pharmacokinetics and pharmacodynamics of low dose aspirin in healthy male volunteers. J Clin Pharmacol 1995;35: 1181–6. [22] Mueller MR, Salat A, Stangl P, Murabito M, Pulaki S, Boehm D, et al. Variable platelet response to low-dose ASA and the risk of limb deterioration in patients submitted to peripheral arterial angioplasty. Thromb Haemost 1997;78:1003–7.
Original article
Aspirin esterase activity — Evidence for skewed distribution in healthy volunteers G.I. Adebayo a,⁎, J. Williams b , S. Healy a a
b
Department of Medicine, Sligo General Hospital, The Mall, Sligo, Ireland 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, p b 0.001) and dibucaine numbers (Spearman's rho = 0.422, p b 0.01). 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, acetylsalicylic 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,
⁎ Corresponding author. E-mail address: [email protected] (G.I. Adebayo).
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
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
300
G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299–303
weight was 67.8 kg (44–114.6 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 μl 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 additionally 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 (33.90–199.00 nmol/ ml/min) were lower than those in males (40.20–222.65 nmol/ ml/min, p b 0.005).
Fig. 1. Aspirin esterase activity (nmol/ml/min at 37 °C) in 107 healthy volunteers expressed as (a) frequency histogram and (b) normal plot.
different studies, as well as to the difficulty in identifying an optimal dose. To assess this possibility, we studied the breakdown 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 14–80 years) and their average
Fig. 2. Relationship between age and aspirin esterase activity in 107 nonmedicated volunteers.
G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299–303
301
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 mean ± 2 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.
In the 41 volunteers so studied, pseudocholinesterase activities were 3.09–10.53 U/ml (mean ± SD, 6.53 ± 1.64 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
Fig. 4. Frequency histogram (a) and normal plot (b) of aspirin esterase activity (nmol/ml/min at 37 °C) in 41 healthy volunteers.
302
G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299–303
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 agedependent, given the lack of a significant relationship between 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 polymorphism 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 association between pseudocholinesterase and aspirin esterase activities revealed a modest but significant correlation (Spearman's rho = 0.593; p b 0.001). Because percentage inhibition by dibucaine, dibucaine number, reveals variants of pseudocholinesterase 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; p b 0.01 – 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 distribution of aspirin esterase activities brings in its trail a consideration 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 thromboxane B2 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 presystemic 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 aggregation, could be significantly reduced and probably contributes 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 thromboxane 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-compliance 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.
G.I. Adebayo et al. / European Journal of Internal Medicine 18 (2007) 299–303
• In this study, we observed an approximately seven-fold difference in the rate of aspirin hydrolysis in 107 nonmedicated volunteers, and with a distribution that deviates from normality. • In seven of our volunteers, a 75-mg daily dose of aspirin could be ineffective as prophylaxis against thromboembolism 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. References [1] Schror K. Aspirin and platelets: the antiplatelet action of aspirin and its role in thrombosis treatment and prophylaxis. Semin Thromb Hemost 1997;23:349–56. [2] Schror K. Antiplatelets drugs. A comparative review. Drugs 1995;50:7–28. [3] Peto R, Gray R, Collins R, Wheatley K, Hennekens C, Jamrozik K, et al. Randomised trial of prophylactic daily aspirin in British male doctors. Br Med J (Clin Res Ed) 1988;296:313–6. [4] Steering Committee of the Physicians' Health Study Research Group. Final report on the aspirin component of the ongoing Physicians' Health Study. N Engl J Med 1989;321:129–35. [5] Collaborative Group of the Primary Prevention Project. Low-dose aspirin and vitamin E in people at cardiovascular risk: a randomised trial in general practice. Lancet 2001;357:89–95. [6] Hansson L, Zanchetti A, Carruthers SG, et al. Effect of intensive blood pressure lowering and low-dose aspirin in patients with hypertension: principal results of the Hypertension Optimal Treatment (HOT) randomised trial. HOT Study Group. Lancet 1998;351:1755–62. [7] The Medical Research Council's General Practice Research Framework. Thrombosis prevention trial: randomised trial of low-intensity oral anticoagulation with warfarin and low-dose aspirin in the primary prevention of ischaemic heart disease in men at risk. Lancet 1998;351: 233–41.
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