| Objective: Aspirin has developed more than 100 years. On the 1970Sf it has been found that aspirin may play the role of anti-platelet aggregation by irreversible inhibition of cyclooxygenase-1 (COX-1) activity. However, in long-term follow-up, it was found that aspirin can not fully inhibit the platelet function for some patients who take aspirin in the treatment. Therefore, in recent years, the concept of aspirin resistance (AR) was put forward. In the process of absorbing and absorbed, aspirin quickly transformed into salicylic acid by aspirin esterase in gastric mucous membrane, plasma, red blood cells and liver. In vivo only the prototype drug: aspirin can play the role of anti-platelet aggregation; Salicylic acid, the hydrolysis products of aspirin had no this effect. Whether aspirin can play the role of antiplatelet effect, the strength of effect and the aspirin resistance could be related to the aspirin esterase in the human body. The purpose of this study is to confirm the correlation and whether the aspirin esterase activity will affect anti-platelet aggregation effect of aspirin, and guide the rationalization clinical administration of aspirin. This study describes a HPLC method to simultaneously determine the concentrations of aspirin (ASA) and salicylic acid (SA) in plasma, use the SA/(ASA+SA) to express the activity of aspirin esterase in plasma. We use the orthogonal design analysis to optimize the incubating conditions and study the aspirin esterase kinetics in Chinese adult human plasma, the determination of aspirin esterase activity in plasma may provide a scientific basis for clinical treatment. we determine the aspirin esterase activity in plasma of 100 healthy Chinese to inspect the distribution of aspirin esterase activity in the Chinese population. Aspirin is often used in combination with other drugs, the determination the difference of effects of these drugs on the activity of aspirin esterase can provide a rational basis for the clinical use of drugs. Aspirin esterase also reside in the liver, red blood cells and gastric, but there is no index reflect the systemic aspirin esterase activity, so we established the index of aspirin esterase activity in vivo to study correlation of the activity of aspirin esterase with the effect of antiplatelet treatment by aspirin and to inspect whether the aspirin esterase activity will affect anticoagulant effect of aspirin and be the cause of aspirin resistance.Methods: A new HPLC method to determine the aspirin and it's metabolites: salicylic acid in the plasma was established. The Shim-pack VP-COD column (4.6mm×150mm, 5μm) and Shim-pack C18 pre-column were used. The mobile phase was acetonitrile and 0.072 mmol· L-1 phosphoric acid (78: 22, v/v). The flow rate was 1mL·min-1, and the column temperature was 30℃. The detective wavelength was at 228nm. Orthogonal experimental design was used to optimize the incubating conditions of aspirin esterase. The concentrations of aspirin and salicylic acid in the plasma were determined by RP-HPLC, use the ratio of salicylic acid (SA) and aspirin (ASA) to express the activity of aspirin esterase. Frequency distribution histogram of aspirin esterase activity of 100 subjects were drawn by calculating the SA/(ASA+SA). The aspirin esterase activity were determine by adding and no adding other drugs usually used in combined with aspirin, to inspect the inhibition of aspirin esterase activity. To develop a GC-MS(SIM) method for the study of pharmacokinetics of aspirin and Salicylic acid in healthy human volunteers. The pharmacokinetic parameters were computed using 3P97 program. The plasma clearance rates of aspirin of 12volunteers were determined. We inspect the correlation of the ratio of SA/ASA at a particular time point with the plasma clearance rate of aspirin to definite the index of aspirin esterase activity in vivo. We study the correlation of aspirin esterase activity with the clinical efficacy of aspirin to inspect whether the aspirin esterase activity isone of the reasons of aspirin resistance. Results:1. A new HPLC method of aspirin and it's metabolite: salicylic acid in the plasma was developed. The method is not only simple and rapid, but also improved the accuracy. The optimal incubating conditions is the A2B2C3D1 using orthogonal design analysis, that is 1mmol·L-1 aspirin and 200 μL plasma were incubated in the pH 7.6 incubating solution for 30 minutes.2. The Michaelis constant of aspirin esterase is Km=429.67nmol ? L-1 and Vmax=2.56 μmol ? min-1 ? mL-1 (plasma) , the optimum reaction temperature is 37 °C and the optimum pH is 7.4. The enzyme is more sensitive to pH changes and is extremely unstable to the heat.3. The drug used along with aspirin in the treatment of cardiovascular disease: nifedipine, diltiazem, verapamil, dipyridamole, captopril, Metoprolol, phenacetium, paracetamol andTMP have significant inhibit effects on the activitiy of aspirin esterase; and phenobarbital, carbamazepine, valproic acid, sodium Danshensu, Lidocaine have significant induce effects; and simvastatin, hydrochlorothiazide, Caffeine have no significant effects on the activity of aspirin esterase.4. The average activity of aspirin esterase of 100 healthy volunteers is 0.2167 ± 0.0683, frequency analysis indicated the normal distribution; The aspirin esterase activity of 67 healthy men and 33 healthy women were (0.2159 ± 0.069 2) and (0.2173 ± 0.0684), there were no significant statistical difference between the two (P < 0.05), the frequency distribution both showed normal distribution.5. After oral administration of aspirin, we make a correlation analysis between the plasma clearance rate and salicylic acid (SA) and aspirin (ASA) ratio of various time points, and a significant correlation were observed between the ratio of the plasma salicylic acid and aspirin of 3.5 hours and aspirin oral clearance rate (r=0.882, P<0.01). So the ratio of the plasma salicylic acid and aspirin of 3.5 hours can reflect aspirin esterase activity. The close correlation that we observed between the activity of aspirin esterase of 12 volunteers, AUCasa, TXB2 inhibition percentage and the reduction of TXB2/6-keto-PGF1α ratio (r=-0.941, P < 0.001; r=-0.821, P < 0.01 和 r=0.804, P< 0.01; r=0.732, P < 0.001 respectively)suggest that the rate of aspirin hydrolysis is a major determinant of its anticoagulant efficacy.Conclusions: The method is simple, accurate and rapid, suitable for the determination of aspirin and salicylic acid in plasma. The method can be used to assay the activity of aspirin esterase in vitro, and open up a new way for carrying out in-depth studies of aspirin-metabolizing enzymes. By analysis, aspirin esterase showed sensitive to the pH change, but extremely unstable to heat. Aspirin is often used in combination with other drugs. The effect of the drugs used along with aspirin clinically on aspirin metabolism could be significant, So we should always be careful while using these drugs with aspirin. The frequency distribution of the activity of aspirin esterase in the Chinese population indicated normal distribution, and there is no significant sex-dependent differences. The activity of aspirin esterase, that is acetylsalicylic acid hydrolysis rate is the key influencing factor of anticoagulant effect of aspirin. The close correlation that we observed between the activity of aspirin esterase of 12 volunteers, AUCasa, TXB2 inhibition percentage and the reduction of TXB2/6-keto-PGF1α ratio suggest that the rate of aspirin hydrolysis is a major determinant of its anticoagulant efficacy. |
