Nocturnal rise of urinary thromboxane B2 in patients with coronary artery disease

Thromb Haemost 2005; 93: 618-9 Dear Sir, The morning peak in the incidence of cardiovascular events can be partly due to a simultaneous surge in platelet function, blood pressure, heart rate, catecholamine and cortisol secretion (1), and to a decrease in plasma fibrinolytic activity (2). Platelet aggregability was evaluated by ex vivo methods, showing an increase only at 9am in both coronary artery disease (CAD) patients (3) and healthy subjects (HS) (4, 5). However, the described peak of myocardial infarction (MI) incidence between 4am and 10am (6) suggests a platelet activation before 9am. In order to further investigate this issue, we analysed the circadian variation of urinary 11-dehydro-thromboxane B2 (11-d-TxB2), a reliable in vivo index of platelet function (7), in CAD patients and HS. CAD patients (15 males and 1 female, mean age 60.8) had luminal diameter stenosis in one or more major coronary arteries and/or in their primary branches greater than 50%, a clinical history of unstable angina (n=9) and/or myocardial infarction (n=7), and no symptom for at least two years before the study. They were assuming aspirin for at least the last 3 years, but discontinued the drug at least 14 days before the study (15±0.6). HS (10 males, 2 females, mean age 59) were not affected by cardiovascular disease. Patients with creatinine clearance lower than 80 ml/min and/or heart failure (as diagnosed by an echocardiographic evaluation) were excluded. The study was based on a 24-hour urinary sampling and a simultaneous ECG monitoring. The 24-hour period was divided into six 4-hour periods, numbered as follows: 10–4am; 24–8am; 38–12am; 412–4pm; 54–8pm; 68–12pm. The urine samples were collected at the end of each period. Patients went to bed at 00.10 am after collection of sample 6, woke up at 4 am to collect sample 1, woke up again at 7am to collect sample 2 at 8am and then started daily activities. From each urine collection, a 20-ml sample was separated, acidified at pH 4 and stored at –30°C until analysis. After Sep-Pak extraction and thin layer chromatography separation, 11-d-TxB2 was measured by an enzyme immunoassay method previously validated by gas chromatography/mass spectrometry (8, 9) and expressed as ng/h (10). The research protocol was approved by the hospital ethical committee and all participants gave their informed consent to the study. For each 4-hour interval, Mann-Whitney U test was used to compare thromboxane excretion between groups. The statistical significance of the overall circadian variation in 11-d-TxB2 was evaluated by Friedman test, while interval-to-interval paired comparisons were performed by the Wilcoxon test. Statistical significance was defined as p<0.05. Treatment(s) included angiotensin-converting enzyme inhibitors (n=7), calcium channel-blocking agents (n=10), diuretics (n=3), statins (n=3), nitrates (n=6), beta-blockers agents (n=3) and sulfonylureas (n= 2). Hypertension only was significantly more prevalent in CAD patients compared to HS. Holter registration showed no sign of myocardial ischemia in all participants. A significant circadian variation in 11-d-TxB2 excretion for the 16 CAD patients (p< 0.0001) and the 12 HS (p< 0.04) was found. In the HS group, the higher level of 11-d-TxB2 was achieved at interval 2 (p< 0.02 versus interval 5 and p< 0.04 versus intervals 1 and 6) and 3 (p< 0.04 versus interval 5 and p< 0.02 versus interval 6). In CAD group, 11-d-TxB2 values measured in samples 1 and 2 were significantly higher than those observed at intervals 3,4,5 and 6 (p< 0.04 ). Eicosanoid excretion was higher in CAD patients than in HS with statistically significant differences observed at intervals 1 and 2 (p< 0.01)(Figure). A similar daily pattern was observed if 11-d-TxB2 was expressed as concentration in urinary volume (ng/ml)(data not shown). In HS our in vivo results confirm the previous ex vivo findings of a raised morning platelet function after awakening and assuming the upright posture (3–5). Accordingly, in our controls we observed an increase in TxB2 biosynthesis particularly at interval 3 (8–12am). In contrast, CAD patients exhibited a different circadian pattern of TxB2 biosynthesis with a nocturnal rise in the excretion rate of the eicosanoid. Thus, 11-d-TxB2 levels of intervals 1 (0–4am) and 2 (4–8am) were significantly increased compared to values measured at the remaining intervals (Figure).