Aspirin Esterase Activity Research Articles

Simple spectrophotometric method for the assessment of aspirin esterase activity

Simple spectrophotometric method for the assessment of aspirin esterase activity

Characterization of aspirin esterase activity in health and disease: In vitro and ex vivo studies

Due to its ability to irreversibly inactivate platelet cyclooxygenase, low-dose aspirin (ASA) is the most widely used antithrombotic agent. Although, studies in specific types of patients with cardiovascular disease (CVD) have shown an incomplete inhibition of platelet’s cyclooxygenase, which may increase the variability in drug response. Some aspects of ASA pharmacokinetics (PK) still need further investigation. In this study, we aimed to characterise the contribution of esterase enzymes to ASA hydrolysis in the peripheral blood and to assess their activity in 36 healthy subjects (Ctrl) and 77 CVD patients. To this aim, an in vitro assay testing esterase activity in parallel to a liquid chromatography-tandem mass spectrometry method simultaneously detecting ASA and its main metabolites salicylic (SA) and gentisic acids, have been developed. Michaelis-Menten constant (Km) calculation, ASA esterase isoforms characterisation, and ASA PK study were then achieved. The calculated Km identified plasma esterases as the enzymes with the higher affinity for the substrate compared to the RBC ones. Both rivastigmine and 4-bis-nitrophenyl phosphate inhibited plasma esterase activities, suggesting that acetylcholinesterase and carboxylesterase largely contribute to ASA hydrolysis. The feasibility of the method here developed has been explored in Ctrl and CVD patients. The effect of ASA treatment on enzyme activity was further evaluated on an age, sex and BMI matched subgroup of patients and Ctrl (n = 10 for each subgroup, on and off ASA). No overall variations were evidenced in both CVD and Ctrl groups, even when the effect of ASA treatment was tested. This result suggests the absence of any influence of disease state, drug treatments, and comorbidities on plasma esterase and the inability of ASA intake to induce esterase function. In conclusion, the method here developed allows a better characterisation of ASA esterase activity and could be helpful to define the PK-related determinants of ASA responsiveness in order to personalise regimen in specific pathological conditions.

Impact of the Chinese herbal medicines on dual antiplatelet therapy with clopidogrel and aspirin: Pharmacokinetics and pharmacodynamics outcomes and related mechanisms in rats

Ethnopharmacological relevanceDual antiplatelet therapy (DAPT) with aspirin (ASA) and clopidogrel (CLP) has been consistently shown clinical effectiveness in patients with coronary artery disease. According to the literature, four traditional Chinese medicine (TCM) herbs effective for prevention cardiovascular diseases, namely Radix Salvia Miltiorrhiza (Red sage root, Danshen), Radix Pueraria Lobata (Kudzu root, Gegen), Radix Angelica Sinensis (Angelica root, Danggui), and Rhizoma Ligusticum chuanxiong (Szehuan lovage rhizome, Chuanxiong), are of high potential to be co-administered during DAPT. Since all these herbs are blood vitalizing medicines and can promote blood circulation and eliminate blood stasis, it was hypothesized that they may potentially alter the clinical outcomes of DAPT with clopidogrel and aspirin. Aim of studyThe current study is proposed aiming to preliminarily evaluate the impact of these four commonly used Chinese medicinal herbs on the pharmacokinetics and pharmacodynamics of the combination therapy with clopidogrel and aspirin and its relevant outcomes and mechanisms. Materials and methodsIn order to mimic the standard dosing regimen for DAPT in human, various Sprague-Dawley rats treatment groups were received a bolus oral dose of DAPT on day 1 followed by DAPT for consecutive 13 days in absence and presence of orally co-administered four TCM herbs (Danshen, Gegen, Danggui and Chuanxiong) at their low and high doses. On day 14, serial blood samples were collected after dosing to obtain the plasma concentrations of ASA, CLP and their corresponding metabolites by LC/MS/MS. At the end of last blood sampling point of each rat, about 4.5 ml of whole blood were collected to estimate the prothrombin time from each treatment groups. After all the blood sampling, the rats were sacrificed followed by collecting their livers for evaluations of enzyme activities and expressions in the related liver microsome preparations and stomach tissues for evaluations of their potential ulcer index. In addition, gene expression and protein levels of related biomarkers (COX-1, COX-2, P2Y12) in rat livers were measured by RT-PCR and Western blot, respectively, and compared among different treatment groups. ResultsCo-administration of Gegen and Danggui significantly altered the pharmacokinetics of ASA and CLP in DAPT with increased systemic exposure of ASA and CLP respectively. Although minimal impact on aspirin esterase activity for all co-administered herbs, significant inhibition on rCyp2c11 and carboxylesterase activities were observed for DAPT with Danshen, Gegen and Danggui co-treatment. In addition, significantly longer PT were found in all DAPT treatment groups. However, a trend of decrease in PT of DAPT in presence of Gegen, Danggui and Chuanxiong was noticed. Nevertheless, all the treatments did not cause detectable changes in COX and P2Y12 mRNA and protein expressions. ConclusionAmong the four studied TCMs, it was demonstrated that co-administration of Gegen and Danggui could lead to altered pharmacokinetics of DAPT with significant inhibition on rCyp2c11 and carboxylesterase activities. Although Gegen, Danggui and Chuanxiong might potentially offset the anticoagulant activity of DAPT, the overall pharmacodynamics outcome was not considered to be harmful due to lack of risk in bleeding, which warrant further verification for its clinical impact.

Impaired liver cytochrome P450 2C11 activity after dual antiplatelet therapy with aspirin and clopidogrel in rats

1. Aspirin (ASA) and clopidogrel (CLP) are used in combination as dual antiplatelet therapy (DAPT) for acute coronary syndrome based on their complementary mechanisms for platelet aggregation inhibition. However, the pharmacokinetics of such drug combination usage has not been thoroughly investigated.2. In the current study, an LC–MS/MS method was developed to simultaneously determine the plasma concentrations of ASA and its metabolite salicylic acid (SA) with CLP and its metabolites, clopidogrel carboxylic acid (CLPM) and clopidogrel active metabolite derivative (CAMD). The pharmacokinetics of ASA, SA, CLP, CLPM and CAMD in rats receiving two-week DAPT with ASA and CLP were then determined.3. After two-week DAPT with ASA and CLP in rats, the activities of aspirin esterase and rCyp2c11, enzymes mediating rat metabolism of ASA and CLP, respectively, in prepared rat liver microsomes were measured followed by further determination of rCyp2c11 mRNA expressions. The results demonstrated that DAPT led to minimal impact on aspirin esterase activity but significant decrease in rCyp2c11 activity and mRNA expression.4. In conclusion, our findings on impairment in rCyp2C11 activity and mRNA expression by DAPT in rats could provide guidance on its safe clinical use with other CYP 2C19 substrates.

Aspirin esterase(s) - Much known, more to learn

Esterases, so called because they catalyze hydrolytic splitting of ester bonds, remain quite intriguing – they partake in the biotransformation of drugs/xenobiotics, but their physiological/biological functions remain unknown. An exception is acetyl cholinesterase that catalyses hydrolysis of the neurotransmitter acetylcholine. Hydrolysis of a xenobiotic/drug can, at times, be catalyzed by different esterases in different tissues or organs. For example, aspirin hydrolysis to salicylate and acetate is catalyzed by carboxylesterases (EC 3.1.1.1) in human gastric mucosa and liver [1], resulting in bioavailability of 65% [2]. Once in the systemic circulation, further hydrolysis is catalyzed by other esterases in the plasma (pseudo cholinesterase, EC 3.1.1.8; and albumin) and red blood cells. Albumin-associated esterase activity is minor, and the main plasma aspirin esterase activity is associated with the plasma cholinesterase [3]. About 80% of aspirin esterase activity in the blood is due to the enzyme in erythrocytes. It is different from that in plasma – there is no significant correlation in levels of the two, and the red cell enzyme is resistant to 1.66 mmol/L physostigmine (in final assay system) that completely inhibits the form in plasma [4]. The suggestion that it is probably ‘non specific’ has been contested by the isolation from human erythrocyte of an esterase with specificity for aspirin by Costello and Green [5]. 2. Factors affecting aspirin esterase activity

Serum aspirin esterase activity in cord blood and in adults

There is scarce information about altered aspirin – esterase (AE) activities in various physiological and pathological states, as well as the putative agents modulating this activity. Since there are no data on AE levels in cord blood, we set up this study to measure its activity in neonates' cord blood and in adults, using an optimized technique developed in our laboratory. We performed a case-control study with 28 newborns, and control adult subjects were selected from a healthy population of hospital workers. Aspirin esterase activity in cord blood was 60 ± 12 nmol/mL/min vs 87 ± 12 nmol/mL/min in adults, p r = 0.86 p

Changes in Plasma Aspirin and Cholinesterase Activity in Patients with Myocardial Infarction or Stroke

Because variations occur in plasma esterase activity depending on the disease state, we analysed plasma esterase activity during the first 24 h and 14 days after acute myocardial infarction or stroke in 59 patients, and compared these findings with those in 52 healthy controls. Plasma aspirin-esterase and cholinesterase activities were significantly increased 24 h and 7 days postinfarction (P<0.01). Both activities were also increased 24 h after stroke (P<0.05). After 14 days both plasma enzyme activities had returned to normal values in both groups of patients. The transitory increase in plasma esterase activity may be related to haemodynamic variations in acute myocardial infarction.

Serum aspirin esterase activity is lower in end-stage renal disease patients than in healthy control subjects and increases after haemodialysis

Studies regarding aspirin metabolism can be important in patients with renal failure who have an increased risk of cardiovascular diseases. We undertook this study to assess the aspirin esterase (AE) status in end-stage renal disease (ESRD) patients. A total of 42 patients on long-term haemodialysis (HD) with a mean dialysis course of 6.1 y were recruited. Serum AE levels were 44% lower and cholinesterase (ChE) levels were 22% lower in ESRD patients before dialysis as compared with control subjects (P = 0.0001). A very strong correlation was found between AE and ChE levels. AE levels increased on average 28% after dialysis with adjustments for age, gender, total cholesterol, triglyceride and high-density lipoprotein cholesterol (P = 0.002). In addition, ChE levels were significantly increased (48%) after dialysis (P = 0.0001). Changes in AE activity were significantly and positively correlated with those of ChE (r = 0.427, P = 0.005). When we adjusted for several confounders, we found that the changes in AE activity operated by dialysis are significant independently of age, gender, aspirin (ASA) intake, cholesterol, triglycerides, high-density lipoprotein cholesterol and ChE. We report that serum AE activity is significantly lower in ESRD and that treatment by HD results in an increase of activity. We confirm that AE is associated with lipid parameters and ChE. Our results show variations in ASA catabolism between the dialysis sessions, suggesting an oscillating pattern in ASA disposal in these patients. The mechanisms for reduced AE activity in uraemia and the effects of HD need further investigation.

Serum aspirin esterase is strongly associated with glucose and lipids in healthy subjects: different association patterns in subjects with type 2 diabetes mellitus

BackgroundAspirin esterase (AE) activity can account for part of aspirin pharmacokinetics in the circulation, possibly being associated with the impairment of aspirin effectiveness as an inhibitor of platelet aggregation.AimsThe study was aimed at investigating the correlations of serum AE activity with cholinesterase (ChE) and metabolic variables in healthy subjects in comparison to subjects with type 2 diabetes mellitus (T2DM).MethodsIn cardiovascular disease-free T2DM subjects and healthy controls, the AE activity levels and/or the correlation patterns between AE and the other variables were analyzed.ResultsNeither AE nor ChE activities were higher in the subjects with T2DM. Serum AE activity strongly correlated with ChE as well as glucose/lipids variables such as total cholesterol and triglyceride in healthy subjects, while the correlations between AE and glucose/lipids variables were not present in T2DM subjects.ConclusionsThese data may reflect the pathophysiological changes between healthy and T2DM subjects. Our data may thus provide the basis for future studies to unravel the mechanisms.

Serum aspirin esterase activity in cord blood and in adults

AbstractThere is scarce information about altered aspirin – esterase (AE) activities in various physiological and pathological states, as well as the putative agents modulating this activity. Since there are no data on AE levels in cord blood, we set up this study to measure its activity in neonates' cord blood and in adults, using an optimized technique developed in our laboratory. We performed a case-control study with 28 newborns, and control adult subjects were selected from a healthy population of hospital workers. Aspirin esterase activity in cord blood was 60 ± 12 nmol/mL/min vs 87 ± 12 nmol/mL/min in adults, p &lt; 0.00001. No correlation of AE activity and HDL cholesterol, HDL/LDL cholesterol or paraoxonase 1 was found. Aspirin esterase correlated significantly with serum cholinesterase: r = 0.86 p &lt; 0.0001, which was also 30% ower in cord blood than in adults. We show here, for the first time, that cord blood serum has a significant aspirin esterase activity, even though is its 40% lower than that found in adults. Our data provide baseline information for future studies on the physiology of aspirin esterase in children and the factors that modulate its activity to reach adult levels.

Serum cholesterol concentration associated with aspirin esterase activity in older people: preliminary data

Metabolism of aspirin (acetylsalicylic acid), commonly used in older people for the prevention of cardiovascular disease, is important to the effectiveness of this drug. Whereas part of aspirin hydrolysis occurs in blood, there is a paucity of information in regards to circulating aspirin esterase activity in various physiological and pathological conditions. High aspirin esterase activity, corresponding to faster aspirin hydrolysis (thus aspirin non-responsiveness), may occur in cardiovascular disease-prone states. The objective of this study was to investigate the effects of cardio-metabolic variables such as cholesterol on serum aspirin esterase activity in older people who participated in an intervention study on physical activity. A total of 18 non-medicated subjects (7 men/11 women, mean age 67.8 years, body mass index = 23.4 +/- 3.3 kg/m(2)), who completed a 3-month interventional program for a mild-to-moderate increase in physical activity, were analyzed. The body mass index, plasma glucose, serum total cholesterol and aspirin esterase activity were measured in the pre- and post-interventional phases of the study. During the interventional period, the changes in aspirin esterase activity correlated significantly and positively with those of total cholesterol concentrations (r = 0.542, P = 0.020; beta = 0.609, P = 0.035 in a multiple linear regression analysis after adjusting for all the measured variables). The results suggest that cholesterol metabolism alterations may be associated with aspirin metabolism in older people.

Reaction of human albumin with aspirin in vitro: Mass spectrometric identification of acetylated lysines 199, 402, 519, and 545

The aspirin esterase activity of human plasma is due to butyrylcholinesterase and albumin. Our goal was to identify the amino acid residues involved in the aspirin esterase activity of albumin. Fatty acid-free human albumin and human plasma were treated with aspirin for 5 min–24 h. Acetylated residues were identified by LC/MS/MS and MALDI-TOF/TOF mass spectrometry of tryptic peptides. Treatment with 0.3 mM aspirin resulted in acetylation of Lys-199, Lys-402, Lys-519, and Lys-545. Treatment with 20 mM aspirin resulted in acetylation of 26 lysines. There was no acetylation of Tyr-411, under any conditions. Acetylated lysine was stable for at least 21 days at pH 7.4, 37 °C. Albumin acetylated by aspirin had reduced esterase activity with β-naphthyl acetate as shown on gels stained for esterase activity. It was concluded that the aspirin esterase activity of albumin is a pseudo-esterase activity in which aspirin stably acetylates lysines and releases salicylate.

Plasma esterases and inflammation in ageing and frailty

Esterases are enzymes of drug metabolism known to be reduced in frail older people and during acute illness. The mechanism for this is unknown. The aim of this study was to examine esterase activity and inflammation in ageing and frailty. Thirty frail patients (mean age 84.9 years) dependent on continuing inpatient care, 40 patients of intermediate frailty attending Day Hospital (84.2 years), 40 fit older controls (82.7 years) and 30 young controls (23.3 years) were studied. Frailty indicators, plasma esterase activities and markers of inflammation were measured. With increasing patient frailty, C-reactive protein (CRP), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) increased significantly and esterase activity, with the exception of aspirin esterase, fell significantly (p < 0.005). There were significant negative correlations between log-transformed IL-6 and acetylcholinesterase (r = -0.354, p < 0.01), butyrylcholinesterase (r = -0.392, p < 0.01) and benzoylcholinesterase activity (r = -0.241, p < 0.05) and significant negative correlations between TNF-alpha and acetylcholinesterase (r = -0.223, p < 0.01), butyrylcholinesterase (r = -0.279, p < 0.01) and benzoylcholinesterase activity (r = -0.253, p < 0.01). Aspirin esterase activity did not correlate with IL-6 or TNF- alpha. Frailty was associated with higher inflammatory markers and lower esterase activity. There was a weak but significant negative correlation between both IL-6 and TNF-alpha and the activity of three of four esterases. The negative correlation between esterase activity and inflammatory markers may have a causal basis, comparable to the inflammatory suppression of cytochrome P-450 enzymes.

Aspirin esterase activity — Evidence for skewed distribution in healthy volunteers

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 < 0.001) and dibucaine numbers (Spearman's rho = 0.422, p < 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.

Increased blood plasma hydrolysis of acetylsalicylic acid in type 2 diabetic patients: A role of plasma esterases

Hydrolysis of acetylsalicylic acid (ASA, aspirin), an antiplatelet drug commonly used in the prevention of stroke and myocardial infarction, seems to play a crucial role in its pharmacological action. Thirty-eight healthy volunteers and 38 type 2 diabetic patients were enrolled to test the hypothesis that the enhanced plasma degradation and lowered bioavailability of ASA in diabetic patients is associated with the attenuation of platelet response. Aspirin esterase activities were tested at pH 7.4 and 5.5. A significantly higher overall aspirin esterase activity was noted at pH 7.4 in the diabetic patients ( P < 0.003), corresponding to faster ASA hydrolysis ( P < 0.006). This increased activity was attributable to butyrylcholinesterase and probably to albumin, because it was effectively inhibited by eserine and 4-bis-nitrophenyl phosphate ( P < 0.01). No significant differences between control and diabetic subjects were found at pH 5.5 in either enzymatic activities or ASA hydrolysis rates. The enhanced plasma ASA degradation in diabetic subjects was significantly associated with the refractoriness of blood platelets to ASA ( P < 0.05) and modulated by plasma cholesterol ( P < 0.01). No direct effects of plasma pH or albumin were observed. In conclusion, higher aspirin esterase activity contributes to the lowered response of diabetic platelets to ASA-mediated antiplatelet therapy.

The effect of community-acquired pneumonia on plasma esterases in older people.

Pneumonia is a major cause of morbidity and mortality in older people. The poor outcome of older pneumonia patients despite treatment is still not understood. The aim of this study was to examine the effect of community-acquired pneumonia on enzymes of drug metabolism in older people. Fifteen patients (median age 67 years) with a clinical and radiological diagnosis of community-acquired pneumonia and 14 healthy volunteers matched for age and gender (median age 75 years) were recruited. Plasma activities of benzoylcholinesterase, butyrylcholinesterase, acetylcholinesterase and aspirin esterase were determined spectrophotometrically at three time points in pneumonia patients--within 24 h of admission to hospital, 2 days later and 10 days later. Monocyte aryl hydrocarbon hydroxylase (AHH) activity was determined spectrofluorimetrically at the same time points. Enzyme activities were measured at one time point in healthy controls. Mean plasma benzoylcholinesterase activity was significantly lower in pneumonia patients on admission to hospital (mean +/- SEM 848 +/- 100) and after 10 days of treatment (mean +/- SEM 925 +/- 114) than in healthy controls (mean +/- SEM 1333 +/- 84, P < 0.05). Similarly, plasma acetylcholinesterase activity was significantly lower in pneumonia patients on admission (P = 0.007) and after 10 days of treatment (P = 0.01) than in controls. Butyrylcholinesterase activity was lower in pneumonia patients on admission (P = 0.029) than in healthy controls, but improved slightly after treatment so there was no longer a significant difference at 10 days compared with controls (P = 0.077). In contrast there were no significant differences in plasma aspirin esterase activity or induced monocyte AHH activity between pneumonia patients and healthy controls. The activities of benzoylcholinesterase (r = -0.536, P = 0.04), butyrylcholinesterase (r = -0.638, P = 0.01), acetylcholinesterase (r = -0.583, P = 0.022) and aspirin esterase (r = -0.624, P = 0.013) correlated inversely with the British Thoracic Society pneumonia poor prognostic index. The activities of several esterases are reduced in older pneumonia patients. Other enzymes including aspirin esterase and induced monocyte AHH activity are unaltered in pneumonia. There was a significant inverse relationship between the activities of all esterases studied and the British Thoracic Society pneumonia poor prognostic index.

Aspirin esterases in North-West Indians: the influence of age and nutrition.

To determine the activity of aspirin esterases in North-West Indian population and to find the effect of age and nutrition on it. The serum albumin, plasma cholinesterase (PChE), aspirin esterase (ASPES) and phenyl acetate esterase (PAE) were determined in 175 subjects: young (< 40 years) and healthy (BMI > 19) = 74; elderly (> 50 years) and healthy (BMI > 19) = 32; young (< 40 years) and emaciated (BMI < 19) = 44; elderly (> 50 years) and emaciated (BMI < 19) = 25). The serum albumin levels significantly decreased with increase in age (r = -0.384, p < 0.01) and with decrease in body mass index (r = 0.457, p < 0.01). When the activity of esterases in four groups was compared, the PAE activity was not found to be affected by age or nutrition and the ASPES and PChE activity were significantly lower only in elderly emaciated (p < 0.01). As elderly emaciated have decreased serum albumin, ASPES and PChE activity, they may need a lower dose of aspirin to achieve the desired antiplatelet and analgesic effect. The young emaciated subjects, in spite of their lower serum albumin levels, may not require a lower dose of aspirin.

Plasma aspirin esterase activity in elderly patients undergoing elective hip replacement and with fractured neck of femur.

Plasma aspirin esterase activity was measured in eight elderly patients undergoing elective hip replacement, and in 11 presenting with femoral-neck fracture. Elective hip surgery was associated with a marked and significant decline in esterase, from 330 +/- 35.9 (SE) (nmol salicylate/ml plasma/min) pre-operatively to 236 +/- 35.6 on the third post-operative day, and returning to normal (359 +/- 24.3) at recovery phase. Patients with fractured neck of femur showed a depressed esterase activity on admission (221 +/- 12.7), rising towards normal (290 +/- 15.4) on recovery. All of these changes were accompanied by a marked acute-phase response, both to injury and surgery. The data suggest that elderly patients experiencing injury or undergoing surgery may have significantly impaired drug metabolism, at least as far as this enzyme is concerned.

Postoperative decline in plasma aspirin-esterase and cholinesterase activity in surgical patients.

We studied preoperative and postoperative plasma hydrolysis of aspirin and plasma cholinesterase activity in surgical patients. Postoperative aspirin esterase and cholinesterase activities fell sharply (119 +/- 32 micrograms ml h-1 and 3746 +/- 1068 U l-1 respectively; P < 0.01) in comparison to basal preoperative values (157 +/- 33 micrograms ml h-1 and 4620 +/- 940 U l-1 respectively, P < 0.01). We suggest that alterations in plasma esterase during the postoperative period may be related to the metabolic response to anesthesia- and surgery-induced stress.

The Association of Age with Aspirin Esterase Activity in Human Liver

The activity of the phase 1 enzyme aspirin esterase was studied in liver tissue from 16 patients, aged 45-88 years. No correlation between age and enzyme activity was found in post-mitochondrial, cytosolic and microsomal fractions. These results provide further evidence that age is not a major determinant of the activity of hepatic drug-metabolizing enzymes in man.