Although less clinical intervention studies have been performed with fibrates than with statins, there are evidences indicating that fibrates may reduce risk of cardiovascular events. The potential clinical benefit of the fenofibrate will be specified by the ongoing Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study, which rationale, methods and aims have been just published.Controlled clinical trials show similar or even greater cardiovascular benefits from statins-based therapy in patient subgroups with diabetes compared with overall study populations. Therefore, statins are the drug of first choice for aggressive lipid lowering actions and reducing risk of coronary artery disease in these patients. However, current therapeutic use of statins as monotherapy is still leaving many patients with mixed atherogenic dyslipidemia at high risk for coronary events. A combination statin/fibrate therapy may be often necessary to control all lipid abnormalities in patients with metabolic syndrome and diabetes adequately, since fibrates provide additional important benefits, particularly on triglyceride and HDL-cholesterol levels. Thus, this combined therapy concentrates on all the components of the mixed dyslipidemia that often occurs in persons with diabetes or metabolic syndrome, and may be expected to reduce cardiovascular morbidity and mortality.Safety concerns about some fibrates such as gemfibrozil may lead to exaggerate precautions regarding fibrate administration and therefore diminish the use of the seagents. However, other fibrates, such as bezafibrate and fenofibrate appear to be safer and better tolerated. We believe that a proper co-administration of statins and fibrates, selected on basis of their safety, could be more effective in achieving a comprehensive lipid control as compared with monotherapy.

Due to their beneficial effects on glucose and lipid metabolism, peroxisome proliferator activated receptors (PPAR's) alpha agonists (fibrates) are good potential candidates for reducing the risk of myocardial infarction (MI) in subjects with metabolic syndrome [ 1 - 3 ]. Although less clinical intervention studies have been performed with fibrates than with statins, there are evidences indicating that fibrates may reduce risk of cardiovascular disease and particularly non-fatal MI [ 4 - 10 ]. Interestingly, reduction of cardiovascular disease with one of the fibric acid derivates – gemfibrozil – was more pronounced in patients displaying baseline characteristics very similar to metabolic syndrome definitions [ 4 , 5 ]. There have been no direct head-to-head comparisons of a statin with a fibrate in any clinical endpoint trial. However, compared with statins, fibrates appear to more selectively target the therapeutic goals in obese individuals with features of insulin resistance and metabolic syndrome (i.e. with near-goal low-density lipoprotein (LDL)-cholesterol and inappropriate high-density lipoprotein (HDL)-cholesterol and triglyceride levels). The primary-prevention trial Helsinki Heart Study showed that treatment with gemfibrozil led to a significant reduction in major cardiovascular events [ 4 ]. Regarding secondary prevention, in the VAHIT study (Veterans Affairs High-density lipoprotein cholesterol Intervention Trial) – which included 30% of diabetic patients – gemfibrozil reduced the occurrence of major cardiovascular events by 22 % [ 5 ]. Similarly, reduction of cardiovascular disease with gemfribrozil was more pronounced in patients displaying above three of the features of metabolic syndrome [ 11 , 12 ]. In two previous small studies bezafibrate decreased the rate of progression of coronary atherosclerosis and decreased coronary event rate [ 6 , 7 ]. In another large trial in 1568 men with lower extremity arterial disease with a relatively short follow-up period, bezafibrate reduced the severity of intermittent claudication for up to three years. [ 8 ]. Ingeneral, the incidence of coronary heart disease in patients on bezafibrate has tended to be lower , but this tendency did not reach statistical significance. However, bezafibrate had significantly reduced the incidence of non-fatal coronary events, particularly in those aged <65 years at entry, in whom all coronary events may also be reduced [ 8 ]. In the Bezafibrate Infarction Prevention (BIP) study an overall trend of a 9.4% reduction of the incidence of primary end point (fatal or non-fatal myocardial infarction or sudden death) was observed. The reduction in the primary end point in 459 patients with high baseline triglycerides (≥200 mg/dL) was significant [ 9 ]. These results are consistent with studies in experimental models showing that pre-treatment of rats with the PPAR-alpha agonist clofibrate causes a significant reduction in induced myocardial infarct size of 43% [ 13 ]. Recently, reduced incidence of type 2 diabetes in patients with impaired fasting glucose level on bezafibrate has been demonstrated [ 14 ]. The potential clinical benefit of the other widespread fibric acid derivative, fenofibrate, on the reduction of cardiovascular disease is still unknown and will be specified by the ongoing Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study, which rationale, methods and aims have been just published [ 15 ]. It will be the largest (approximately 10000 patients) ever conducted fibrate-based controlled clinical trial in diabetic patients. The results are expected for 2005. An added strength of this trial is its ability to examine important clinical outcomes across diverse ethnic and gender subgroups. The results of this study will clarify whether the beneficial lipid-modifying effects of micronised fenofibrate lead to a reduction of cardiovascular morbidity and mortality. Despite increasing use of statins, a significant number of coronary events still occur and many of such events take place in patients presenting with the metabolic syndrome. Whereas statins remain the drug of choice for patients who need to achieve the LDL-cholesterol goal, fibrate therapy may represent the alternative intervention for subjects with atherogenic dyslipidemia typical for metabolic syndrome and an LDL-cholesterol already close to goal values. In addition, the concomitant use of fibrates seems to be attractive in patients whose LDL-cholesterol is controlled by statin therapy but whose HDL-cholesterol and/or triglyceride levels are still inappropriate [ 16 - 19 ]. This strategy will be tested in the ongoing Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial [ 20 ]. The factor that dominates in overweight-related metabolic syndrome is the permanent elevation of plasma free fatty acids (FFA) and the predominant utilization of lipids by the muscle inducing a diminution of glucose uptake and insulin resistance. Currently, an insulin-resistant state – as the key phase of metabolic syndrome – constitutes the major risk factor for development of macrovascular complications [ 21 - 23 ]. On the basis of the current concept of the evolution of adipogenesis via PPAR modulation toward insulin resistance and atherothrombotic macrovascular complications (including MI), the decreasing of plasma FFA and improving of insulin sensitization by PPAR agonists seems to be a logical and valuable goal for therapy. It is important to note that on a whole-body level, lipid and glucose metabolisms interact intimately. Briefly, PPAR alpha is activated by fibric acids (e.g. bezafibrate) and form heterodimers with the 9-cis retinoic acid receptor (RXR). These heterodimers bind to peroxisome proliferator response elements, which are located in numerous gene promoters and increase the level of the expression of mRNAs encoded by PPAR alpha target genes. Bezafibrate reduces triglyceride plasma levels through increases in the expression of genes involved in fatty acid-beta oxidation and by decrease in apolipoprotein C-III gene expression. Fibric acids increase HDL-cholesterol partly by increasing apolipoprotein A-I and apolipoprotein A-II gene expression. Their triglyceride-lowering and HDL-cholesterol raising effects lead to decreased systemic availability of fatty acid, diminished fatty acid uptake by muscle with improvement of insulin sensitization and reduced plasma glucose level [ 24 - 28 ]. Evidence also suggests that there is a 'fibrate effect' that mediates the reduction in CHD risk beyond the favorable impact of these agents on HDL-cholesterol levels. This last notion is consistent with the pleiotropic effects of fibrates which are known to be related to their mechanisms of action [ 29 ]. Being PPAR alpha ligands, fibrates have a significant impact on the synthesis of several apolipoproteins (apo) and enzymes of lipoprotein metabolism as well as on the expression of several genes involved in fibrinolysis and inflammation. Such changes contribute to improve the catabolism of triglyceride-rich lipoproteins, leading to a substantial increase in HDL-cholesterol levels accompanied by a shift in the size and density of LDL particles (from small, dense LDL particles to larger, more buoyant cholesteryl ester-rich LDL). Controlled clinical trials show similar or even greater cardiovascular benefits from statins-based therapy in patient subgroups with diabetes, impaired fasting glucose, and metabolic syndrome, compared with overall study populations. Therefore, statins are the drug of first choice for aggressive lipid lowering actions and reducing risk of coronary artery disease in these patients. However, current therapeutic use of statins as monotherapy is still leaving many patients with mixed atherogenic dyslipidemia at high risk for coronary events. A combination statin/fibrate therapy may be often necessary to control all lipid abnormalities in patients with metabolic syndrome and diabetes adequately, since fibrates provide additional important benefits, particularly on triglyceride and HDL-C levels. Thus, this combined therapy concentrates on all the components of the mixed dyslipidemia that often occurs in persons with diabetes or metabolic syndrome, and may be expected to reduce cardiovascular morbidity and mortality. Safety concerns about some fibrates such as gemfibrozil may lead to exaggerate precautions regarding fibrate administration and therefore diminish the use of the seagents. However, other fibrates such as bezafibrate and fenofibrate appear to be safer and better tolerated [ 30 - 36 ]. We believe that a proper co-administration of statins and fibrates, selected on basis of their safety, could be more effective in achieving a comprehensive lipid control as compared with monotherapy.

Due to their beneficial effects on glucose and lipid metabolism, peroxisome proliferator activated receptors (PPAR's) alpha agonists (fibrates) are good potential candidates for reducing the risk of myocardial infarction (MI) in subjects with metabolic syndrome [ 1 - 3 ]. Although less clinical intervention studies have been performed with fibrates than with statins, there are evidences indicating that fibrates may reduce risk of cardiovascular disease and particularly non-fatal MI [ 4 - 10 ]. Interestingly, reduction of cardiovascular disease with one of the fibric acid derivates – gemfibrozil – was more pronounced in patients displaying baseline characteristics very similar to metabolic syndrome definitions [ 4 , 5 ].There have been no direct head-to-head comparisons of a statin with a fibrate in any clinical endpoint trial. However, compared with statins, fibrates appear to more selectively target the therapeutic goals in obese individuals with features of insulin resistance and metabolic syndrome (i.e. with near-goal low-density lipoprotein (LDL)-cholesterol and inappropriate high-density lipoprotein (HDL)-cholesterol and triglyceride levels).The primary-prevention trial Helsinki Heart Study showed that treatment with gemfibrozil led to a significant reduction in major cardiovascular events [ 4 ]. Regarding secondary prevention, in the VAHIT study (Veterans Affairs High-density lipoprotein cholesterol Intervention Trial) – which included 30% of diabetic patients – gemfibrozil reduced the occurrence of major cardiovascular events by 22 % [ 5 ]. Similarly, reduction of cardiovascular disease with gemfribrozil was more pronounced in patients displaying above three of the features of metabolic syndrome [ 11 , 12 ].In two previous small studies bezafibrate decreased the rate of progression of coronary atherosclerosis and decreased coronary event rate [ 6 , 7 ]. In another large trial in 1568 men with lower extremity arterial disease with a relatively short follow-up period, bezafibrate reduced the severity of intermittent claudication for up to three years. [ 8 ]. Ingeneral, the incidence of coronary heart disease in patients on bezafibrate has tended to be lower , but this tendency did not reach statistical significance. However, bezafibrate had significantly reduced the incidence of non-fatal coronary events, particularly in those aged <65 years at entry, in whom all coronary events may also be reduced [ 8 ]. In the Bezafibrate Infarction Prevention (BIP) study an overall trend of a 9.4% reduction of the incidence of primary end point (fatal or non-fatal myocardial infarction or sudden death) was observed. The reduction in the primary end point in 459 patients with high baseline triglycerides (≥200 mg/dL) was significant [ 9 ]. These results are consistent with studies in experimental models showing that pre-treatment of rats with the PPAR-alpha agonist clofibrate causes a significant reduction in induced myocardial infarct size of 43% [ 13 ].Recently, reduced incidence of type 2 diabetes in patients with impaired fasting glucose level on bezafibrate has been demonstrated [ 14 ]. The potential clinical benefit of the other widespread fibric acid derivative, fenofibrate, on the reduction of cardiovascular disease is still unknown and will be specified by the ongoing Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study, which rationale, methods and aims have been just published [ 15 ]. It will be the largest (approximately 10000 patients) ever conducted fibrate-based controlled clinical trial in diabetic patients. The results are expected for 2005. An added strength of this trial is its ability to examine important clinical outcomes across diverse ethnic and gender subgroups. The results of this study will clarify whether the beneficial lipid-modifying effects of micronised fenofibrate lead to a reduction of cardiovascular morbidity and mortality.Despite increasing use of statins, a significant number of coronary events still occur and many of such events take place in patients presenting with the metabolic syndrome. Whereas statins remain the drug of choice for patients who need to achieve the LDL-cholesterol goal, fibrate therapy may represent the alternative intervention for subjects with atherogenic dyslipidemia typical for metabolic syndrome and an LDL-cholesterol already close to goal values. In addition, the concomitant use of fibrates seems to be attractive in patients whose LDL-cholesterol is controlled by statin therapy but whose HDL-cholesterol and/or triglyceride levels are still inappropriate [ 16 - 19 ]. This strategy will be tested in the ongoing Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial [ 20 ].The factor that dominates in overweight-related metabolic syndrome is the permanent elevation of plasma free fatty acids (FFA) and the predominant utilization of lipids by the muscle inducing a diminution of glucose uptake and insulin resistance. Currently, an insulin-resistant state – as the key phase of metabolic syndrome – constitutes the major risk factor for development of macrovascular complications [ 21 - 23 ]. On the basis of the current concept of the evolution of adipogenesis via PPAR modulation toward insulin resistance and atherothrombotic macrovascular complications (including MI), the decreasing of plasma FFA and improving of insulin sensitization by PPAR agonists seems to be a logical and valuable goal for therapy.It is important to note that on a whole-body level, lipid and glucose metabolisms interact intimately. Briefly, PPAR alpha is activated by fibric acids (e.g. bezafibrate) and form heterodimers with the 9-cis retinoic acid receptor (RXR). These heterodimers bind to peroxisome proliferator response elements, which are located in numerous gene promoters and increase the level of the expression of mRNAs encoded by PPAR alpha target genes. Bezafibrate reduces triglyceride plasma levels through increases in the expression of genes involved in fatty acid-beta oxidation and by decrease in apolipoprotein C-III gene expression. Fibric acids increase HDL-cholesterol partly by increasing apolipoprotein A-I and apolipoprotein A-II gene expression. Their triglyceride-lowering and HDL-cholesterol raising effects lead to decreased systemic availability of fatty acid, diminished fatty acid uptake by muscle with improvement of insulin sensitization and reduced plasma glucose level [ 24 - 28 ].Evidence also suggests that there is a 'fibrate effect' that mediates the reduction in CHD risk beyond the favorable impact of these agents on HDL-cholesterol levels. This last notion is consistent with the pleiotropic effects of fibrates which are known to be related to their mechanisms of action [ 29 ]. Being PPAR alpha ligands, fibrates have a significant impact on the synthesis of several apolipoproteins (apo) and enzymes of lipoprotein metabolism as well as on the expression of several genes involved in fibrinolysis and inflammation. Such changes contribute to improve the catabolism of triglyceride-rich lipoproteins, leading to a substantial increase in HDL-cholesterol levels accompanied by a shift in the size and density of LDL particles (from small, dense LDL particles to larger, more buoyant cholesteryl ester-rich LDL).Controlled clinical trials show similar or even greater cardiovascular benefits from statins-based therapy in patient subgroups with diabetes, impaired fasting glucose, and metabolic syndrome, compared with overall study populations. Therefore, statins are the drug of first choice for aggressive lipid lowering actions and reducing risk of coronary artery disease in these patients. However, current therapeutic use of statins as monotherapy is still leaving many patients with mixed atherogenic dyslipidemia at high risk for coronary events.A combination statin/fibrate therapy may be often necessary to control all lipid abnormalities in patients with metabolic syndrome and diabetes adequately, since fibrates provide additional important benefits, particularly on triglyceride and HDL-C levels. Thus, this combined therapy concentrates on all the components of the mixed dyslipidemia that often occurs in persons with diabetes or metabolic syndrome, and may be expected to reduce cardiovascular morbidity and mortality.Safety concerns about some fibrates such as gemfibrozil may lead to exaggerate precautions regarding fibrate administration and therefore diminish the use of the seagents. However, other fibrates such as bezafibrate and fenofibrate appear to be safer and better tolerated [ 30 - 36 ]. We believe that a proper co-administration of statins and fibrates, selected on basis of their safety, could be more effective in achieving a comprehensive lipid control as compared with monotherapy.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.


In  PLoS Biology , volume 2, issue 6:In  PLoS Biology , volume 2, issue 6:PLoS BiologyIntegrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA ClonesTadashi Imanishi, Takeshi Itoh, Yutaka Suzuki, Claire O'Donovan, Satoshi Fukuchi, et al.Tadashi Imanishi, Takeshi Itoh, Yutaka Suzuki, Claire O'Donovan, Satoshi Fukuchi, et al.DOI: 10.1371/journal.pbio.00201621. The abbreviation FLJ was expanded incorrectly (as full-length long Japan) in the abbreviations list and in the first paragraph of the Results/Discussion section. FLJ stands for full-length cDNA Japan.2. In Table 1, the number of cDNAs and the number of library origins for two of the cDNA sources were incorrect. The correct numbers are as follows:


Background Apolipoprotein A-V (Apo A-V) gene has recently been identified as a new apolipoprotein involved in triglyceride metabolism. A single nucleotide polymorphism (SNP3) located in the gene promoter (-1131) was associated with triglyceride variation in healthy subjects. In type 2 diabetes the triglyceride level increased compared to healthy subjects. Hypertriglyceridemia is a risk factor for coronary artery disease. We aimed to examine the interaction between SNP3 and lipid profile and coronary artery disease (CAD) in Tunisian type 2 diabetic patients.
Background
Apolipoprotein A-V (Apo A-V) gene has recently been identified as a new apolipoprotein involved in triglyceride metabolism. A single nucleotide polymorphism (SNP3) located in the gene promoter (-1131) was associated with triglyceride variation in healthy subjects. In type 2 diabetes the triglyceride level increased compared to healthy subjects. Hypertriglyceridemia is a risk factor for coronary artery disease. We aimed to examine the interaction between SNP3 and lipid profile and coronary artery disease (CAD) in Tunisian type 2 diabetic patients.Results The genotype frequencies of T/T, T/C and C/C were 0.74, 0.23 and 0.03 respectively in non diabetic subjects, 0.71, 0.25 and 0.04 respectively in type 2 diabetic patients. Triglyceride level was higher in heterozygous genotype (-1131 T/C) of apo A-V (p = 0.024). Heterozygous genotype is more frequent in high triglyceride group (40.9%) than in low triglyceride group (18.8%) ; p = 0.011. Despite the relation between CAD and hypertriglyceridemia the SNP 3 was not associated with CAD.
Results
The genotype frequencies of T/T, T/C and C/C were 0.74, 0.23 and 0.03 respectively in non diabetic subjects, 0.71, 0.25 and 0.04 respectively in type 2 diabetic patients. Triglyceride level was higher in heterozygous genotype (-1131 T/C) of apo A-V (p = 0.024). Heterozygous genotype is more frequent in high triglyceride group (40.9%) than in low triglyceride group (18.8%) ; p = 0.011. Despite the relation between CAD and hypertriglyceridemia the SNP 3 was not associated with CAD.Conclusion In type 2 diabetic patients SNP3 is associated with triglyceride level, however there was no association between SNP3 and coronary artery disease.
Conclusion
In type 2 diabetic patients SNP3 is associated with triglyceride level, however there was no association between SNP3 and coronary artery disease.

Background Dyslipidemia in type 2 diabetes are most frequently characterized by elevation of total serum triglycerides, of very low density lipoprotein-triglyceride (VLDL-TG) and low level of high density lipoprotein-cholesterol (HDL-C) [ 1 ]. Hypertriglyceridemia is an independent risk factor for coronary artery disease (CAD) in type 2 diabetes [ 2 ]. Triglyceridemia is modulated by environmental and genetic factors. A new identified gene associated with triglyceride level was the gene encoding for apo A-V located at the chromosome 11 (11q23), in the vicinity of apoA-I/C-III/A-IV cluster [ 3 ]. Studies on transgenic mice overexpressing human apo A-V showed a decreased level of triglyceride, whereas knock-out mice showed an increased level of triglyceride [ 3 ]. These results prove the regulator effect of apo A-V on triglyceride metabolism. Moreover apo A-V regulates levels of circulating triglyceride and cholesterol [ 4 ]. Four neighboring single nucleotide polymorphisms (SNP1 to SNP4) within apo A-V were identified by Pennachio et al [ 3 ]. The first three of the SNPs (SNPs1–3) were in significant linkage disequilibrium suggesting the existence of a common haplotype in apo A-V gene. The minor allele of each SNP was associated with high triglyceride level. In other study, the SNP3 (T/C polymorphism) was also associated with HDL-C concentration [ 5 ]. This suggests that genetic variability of the apo A-V gene is likely to also have an impact on the lipid profile of type 2 diabetic patients, but reports on the subjects are few [ 6 ]. We have addressed the issue to examine the interaction between SNP3 and lipid profile and coronary artery disease (CAD) in type 2 diabetic patients compared to controls in Tunisian population.
Background
Dyslipidemia in type 2 diabetes are most frequently characterized by elevation of total serum triglycerides, of very low density lipoprotein-triglyceride (VLDL-TG) and low level of high density lipoprotein-cholesterol (HDL-C) [ 1 ]. Hypertriglyceridemia is an independent risk factor for coronary artery disease (CAD) in type 2 diabetes [ 2 ]. Triglyceridemia is modulated by environmental and genetic factors. A new identified gene associated with triglyceride level was the gene encoding for apo A-V located at the chromosome 11 (11q23), in the vicinity of apoA-I/C-III/A-IV cluster [ 3 ]. Studies on transgenic mice overexpressing human apo A-V showed a decreased level of triglyceride, whereas knock-out mice showed an increased level of triglyceride [ 3 ]. These results prove the regulator effect of apo A-V on triglyceride metabolism. Moreover apo A-V regulates levels of circulating triglyceride and cholesterol [ 4 ]. Four neighboring single nucleotide polymorphisms (SNP1 to SNP4) within apo A-V were identified by Pennachio et al [ 3 ]. The first three of the SNPs (SNPs1–3) were in significant linkage disequilibrium suggesting the existence of a common haplotype in apo A-V gene. The minor allele of each SNP was associated with high triglyceride level. In other study, the SNP3 (T/C polymorphism) was also associated with HDL-C concentration [ 5 ]. This suggests that genetic variability of the apo A-V gene is likely to also have an impact on the lipid profile of type 2 diabetic patients, but reports on the subjects are few [ 6 ].We have addressed the issue to examine the interaction between SNP3 and lipid profile and coronary artery disease (CAD) in type 2 diabetic patients compared to controls in Tunisian population.Results Description of the participating groups Type 2 diabetic patients have a BMI values more important than non diabetic subjects. Whereas the WHR in lower in controls. Males and smokers are more frequent in patients with CAD (Table  1 ). To compare lipid parameters, we take into consideration the lipid lowering drugs use, then patients who are taking lipid lowering drugs were excluded (Table  2 ). Plasma total cholesterol and total triglyceride concentrations were significantly higher in type 2 diabetic patients than in the non diabetic patients. Subjects with CAD had lower concentration of HDL-C and higher concentration of triglyceride as compared to those without CAD (Table  2 ). Table 1 Clinical characteristics of the four studied groups Diabetes- CAD- Diabetes+ CAD- Diabetes+ CAD+ Diabetes- CAD+ Diabetes vs non Diabetes CAD vs non CAD Number 99 78 74 57 Sex (% of men) 50.5 49.4 75.4 84.2 ns < 0.001 Age (years) 52.9 ± 8.7 51.5 ± 7.3 55.7 ± 6.8 59.0 ± 6.8 ns < 0.001 BMI (Kg/m 2 ) 27.6 ± 4.0 28.8 ± 4.6 28.1 ± 3.8 26.6 ± 4.8 0.029 ns Smokers (%) 42 53 86 80 0.003 < 0.001 SBP (mmHg) 12.5 ± 1.3 13.2 ± 1.4 13.0 ± 1.8 12.4 ± 2.1 0.001 ns DBP (mmHg) 7.5 ± 0.7 7.8 ± 0.9 7.6 ± 1.0 7.1 ± 1.1 0.006 0.018 WHR 0.82 ± 0.09 0.92 ± 0.07 0.95 ± 0.07 0.92 ± 0.06 ns 0.016 Glucose (mmol/l) 5.2 ± 0.4 10.4 ± 3.4 11.7 ± 3.5 6.8 ± 3.6 < 0.001 < 0.001 HbA1c (%) 4.5 ± 0.9 10.4 ± 3.4 9.4 ± 2.4 11.3 ± 4.1 0.009 < 0.001 Diabetes duration (years) 6.8 ± 5.2 7.9 ± 6.1 Lipid lowering drug use (%) 11.1 0 16.2 70.2 SBP : systolic blood pressure DBP : diastolic blood pressure Table 2 lipid profiles of the four studied groups Diabetes- CAD- Diabetes+ CAD- Diabetes+ CAD+ Diabetes- CAD+ Diabetes vs non Diabetes CAD vs non CAD Number 88 78 62 17 Cholesterol (mmol/l) 4.70 ± 1.22 5.16 ± 1.14 5.13 ± 1.12 4.06 ± 1.22 < 0.001 ns HDL-C (mmol/l) 0.94 ± 0.24 0.96 ± 0.38 0.79 ± 0.22 0.72 ± 0.21 ns < 0.001 Total TG (mmol/l) 1.39 ± 0.75 2.11 ± 1.48 2.27 ± 1.77 2.81 ± 2.53 < 0.001 < 0.001 Heterozygous genotype had the high triglyceride level According to SNP3 of the apo A-V gene, variation of lipid parameters in diabetic or non diabetic patients are shown in Table  3 . Non diabetic subjects having the heterozygous genotype (T/C) showed an increased triglyceride level and decreased HDL-C concentration. However these variations were not significant. In type 2 diabetic patients, triglyceride level increased significantly in C/T genotype in association with non significant decrease in HDL-C concentration. The genotype frequencies of T/T, T/C and C/C were 0.74, 0.23 and 0.03 respectively in non diabetic subjects, 0.71, 0.25 and 0.04 respectively in type 2 diabetic patients. The SNP3 was shown to be in Hardy-Weinberg equilibrium. It is clear that there was no difference in genotype distribution between diabetic and non diabetic subjects. The type 2 diabetic population was classified further into those with high and low triglyceride concentration (cut point 2.2 mmol/l which was more than 90 percentile level in the healthy population). The SNP3 frequencies for T/T, T/C and C/C genotypes in the low triglyceride groups were 77.1 %, 18.8 % and 4.2 % respectively, and those in the high triglyceride group were 59.1 %, 40.9 % and 0 % respectively. The difference in genotype frequencies between low and high triglyceride groups were significant (p = 0.011) (Table  4 ). Table 3 Clinical Characteristics and lipid profile according to SNP3 of apo A-V in type 2 diabetic and in non diabetic subjects Type 2 Diabetes Non Diabetes Characteristics Genotypes TT TC TT TC Number 100 36 76 25 Sex (% men) 61 50 55.8 60 Smokers (%) 67.3 81.3 44.4 46.2 Age (years) 52.8 ± 7.6 54.8 ± 6.2 53.3 ± 8.8 51.5 ± 7.5 BMI (Kg/m 2 ) 28.5 ± 4.3 28.7 ± 3.9 27.3 ± 3.0 28.1 ± 5.0 WHR 0.94 ± 0.06 0.93 ± 0.08 0.93 ± 0.08 0.9 ± 0.08 Cholesterol (mmol/l) 5.17 ± 1.15 5.17 ± 1.07 4.61 ± 1.32 4.53 ± 0.99 HDL-C (mmol/l) 0.90 ± 0.32 0.83 ± 0.34 0.92 ± 0.25 0.83 ± 0.26 Total TG (mmol/l) 2.05 ± 1.61 2.62 ± 1.59* 1.6 ± 1.42 1.75 ± 1.07 * significant difference between TT and TC genotypes (p = 0.016). Table 4 Distribution of different apo A-V genotypes in type 2 diabetes between low and high triglyceride groups Low triglyceride group ≤ 2.2 mmol/l High triglyceride group > 2.2 mmol/l Polymorphism Number % Number % T/T 74 77.1 26 59.1 T/C 18 18.8 18 40.9 C/C 4 4.2 nd nd Total 96 100 44 100 nd : not detected X2 = 8.962, p = 0.011, degree of freedom = 2. No association between SNP3 and CAD To investigate the relation of SNP3 polymorphism with coronary artery disease we studied the association in all subjects (those who are taking lipid lowering drugs are included). There was no association between coronary artery disease and SNP3 either in non diabetic subjects or in type 2 diabetic patients (Table  5 ). Table 5 Association between SNP3 of apo A-V and risk for coronary artery disease in diabetic and non diabetic subjects Genotypes CAD- CAD + Diabetes- T/T 74 (74.7) 41 (71.9) P = 0.514 OR = 1.29 (0.6–2.77) T/C 21 (21.1) 15 (26.3) C/C 4 (4.0) 1 (1.8) Diabetes+ T/T 56 (71.8) 52 (70.3) P = 0.717 OR = 0.87 (0.41–1.83) T/C 21 (26.9) 17 (23.0) C/C 1 (1.3) 5 (6.7) OR was calculated using T/T and T/C genotypes only.
Results
Description of the participating groups Type 2 diabetic patients have a BMI values more important than non diabetic subjects. Whereas the WHR in lower in controls. Males and smokers are more frequent in patients with CAD (Table  1 ). To compare lipid parameters, we take into consideration the lipid lowering drugs use, then patients who are taking lipid lowering drugs were excluded (Table  2 ). Plasma total cholesterol and total triglyceride concentrations were significantly higher in type 2 diabetic patients than in the non diabetic patients. Subjects with CAD had lower concentration of HDL-C and higher concentration of triglyceride as compared to those without CAD (Table  2 ). Table 1 Clinical characteristics of the four studied groups Diabetes- CAD- Diabetes+ CAD- Diabetes+ CAD+ Diabetes- CAD+ Diabetes vs non Diabetes CAD vs non CAD Number 99 78 74 57 Sex (% of men) 50.5 49.4 75.4 84.2 ns < 0.001 Age (years) 52.9 ± 8.7 51.5 ± 7.3 55.7 ± 6.8 59.0 ± 6.8 ns < 0.001 BMI (Kg/m 2 ) 27.6 ± 4.0 28.8 ± 4.6 28.1 ± 3.8 26.6 ± 4.8 0.029 ns Smokers (%) 42 53 86 80 0.003 < 0.001 SBP (mmHg) 12.5 ± 1.3 13.2 ± 1.4 13.0 ± 1.8 12.4 ± 2.1 0.001 ns DBP (mmHg) 7.5 ± 0.7 7.8 ± 0.9 7.6 ± 1.0 7.1 ± 1.1 0.006 0.018 WHR 0.82 ± 0.09 0.92 ± 0.07 0.95 ± 0.07 0.92 ± 0.06 ns 0.016 Glucose (mmol/l) 5.2 ± 0.4 10.4 ± 3.4 11.7 ± 3.5 6.8 ± 3.6 < 0.001 < 0.001 HbA1c (%) 4.5 ± 0.9 10.4 ± 3.4 9.4 ± 2.4 11.3 ± 4.1 0.009 < 0.001 Diabetes duration (years) 6.8 ± 5.2 7.9 ± 6.1 Lipid lowering drug use (%) 11.1 0 16.2 70.2 SBP : systolic blood pressure DBP : diastolic blood pressure Table 2 lipid profiles of the four studied groups Diabetes- CAD- Diabetes+ CAD- Diabetes+ CAD+ Diabetes- CAD+ Diabetes vs non Diabetes CAD vs non CAD Number 88 78 62 17 Cholesterol (mmol/l) 4.70 ± 1.22 5.16 ± 1.14 5.13 ± 1.12 4.06 ± 1.22 < 0.001 ns HDL-C (mmol/l) 0.94 ± 0.24 0.96 ± 0.38 0.79 ± 0.22 0.72 ± 0.21 ns < 0.001 Total TG (mmol/l) 1.39 ± 0.75 2.11 ± 1.48 2.27 ± 1.77 2.81 ± 2.53 < 0.001 < 0.001
Description of the participating groups
Type 2 diabetic patients have a BMI values more important than non diabetic subjects. Whereas the WHR in lower in controls. Males and smokers are more frequent in patients with CAD (Table  1 ). To compare lipid parameters, we take into consideration the lipid lowering drugs use, then patients who are taking lipid lowering drugs were excluded (Table  2 ). Plasma total cholesterol and total triglyceride concentrations were significantly higher in type 2 diabetic patients than in the non diabetic patients. Subjects with CAD had lower concentration of HDL-C and higher concentration of triglyceride as compared to those without CAD (Table  2 ).Clinical characteristics of the four studied groupsDiabetes- CAD-Diabetes+ CAD-Diabetes+ CAD+Diabetes- CAD+Diabetes vs non DiabetesCAD vs non CADSBP : systolic blood pressureDBP : diastolic blood pressurelipid profiles of the four studied groupsDiabetes- CAD-Diabetes+ CAD-Diabetes+ CAD+Diabetes- CAD+Diabetes vs non DiabetesCAD vs non CADHeterozygous genotype had the high triglyceride level According to SNP3 of the apo A-V gene, variation of lipid parameters in diabetic or non diabetic patients are shown in Table  3 . Non diabetic subjects having the heterozygous genotype (T/C) showed an increased triglyceride level and decreased HDL-C concentration. However these variations were not significant. In type 2 diabetic patients, triglyceride level increased significantly in C/T genotype in association with non significant decrease in HDL-C concentration. The genotype frequencies of T/T, T/C and C/C were 0.74, 0.23 and 0.03 respectively in non diabetic subjects, 0.71, 0.25 and 0.04 respectively in type 2 diabetic patients. The SNP3 was shown to be in Hardy-Weinberg equilibrium. It is clear that there was no difference in genotype distribution between diabetic and non diabetic subjects. The type 2 diabetic population was classified further into those with high and low triglyceride concentration (cut point 2.2 mmol/l which was more than 90 percentile level in the healthy population). The SNP3 frequencies for T/T, T/C and C/C genotypes in the low triglyceride groups were 77.1 %, 18.8 % and 4.2 % respectively, and those in the high triglyceride group were 59.1 %, 40.9 % and 0 % respectively. The difference in genotype frequencies between low and high triglyceride groups were significant (p = 0.011) (Table  4 ). Table 3 Clinical Characteristics and lipid profile according to SNP3 of apo A-V in type 2 diabetic and in non diabetic subjects Type 2 Diabetes Non Diabetes Characteristics Genotypes TT TC TT TC Number 100 36 76 25 Sex (% men) 61 50 55.8 60 Smokers (%) 67.3 81.3 44.4 46.2 Age (years) 52.8 ± 7.6 54.8 ± 6.2 53.3 ± 8.8 51.5 ± 7.5 BMI (Kg/m 2 ) 28.5 ± 4.3 28.7 ± 3.9 27.3 ± 3.0 28.1 ± 5.0 WHR 0.94 ± 0.06 0.93 ± 0.08 0.93 ± 0.08 0.9 ± 0.08 Cholesterol (mmol/l) 5.17 ± 1.15 5.17 ± 1.07 4.61 ± 1.32 4.53 ± 0.99 HDL-C (mmol/l) 0.90 ± 0.32 0.83 ± 0.34 0.92 ± 0.25 0.83 ± 0.26 Total TG (mmol/l) 2.05 ± 1.61 2.62 ± 1.59* 1.6 ± 1.42 1.75 ± 1.07 * significant difference between TT and TC genotypes (p = 0.016). Table 4 Distribution of different apo A-V genotypes in type 2 diabetes between low and high triglyceride groups Low triglyceride group ≤ 2.2 mmol/l High triglyceride group > 2.2 mmol/l Polymorphism Number % Number % T/T 74 77.1 26 59.1 T/C 18 18.8 18 40.9 C/C 4 4.2 nd nd Total 96 100 44 100 nd : not detected X2 = 8.962, p = 0.011, degree of freedom = 2.
Heterozygous genotype had the high triglyceride level
According to SNP3 of the apo A-V gene, variation of lipid parameters in diabetic or non diabetic patients are shown in Table  3 . Non diabetic subjects having the heterozygous genotype (T/C) showed an increased triglyceride level and decreased HDL-C concentration. However these variations were not significant. In type 2 diabetic patients, triglyceride level increased significantly in C/T genotype in association with non significant decrease in HDL-C concentration. The genotype frequencies of T/T, T/C and C/C were 0.74, 0.23 and 0.03 respectively in non diabetic subjects, 0.71, 0.25 and 0.04 respectively in type 2 diabetic patients. The SNP3 was shown to be in Hardy-Weinberg equilibrium. It is clear that there was no difference in genotype distribution between diabetic and non diabetic subjects. The type 2 diabetic population was classified further into those with high and low triglyceride concentration (cut point 2.2 mmol/l which was more than 90 percentile level in the healthy population). The SNP3 frequencies for T/T, T/C and C/C genotypes in the low triglyceride groups were 77.1 %, 18.8 % and 4.2 % respectively, and those in the high triglyceride group were 59.1 %, 40.9 % and 0 % respectively. The difference in genotype frequencies between low and high triglyceride groups were significant (p = 0.011) (Table  4 ).Clinical Characteristics and lipid profile according to SNP3 of apo A-V in type 2 diabetic and in non diabetic subjectsType 2 DiabetesNon DiabetesGenotypesTTTCTTTC* significant difference between TT and TC genotypes (p = 0.016).Distribution of different apo A-V genotypes in type 2 diabetes between low and high triglyceride groupsLow triglyceride group ≤ 2.2 mmol/lHigh triglyceride group > 2.2 mmol/lNumber%Number%nd : not detectedX2 = 8.962, p = 0.011, degree of freedom = 2.No association between SNP3 and CAD To investigate the relation of SNP3 polymorphism with coronary artery disease we studied the association in all subjects (those who are taking lipid lowering drugs are included). There was no association between coronary artery disease and SNP3 either in non diabetic subjects or in type 2 diabetic patients (Table  5 ). Table 5 Association between SNP3 of apo A-V and risk for coronary artery disease in diabetic and non diabetic subjects Genotypes CAD- CAD + Diabetes- T/T 74 (74.7) 41 (71.9) P = 0.514 OR = 1.29 (0.6–2.77) T/C 21 (21.1) 15 (26.3) C/C 4 (4.0) 1 (1.8) Diabetes+ T/T 56 (71.8) 52 (70.3) P = 0.717 OR = 0.87 (0.41–1.83) T/C 21 (26.9) 17 (23.0) C/C 1 (1.3) 5 (6.7) OR was calculated using T/T and T/C genotypes only.
No association between SNP3 and CAD
To investigate the relation of SNP3 polymorphism with coronary artery disease we studied the association in all subjects (those who are taking lipid lowering drugs are included). There was no association between coronary artery disease and SNP3 either in non diabetic subjects or in type 2 diabetic patients (Table  5 ).Association between SNP3 of apo A-V and risk for coronary artery disease in diabetic and non diabetic subjectsGenotypesCAD-CAD +OR was calculated using T/T and T/C genotypes only.Discussion Elevated serum lipid levels are an important risk factor for atherosclerosis. Both environmental and genetic factors contribute to variability in serum lipid levels [ 5 ]. In this study, we choose the apo A-V gene as a genetic factor that predispose to elevated triglyceride level. The dynamic interfacial properties of apo A-V are consistent with the hypothesis that apo A-V impedes triglyceride particle assembly [ 7 ]. Thus, the effect of apo A-V on triglyceride level can be attributed to the intracellularly function of apo A-V to modulate hepatic VLDL synthesis and/or secretion. Also apo A-V can lower plasma triglyceride by activating the lipoprotein lipase (LPL)[ 8 ]. In our study we showed that SNP3 was significantly associated with hypertriglyceridemia especially in type 2 diabetic patients. This finding confirmed the idea that the effect of this SNP on triglyceride metabolism was not influenced by ethnic background [ 9 ]. However, it has been reported that the major and minor allele frequencies differed between populations such as : 0.06, 0.09 and 0.34 for the C allele in UK, Caucasian and Japanese respectively [ 10 , 3 , 9 ]. In our population, the minor allele frequency (0.13) is almost the same as in Caucasian [ 3 ] but lower than in Japanese population [ 9 ]. However, the triglyceride level is higher in Tunisian than in Japanese population. This paradoxical observation confirms that triglyceride level is influenced by environmental factors and other genetic factors (apo CIII...). In type 2 diabetes the triglyceride level is increased, which is due to multiple factors related to insulin and carbohydrate metabolism, LPL activity, CETP activity... The absence of an unusual allele frequency of SNP3 in type 2 diabetic patients compared to non diabetic subjects, in spite of triglyceride level variation, shows that there is no association between apo A-V SNP3 polymorphism and the presence or absence of diabetes. Diabetic homozygous for the major allele are more frequent in low triglyceride group, showing that SNP3 is associated with triglyceride variation in type 2 diabetic patients. Contrary to Esteve et al., who have reported no significant difference in triglyceride concentration with the apo A-V polymorphism in type 2 diabetic patients [ 6 ], our results showed, then, that SNP3 is associated with triglyceride level. The relationship between SNP3 and coronary artery disease is under discussion. The LOCAT study showed that there is no association between SNP3 and progression of coronary heart disease [ 11 ]. Our results find no association between SNP3 of apo A-V and development of coronary artery disease either in diabetic patients or in non diabetic subjects. This suggests that the high triglyceride level in T/C genotype alone was not a good discriminator of coronary heart disease. In contrast ; Szalai et al. showed an association between SNP3 and an increased risk for severe coronary artery disease [ 12 ]. Identifying genetic and environmental factors that influence plasma lipid levels represents a key step towards developing strategies for preventing and treating CAD. Usually, in the case of type 2 diabetes, patients are taking lipid lowering drugs in order to ameliorate their lipid profile, namely lower triglyceride level and increase HDL-C concentration. Fibrates represent a commonly used therapy for lowering plasma triglyceride, its mechanism of action involves the activation of the nuclear receptor peroxisome proliferator activated receptor alpha (PPARα). The apo A-V is a highly responsive PPAR  target gene [ 13 ]. While SNP3 is located in promoter region, it should be interesting to study the interaction between this polymorphism and lipid lowering drugs response in different population.
Discussion
Elevated serum lipid levels are an important risk factor for atherosclerosis. Both environmental and genetic factors contribute to variability in serum lipid levels [ 5 ]. In this study, we choose the apo A-V gene as a genetic factor that predispose to elevated triglyceride level. The dynamic interfacial properties of apo A-V are consistent with the hypothesis that apo A-V impedes triglyceride particle assembly [ 7 ]. Thus, the effect of apo A-V on triglyceride level can be attributed to the intracellularly function of apo A-V to modulate hepatic VLDL synthesis and/or secretion. Also apo A-V can lower plasma triglyceride by activating the lipoprotein lipase (LPL)[ 8 ]. In our study we showed that SNP3 was significantly associated with hypertriglyceridemia especially in type 2 diabetic patients. This finding confirmed the idea that the effect of this SNP on triglyceride metabolism was not influenced by ethnic background [ 9 ]. However, it has been reported that the major and minor allele frequencies differed between populations such as : 0.06, 0.09 and 0.34 for the C allele in UK, Caucasian and Japanese respectively [ 10 , 3 , 9 ]. In our population, the minor allele frequency (0.13) is almost the same as in Caucasian [ 3 ] but lower than in Japanese population [ 9 ]. However, the triglyceride level is higher in Tunisian than in Japanese population. This paradoxical observation confirms that triglyceride level is influenced by environmental factors and other genetic factors (apo CIII...).In type 2 diabetes the triglyceride level is increased, which is due to multiple factors related to insulin and carbohydrate metabolism, LPL activity, CETP activity... The absence of an unusual allele frequency of SNP3 in type 2 diabetic patients compared to non diabetic subjects, in spite of triglyceride level variation, shows that there is no association between apo A-V SNP3 polymorphism and the presence or absence of diabetes. Diabetic homozygous for the major allele are more frequent in low triglyceride group, showing that SNP3 is associated with triglyceride variation in type 2 diabetic patients. Contrary to Esteve et al., who have reported no significant difference in triglyceride concentration with the apo A-V polymorphism in type 2 diabetic patients [ 6 ], our results showed, then, that SNP3 is associated with triglyceride level.The relationship between SNP3 and coronary artery disease is under discussion. The LOCAT study showed that there is no association between SNP3 and progression of coronary heart disease [ 11 ]. Our results find no association between SNP3 of apo A-V and development of coronary artery disease either in diabetic patients or in non diabetic subjects. This suggests that the high triglyceride level in T/C genotype alone was not a good discriminator of coronary heart disease. In contrast ; Szalai et al. showed an association between SNP3 and an increased risk for severe coronary artery disease [ 12 ]. Identifying genetic and environmental factors that influence plasma lipid levels represents a key step towards developing strategies for preventing and treating CAD. Usually, in the case of type 2 diabetes, patients are taking lipid lowering drugs in order to ameliorate their lipid profile, namely lower triglyceride level and increase HDL-C concentration. Fibrates represent a commonly used therapy for lowering plasma triglyceride, its mechanism of action involves the activation of the nuclear receptor peroxisome proliferator activated receptor alpha (PPARα). The apo A-V is a highly responsive PPAR  target gene [ 13 ]. While SNP3 is located in promoter region, it should be interesting to study the interaction between this polymorphism and lipid lowering drugs response in different population.Conclusion In summary, the apo A-V SNP3 is associated with triglyceride level in Tunisian type 2 diabetic patients. However, this SNP is unlikely to be associated with the presence of diabetes. Although SNP3 is associated with hypertrigyceridemia, there was no relationship between this polymorphism and coronary artery disease. Further investigations were needed to determine the effect of SNP3 on lipid lowering drug response.
Conclusion
In summary, the apo A-V SNP3 is associated with triglyceride level in Tunisian type 2 diabetic patients. However, this SNP is unlikely to be associated with the presence of diabetes. Although SNP3 is associated with hypertrigyceridemia, there was no relationship between this polymorphism and coronary artery disease. Further investigations were needed to determine the effect of SNP3 on lipid lowering drug response.Methods Subjects Three hundred and eight subjects, aged 45–70 years, participated in this study. They belong to four groups. The first group contained 74 type 2 diabetic patients with CAD, among whom 12 are taking lipid lowering drugs. The second group contained 78 type 2 diabetic patients without CAD, all of them did not take lipid lowering drugs. The third group contained 57 patients with CAD and without diabetes, 70% of the patients are taking lipid lowering drugs. The last group contained 99 controls without diabetes nor CAD, 11 subjects are taking lipid lowering drugs. The clinical and biological characteristics of each group are summarized in Table  1 . All participants were recruited in the departments of internal medicine and cardiology in Monastir university hospital. Written or verbal informed consent were obtained from all patients and controls before the study. All written informed consent were not possible because the majority of eligible subjects in our study were illiterate. The diagnosis of diabetes was based on a previous history of diabetes according to American Diabetes Association criteria [ 14 ]. The patients with CAD were defined as clinical history of stable angina pectoris, previous acute coronary syndromes with or without ST segment elevation. This CAD was confirmed by coronary angiography. Exclusion criteria were taking insulin, having a renal or liver failure or thyroid disease, alcohol consumption before 3 days or less, Body Mass Index (BMI) more than 35 Kg/m 2  and glycosylated hemoglobin (Hb A1c) more than 12%. Post menopausal women had no hormone replacement therapy. BMI was calculated using the formula : weight (Kg)/height 2  (m 2 ). Obesity was defined as BMI>30 kg/m 2 . Waist-to-hip ratio (WHR) was calculated from measurements of the waist circumference taken at the mid point between umbilicus and xiphoid and hip circumference, at the widest point around the hips, respectively. Blood samples were drawn after subjects had fasted overnight (12 hours) into tubes containing EDTA. Plasma was immediately separated by centrifugation. Laboratory Analysis After DNA extraction, the single nucleotide polymorphism SNP3 within the apo A-V gene (-1311 T/C) was determined by PCR-RFLP analysis using MseI restriction endonuclease as described previously [ 3 ]. Plasma glucose, glycosylated haemoglobin (HbA1c), lipids and lipoproteins were determined as described by Smaoui et al. [ 15 ]. Statistical Analyses Data management and statistical analysis were performed using SPSS 10.0 software. Results are summarized as mean ± SD. Since triglyceride levels were not normally distributed, logarithmic transformation of triglyceride concentration was performed before the statistical analysis. Student's test was used to compare continuous variables and Chi square (χ 2 ) test was used to examine distribution of categorical variables. A value of p < 0.05 was considered significant.
Methods
Subjects Three hundred and eight subjects, aged 45–70 years, participated in this study. They belong to four groups. The first group contained 74 type 2 diabetic patients with CAD, among whom 12 are taking lipid lowering drugs. The second group contained 78 type 2 diabetic patients without CAD, all of them did not take lipid lowering drugs. The third group contained 57 patients with CAD and without diabetes, 70% of the patients are taking lipid lowering drugs. The last group contained 99 controls without diabetes nor CAD, 11 subjects are taking lipid lowering drugs. The clinical and biological characteristics of each group are summarized in Table  1 . All participants were recruited in the departments of internal medicine and cardiology in Monastir university hospital. Written or verbal informed consent were obtained from all patients and controls before the study. All written informed consent were not possible because the majority of eligible subjects in our study were illiterate. The diagnosis of diabetes was based on a previous history of diabetes according to American Diabetes Association criteria [ 14 ]. The patients with CAD were defined as clinical history of stable angina pectoris, previous acute coronary syndromes with or without ST segment elevation. This CAD was confirmed by coronary angiography. Exclusion criteria were taking insulin, having a renal or liver failure or thyroid disease, alcohol consumption before 3 days or less, Body Mass Index (BMI) more than 35 Kg/m 2  and glycosylated hemoglobin (Hb A1c) more than 12%. Post menopausal women had no hormone replacement therapy. BMI was calculated using the formula : weight (Kg)/height 2  (m 2 ). Obesity was defined as BMI>30 kg/m 2 . Waist-to-hip ratio (WHR) was calculated from measurements of the waist circumference taken at the mid point between umbilicus and xiphoid and hip circumference, at the widest point around the hips, respectively. Blood samples were drawn after subjects had fasted overnight (12 hours) into tubes containing EDTA. Plasma was immediately separated by centrifugation.
Subjects
Three hundred and eight subjects, aged 45–70 years, participated in this study. They belong to four groups. The first group contained 74 type 2 diabetic patients with CAD, among whom 12 are taking lipid lowering drugs. The second group contained 78 type 2 diabetic patients without CAD, all of them did not take lipid lowering drugs. The third group contained 57 patients with CAD and without diabetes, 70% of the patients are taking lipid lowering drugs. The last group contained 99 controls without diabetes nor CAD, 11 subjects are taking lipid lowering drugs. The clinical and biological characteristics of each group are summarized in Table  1 .All participants were recruited in the departments of internal medicine and cardiology in Monastir university hospital. Written or verbal informed consent were obtained from all patients and controls before the study. All written informed consent were not possible because the majority of eligible subjects in our study were illiterate. The diagnosis of diabetes was based on a previous history of diabetes according to American Diabetes Association criteria [ 14 ]. The patients with CAD were defined as clinical history of stable angina pectoris, previous acute coronary syndromes with or without ST segment elevation. This CAD was confirmed by coronary angiography. Exclusion criteria were taking insulin, having a renal or liver failure or thyroid disease, alcohol consumption before 3 days or less, Body Mass Index (BMI) more than 35 Kg/m 2  and glycosylated hemoglobin (Hb A1c) more than 12%. Post menopausal women had no hormone replacement therapy. BMI was calculated using the formula : weight (Kg)/height 2  (m 2 ). Obesity was defined as BMI>30 kg/m 2 . Waist-to-hip ratio (WHR) was calculated from measurements of the waist circumference taken at the mid point between umbilicus and xiphoid and hip circumference, at the widest point around the hips, respectively. Blood samples were drawn after subjects had fasted overnight (12 hours) into tubes containing EDTA. Plasma was immediately separated by centrifugation.Laboratory Analysis After DNA extraction, the single nucleotide polymorphism SNP3 within the apo A-V gene (-1311 T/C) was determined by PCR-RFLP analysis using MseI restriction endonuclease as described previously [ 3 ]. Plasma glucose, glycosylated haemoglobin (HbA1c), lipids and lipoproteins were determined as described by Smaoui et al. [ 15 ].
Laboratory Analysis
After DNA extraction, the single nucleotide polymorphism SNP3 within the apo A-V gene (-1311 T/C) was determined by PCR-RFLP analysis using MseI restriction endonuclease as described previously [ 3 ]. Plasma glucose, glycosylated haemoglobin (HbA1c), lipids and lipoproteins were determined as described by Smaoui et al. [ 15 ].Statistical Analyses Data management and statistical analysis were performed using SPSS 10.0 software. Results are summarized as mean ± SD. Since triglyceride levels were not normally distributed, logarithmic transformation of triglyceride concentration was performed before the statistical analysis. Student's test was used to compare continuous variables and Chi square (χ 2 ) test was used to examine distribution of categorical variables. A value of p < 0.05 was considered significant.
Statistical Analyses
Data management and statistical analysis were performed using SPSS 10.0 software. Results are summarized as mean ± SD. Since triglyceride levels were not normally distributed, logarithmic transformation of triglyceride concentration was performed before the statistical analysis. Student's test was used to compare continuous variables and Chi square (χ 2 ) test was used to examine distribution of categorical variables. A value of p < 0.05 was considered significant.List of abbreviations Apo : apolipoprotein BMI : Body Mass Index CAD : Coronary Artery Disease HDL : High Density Lipoproteins HDL-C : HDL cholesterol PCR-RFLP : Polymerase Chain Reaction-Restriction Fragment Length Polymorphism SNP : Single Nucleotide Polymorphism TG : Triglyceride VLDL : Very Low Density Lipoproteins WHR : Waist-to-Hip Ratio
List of abbreviations
Apo : apolipoproteinBMI : Body Mass IndexCAD : Coronary Artery DiseaseHDL : High Density LipoproteinsHDL-C : HDL cholesterolPCR-RFLP : Polymerase Chain Reaction-Restriction Fragment Length PolymorphismSNP : Single Nucleotide PolymorphismTG : TriglycerideVLDL : Very Low Density LipoproteinsWHR : Waist-to-Hip RatioAuthors' contributions R.Ch and N.A: carried out the molecular studies, participated in the design of the study and drafted the manuscript ; M.S : carried out the biochemical essay ; S.H and Sy.M : interested in the clinical aspect ; M.H : conceived of the study, and participated in its design and coordination and helped to draft the manuscript and revised it critically for important intellectual content and have given final approval of the version to be published; MS.M : revised the article critically for important intellectual content and have given final approval of the version to be published.
Authors' contributions
R.Ch and N.A: carried out the molecular studies, participated in the design of the study and drafted the manuscript ; M.S : carried out the biochemical essay ; S.H and Sy.M : interested in the clinical aspect ; M.H : conceived of the study, and participated in its design and coordination and helped to draft the manuscript and revised it critically for important intellectual content and have given final approval of the version to be published; MS.M : revised the article critically for important intellectual content and have given final approval of the version to be published.


Percutaneous coronary intervention can be associated with distal embolization of thrombotic material causing myocardial necrosis and infarction.We discuss the role of intravascular imaging to guide the use of a distal protection device by describing the outcome of a young woman presenting with non-ST elevation myocardial infarction. Coronary angiography demonstrated an isolated minor stenosis in the proximal left anterior descending coronary artery with slight haziness beyond the lesion. Intravascular ultrasound confirmed an extensive thrombus overlying a bulky atherosclerotic plaque. A distal filter wire was therefore successfully used to reduce the risk of distal embolization.The use of intravascular ultrasound in patients presenting with acute coronary syndrome may reveal large thrombi that are difficult to image using conventional angiographic techniques. Intravascular ultrasound can therefore be used as a tool to select lesions requiring distal protection.

Background Patients with acute coronary syndromes (ACS) are increasingly treated with an early invasive strategy. During percutaneous coronary interventions (PCI) interventionalists have often to deal with thrombus-laden lesions in native coronary vessels. This poses the serious problem of preventing macroscopic and microscopic distal embolization [ 1 ]. There have been many recent advances in this field, and new tools are available for shielding the distal microvasculature, including occlusive systems and basket filters. In the very emboli-prone saphenous vein grafts interventions, for example, both techniques have been associated with favourable results [ 2 , 3 ]. In native vessels cost and efficacy [ 4 ] considerations prompt a careful use of these devices, and a sensible approach could be selecting lesion and patients at high risk of distal embolization. For example, preventing distal embolization is particularly important in young patients presenting with their first coronary event with a large thrombotic burden [ 5 , 6 ]. Angiography alone is known to underestimate the risk of distal embolization: for example, only overt signs of massive thrombus burden are predictive of no-reflow in myocardial infarction patients treated with primary PCI [ 7 ]. Intravascular ultrasound (IVUS) has the potential to overcome many of the limitations of angiography, including lesion characterization and assessment of plaque rupture and thrombus [ 8 - 11 ], but its use is still restricted. We present a case of successful prevention of macroscopic distal embolism in a young patient with ACS obtained combining IVUS and distal filter protection.
Background
Patients with acute coronary syndromes (ACS) are increasingly treated with an early invasive strategy. During percutaneous coronary interventions (PCI) interventionalists have often to deal with thrombus-laden lesions in native coronary vessels. This poses the serious problem of preventing macroscopic and microscopic distal embolization [ 1 ]. There have been many recent advances in this field, and new tools are available for shielding the distal microvasculature, including occlusive systems and basket filters. In the very emboli-prone saphenous vein grafts interventions, for example, both techniques have been associated with favourable results [ 2 , 3 ].In native vessels cost and efficacy [ 4 ] considerations prompt a careful use of these devices, and a sensible approach could be selecting lesion and patients at high risk of distal embolization. For example, preventing distal embolization is particularly important in young patients presenting with their first coronary event with a large thrombotic burden [ 5 , 6 ].Angiography alone is known to underestimate the risk of distal embolization: for example, only overt signs of massive thrombus burden are predictive of no-reflow in myocardial infarction patients treated with primary PCI [ 7 ]. Intravascular ultrasound (IVUS) has the potential to overcome many of the limitations of angiography, including lesion characterization and assessment of plaque rupture and thrombus [ 8 - 11 ], but its use is still restricted.We present a case of successful prevention of macroscopic distal embolism in a young patient with ACS obtained combining IVUS and distal filter protection.Case presentation A 41-year-old lady presented with a short history of cardiac sounding chest pain. She was a smoker, had hypertension and had a strong family history of premature ischaemic heart disease. The ECG showed widespread anterior T wave inversion. Conventional treatment was started immediately for ACS including intravenous nitrates, low molecular weight heparin, aspirin and clopidogrel. The patient remained pain-free following admission and serial biochemistry demonstrated a rise in cardiac troponin I to 24 mmol/l at 24 hours. There was no evidence of Q waves on the ECG and left ventricular function was normal on echocardiography. Coronary angiography was undertaken four days after admission. This demonstrated mural thickening of the proximal left anterior descending (LAD) coronary artery with a possible filling defect at the distal end of the lesion but no evidence of coronary atherosclerosis elsewhere (Fig.  1B ). Interrogation of the proximal LAD stenosis with IVUS (Galaxy II system; Atlantis 40 MHz Catheter, Boston Scientific/Scimed, Inc., Maple Grove, Minnesota) showed a large intracoronary thrombus (Fig.  1C ) adherent to a mural atheromatous plaque starting at the ostium of LAD (Fig.  1A ). The thrombus had a lobulated appearance with no evidence of internal blood flow or speckling. It contained echolucent areas indicating cavitation and consistent with ongoing thrombus organization. No evidence of complete plaque rupture (intraplaque cavity communicating with the lumen) was seen. Figure 1 Composite image showing an angiographic right anterior oblique projection of the left coronary system (Panel B) and two intravascular ultrasound images (Panels A&C) before the intervention. Dotted line shows the approximate location of the IVUS slices. An eccentric plaque is visible in the proximal left anterior descending artery (Panel B), with some haziness at the distal end. IVUS confirms the presence of the plaque (Panel A) and shows a bulky, partially organized, thrombus (Panel C) loosely attached to the distal end of the plaque. No signs of plaque rupture are visible. Percutaneous coronary intervention was performed using distal filter protection with the EZ Filterwire (Boston Scientific, Natick, MA, USA). The GpIIb/IIIa antagonist Abciximab was administered. A 4.0 × 15 mm stent was placed directly with good final angiographic result (Fig.  2B ) and no evidence of macroscopic distal embolisation. Repeat IVUS confirmed good apposition of the stent and the absence of residual thrombus (Fig.  2A & 2C ). At retrieval, the Filterwire contained a small but significant amount of pink thrombotic material and yellowish plaque debris. Equivalent chest x-ray radiation dose (assuming a single posteroanterior projection chest x-ray is eight centi-Gray/cm2) was 380. Figure 2 Composite image showing an angiographic right anterior oblique projection of the left coronary system (Panel B) and two intravascular ultrasound images (Panels A&C) after the intervention. Dotted line shows the approximate location of the IVUS slices. An excellent angiographic result is visible on the left anterior descending (Panel A). IVUS confirms good apposition of the stent (Panel A&B), with no residual or prolapsed thrombus.
 No further Troponin I elevation occurred; the patient was discharged home the next day, and remains asymptomatic at two months.
Case presentation
A 41-year-old lady presented with a short history of cardiac sounding chest pain. She was a smoker, had hypertension and had a strong family history of premature ischaemic heart disease. The ECG showed widespread anterior T wave inversion. Conventional treatment was started immediately for ACS including intravenous nitrates, low molecular weight heparin, aspirin and clopidogrel. The patient remained pain-free following admission and serial biochemistry demonstrated a rise in cardiac troponin I to 24 mmol/l at 24 hours. There was no evidence of Q waves on the ECG and left ventricular function was normal on echocardiography.Coronary angiography was undertaken four days after admission. This demonstrated mural thickening of the proximal left anterior descending (LAD) coronary artery with a possible filling defect at the distal end of the lesion but no evidence of coronary atherosclerosis elsewhere (Fig.  1B ). Interrogation of the proximal LAD stenosis with IVUS (Galaxy II system; Atlantis 40 MHz Catheter, Boston Scientific/Scimed, Inc., Maple Grove, Minnesota) showed a large intracoronary thrombus (Fig.  1C ) adherent to a mural atheromatous plaque starting at the ostium of LAD (Fig.  1A ). The thrombus had a lobulated appearance with no evidence of internal blood flow or speckling. It contained echolucent areas indicating cavitation and consistent with ongoing thrombus organization. No evidence of complete plaque rupture (intraplaque cavity communicating with the lumen) was seen.Percutaneous coronary intervention was performed using distal filter protection with the EZ Filterwire (Boston Scientific, Natick, MA, USA). The GpIIb/IIIa antagonist Abciximab was administered. A 4.0 × 15 mm stent was placed directly with good final angiographic result (Fig.  2B ) and no evidence of macroscopic distal embolisation. Repeat IVUS confirmed good apposition of the stent and the absence of residual thrombus (Fig.  2A & 2C ). At retrieval, the Filterwire contained a small but significant amount of pink thrombotic material and yellowish plaque debris. Equivalent chest x-ray radiation dose (assuming a single posteroanterior projection chest x-ray is eight centi-Gray/cm2) was 380.No further Troponin I elevation occurred; the patient was discharged home the next day, and remains asymptomatic at two months.Discussion Acute coronary syndromes with extensive thrombosis in the absence of widespread coronary atheroma are detected most frequently in young smokers [ 5 , 6 ]. The necessity of preventing distal embolic during PCI is becoming increasing recognised, as traditional angioplasty may be associated with distal myocardial necrosis [ 1 ]. In our case, angiographic appearance was not suggestive of either obstructive coronary disease or a high embolic risk: none of the criteria set up by Yip et al [ 7 ] (cutoff pattern of occlusion, accumulated thrombus > 5 mm proximal to the occlusion, presence of floating thrombus, persistent dye stasis distal to the obstruction, reference lumen diameter of the culprit artery > or = 4 mm, and incomplete obstruction with presence of accumulated thrombus more than three times the reference lumen diameter of culprit artery) was met, and the patient had already received prolonged antiplatelet and anticoagulant therapy. However, we suspected the presence of a significant thrombotic mass, mainly on the clinical grounds of widespread ECG change and Troponin I elevation, and performed IVUS examination. The importance of the IVUS results is clear: the visualization of a lobulated thrombus, loosely attached to an eroded, non-ruptured plaque, prompted us to use distal protection, which in turn may have prevented significant embolism. IVUS proved also useful in ensuring full coverage of the lesion, resolving the relation of the lesion with the LAD ostium, correctly sizing the stent, and ruling out thrombus or plaque prolapse through stent struts.
Discussion
Acute coronary syndromes with extensive thrombosis in the absence of widespread coronary atheroma are detected most frequently in young smokers [ 5 , 6 ].The necessity of preventing distal embolic during PCI is becoming increasing recognised, as traditional angioplasty may be associated with distal myocardial necrosis [ 1 ].In our case, angiographic appearance was not suggestive of either obstructive coronary disease or a high embolic risk: none of the criteria set up by Yip et al [ 7 ] (cutoff pattern of occlusion, accumulated thrombus > 5 mm proximal to the occlusion, presence of floating thrombus, persistent dye stasis distal to the obstruction, reference lumen diameter of the culprit artery > or = 4 mm, and incomplete obstruction with presence of accumulated thrombus more than three times the reference lumen diameter of culprit artery) was met, and the patient had already received prolonged antiplatelet and anticoagulant therapy.However, we suspected the presence of a significant thrombotic mass, mainly on the clinical grounds of widespread ECG change and Troponin I elevation, and performed IVUS examination. The importance of the IVUS results is clear: the visualization of a lobulated thrombus, loosely attached to an eroded, non-ruptured plaque, prompted us to use distal protection, which in turn may have prevented significant embolism.IVUS proved also useful in ensuring full coverage of the lesion, resolving the relation of the lesion with the LAD ostium, correctly sizing the stent, and ruling out thrombus or plaque prolapse through stent struts.Conclusion We present a case of successful management of an intracoronary thrombus, which was accomplished combining IVUS data and a distal protection device. IVUS proved invaluable in the diagnosis and treatment of this challenging case. We believe IVUS guidance should be considered for assessing thrombotic burden and embolism potential particularly in young patients with ACS when angiographic data are inconclusive.
Conclusion
We present a case of successful management of an intracoronary thrombus, which was accomplished combining IVUS data and a distal protection device.IVUS proved invaluable in the diagnosis and treatment of this challenging case. We believe IVUS guidance should be considered for assessing thrombotic burden and embolism potential particularly in young patients with ACS when angiographic data are inconclusive.Competing interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions IP and AM have written the first draft of the manuscript. AB and IP have performed the coronary intervention. IP, AM, VA and AB participated in the design and coordination of the final manuscript. All authors have read and approved the final manuscript.
Authors' contributions
IP and AM have written the first draft of the manuscript. AB and IP have performed the coronary intervention. IP, AM, VA and AB participated in the design and coordination of the final manuscript. All authors have read and approved the final manuscript.