Cysteine and related aminothiols in cardiovascular disease, obesity and insulin resistance.

Cysteine, homocysteine, glutathione and cysteinylglycine are metabolically related low molecular weight aminothiols involved in antioxidant defense and in thiol redox control in many cellular and extracellular processes. Alterations in plasma aminothiol profiles are evident in pathologic conditions associated with oxidative stress and accordingly can be considered potential biomarkers. More recently, cysteine has received special attention on this regard and several methodologies for the separation and measurement of cysteine as well as other related aminothiols have been developed and refined. In this review, we provide insight into characteristics, biologic functions, and metabolic interactions of aminothiols (including their involvement in thiol redox status). We highlight cysteine and review methodologic approaches to elucidate its potential role as a risk factor as well as a biomarker in cardiovascular disease, obesity and insulin resistance.

Circulating antioxidant vitamins and copper in Azorean coronary artery disease patients under preventive medication - A case study.

BACKGROUND AND AIM: Oxidative stress and inflammation are conditions that are deeply involved in atherosclerosis and consequent coronary artery disease (CAD). Therefore, the aim of this study was to assess the relationship among circulating antioxidant vitamins (C, A, E), copper, and other pro- or antioxidant/inflammation markers in patients with and without CAD under preventive medication. SUBJECTS AND METHODS: 174 Azorean subjects symptomatic for CAD (age 56 ± 9y; 68 % men) submitted to coronary angiography were split into 2 groups: one formed by CAD patients (≥50 % stenosis in at least one major coronary vessel) and the other by non-CAD patients (<50 % stenosis). Both groups were age-, sex- and BMI-matched. Plasma levels of vitamins or copper were measured by HPLC and AAS, respectively. RESULTS AND CONCLUSIONS: Lower vitamin C levels were observed in CAD patients (mainly in women, who exhibited a high rate of diabetes mellitus) as compared to the non-CAD ones. Also, CAD patients (mainly men) exhibited significantly higher concentrations of plasma copper than their non-CAD counterparts (1.17 ± 0.3 mg/L vs. 1.09 ± 0.3 mg/L, p = 0.030). In bivariate analysis, plasma copper levels were positively associated with serum LDL-cholesterol (r=0.22; p = 0.004) and chiefly with C-reactive protein (r=0.40; p < 0.001). Furthermore, they were significantly lower in recurrent vs. non recurrent CAD patients (1.07±0.2 vs. 1.24±0.3 mg/L, p = 0.004). ROC analysis showed that plasma copper, whenever >1.06 mg/L, was an independent risk factor for CAD in primary prevention for men, which suggests that its levels can fluctuate with medical therapy (such as anti-inflammatory), thus indicating that copper is not a reliable marker for CAD. Moreover, plasma copper concentration was not associated with CAD severity. Yet, results do suggest that, even within its reference concentration range, it could be useful as an acute inflammation marker in CAD management.

Rapid, sensitive and simultaneous determination of ascorbic and uric acids in human plasma by ion-exclusion HPLC-UV.

OBJECTIVES: Ascorbic (AA) and uric (UA) acids act as antioxidants and are capable to react with biologically relevant oxidants. We aimed to developed a simple, rapid, sensitive, and accurate ion-exclusion HPLC-UV methodology for the simultaneously determination of AA and UA in human plasma. METHODS: Analytical pre-requisites, such as the use of heparin as an anticoagulant and meta-phosphoric acid as a stabilizer were added for accurate and reliable measurements. Chromatographic separation was achieved by an isocratic elution on a HEMA-BIO 1000 SB analytical column using a phosphate buffer, pH 2.4, as a mobile phase. RESULTS: Results indicated an excellent linearity with correlation coefficients (r(2))≥0.999. The LOD of AA and UA was 1.02 and 1.42nmol/mL, respectively, while LOQ ranged from 0.306 to 0.426nmol/mL. A great repeatability for both antioxidants was found, where the CV (%) values for intra-day were lower than 1.8% and under 6.5% for the inter-day assay. The recovery of AA ranged from 92% to 96% and from 99% to 100% for UA. CONCLUSION: This validated method allows the determination of both antioxidants within 10min, and is well suited to routine measurements and/or high-throughput clinical analysis. The methodology was applied to assess the antioxidant status of a group of Azorean subjects.

Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC.

Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3µm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r(2)) ≥0.99. The LOD for the four plasma thiols was lower than 0.10µmol/L, while LOQ varied from 0.5 to 15µmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily.

Cross-sectional study of risk factors for atherosclerosis in the Azorean population.

BACKGROUND: Atherosclerosis-a major cause of vascular disease, including ischemic heart disease (IHD), is a pathology that has a two-fold higher mortality rate in the Azorean Islands compared to mainland Portugal. AIM: This cross-sectional study investigated the role of genetic variation in the prevalence of atherosclerosis in this population. SUBJECTS AND METHODS: A total of 305 individuals were characterized for polymorphisms in eight susceptibility genes for atherosclerosis: ACE, PAI1, NOS3, LTA, FGB, ITGB3, PON1 and APOE. Data were analysed with respect to phenotypic characteristics such as blood pressure, lipid profile, life-style risk factors and familial history of myocardial infarction. RESULTS: In the total sample, frequencies for hypercholestrolemic, hypertensive and obese individuals were 63.6%, 39.3% and 23.3%, respectively. The genetic profile was similar to that observed in other European populations, namely in mainland Portugal. No over-representation of risk alleles was evidenced in this sample. CONCLUSIONS: One has to consider the possibility of an important non-genetic influence on the high cholesterolemia present in the Azorean population. Since diet is the most important life-style risk factor for dyslipidemia, studies aiming to evaluate the dietary characteristics of this population and its impact on serum lipid levels will be of major importance.

Influence of dyslipidemia and smoking on redox markers in humans--a critical study.

UNLABELLED: Experimental research indicates that oxidative processes play a role in susceptibility to a large number of diseases. A better understanding of the parameters affecting redox balance could delay and even prevent such processes. OBJECTIVE: The present study aims to investigate blood parameters associated with antioxidant systems in a Portuguese population for the first time, taking into consideration gender, age range, lipid profile and smoking habits as influencing factors. DESIGN AND PARTICIPANTS: One hundred and eighty-three healthy Portuguese subjects of both genders were recruited from the metropolitan area of Lisbon. The group consisted of individuals aged from 20 to 70 years, who gave their informed consent before participating in the study. All subjects were screened to determine eligibility, which was based on a clinical report. Subjects were considered eligible if they had no acute or chronic illness and were not taking any drugs or dietary supplements that could compromise the values of the studied parameters. The subjects were then divided into different subgroups according to gender, age range, lipid profile and smoking habits. METHODS: Whole blood glutathione peroxidase activity and serum albumin, transferrin and uric acid were determined using commercially available kits. Superoxide dismutase activity in erythrocytes and thiobarbituric acid reactive substances in serum were measured using methods published elsewhere. RESULTS: Glutathione peroxidase activity was not affected by any of the studied variables, but superoxide dismutase activity decreased with smoking. Albumin levels remained unchanged under all conditions. Hyperlipidemia was associated with higher lipid peroxidation as well as higher uric acid levels. Gender was the strongest predictor for transferrin, total iron binding capacity and uric acid variations. Finally, a multivariate statistical model clearly separated genders and lipid profile and genders and smoking. CONCLUSIONS: The present study suggests that hyperlipidemia and smoking should be considered important selection criteria in epidemiological studies focusing on oxidative stress and on the atherosclerotic process.

Comparison of apolipoprotein E polymorphism and other atherosclerotic risk factors in two portuguese populations: the influence of insularity

The main objective of this study was to investigate the apolipoprotein (apo) E genotypes in Portuguese populations from mainland (Lisbon city) and from San Miguel Island, Azores' Archipelago (Ponta Delgada city) and to look for differences between these particular sites in apparently healthy subjects. Also, subjects with clinical diagnosis of atherosclerotic disease were investigated in San Miguel Island. In Lisbon, the genotypes distribution was: epsilon3/epsilon 3 > epsilon 3/epsilon 4 > epsilon 2/epsilon 3 > epsilon 4/epsilon 4 while that, for Ponta Delgada and regardless the health condition, was: epsilon 3/epsilon 3 > epsilon 2/epsilon 3 > epsilon 3/epsilon 4. Within Ponta Delgada control group, females and males had distinct genotype frequencies. The most common atherosclerotic risk factors as body mass index, blood hypertension and serum lipid parameters, presented some differences among the allelic subgroups of apo E. The major conclusions were: 1) an apparent influence of insularity in apo E polymorphism was observed; 2) both the high risk genotypes epsilon 2/epsilon 2 and epsilon 2/epsilon 4 were not found, even in patients; 3) curiously, the genotypes proportion in females was not homogenous among the three groups.

O principal objectivo deste estudo é o de pesquisar o efeito da insularidade nos polimorfismos da apolipoproteína (apo) E em indivíduos saudáveis do continente (Lisboa) e de Ponta Delgada (Ilha de S. Miguel, Arquipélago dos Açores). Adicionalmente, estudar a distribuição dos seus genótipos em doentes com aterosclerose da Ilha de S. Miguel. Em Lisboa, a distribuição dos genótipos da apo E foi a seguinte: épsilon3/épsilon3 > épsilon3/épsilon4 > épsilon2/épsilon3 > épsilon4/épsilon 4, ao passo que em Ponta Delgada e independentemente da condição fisiológica foi: épsilon 3/épsilon 3 > épsilon 2/épsilon 3 > épsilon 3/épsilon 4. Distintas frequências genotípicas foram observadas entre homens e mulheres no grupo saudável de Ponta Delgada. O índice de massa corporal, hipertensão arterial e perfil lipídico, factores de risco associados ao processo aterosclerótico, revelaram algumas diferenças quando avaliados em função dos grupos alélicos. Neste estudo, os genótipos de risco da apo E, épsilon2/épsilon2 e épsilon2/épsilon4, não foram contabilizados. Curiosamente a proporção dos genótipos nas mulheres foi heterogénea nos 3 grupos estudados.

Polymorphism of the APOE locus in the Azores Islands (Portugal).

Our aim in this study is to report on the polymorphism of the APOE gene in the Azores Islands (Portugal) to obtain a population baseline of the existing variation in this locus, known to be one of the genetic determinants of plasma lipid levels. One hundred twenty-six Azorean individuals were typed for the APOE polymorphism using standard PCR-RFLP. Allele frequencies obtained for APOE*2, APOE*3, and APOE*4 were 6.75%, 83.73%, and 9.52%, respectively. The APOE*3/*3 genotype presented the highest frequency (69.84%), and the APOE*4/*4 genotype had the lowest frequency (0.79%). Genotype frequencies were in conformity with Hardy-Weinberg expectations. The observed genotype and allele frequencies were similar to those reported for other Iberian samples. Furthermore, Nei's gene diversity (H = 0.2864 +/- 0.0351) was similar to that reported for samples from mainland Portugal. The data generated from this study will be of importance in the context of ongoing studies concerning the factors that influence lipid levels in the Azorean population.