KAsH: A new tool to predict in-hospital mortality in patients with myocardial infarction.

INTRODUCTION: Complex risk scores have limited applicability in the assessment of patients with myocardial infarction (MI). In this work, the authors aimed to develop a simple to use clinical score to stratify the in-hospital mortality risk of patients with MI at first medical contact. METHODS: In this single-center prospective registry assessing 1504 consecutively admitted patients with MI, the strongest predictors of in-hospital mortality were selected through multivariate logistic regression. The KAsH score was developed according to the following formula: KAsH=(Killip class×Age×Heart rate)/systolic blood pressure. Its predictive power was compared to previously validated scores using the DeLong test. The score was categorized and further compared to the Killip classification. RESULTS: The KAsH score displayed excellent predictive power for in-hospital mortality, superior to other well-validated risk scores (AUC: KAsH 0.861 vs. GRACE 0.773, p<0.001) and robust in subgroup analysis. KAsH maintained its predictive capacity after adjustment for multiple confounding factors such as diabetes, heart failure, mechanical complications and bleeding (OR 1.004, 95% CI 1.001-1.008, p=0.012) and reclassified 81.5% of patients into a better risk category compared to the Killip classification. KAsH's categorization displayed excellent mortality discrimination (KAsH 1: 1.0%, KAsH 2: 8.1%, KAsH 3: 20.4%, KAsH 4: 55.2%) and better mortality prediction than the Killip classification (AUC: KAsH 0.839 vs. Killip 0.775, p<0.0001). CONCLUSION: KAsH, an easy to use score calculated at first medical contact with patients with MI, displays better predictive power for in-hospital mortality than existing scores.

A new and fast methodology to assess oxidative damage in cardiovascular diseases risk development through eVol-MEPS-UHPLC analysis of four urinary biomarkers.

In this work, a new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (eVol(®)-MEPS) followed by ultra-high pressure liquid chromatography (UHPLC) analysis with photodiodes (PDA) detection, was developed to establish the urinary profile levels of four putative oxidative stress biomarkers (OSBs) in healthy subjects and patients evidencing cardiovascular diseases (CVDs). This data was used to verify the suitability of the selected OSBs (uric acid-UAc, malondialdehyde-MDA, 5-(hydroxymethyl)uracil-5-HMUra and 8-hydroxy-2'-deoxyguanosine-8-oxodG) as potential biomarkers of CVDs progression. Important parameters affecting the efficiency of the extraction process were optimized, particularly stationary phase selection, pH influence, sample volume, number of extraction cycles and washing and elution volumes. The experimental conditions that allowed the best extraction efficiency, expressed in terms of total area of the target analytes and data reproducibility, includes a 10 times dilution and pH adjustment of the urine samples to 6.0, followed by a gradient elution through the C8 adsorbent with 5 times 50 µL of 0.01% formic acid and 3×50 µL of 20% methanol in 0.01% formic acid. The chromatographic separation of the target analytes was performed with a HSS T3 column (100 mm × 2.1 mm, 1.7 µm in particle size) using 0.01% formic acid 20% methanol at 250 µL min(-1). The methodology was validated in terms of selectivity, linearity, instrumental limit of detection (LOD), method limit of quantification (LOQ), matrix effect, accuracy and precision (intra-and inter-day). Good results were obtained in terms of selectivity and linearity (r(2)>0.9906), as well as the LOD and LOQ, whose values were low, ranging from 0.00005 to 0.72 µg mL(-1) and 0.00023 to 2.31 µg mL(-1), respectively. The recovery results (91.1-123.0%), intra-day (1.0-8.3%), inter-day precision (4.6-6.3%) and the matrix effect (60.1-110.3%) of eVol(®)-MEPS/UHPLC-PDA method were also very satisfactory. Finally, the application of the methodology to the determination of target biomarkers in normal subjects and CVDs patients' revealed that the DNA adducts 5-HMUra and 8-oxodG levels are much more abundant in CVDs patients while no statistic differences were obtain for MDA and UAc. This result points to the importance of 5-HMUra and 8-oxodG as biomarkers of CVDs risk progression and further epidemiological studies are needed to explore the importance of this correlation.

Independent association of the variant rs1333049 at the 9p21 locus and coronary heart disease.

INTRODUCTION: Recent genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) at the 9p21 locus as risk factors for coronary artery disease (CAD). Among them, the SNP rs1333049 has demonstrated a consistent association with CAD, which has been successfully replicated in several populations. AIM: To investigate whether the SNP rs1333049 located on the 9p21 chromosome is an independent risk factor for CAD in a Portuguese population. METHODS: We performed a case-control study which included 1406 individuals, 723 consecutive coronary patients (mean age 53.71 +/- 8.9 years, 79.9% male and 683 controls without coronary disease (mean age 53.3 +/- 10.5 years, 73.9% male). Cases and controls were selected so as not to be significantly different in terms of gender and age. We studied the SNP rs1333049 at the 9p21 locus in all individuals, using standard PCR combined with the TaqMan technique (Applied Biosystems). The allelic and genotype distribution (C/G), odds ratios and corresponding confidence intervals for CAD risk were determined. A forward Wald logistic regression analysis model was constructed, adjusted for age, gender, conventional risk factors, biochemical markers and the genotypes under study, in order to determine which variables were linked significantly and independently with CAD. RESULTS: The C allele was found in 60% of the CAD patients and 53% of the controls, with OR = 1.33; p = 0.0002. The CC genotype appeared in 35.7% of CAD patients, with OR = 1.34, p = 0.010. The heterozygous CG genotype was present in 48.1% of the CAD patients and 47% of the controls, and did not present vascular risk (OR = 1.05, p = 0.670). After logistic regression analysis, the CC genotype remained in the equation with OR = 1.7; p = 0.018 and CG with OR = 1.5, p = 0.048. CONCLUSION: In the present study we replicated the coronary risk linked to the recently discovered variant rs1333049 on the 9p21 chromosome in a Portuguese population. Although the mechanism underlying the risk is still unknown, the robustness of this risk allele in risk stratification for CAD has been consistent, even in very different populations. The presence of the CC or CG genotype may thus prove to be useful for predicting the risk of developing CAD in the Portuguese population.

RAS gene polymorphisms, classical risk factors and the advent of coronary artery disease in the Portuguese population.

BACKGROUND: Several polymorphisms within the renin-angiotensin system cluster of genes have been associated with the advent of coronary artery disease (CAD) or related pathologies. We investigated the distribution of 5 of these polymorphisms in order to find any association with CAD development and distinguish if any of the biochemical and behavioural factors interact with genetic polymorphisms in the advent of the disease. METHODS: ACE I/D (rs4340), ACE A11860G (rs4343), AT1R A1166C (rs5186), AGT T174M (rs4762) and AGT M235T (rs699) gene polymorphisms were PCR-RFLP analysed in 298 CAD patients and 510 controls from Portugal. Several biochemical and behavioural markers were obtained. RESULTS: ACE I/D DD and ACE11860 GG genotypes are risk factors for CAD in this population. The simultaneous presence of ACE I/D I and ACE11860 A alleles corresponds to a significant trend towards a decrease in CAD incidence. We found several synergistic effects between the studied polymorphisms and classical risk factors such as hypertension, obesity, diabetes and dyslipidaemia: the presence of the DD genotype of ACE I/D (and also ACE11860 GG) increases the odds of developing CAD when associated to each one of these classical risk factors, particularly when considering the male and early onset CAD subgroup analysis; AGT235 TT also increases the CAD risk in the presence of hypertension and dyslipidaemia, and AT1R1166 interacts positively with hypertension, smoking and obesity. CONCLUSION: ACE polymorphisms were shown to play a major role in individual susceptibility to develop CAD. There is also a clear interaction between RAS predisposing genes and some biochemical/environmental risk factors in CAD onset, demonstrating a significant enhancement of classical markers particularly by ACE I/D and ACE11860.

Methylenetetrahydrofolate reductase gene, homocysteine and coronary artery disease: the A1298C polymorphism does matter. Inferences from a case study (Madeira, Portugal).

Elevated levels of plasma homocysteine, an independent risk factor and a strong predictor of mortality in patients with coronary artery disease (CAD), can result from nutritional deficiencies or genetic errors, including methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms. The contribution of these polymorphisms in the development of CAD remains controversial. We analysed the impact of MTHFR C677T and A1298C on fasting homocysteine and CAD in 298 CAD patients proved by angiography and 510 control subjects from the Island of Madeira (Portugal). After adjustment for other risk factors, plasma homocysteine remained independently correlated with CAD. Serum homocysteine was significantly higher in individuals with 677TT and 1298AA genotypes. There was no difference in the distribution of MTHFR677 genotypes between cases and controls but a significant increase in 1298AA prevalence was found in CAD patients. In spite of the clear effect of C677T mutation on elevated homocysteine levels we only found an association between 1298AA genotype and CAD in this population. The simultaneous presence of 677CT and 1298AA genotypes provides a significant risk of developing the disease, while the 1298AC genotype, combined with 677CC, shows a significant trend towards a decrease in CAD occurrence. The data shows an independent association between elevated levels of homocysteine and CAD. Both MTHFR polymorphisms are associated with increased fasting homocysteine (677TT and 1298AA genotypes), but only the 1298AA variant shows an increased prevalence in CAD group. Odds ratio seem to indicate that individuals with the MTHFR 1298AA genotype and the 677CT/1298AA compound genotype had a 1.6-fold increased risk for developing CAD suggesting a possible association of MTHFR polymorphisms with the risk of CAD in Madeira population.

[Angiotensin converting enzyme gene polymorphisms and coronary risk in a Portuguese population].

BACKGROUND: A family history of coronary heart disease (CHD) is a strong risk marker for the disease, independently of classical risk factors. It could be decoded by recognizing the polymorphisms associated with increased risk. Renin-angiotensin system genes are candidate genes in CHD and the deletion allele of the angiotensin converting enzyme (ACE) has been reported as deleterious. However, there is disagreement as to the role of the insertion/deletion polymorphism of the ACE gene in coronary risk. AIM: To evaluate whether ACE gene polymorphisms constitute a CHD risk factor. METHODS: We conducted a population-based case-control study of 301 subjects with a history of myocardial infarction or angiographic evidence of coronary heart disease and 510 age- and gender-matched controls, without CHD, living in a region with high CHD mortality rates. Blood samples were taken, DNA extracted and genotypes determined by the polymerase chain reaction (PCR). Amplification products were identified by agarose gel electrophoresis. STATISTICAL ANALYSIS: The Data were evaluated by SPSS for Windows, using the Student's t test, the chi-square test, odds ratios and 95% confidence intervals. RESULTS: The prevalence of the DD, ID and II genotype was 41.2%, 46.3%, 12.5% in the cases and 28.1%, 55.2% and 16.7% in the control group. The frequency of the DD genotype was significantly higher in the cases than in the controls (41.2% vs. 28.1%, odds ratio 1.79, 95% CI 1.31 to 2.4, p < 0.0001). By contrast, the ID and II genotypes' prevalence was higher in the control group (55.2% vs. 46.3%, p = 0.002 and 16.7 vs. 12.5%, p = NS, respectively) compared to the case group. CONCLUSIONS: This study clearly shows that the ACE DD polymorphism is strongly linked to CHD, and if our data are confirmed in a larger population sample, more aggressive vascular prevention could be justified in patients carrying the DD genotype.

Polymorphism of the ACE gene is associated with extent and severity of coronary disease.

BACKGROUND: The progression and extent of coronary heart disease (CHD) are extremely variable and in many instances independent of conventional risk factors. The differences may be partly explained by less favorable genetic polymorphisms that are associated with them. The polymorphisms of the angiotensin I converting enzyme (ACE) gene have been thoroughly evaluated, but the connection between them and the extent of CHD is unknown. AIMS: Our study is aimed at determining whether any or all of the polymorphisms of the ACE gene are markers of the extent and severity of CHD. METHODS: This was a descriptive study of 296 patients with a history of myocardial infarction or with coronary disease confirmed by coronary angiography. The severity of CHD was quantified according to Leaman's score (based on the number of arteries with more than 75% reduction in diameter and the number of affected coronary segments). The ACE genotypes were determined by specific polymerase chain reaction amplification and the segments were subjected to polyacrylamide gel electrophoresis. The mean coronary score and standard deviation of the three polymorphisms were calculated and the values statistically compared using the Student's t test for independent samples. RESULTS: 296 patients with a mean age of 55.103 years, 234 male, were evaluated. CONCLUSION: The study clearly shows that the DD genotype is linked to the extent of CHD, with a high level of significance. If this is confirmed, careful secondary prevention is indicated in patients with this genotype.