Volume 43, Issue 7

Cardiovascular diseases are a real public health problem and have multifactorial origin. Full comprehension of these diseases is very difficult because of their clinical and biological heterogeneity. The best way to understand the development of these diseases is to first investigate each biological system involved in the diseases and secondly, interactions between them. Studying intermediate phenotype variation for these biological systems is a new and promising approach for their elucidation. Among these metabolic systems, inflammation is known to be implicated in the occurrence and worsening of cardiovascular diseases. For this review, we chose to report the most important results concerning environmental, genetic and epigenetic determinants that influence intermediate phenotypes (protein and RNA levels) of inflammation. Indeed, numerous studies have investigated these determinants in healthy populations, in other words, without the influence of either disease or medication. This new approach will help to better understand the regulation of these intermediate phenotypes and to identify a panel of risk markers, which may be useful in predicting and warning those at risk and in giving adequate treatment to each patient.

Coeliac disease (CD) is an inflammatory disorder of the small intestine characterised by a permanent intolerance to gluten-derived peptides. When gluten-derived peptides reach the lamina propria in CD patients, they provoke specific changes in the mucosa of their small intestine. Although the susceptibility to CD is strongly determined by environmental gluten, it is clearly a common genetic disorder. Important genetic factors for CD are the HLA-DQ genes located in the MHC region on chromosome 6 [ HLA-DQ2 (95%) or HLA-DQ8 (∼5%) heterodimers]. So far, the only treatment for CD consists of a life-long gluten-free diet. A key question in CD is why the gluten-derived peptides are resistant to further breakdown by endogenous proteases and how, in turn, they can activate a harmful immune response in the lamina propria of genetically predisposed individuals. Four mechanisms, namely apoptosis, oxidative stress, matrix metalloproteinases and dysregulation of proliferation and differentiation, are thought to play a role in the pathophysiology of CD. Whether the genes involved in these four mechanisms play a causative role in the development of the villous atrophy or are, in fact, a consequence of the disease process is unknown. In this review we summarise these mechanisms and discuss their validity in the context of current insights derived from genetic, genomic and molecular studies. We also discuss future directions for research and the therapeutic implications for patients.

Gene expression profiling studies in human diseases have allowed better understanding of pathophysiological processes. In addition, they may lead to the development of new clinical tools to improve diagnosis and prognosis of patients. Most of these studies have been successfully performed for human cancers. Inspired by these results, researchers in the cardiovascular field have also started using large-scale transcriptional analysis to better understand and classify human cardiovascular disease. Here we provide an overview of the literature revealing new cardiac disease markers and encouraging results for further development of the expression profiling strategy for future clinical applications in cardiology.

Cytochrome P450 (CYP) is a multigene family of enzymes involved in important life functions; some of these genes are inducible and are implicated in the oxidative metabolic activation and detoxification of many endogenous and exogenous compounds. CYP1B1 codes for an enzyme that catalyses the production of a 2- and 4-hydroxyl group in estrone and estradiol, while CYP1A1 catalyzes the 2-hydroxylation of estradiol in endometrium. The two genes were evaluated in a cohort of 150 subjects: African-American women had significantly lower 2-hydroxyl estrone/16-hydroxyl estrone (2-OHE1/16-OHE1) urinary metabolite ratios than Caucasian women (2.06±1.05 vs. 1.43±0.56; p=0.0002). A common polymorphism in the CYP1B1 gene (leucine to valineat codon 432) was associated with changes in urinary estrogen levels: both Caucasian and African-American women carrying the variant allele showed higher urinary metabolite ratios than women with the wild-type allele. No effect of the CYP1A1 MspI was observed. The 4-OHE1/2-OHE1 ratio was lower in subjects carrying the variant allele (Leu). The percentage change in 2-OHE1/16-OHE1 urinary ratio after indole treatment was significant in both Caucasian and African-American women carrying the wild-type CYP1B1 genotype, although it was more evident in African-Americans than in Caucasians. These results suggest that the Leu/Val CYP1B1 polymorphism may modify estradiol metabolism.

DNA topoisomerase I (Topo I) is involved in DNA replication, transcription, recombination and repair. Clinical interest has focused on Topo I as it is the molecular target of camptothecin (CPT), used in first and second lines of treatment for different cancer types. Furthermore, it is well demonstrated that the patients who best responded to CPT-based chemotherapy were generally those with the greatest tumoral Topo I expression and/or activity. We developed a sensitive, simple and reproducible method to measure Topo I mRNA expression in human cancer samples. Experiments were performed in two steps. First, we checked the accuracy of the reverse transcription-polymerase chain reaction (RT-PCR) method by testing intra- and interassay reproducibility of Topo I and G6PDH gene amplification in different cell types. We observed that crossing-points (Cps) were different, depending on the cell type, dilution or cDNA concentration, but that the intra- and interassay Cp standard deviation (SD) never exceeded 0.77% and 1.39% for Topo I amplification, or 1.63% and 2.9% for G6PDH amplification, respectively. Secondly, we used our method to measure Topo I mRNA levels in primary tumor samples obtained from 27 patients with advanced colorectal cancer and 10 patients with pharyngeal/laryngeal cancer. The accuracy of G6PDH as a housekeeping gene was tested by analyzing its correlation with the mRNA level of a second housekeeping gene, porphobilinogen deaminase ( PBG-D ) in the tumoral samples. We found that the normalized Topo I/G6PDH mRNA ratios were significantly correlated with that of Topo I/PBGD in colorectal tumors (r 2 =0.47, p=0.02) but not in pharyngeal/laryngeal tumors (r 2 =0.35, p=0.3). Neither ratio showed any significant association with clinicopathological parameters, such as gender, age, tumor size, or grade and lymph node status. We believe that RT-PCR is a reliable and highly reproducible technique. However, the choice of the reference gene is an important point and must be defined based on the samples studied.

We examined insulin sensitivity and secretion, together with the levels of selected glucoregulatory hormones, in 15 female patients with severe hypothyroidism (H) and during subsequent thyroid hormone replacement therapy (HRT) using the euglycaemic hyperinsulinaemic clamp technique. Insulin action, as evaluated by glucose disposal, the insulin sensitivity index, and fasting post-hepatic insulin delivery rate were established. The basal levels of insulin, C-peptide and counter-regulatory hormones were measured in basal condition. In H, glucose disposal (p<0.01), the insulin sensitivity index (p<0.01) and post-hepatic insulin delivery rate (p<0.05) were significantly lower than during HRT. No significant changes in the levels of fasting insulin and C-peptide were observed. The levels of counter-regulatory hormones in patients with H were significantly higher than during HRT (glucagon, p<0.05; epinephrine, p<0.01; cortisol, p<0.05; growth hormone, p<0.05). In H, an inverse correlation between insulin sensitivity and insulin secretion was observed (p<0.05). Cortisol was the most important factor affecting the variability of insulin sensitivity values, regardless of thyroid function (p=0.0012). In conclusion, H altered both insulin sensitivity and the levels of selected counter-regulatory hormones. The situation was restored by HRT, as manifested not only by normalisation of insulin sensitivity, secretion and levels of glucoregulatory hormones, but also by improvement of their relationships.

We studied the linearity and detection limits of the capillary zone electrophoresis system Capillarys ® in the measurement of serum monoclonal protein. Three monoclonal proteins with different isotypes and electrophoretic migrations were diluted with a hypo-γ-globulinemic polyclonal serum pool. Mathematical linearity was observed for all monoclonal protein isotypes in the ranges studied without removing the polyclonal γ-globulin background. Theoretical concentrations of 0.43, 0.89 and 0.33g/L for monoclonal proteins immunoglobulin (Ig)G, IgA and IgM, respectively, gave a discernible spike by Capillarys ® , although they were measured as 0.76, 1.09 and 0.76g/L, respectively. We observed overestimation of monoclonal protein inversely correlated to concentrations below 15g/L. All these limitations have to be taken into account when monitoring monoclonal proteins, because the loss of linearity and the protein background may hide an increase in concentration at low levels.

Background: Recently, automated urine test strip readers became available that can report quantitative data. We explored the possibility of measuring all ketone bodies (acetone, acetoacetate, 3-hydroxybutyrate) in urine with these test strips. Monitoring urinary ketone concentrations could offer the advantages of measuring higher values (due to the low renal thresholds) and being less sensitive to fluctuations. Methods: We evaluated URISYS 2400 (Roche) quantitative reflectance data for the ketone reflectance field and compared it with biochemical data from urine samples. Using an easy sample pre-treatment with 3-hydroxybutyrate dehydrogenase, we were able to assay 3-hydroxybutyrate as well, which normally does not react on urine test strips. Results: Within- and between-run reproducibility of the reflectance signal for high- and low-concentration urine pools was 11.0–3.6% and 11.0–5.8% for aceto-acetate, 8.2–9.2% and 10.4–16.1% for acetone, and 5.1–3.0% and 5.6–3.5% for 3-hydroxybutyrate, respectively. The lower limit of detection for acetoacetate was 0.13mmol/L (CV=3.6%). Fair agreement was obtained between test strip data for ketones andcolorimetrically determined acetoacetate values (r=0.90). Conclusions: In urine test strip analysis, quantitative ketone reflectance data allow a simple and fast analysis, offering affordable screening for the detection of ketone body production in diabetes, especially in emergency settings.

A convenient liquid chromatography-tandem mass spectrometry method for the quantification of the triazole antifungal agent voriconazole in plasma samples is described. Fenbuconazole is used as an internal standard. After protein precipitation, automated solid-phase extraction is applied. Electrospray ionization in the positive mode is used and the following mass transitions are recorded: voriconazole, 350→127; and fenbuconazol, 337→125. The analytical run time is 4min. The response was linear from 78 to 5000μg/L. The total coefficient of variation (n=16) was 12.6% for a low-concentration pool (143μg/L), 4.7% for a medium-concentration pool (419μg/L), and 5.0% for a high-concentration pool (4304μg/L). The method is proposed for future investigations that should be performed to test the hypothesis that therapeutic drug monitoring of voriconazole is clinically useful.

Background: A vast amount of scientific research is directed towards the beneficial effects of antioxidants on health. For this reason, several assays have been developed to determine the total antioxidant capacity of blood. Methods: In this study two procedures based on the use of the green-blue 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS •+ ) were compared. In the first (commercially available) procedure, ABTS •+ was generated in the presence of the blood sample. In the second procedure, referred to as the decolorization assay, antioxidants react with preformed ABTS •+ . Results: It was found that the first procedure leads to greater underestimation of the actual antioxidant capacity and is more prone to artifacts than the second procedure. Therefore, only the latter procedure was evaluated in detail and it appeared that (i) plasma is preferred over serum, (ii) the high background produced by albumin can be circumvented by deproteination, (iii) samples can be stored at −80°C for 12months, and (iv) the assay has high precision. Due to poor linearity, the procedure has to be standardized to allow sample comparison. Conclusions: The decolorization assay is a reliable and robust assay that can be applied routinely to predict the antioxidant capacity of blood.

The measurement of cardiac troponins has emerged as the biochemical “gold standard” for the diagnosis and management of patients with acute chest pain. However, earlier markers should support investigation strategies, as several patients with acute coronary syndrome might present with non-diagnostic concentrations. Ischemia-modified albumin (IMA), measured by the albumin cobalt binding (ACB) assay, was recently proposed for early detection of myocardial ischemia. To establish the potential influence of endurance training on the diagnostic approach to patients with suspected myocardial injury, cardiac troponin T (cTnT), creatine kinase isoenzyme MB (CK-MB), myoglobin and IMA were evaluated in healthy individuals subjected to different aerobic workloads. The concentrations of both IMA and CK-MB were significantly increased in athletes subjected to high-workload endurance training, whereas the concentration of cTnT and myoglobin was not influenced by physical exercise in the medium term. Taken together, our results demonstrate that demanding aerobic physical activity might influence the generation of IMA, which might be increased in the medium term following high-workload endurance training, while the concentration of other conventional markers of myocardial injury remains non-diagnostic.

Steinert's disease (myotonic dystrophy type 1; MD) is caused by a CTG trinucleotide expansion on 19q13.3. Although the pathogenic mechanism underlying multisystem involvement in MD is still unclear, a role of oxidative stress in this disease has been suggested. We investigated 39 MD patients to assess the plasma concentration of advanced oxidation protein products (AOPPs) and γ-glutamyltransferase (GGT) and related them to clinical severity scores. Plasma AOPP levels (p=0.021), total serum GGT activity (p=0.0005) and GGT activity associated with low-density lipoprotein (p=0.0021) were significantly higher in patients than in controls. There was significant correlation between serum GGT levels and AOPPs (r=0.5831; p=0.0022). A statistically significant increase in serum GGT with age was found in MD patients (p=0.0193). Receiver operating characteristic curve analysis showed that higher AOPP levels were significantly associated with extra-muscular signs of the disease, i.e., cataracts and heart involvement (area under the curve±SE=0.908±0.083), but not with muscular involvement. The concomitant increment in GGT and AOPPs indicates a possible role of oxidative stress in the pathogenesis of MD type 1, while the association of increased AOPP levels with extra-muscular signs of the disease suggests that individual susceptibility to oxidative stress can modulate the extra-muscular phenotype of the disease.

Methods for the measurement of autoantibodies frequently provide controversial results. The objective of the present study was to evaluate the performance of Spanish Clinical Laboratories in the measurement of anti-Sm antibodies. A total of 23 laboratories participated, analysing 30 serum samples from patients with systemic lupus erythematosus and other autoimmune and non-autoimmune diseases. The laboratories used four extractable nuclear antigen screens, eight enzyme-linked immunosorbent assays (ELISAs) specific for anti-Sm, one line-blot, one dot-blot and one double immunodiffusion assay, from 15 different manufacturers. A total of 871 results were obtained. In general, very good sensitivity was obtained (95–100%), but specificity was moderate (52–86%) and must be improved. Most ELISAs and the line-blot were valid assays for anti-Sm detection and could serve as tests both for analysis and/or confirmation. The likelihood ratios indicated that both methods can be considered very useful or useful for the determination of anti-Sm antibodies. Nevertheless, the analytical quality of the methods for the measurement of anti-Sm antibodies could probably be improved by standardisation of the methods and the participation of laboratories in external quality control programs.

Background: In peripheral blood, chemotaxis, phagocytosis, and oxidative burst of polymorphonuclear cells (PMNs) can be assessed by flow cytometry, whereas function tests, i.e., quality control in PMN concentrates designed for neutropenia therapy, are lacking. Methods: PMN concentrates (n=6) harvested from healthy donors who had been premedicated with granulocyte colony-stimulating factor (G-CSF) and dexamethasone were stored undiluted (control, C; n=6) and diluted 1:4 (D; n=6) with autologous plasma for 72h. Commercial flow cytometry function tests were performed to quantify changes in chemotaxis, phagocytosis, and oxidative burst of PMNs over time. Results: Median levels of phagocytosis and oxidative burst levelled at 86% (82–94) and 98% (83–100) in C on the day of apheresis, respectively, but deteriorated to 15% (0–24) and 0% within 72h; in D these parameters remained close to 90%. Median levels of chemotaxis were comparable in C (69%, 65–74) and D (74%, 70–84) at baseline. No migration was detected in C after 72h; however, D retained approximately 63% (13–76) migration capacity. Conclusion: Quality control in PMN concentrates is practical using flow cytometry and commercial test kits. While phagocytosis and oxidative burst may be maintained for 72h in vitro, chemotaxis of apheresed PMNs is already reduced on the day of apheresis.

We examined the circadian variation of holo-transcobalamin (holo-TC), total transcobalamin (total TC) and plasma cobalamins, and the association between food intake and fluctuations in the biochemical markers. A total of 17 healthy women (mean age 33years; range 24–40) participated. The subjects were admitted at 08:00h after an overnight fast, and blood samples were obtained every 20min for 24h from 12:00h. Holo-TC and total TC were measured by an ELISA, and plasma cobalamins were measured by a routine method (Centaur, Bayer). All subjects, except one, had 12:00h levels within the reference interval for all variables studied, and all subjects had normal renal function as judged from plasma creatinine. We found a small intra-individual variation (12%, 10%, 10%) and a considerable inter-individual variation (56%, 26%, 43%) in the level of holo-TC, total TC and plasma cobalamins, respectively. During the night, the absolute values of all components decreased, as did plasma albumin. There was no systematic association between intake of food and fluctuation of the biochemical markers. In conclusion, we found no significant circadian variation in serum holo-TC in healthy vitamin-replete subjects on a standard diet. This supports the view that holo-TC is a marker of long-term vitamin B 12 status, and that samples may also be obtained from non-fasting subjects.

Background: Low levels of plasma homocysteine have been found in children and adult populations living in Burkina Faso in association with a low prevalence of coronary heart disease. Methods: Based on this finding, the levels of plasma homocysteine and other thiols (cysteine, cysteinylglycine, glutathione) in postmenopausal women living in Burkina Faso were evaluated with the aim of investigating whether age and life conditions influence plasma homocysteine and other thiol levels. Results: It was found that in older postmenopausal women the mean level of homocysteine was higher (16.4±6.6 μmol/L) than in fertile women (6.8±1.2μmol/L) and that thisincrease was correlated with cysteine levels (166.6±44.6μmol/L). While the glutathione level in postmenopausal women was lower (3.6±2.3μmol/L) compared with fertile women (7.0±1.7μmol/L), cysteinylglycine levels were within the normal range (29.9±9.3μmol/L). No correlation was found between homocysteine levels and serum folate, vitamin B 12 , vitamin B 6 , cystatin C and serum creatinine levels. The older the women were, the higher were their plasma homocysteine levels: levels up to 20.2±9.1μmol/L were found in those >70years old. Conclusions: The elevated levels of homocysteine in the postmenopausal women of Burkina Faso must be viewed as a characteristic of older age and its metabolic consequences.

Purpose: We aimed to evaluate the diagnostic performance of the IMMULITE 2000 Allergy System from Diagnostic Products Corporation (DPC) for the detection of inhalant and food allergies, focusing on inhalant and food screens, mixes and single allergens. Methods: Serum samples were collected from new, unselected patients who were referred to the allergist for a suspected allergy. Patients were classified as study diagnosis-positive for inhalant (food) allergy if they had both a positive clinical examination/history and a positive skin test for inhalant (food) allergy; otherwise – failing one or both of these criteria – they were classified as study diagnosis-negative. Classification and testing of the serum samples was carried out in a blinded fashion. Values greater than 0.35 kU/L were considered positive. Results: Of the 118 patients included, 63 were considered study diagnosis-positive for inhalation and/or food allergy. DPC inhalation screening showed 82% total agreement (TA) and 91% sensitivity relative to the study diagnoses. The DPC food panel showed 96% TA and 98% specificity relative to the study diagnoses. Relative to specific intracutaneous testing (ICT), the DPC D1, E1 and E5 assays had sensitivity of 82–90%; tree and grass panels had sensitivity of 74% and 95%. The DPC weed panel and initial lots of DPC E5 had poor sensitivity (<40%); mold panel sensitivity was equally low for both DPC and the routinely used Pharmacia assay (36%). Relative to skin prick testing (SPT), specific food allergens had TA of 94–98% and specificity of 95–100%. Conclusion: In patients classified by the combination of clinical examination/history and skin test results, the DPC IMMULITE 2000 Allergy System generally demonstrated acceptable sensitivity, specificity and TA compared to the study diagnoses, both at the screening level and at the level of panels and single allergens.

The performance of the ABX Pentra 400, a new multiparametric analyser available for routine and specialised clinical chemistry analyses, was evaluated according to the National Committee for Clinical Laboratory Standards (NCCLS) and Valtec protocols. Substrate concentrations and enzyme activities were determined by spectrophotometric measurement after coloured reaction or UV detection-based reactions; electrolyte concentrations were determined with ion-selective electrodes, and specific protein concentrations were assayed by immunoturbidimetry. In total, 32 of the most common clinical chemistry parameters were evaluated under simulated routine conditions. The analytical performance of the system and the quality of the ABX Pentra reagent line were both examined in a single-site study. The ABX Pentra 400 was compared with the Olympus AU640, Konelab-Konepro, Beckman Coulter-Immage and Beckman Coulter-Access 2, depending on the tests available on these analysers. The coefficient of variation (CV) values were within expected values. Coefficients of correlation showed high correlation between the analysers compared. The influence of interfering substances was moderate. The practicability of the system was good. We conclude that good reliability and practicability make the ABX Pentra 400 system suitable for laboratories with various needs.