Urinary 11-dehydro thromboxane B2 levels in type 2 diabetic patients before and during aspirin intake

Clinica Chimica Acta 412 (2011) 1366–1370

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Urinary 11-dehydro thromboxane B2 levels in type 2 diabetic patients before and during aspirin intake Lillian Harboe Gonçalves a,b, Luci Maria Sant'Ana Dusse a, Ana Paula Fernandes a, Karina Braga Gomes a, Mirelle Oliveira Sóter a, Michelle Teodoro Alves a, Kathryna Fontana Rodrigues a, Fernanda Rocha Freitas a, Flavia Komatsuzaki a, Marinez Oliveira Sousa a, Adriana Aparecida Bosco c, Gérson Antônio Pianett d, Maria das Graças Carvalho a,⁎ a

Department of Clinical and Toxicological Analysis-Faculty of Pharmacy-Federal University of Minas Gerais, Belo Horizonte, Brazil Rede Sarah de Hospitais de Reabilitação, Belo Horizonte, Brazil c Santa Casa Hospital, Belo Horizonte, Brazil d Department of Pharmaceutical Products, Brazil b

a r t i c l e

i n f o

Article history: Received 15 September 2010 Received in revised form 6 April 2011 Accepted 7 April 2011 Available online 13 April 2011 Keywords: Aspirin Type 2 diabetes 11-Dehydro thromboxane B2 Primary prevention

a b s t r a c t Background: Diabetic patients commonly present an increased risk for cardiovascular events, for which aspirin is the most frequently used medication for primary prevention. Urinary 11-dehydro thromboxane (11dhTXB2) concentrations assess the effect of aspirin on platelets and identify patients who are at risk of cardiovascular events. The present study investigated whether or not type 2 diabetic patients who took a daily dose of 100 mg of aspirin had a significant reduction in urinary 11-dhTXB2 concentrations and whether these results were associated with clinical and laboratory variables. Methods: Eighty-one type 2 diabetic patients were enrolled in the study. Laboratory tests included the determination of lipidic profile, glycated hemoglobin, platelets count, molecular analysis for both GPIIbIIIa and COX-1 polymorphisms, and urinary 11-dhTXB2. Results: Patients' median value for urinary 11-dhTXB2 before aspirin intake was 179 pg/mg of creatinine. After 15 days taking aspirin, the patients presented median of 51 pg/mg of creatinine, thus revealing a significant difference between medians (p = 0.00). A reduction of 95% in urinary 11-dhTXB2 concentrations could only be identified in 4 patients (5%). A BMI of ≥ 26 presented a significant association with a reduction of urinary 11-dhTXB2 concentrations (p = 0.010), as shown by the multiple logistic regression model. Other clinical and laboratory variables showed no association. Conclusions: Regardless of the mechanisms related to aspirin non-responsiveness, most patients enrolled in the present study also presented a reduced or minimal response to low-dose aspirin therapy, thereby indicating a clear variability related to aspirin effectiveness. Moreover, BMI appears to be independently associated to the reduction of urinary 11-dhTXB2 concentrations in type 2 diabetic patients taking aspirin. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Type 2 diabetic patients commonly present an increased risk for cardiovascular events. Aspirin therapy was jointly recommended in 2007 by the American Diabetes Association (ADA) and the American Heart Association (AHA) for the primary prevention in type 2 diabetes mellitus patients with increased cardiovascular risk [1]. Given that it inhibits thromboxane A2 (TXA2) synthesis, aspirin is the most frequently used antithrombotic medication and reduces the risk of cardiovascular events by N25% in a broad category of patients [2,3]. It ⁎ Corresponding author at: Faculty of Pharmacy of the Federal University of Minas Gerais-Avenida Antônio Carlos, 6627, Pampulha, Belo Horizonte, Minas Gerais, 31270910, Brazil. Tel.: + 55 3134096881. E-mail address: [email protected] (M.G. Carvalho). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.04.006

exerts its major antithrombotic effect by irreversibly acetylating platelet cyclooxygenase-1 (COX-1), thereby blocking the formation of TXA2. Urinary concentrations of 11-dehydro thromboxane (11dhTXB2), a stable metabolite of TXA2, can indicate the extent of inhibition of TXA2 generation as well as platelets activation [4–6]. High concentrations of urinary 11-dhTXB2 when undergoing aspirin therapy indicate an incomplete inhibition of TXA2 synthesis and has been associated with an increased risk of cardiovascular events [7]. Recent studies suggest that some patients do not benefit from aspirin's effect on TXA2 synthesis as a means of primary prevention, especially in diabetic patients. The meta-analysis study of the Antithrombotic Trialist Collaboration found that low aspirin doses for the primary prevention of serious vascular events did not benefit patients in the same manner that it did for secondary prevention [6]. In another meta-analysis study [8], it was reported that aspirin

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therapy in diabetic patients for primary prevention does not significantly reduce the risk of cardiovascular events. Data from clinical trials have shown that the aspirin effect may vary in an individual manner, and that obesity and poor glycemic control may influence platelet reactivity [9,10] and are associated with a poor response to aspirin antiplatelet effects [11,12]. Aspirin responsiveness has also been associated with genetic polymorphisms of the platelet glycoprotein (GP) IIbIIIa gene, a Leu (PlA1) to Pro (PlA2) substitution at position 33 of the GP IIIa subunit [13–15] and of cyclooxygenase-1 gene, a two single-nucleotide polymorphisms, A-842G and C50T, which are in complete linkage disequilibrium [16–18]. According to Patrono et al. [19], acetylation of the COX-1 platelet does not attain functional relevance until the maximal capacity to generate thromboxane A2 is reduced by at least 95%. This reduction can be assessed by urinary 11-dhTXB2 measurements. Therefore, this study aimed to measure the urinary 11-dhTXB2 concentrations of type 2 diabetic patients before and during aspirin intake to verify whether or not a 95% reduction had in fact been achieved. Considering that urinary 11-dhTXB2 concentrations have been used to assess the effect of aspirin on platelets and identify patients who are at risk of cardiovascular events, the aim of this study was to investigate the aspirin responsiveness in type 2 diabetes patients by measuring the urinary 11-dhTXB2 concentrations and by determining whether or not these results can be affected by clinical and molecular variables. As aspirin still continues to be prescribed to millions of individuals worldwide as a useful tool for the prevention of thrombotic events, it was deemed pertinent to investigate how asymptomatic type 2 diabetic patients from our geographic region respond to the administration of aspirin in low doses, considering that prior literature reveals a wide range of variability in the effectiveness of aspirin [20–22].

and on the fifteenth day after taking 100 mg of daily. A sample was drawn in one test tube with no anticoagulant to obtain serum on both days. Another sample was drawn in a second tube with EDTA (Sarsted®) to obtain platelet count, glycated hemoglobin (HbA1c) and a molecular analysis on the first blood collection day only. All participants were asked to provide a first morning urine specimen on both days of blood collection. Serum and urine samples were divided into aliquots and stored at − 80 °C until analysis. Platelets count and HbA1c dosage were performed on the same day of blood collection. Genomic DNA was extracted from peripheral white blood cells on the following day using “Wizard Genomic DNA Purification” (Promega Inc., Madison, WI), according to standard protocols. Stored urine and serum samples were thawed at the moment of analysis. Urinary 11-dhTXB2 was quantitatively measured using a commercially available enzyme immunoassay (Cayman Chemical Co, Ann Arbor MI 11-dhTXB2 EIA kit) with interassay and intra-assay coefficients of variation of 12.2% and 10%, respectively. Urinary creatinine was measured using colorimetric method. Total and HDL cholesterol as well as triglycerides were analysed by enzimatic colorimetric method. LDL cholesterol was calculated using Friedwald's equation. HbA1c was measured using ion exchange chromatography method. Platelets count was obtained using Coulter T-890®. GPIIbIIIa (PIA) gene polymorphism was studied using a polymerase chain reaction–restriction fragment length polymorphism (PCRRFLP) technique [14,24]. The C50T COX-1 polymorphism (with C50T in complete linkage disequilibrium with A-842G) was detected using a nested polymerase chain reaction–restriction fragment length polymorphism technique [25]. All assays were performed by laboratory staff blinded to sample identification. Compliance to treatment was self-reported.

2. Materials and methods

2.2. Statistical analysis

A single center study was conducted on a cohort of type 2 diabetic patients attending the outpatient clinic of the Santa Casa Hospital in Belo Horizonte, Brazil. A total of 81 type 2 diabetic patients (58 females and 23 males) with a mean age of 57.20 ± 9.85 years (ranging from 30 to 81 years) and who were taking 100 mg of aspirin daily (distributed for free by the Brazilian Public Health Service), were enrolled in the study. Laboratory and statistical analyses were performed at the Faculty of Pharmacy from the Federal University of Minas Gerais (UFMG), Brazil. The study was conducted between June 2008 and June 2010. Local institutional ethics committees approved this study and informed consent was obtained from all participants. Patients were selected sequentially following inclusion and exclusion criteria and type 2 diabetes diagnosis was defined according to the criteria of American Diabetes Association [23]. Only patients who declared no use of aspirin for at least two weeks prior to blood collection and with a medical prescription for a daily intake of 100 mg of aspirin were included. Exclusion criteria were therapy with other antiplatelet agents, unfractionated or low molecular weight heparin, antithrombotic and nonsteroidal anti-inflammatory drugs, chronic or acute inflammatory diseases, hepatic or renal disease, surgery at any time during the last six months and pregnancy. Descriptive data as well as body weight and height were obtained during patient interviews and from health records taken on the day of the invitation.

All data were checked for normality using the Shapiro Wilk test. Results were represented as mean and standard deviation (SD) when data were normally distributed. When abnormally distributed, data were presented as median and interquartile range. Statistical analysis was initially performed usingPearson Chi-square, Mann Whitney, Kruskal Wallis, t test and Wilcoxon tests and was considered significant when p b 0.05. To examine the association between baseline patients characteristics and the reduction of urinary 11-dhTXB2 concentrations, variables significant to 0.20 concentration were selected by a univariate analysis to be submited to a multiple logistic regression model. Goodness of fit for this model was assessed using the Hosmer and Lemeshow test. A p b 0.05 was considered statistically significant in the final multiple logistic regression. All analyses were performed using the Statistical Package of the Social Sciences software (SPSS), ver 13.0.

2.1. Laboratory investigation Blood laboratory tests included the determination of total and HDL cholesterol, triglycerides, glycated hemoglobin (HbA1c), platelets count, and molecular analysis of GPIIbIIIa (PIA) and COX-1 polymorphisms. Urine analysis included creatinine and 11-dhTXB2. Blood samples were obtained by clean venipuncture after 12 h of overnight fasting on the day before beggining treatment with aspirin

3. Results Sixty eight patients presented a diagnosis of hypertension, 9 of whom were current smokers while 18 were prior smokers. Six patients had no prior history of medication to control diabetes. Oral hypoglycemic medication was used by 66 patients, while 50 used insulin. Statin therapy was used by 45 patients. Patients presented a median body mass index (BMI) of 30 kg/m2 (interquartile range: 25– 32 kg/m2), a mean platelets count of 235 ± 57/mm3 and a median HbA1c of 8.0% (interquartile range: 6.7–8.8%). The lipidic profile of patients showed a median total cholesterol of 189 mg/dl (interquartile range: 160–214 mg/dl), HDL cholesterol of 49 mg/dl (interquartile range: 42–58 mg/dl), LDL cholesterol of 107 mg/dl (interquartile range: 90–131 mg/dl) and triglycerides of 130 mg/dl (interquartile range: 93–178 mg/dl). Patients' median value for urinary 11-dhTXB2 immediately before aspirin intake was 179 pg/mg creatinine (interquartile range: 117–

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Table 1 Urinary 11-dhTXB2 levels before and after 15 days of aspirin intake, according to participants' characteristics. Participants' characteristics

Median (IQR) 11-dh TXB2 before aspirin intake (pg/mg creatinine)

Median (IQR) 11-dh TXB2 after aspirin intake (pg/mg creatinine)

p value*

% reduction

Male (n = 23) Female (n = 58) p value** Hypertensive (n = 68) Nonhypertensive (n = 13) p value** Current smoker (n = 9) Prior smoker (n = 18) Non smoker (n = 54) p value*** BMI ≤ 25 kg/m2 (n = 18) BMI N 25 ≤ 30 kg/m2 (n = 21) BMI N 30 kg/m2 (n = 42) p value*** Total cholesterol ≤ 200 mg/dL(n = 52) Total cholesterol N 200 mg/dL (n = 29) p value** LDL cholesterol ≤ 130 mg/dL(n = 60) LDL cholesterol N 130 mg/dL(n = 21) p value** Triglycerides ≤ 150 mg/dL (n = 49) Triglycerides N 150 mg/dL (n = 32) p value** HbA1c ≤ 7.0% (n = 24) HbA1c N 7 b10% (n = 40) HbA1c ≥ 10.0% (n = 17) p value*** GP IIbIIIa polymorphism absent (n = 60) GP IIbIIIa polymorphism present (n = 21) p value** COX-1 polymorphism absent (n = 65) COX-1 polymorphism present (n = 16) p value**

151 (215–104) 204 (320–131) 0.107 179 (284–117) 179 (315–61) 0.852 268 (477–215) 179 (277–97.5) 164 (284–117) 0.165 163 (211–82) 174 (335–101) 208 (326–147.5) 0.173 176 (290–111) 179 (296–149) 0.708 188 (306–116) 160 (253–139) NS 174 (284–117) 179 (371–118) NS 174 (239–119) 165 (298–117) 219 (361–103) NS 179 (326–120) 156 (216–116) NS 179 (284–124) 180 (316–100) NS

52 (70–33) 51 (80–32) 0.814 52 (81–33.5) 46 (77–23) 0.563 68 (161–34) 64 (88–43) 45 (72–32) 0.201 63 (99–32) 51 (70–34) 48 (79–30) 0.591 51 (70–31) 54.00 (89–36) 0.183 51 (72–31) 54 (99–36) NS 46 (74–31) 52 (88–40) NS 51 (66–23) 50 (81–36) 65 (90–32) NS 55 (88–33) 36 (57–30) 0.034 46 (85–31) 53 (72–48) NS

0.00 0.00

66 75

0.00 0.015

71 74

0.021 0.001 0.00

75 64 72

0.005 0.00 0.00

61 71 77

0.00 0.00

71 70

0.00 0.00

73 66

0.00 0.00

74 71

0.00 0.00 0.025

71 69 70

0.00 0.00

69 77

0.000 0.001

74 71

IQR = interquartile range; BMI = body mass index; * Wilcoxon test; **Mann Whitney test; ***Kruskal Wallis test.

289 pg/mg creatinine). After 15 days of taking aspirin patients presented a median urinary 11-dhTXB2 of 51 pg/mg creatinine (interquartile range: 32–79 pg/mg creatinine). Thus, a significant difference between medians could be observed (p= 0.00; Wilcoxon test). GPIIbIIIa (PIA) polymorphism studies showed that the allele PIA1A2 was present in 19 patients (heterozygosity) while the allele PIA2A2 was found in 2 patients (homozygosity). Among the 81 patients studied, 10 were heterozygous for the C50T polymorphism and 6 were homozygous . The expected reduction of 95% in urinary 11-dhTXB2 concentrations after aspirin intake [26] could only be identified in 4 patients (5%) while a reduction of 90% was found in 10 patients (12%). Reductions in urinary 11-dhTXB2 concentrations of 76–100% could be observed in 36 patients, of 51–75% in 24 patients and of 26–50% in 9 patients. Three patients presented a reduction of 1–25%. One patient presented no change in urinary 11-dhTXB2 concentrations and 8 presented higher concentrations after 15 days taking 100 mg of aspirin most likely due to an inflammatory state during this period. Table 1 shows a comparison focusing on the percentage of reduction in urinary 11-dhTXB2 concentrations according to patients' characteristics. From all variables analysed only GPIIb/IIIa polymorphism (PIA1A2+ PIA2A2), as compared to those with wild type (PIA1A1), appears to show a favorable effect on the urinary 11-dhTXB2 concentrations, in turn leading to a significant reduction in the concentrations of this marker (p = 0.034). Considering a reduction of over 90% in urinary 11-dhTXB2 concentrations as an independent variable, no studied variable could be included in the multiple logistic regression model, given that no variable reached the limit of p≤0.20 in an univariate analysis. This is most likely due to the fact that this reduction could only be identified in 14 patients (17%). Considering a reduction of 50–100% in urinary 11-dhTXB2 concentrations

as an independent variable, only BMI and oral hypoglycemic use reached the limit of p≤0.20. Hence, these variables could be included in the multiple logistic regression. According to this model a reduction of ≥50– 100% in urinary 11-dhTXB2 concentrations proved to be significantly associated with BMI only as shown by the multiple logistic regression model (Table 2). Thus, patients with a BMI of N25 kg/m2 and ≤30 kg/m2 present a nearly 7 times greater chance of lower concentrations of urinary 11-dhTXB2, (OR=6.939; CI=1.592–30.243; p=0,010), i.e., a higher percentage of reduction in the concentrations of this marker, as compared to patients with BMI≤25 kg/m2, while patients with a BMI of N30 kg/m2 tend to present a nearly 5.5 times greater chance (OR = 5.510; CI=1.531–19.836; p=0.009). 4. Discussion It is widely recognised that regional and demographic characteristics may affect clinical and lab variables in different manners. As physicians must be aware of the variability of aspirin effectiveness in Table 2 Multiple logistic regression model using a reduction of 50–100% in urinary 11-dhTXB2 levels as a response variable. Variable

Reduction ≥ 50–100%

Reduction b 50–100%

N(%)

N(%)

OR

CI 95% OR

p value

1 6.939 5.510

[1.592;30.243] [1.531;19.836]

0.010 0.009

2

BMI (kg/m ) ≤ 25 10 (50.0) N 25 ≤ 30 21 (84.0) N 30 30 (83.3)

10 (50.0) 4 (16.0) 6 (16.7)

BMI = body mass index; OR = Odds ratio; CI = Confidence Interval; Goodness-of-fit logistic regression by Hosmer and Lemeshow test (p = 0.338).

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general, it is essential to assess individual responses to verify whether or not aspirin is working properly in type 2 diabetes patients, in this case, those selected from a Brazilian population sample. Although the expected 95% of reduction in urinary 11-dhTXB2 concentrations was not achieved, median values of 11-dhTXB2 before and after aspirin intake were statistically different (p = 0.00). Zisman et al. [27] studied healthy individuals and aspirin-treated patients and found that urine concentrations of 11-dhTXB2 were approximately three times lower with aspirin treatment than without, although in two patients concentrations were higher with aspirin. Our results show that median urinary 11-dhTXB2 concentrations were more than three times lower after aspirin intake in most patients. Carrol et al. [28] assayed 11-dhTXB2 concentrations in the first voided urine samples collected 2–8 h after interventional cardiology procedures performed on aspirin resistant and aspirin sensitive patients. Values were significantly higher (p = 0.008) in the aspirin resistant group (845 ± 614 pg/mg creatinine, n = 14) as compared to the aspirin inhibited group (493 ± 348 pg/mg creatinine, n = 46). These values are much higher than those of the present study, as they reflect the inflammatory state triggered by interventional cardiology procedures. Our study population presented basic characteristics which can be found in the realm of patients who attend specialized public health care clinics in Brazil in an attempt to control diabetes. Women tended to be the predominant patients. In addition, only 11% reported being current smokers, most likely because the patients had become conscious of the risks that tobacco brings to diabetic patients. Almost all patients presented reasonable total and LDL cholesterol concentrations probably because those who presented hypercholesterolemia were taking statin. However, patients' HbA1c concentrations proved to be undesirable, given that the mean value found was 8% whereas the limit value that indicates a good glycemic control is 7%. Median body mass index found for the group (30 kg/m2) also indicated a poor weight control and nearly obese patients. Analysing these data as a whole may partly explain why patients with usual doses of aspirin showed an incomplete suppression of urinary 11-dhTXB2 suggesting an increased risk of subsequent vascular events. Undas et al. [29] studied GPIIbIIIa (PIA) polymorphism and found that the allele PIA2 is expressed in platelets of approximately 25% of all caucasians, while Pamukcu et al. [14] found 22% with the PIA1A2 allele but no one with the PIA2A2 in a population of 94 patients. The present study found that the GPIIbIIIa (PIA) polymorphism was not correlated to a reduction in urinary 11-dhTXB2 concentrations. Pamukcu et al. [14] found no statistically significant relation between aspirin resistance and GPIIbIIIa (PIA) polymorphism while Macchi et al. [13] concluded that homozygous platelets (PIA2A2 allele) appeared to be less sensitive to inhibitory action of aspirin administered in low doses. Surprisingly, in the present study, lower concentrations of urinary 11-dhTXB2 in patients presenting the GPIIbIIIa (PIA) polymorphism (PIA1A2 and PIA2A2), as compared to those with the wild type (p= 0.034; Table 1), could be observed. However, the small number of patients assayed as a whole and consequently the very small subset of patients presenting the PIA2 allele (2 homozygous and 19 heterozygous) preclude any conclusive comment on its association with 11-dhTXB2 concentrations in patients taking aspirin. At the present study, among the 81 patients studied, 12.35% were heterozygous for the C50T polymorphism and 7.40% were homozygous. However, COX-1 polymorphism did not affect the reduction in urinary 11-dhTXB2 concentrations, which is in agreement with the results found by Pettinella et al. [30]. Eikelboom et al. [7] found that increasing age, female sex, current smoking, and oral hypoglycemic or angiotensin-converting enzyme inhibitor therapy were independently associated with higher concentrations of 11-dhTXB2 in patient urine, whereas statin treatment was associated with lower concentrations. The present study found no association between these variables and a reduction in urinary 11dhTXB2 concentrations. By contrast, it was observed that only the

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body mass index presented a significant association with the reduction in urinary 11-dhTXB2 concentrations. This result suggests that aspirin resistance is negatively associated with obesity in patients with type 2 diabetes, given that the reduction of urinary 11-dhTXB2 concentrations was higher in patients with BMI N25 kg/m2. The association of BMI and reduction of the aspirin effect on urinary 11dhTXB2 concentrations were comparable in subjects with BMI of N25 and ≤30 kg/m2 and subjects with BMI of N30 kg/m2 (Table 2). A small size of the subject population may explain the unexpected results related to a higher odds ratio for BMI N25 and ≤30 kg/m2 compared to the odds ratio for BMI N 30 kg/m2 and this finding constitutes a limitation of this study. Opposite findings have recently been reported in studies by Cohen et al. and Ertugrul et al., using platelet aggregation as a method for evaluating COX-1 inhibition by aspirin. On the other hand, a recent study by Frelinger et al. showed that serum thromboxane B2 concentrations proved to be higher in aspirin-treated patients with obesity than in those without obesity. Comparing our results with those found by these investigators, from the viewpoints of methodology, the discrepancies may be explained by different doses of drug, the type of assay used, patient population and studied size sample, among other factors, such as, statin and antidiabetic drugs used in parallel, the duration of the diabetes, glycemic control, etc. The measurement of urinary 11-dhTXB2 concentrations is more direct and sensitive for evaluating COX-1 activity than is platelet aggregation. Thus, our assay evaluated the generation of urinary 11-dhTXB2, which depends on in vivo platelet activation, which may be a reflection of the total atherothrombotic burden, among other factors. It is known that urinary 11-dhTXB2 may be derived, in part, from nonplatelet sources. Therefore, it can be concluded that, in the Brazilian aspirin-treated patient population, the variable response to this drug was due in part to incomplete COX-1 inhibition as well as to COX-1-independent platelet hyperreactivity. Whether aspirin response in patients with diabetes type 2 is associated to BMI warrants further investigation involving a larger number of individuals in an attempt to clarify these conflicting findings. Emerging evidence of variable response to the administration of aspirin in low doses was confirmed in the present study in a small set of Brazilian asymptomatic type 2 diabetes patients who showed biochemical evidence of persistent thromboxane-dependent platelet activation supported by high concentrations of 11-dhTXB2 in their urine samples. Potential mechanisms involved may include accelerated platelet turnover, drug bioavailability, competitive interference by other NSAID's, platelet polymorphisms, hyperlipidemia, diet and lifestyle, reduced COX-1 binding capacity by aspirin due to COX-1 hyperglication and enhanced thromboxane production by COX-2, among others. Despite the small number of investigated cases, our results from the local area of Belo Horizonte, Brazil, confirm previous reports developed in other regions of the world which focus on an insufficient suppression of thromboxane generation with low-dose aspirin intake. Considering that as much as a 95% redution in the urinary 11-dhTXB2 concentrations is essential to prevent cardiovascular events [19], clinical cointerventions to minimize the effect of modifiable variables may well represent an effective strategy for the management of patients showing a limited antiagregant response to low-dose aspirin intake. Therefore, the lack of aspirin responsiveness should be the basis for other alternatives measures such as other antiplatelet therapies and clinical cointerventions in addition to diet and lifestyle changes, in order to reduce the risk of cardiovascular events. Finally, another important aspect to be considered is that urinary 11-dhTXB2 is not subjected to the preanalytical variables associated with other blood-based indirect measures of platelet activation. Urinary 11-dhTXB2 constitutes a readily available marker for in vivo platelet activation and its quantification may be very useful for

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monitoring type 2 diabetic patients taking low-dose aspirin, considering that an ineffective response may lead to an increased risk of death. Thus, the correct identification of patients who are not adequately protected despite their usual prescribed daily dose of aspirin is warranted. In addition, facilities for running 11-dhTXB2 assays may encourage lab professionals to introduce this procedure into the clinical routine. 5. Conclusion Regardless of the mechanisms related to aspirin non-responsiveness, most patients enrolled in the present study also presented a reduced or minimal response to low-dose aspirin therapy, thereby indicating a clear variability related to aspirin effectiveness. Within the present study's conditions, gender, age, hypertension, smoking habits, insulin use, oral hypoglycemic use, statin intake, as well as platelet count, total and HDL cholesterol, triglycerides and HbA1c concentrations, and GPIIbIIIa and COX-1 polymorphisms did not affect urinary 11-dhTXB2 concentrations. However, higher body mass index appeared to be independently associated with the reduction in urinary 11-dhTXB2 concentrations in Brazilian asymptomatic type 2 diabetic patients taking aspirin. List of abreviations 11-dhTXB2 11-dehydro thromboxane B2 COX-1 Cyclooxygenase-1 GP Platelet glycoprotein TXA2 Thromboxane A2

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