Volume 40, Issue 5

An endothelial nitric oxide synthase gene ( NOS3 ) polymorphism in exon 7 (G894T), resulting in Glu298Asp substitution at protein level, has been associated with myocardial infarction, hypertension and coronary atherosclerosis in some populations. This polymorphism is usually identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, the procedures described to date do not eliminate the possibility of misclassification and either require confirmation by DNA sequencing or are timeconsuming. In this study, a PCR-RFLP procedure to detect the G894T polymorphism at the NOS3 was optimized by the introduction of a constitutive cleavage site in the amplification product. This cleavage site provides an internal control for enzymatic activity to avoid mistyping. The method was validated by the study of 35 white unrelated individuals with familial hypercholesterolemia and 70 controls. The frequency of the variant allele (T) was similar between both groups (27% vs . 22%, NS), and comparable to the frequency found in other white populations. However, future studies are necessary to confirm these data. In summary, the optimized procedure for detection of the G894T NOS3 polymorphism is rapid, simple, and does not require confirmatory tests. Using this method, we found no association between this polymorphism and familial hypercholesterolemia.

Mutations in the low-density lipoprotein receptor ( LDLR ) gene cause familial hypercholesterolemia (FH), one of the most common single gene disorders. It is thought that FH affects approximately 1 of 500 individuals in most populations. Single-strand conformation polymorphism (SSCP) analysis is widely used to detect mutations in the LDLR gene. However, several factors such as temperature, pH, running time, gel composition and size of the DNA fragments can influence its sensitivity. We have optimized the electrophoretic conditions to screen mutations in the promoter region and exons 1–18 of the LDLR gene by varying temperature (5 °C, 8 °C, 12 °C and 15 °C), voltage (300 to 600 V), and running time (1 to 4 hours) in the semi-automated GenePhor™ system (Amersham Biosciences). The efficiency of the method was evaluated by using 30 positive controls (DNA samples with mutations and polymorphisms in the LDLR gene, previously characterized) and DNA samples from 90 Brazilian patients with FH. Our results show that the use of two temperatures (5 °C and 15 °C) in combination with other optimized conditions resulted in high mutation detection rate (97%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for the diagnosis of genetic disorders, cancer, and for pharmacogenetic studies.

Postmenopausal women run the same risks of coronary heart disease as men. The lipid alterations observed at this time reflect increased blood levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a), and reduced high-density lipoprotein cholesterol (HDL-C) levels. These changes lead to a higher risk of coronary artery disease, and hormonal therapy has a favorable effect on lipid metabolism. In this paper we review the literature on hormone replacement therapy (HRT) in postmenopausal women with the emphasis on the role of lipids in the pathogenesis of coronary heart disease, and on the action of estrogens and their correlation with progestogens, as well as routes of HRT administration. We conclude that the HRT changes the lipid profile in a potentially anti-atherogenic direction, usually reducing LDL-C and increasing HDL-C and triglycerides. Otherwise, for postmenopausal women with established coronary disease HRT is not recommended.

Individuals infected with the human immunodeficiency virus (HIV-1) present with decreased CD4, a progressive increase in viral load, compromised cell immune defense, and hematologic alterations. The aim of this study was to assess the serum viral load, CD4, CD8, lymphocyte count and hematocrit at the beginning of antiretroviral therapy in individuals who were supplemented with N -acetylcysteine (NAC). Twenty volunteers participated in this double-blind, placebocontrolled 180-day study. Ten participants received 600 mg of NAC per day (NAC group) and the other ten serving as a control group received placebo. The above mentioned parameters were determined before treatment, and after 60, 120 and 180 days. In NAC-treated patients hematocrit remained stable and an increase in CD4 cell count took place earlier than that in the control group.

In HIV-infected patients, an increase in the production of oxygen-reactive species (ROS) is observed, with a consequent reduction of plasma levels of antioxidants such as α-tocopherol. The nuclear transcription factor-κB (NF-κB) is activated by a prooxidant state in the infected T cells through the release of its inhibitory subunit I-κB. The aim of the present work was to evaluate the behavior of hematological parameters and markers of anemia in HIV-infected patients who underwent antiretroviral therapy associated with 800 mg/day αtocopherol supplementation. Blood samples were collected from supplemented (n=9) and not-supplemented (n=9) HIV-seropositive patients (n=18). We observed a decreased viral load in the α-tocopherol-supplemented group (p<0.05); other changes, such as an increase in the CD4/CD8 ratio, in the hematocrit and in the hemoglobin concentration were also observed, though lacking statistical significance. We conclude that antiretroviral therapy in association with αtocopherol (800 mg/day) supplementation is more effective in reducing viral load levels and also, possibly, in recovering other hematological parameters after a 60-day period of use.

Twenty-five teenagers, 13 males and 12 females, some obese and others overweight, aged between 12 and 18 years, were studied over 8 months, under the supervision of a multidisciplinary team. Effects of re-education in eating habits and physical activity on the lipid profile were evaluated. Dyslipidaemia characterised by increased levels of total cholesterol, low-density lipoprotein cholesterol and triglycerides was obeserved in 64%, 12% and 44% of the teenagers, respectively. Whereas decreased levels of high-density lipoprotein cholesterol were observed in 28%, tendency to hypertension has been observed in 36% of the teenagers. After 8 months, the number of teenagers with total hypercholesterolaemia and hypertriglyceridaemia decreased to 32% and 24%, respectively. Low-density lipoprotein cholesterol levels did not vary significantly. High-density lipoprotein-cholesterol levels increased in 17% of participants. Reduction of blood pressure occurred in most teenagers. These data suggest that reeducation programmes in eating habits associated with changes in behaviour and physical activity can benefit obese teenagers and prevent various diseases.

Oxidation of proteins occurs both as a side-effect of aerobic energy metabolism and as an effect of specific metabolism of phagocytic polymorphonuclear granulocytes producing O 2 − · and H 2 O 2 . In contrast to other cells, which control their H 2 O 2 level by degrading it to O 2 and H 2 O, polymorphonuclear neutrophilic leukocytes (PMN) use H 2 O 2 as a substrate for oxidizing chloride ions to HOCl which rapidly react with all neighboring thiol, disulfide and amino residues. Chloramines, which are the most abundant HOCl reaction products, react with proteins, modifying only certain exposed methionine and cysteine residues. This may account for selective inactivation of a number of enzymes, carrier proteins and peptide mediators, including the α 1 -proteinase inhibitor, α 2 -macroglobulin and plasminogen activator inhibitor. Inactivaton of plasma proteinase inhibitors protects PMN elastase, collagenase, cathepsin G and other serine proteases in the inflammatory foci. This promotes proteolytic degradation of damaged tissue, removal of bacterial debris and wound healing, as well as tissue remodeling related to the inflammatory processes. Oxidative control of protease-anti-protease balance affects the development of the inflammatory processes. Moreover, inactivation of plasma proteinase inhibitors facilitates primary antigen processing, upregulates lymphocyte proliferative response and activates the local immune response. Oxidation produces a specific protein tagging which attracts and stimulates immune active cells. Therefore, humoral response against oxidatively modified proteins occurs more effectively than that of the native proteins. The effect is dose-dependent with respect to the amount of oxidant employed. Glycol aldehyde, which is the serine chloramine spontaneous decay product, in mice immunized with glycol aldehydemodified egg-white albumin, yields specific IgG production manifold higher than that in mice immunized with native albumin. Immunopotentiation is produced by proliferation expansion of the same immunocompetent clones. Oxidative tagging of proteins may also affect the autoimmune-type reaction. Thus, a growing body of data suggest that the specific role of protein oxidation by activated PMN is oxidative protein tagging facilitating further development of the immune reaction.

We discovered a hitherto undescribed insertion/deletion (I/D) polymorphism of 7 base pairs in intron 4 of the HP1 allele and intron 4 and 6 of the HP2 allele. Genotyping was performed in 311 Belgian subjects. The association between serum haptoglobin (Hp) and lipid concentrations and the Hp I/D genotypes was investigated. Genotype distribution in 103 Hp 1–1 phenotypes (50.5% DD, 39.8% DI, 9.7% II) and D allele frequency (0.70) were in close agreement with the Hardy-Weinberg equilibrium. No association between Hp concentrations and the Hp I/D genotypes could be found. Apolipoprotein (apo)A2, apoB and cholesterol concentrations were slightly lower in Hp II compared to DI and DD. Low-density lipoprotein (LDL)-cholesterol and ultrasensitive C-reactive protein (CRP) concentrations and the apoA1/apoA2 ratios differed significantly between Hp D/I genotypes. We added a further component to the molecular heterogeneity of Hp by the detection of an I/D polymorphism. Studies on the Hp I/D polymorphism in various populations open perspectives for further investigation of the distribution pattern of the human Hp gene. The association of the Hp I/D polymorphism with various lipid parameters might add a further component to the complex and multifaceted lipid metabolism.

The primary genetic cause of type III hyperlipoproteinemia is the homozygous presence of the apolipoprotein E2 allele. However, only approximately 1% of subjects with the apolipoprotein E2/E2 genotype develop type III hyperlipoproteinemia. Other factors are therefore necessary to express type III hyperlipoproteinemia. Two individuals were identified as having type III hyperlipoproteinemia (triglyceride to very low-density lipoprotein (VLDL) cholesterol ratio >0.3). However, in contrast to unchanged or slightly decreased low-density lipoprotein (LDL)-cholesterol levels typically observed in type III patients, elevated LDL-cholesterol levels were observed. The expected apolipoprotein E2/E2 isoform was confirmed by genetic analysis. To explain the elevated LDL-cholesterol level, single strand conformation polymorphism analysis was performed to screen for mutations in the LDL receptor gene. In both individuals, mutations causing an impaired LDL receptor function (2 bp insertion in exon 3 and Glu 119 →Gly mutation in exon 4) were identified. In six more unrelated individuals, these mutations combined with the common apolipoprotein E3/E3 genotype, resulted in an isolated, severe LDLcholesterol elevation. Our results indicate that the level of LDL receptors plays an important role in remnant clearance, and that the combination of the binding-defective apolipoprotein E2 with a defective LDL receptor precipitate type III hyperlipoproteinemia.

Circulating human thyroglobulin (hTG) measurement has a pivotal role in the management of patients affected by differentiated thyroid cancer (DTC). Generally, hTG serum concentration less than 1 ng/ml is considered a marker of complete remission after total thyroid ablation. Recently, high-sensitivity immunoradiometric assays (IRMA) have been developed to detect very low hTG serum concentrations. The present study was undertaken to test a newly developed high-sensitivity hTG IRMA and to evaluate its diagnostic performance and reproducibility in the follow-up of patients affected by DTC. We retrospectively selected 156 patients without signs of recurrence and 39 patients with DTC recurrence. Serum samples were collected during L-thyroxine (T4) suppressive therapy (ONT4) and 4 weeks after T4 withdrawal (OFFT4), and hTG was measured by a specific high-sensitivity IRMA (DYNOtest ® Tg-plus, BRAHMS Diagnostica GmbH, Berlin, Germany). Sera showing the presence of antibodies against hTG (AbhTG) or hTG-recovery less than 80% were excluded from the study. The receiver operator characteristic (ROC) curve analysis was performed to select the best cut-off levels, and diagnostic performance of the marker was evaluated. By using ONT4 cut-off level of 0.2 ng/ml and OFFT4 cut-off level of 0.5 ng/ml we obtained a sensitivity/specificity/accuracy profile of 0.92/0.98/0.97 and 0.97/0.98/0.98, respectively. We found false-negative results in three (12%) and one (4%) out of 24 patients with cervical recurrence by using 0.2 and 0.5 ng/ml cut-off levels, respectively. However, we found false-negative results in 13 (54%) and six (25%) patients when 1.0 ng/ml cutoff level was used. Finally, DYNOtest ® Tg-plus showed a very satisfactory intra- and inter-assay reproducibility in the very low hTG concentration range. Based on our data, we conclude that DYNOtest ® Tg-plus assay is effective and accurate in evaluation of patients with DTC.

Serology markers of coeliac disease (CD)–antigliadin IgA/IgG antibodies (AGA/AGG) with purified α-gliadin, antiendomysium IgA antibodies (EmA) and anti-tissue transglutaminase (atTG) IgA/IgG antibodies–determined in 1451 serum samples, were analysed with respect to different screening algorithms. Determination of atTG using five ELISA methods was compared taking into account the impact of human recombinant antigen and IgG class of atTG. A subgroup of 119 patients undergoing small intestinal biopsy was used to calculate sensitivity and specificity of CD markers. The highest sensitivity (94%) was obtained for AGG, and the highest specificity (93.5%) was obtained for EmA. All coeliac disease patients were detected using the combination of all four CD markers, resulting in 100% sensitivity. CD and type 1 diabetes mellitus autoantigens were determined in 139 diabetic patients. The atTG IgA mean value (16.7 IU/ml) was higher in the antiglutamate dehydrogenase antibody (GAD)-positive subgroup, where at least one CD marker was positive in 83.6% subjects. In the GAD-negative subgroup atTG IgA was 8.73 IU/ml and at least one CD marker was positive in 57.4% subjects. atTG in IgA and IgG classes could be recommended as valuable serological markers of CD in the differential diagnosis of malabsorption as well as in various screening algorithms. ELISA determination of atTG with human antigen could increase the specificity, especially in patients with other autoimmune diseases.

We evaluated serum total adenosine deaminase, its isoenzymes adenosine deaminase-1 and adenosine deaminase-2, and cytidine deaminase activities in 24 patients with active systemic lupus erythematosus, and in 26 healthy control subjects, and found the means±SD values to be 21.38±5.96 IU/l, 3.74±2.12 IU/l, 17.72±5.02 IU/l and 17.89±4.62 IU/l, respectively in the patients, and 14.97±4.71 IU/l, 4.01±1.35 IU/l, 10.91±3.91 IU/l and 7.39±3.97 IU/l, respectively in the control subjects. When compared to the healthy controls, serum total adenosine deaminase, adenosine deaminase-2 and cytidine deaminase levels were significantly higher (p<0.001) in systemic lupus erythematosus patients, but the decrease of adenosine deaminase-1 level was not statistically significant (p>0.05). The increased adenosine deaminase-2 may be of macrophage origin. It closely correlated with clinical signs of active systemic lupus erythematosus. The membranes of polymorphonuclear neutrophils may be damaged, and cytidine deaminase may be released into serum. In conclusion, serum total adenosine deaminase, adenosine deaminase-2 and cytidine deaminase activities may serve as useful indicators for evaluating disease activity in patients with active systemic lupus erythematosus.

Oral N-acetylcysteine supplementation in nine young healthy females induced a quick and highly significant decrease in plasma homocysteine levels and an increase in whole blood concentration of the antioxidant glutathione. N -acetylcysteine impresses as an efficient drug in lowering homocysteine concentration and might be beneficial for individuals with hyperhomocysteinemia who are at increased risk of cardiovascular disease.

One can compare the difference between two sequential values with the biological variation. Biological variation is a measure of the random disturbances of an analyte's value, measured at different times. When the difference > Z √2̅ SD BV then the difference is due to an underlying disease process or physiologic change. A Z value of 1.96 yields a 95% confidence limit. When using multiple sequential values or time periods exceeding that for the empirically derived biological variance, a random walk model allows one to estimate the spread of the variance. For a difference, (Δ) to be significant, Δ> Z √2̅ n̅SD BV , where n is the ratio of time reflecting the longer time period divided by the shorter time period. Not all variances grow to this degree over time, because restoring forces diminish the extent of random disturbances. The relationship between a biological variance measured over a longer time period to the one measured over a shorter period can be expressed in terms of this restoring force as where n is the ratio of time periods. One can calculate Ф using this formula and a spreadsheet. From Ф one can calculate the biological variance for any time period, within experimental limits, and compare the difference in sequential values with it. Test intervals can be calculated based on these biological variances.

Uric acid, which is the final product of purine nucleoside metabolism, is a strong peroxynitrite scavenger. Several studies report on lower serum uric acid levels in multiple sclerosis. In this study, we investigated serum uric acid levels before and after high-dose methylprednisolone treatment (intravenous 1 g/day/5 days) in multiple sclerosis patients. Blood samples from 25 definite multiple sclerosis patients (11 male and 14 female) before and after methylprednisolone treatment (days 0, 6 and 30) and from 20 healthy donors (9 male and 11 female) were analyzed. Serum uric acid levels were measured using a quantitative enzymatic assay (Elitech diagnostics, Sees, France) according to the manufacturer's protocol, and the results were standardized using a commercial uric acid standard solution. We observed significantly increased serum uric acid levels 1 day after the termination of the therapy (day 6). These differences were sustained for 30 days after starting treatment (during remission period). Mean serum uric acid levels were significantly higher in the control group. These results suggest that increasing the uric acid concentration may represent one of the possible mechanisms of action of methylprednisolone in multiple sclerosis.

The comparison of analytical results calls for validation of the assays used, i.e. for the documentation of accuracy (trueness and precision), linearity and specificity. In addition, there is a growing demand for evaluation and documentation of traceability and uncertainty of analytical results. However, models for establishing the traceability and uncertainty of immunoassay results are lacking. Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for determination of the human cytokines interleukin-4 (IL-4), interleukin-5 (IL-5), interferon-γ (IFN-γ) and tumor necrosis factor-α(TNF-α. The accuracy of each of the assays was evaluated in the ranges of 1–15 μg/l (IL-4), 0.001–1 μg/l (IL-5), 0.5–2.5 μg/l (IFN-γ) and 0.14–2.2 μg/l (TNF Other evaluated performance characteristics were the limit of detection (LOD), immunological specificity, and robustness. Traceability was ensured by the use of World Health Organization International Standards (WHO IS). An uncertainty budget, which combined the contribution from all known uncertainty components, was established for each cytokine ELISA. The between-run relative analytical standard deviation (RsD A ) of the assessed ELISAs was found to be in the range of 11–18%, except for IL-5 where RSD A increased at decreasing concentrations. The LOD was 0.12 μg/l, 0.0077 μg/l, 0.0069 μg/l and 0.0063 μg/l for IL-4, IL-5, IFN-γ and TNF-α respectively. Traceability to the WHO IS was established for each of the cytokines. The combined relative standard uncertainty (u result /C result ) was 28%, 22–62%, 28% and 24% for the IL-4, IL-5, IFN-γ and TNF-α results, respectively. The major contributions to uncertainty came from the relative analytical standard deviation and from the uncertainty of the mass concentration of the WHO IS. The uncertainty of the WHO IS was not stated in the accompanying certificate and was evaluated by other means. The largest sources of uncertainty were located outside our laboratory. This means that the possibilities to improve the reliability of the results produced by the ELISAs are very limited. The task of evaluating measurement uncertainty would be much easier if producers of international reference standards reported the uncertainty of the value of standards. The model for evaluating uncertainty presented in this paper is applicable to other types of assays and to most analytical methods.

Many laboratories rely on dedicated nephelometers or turbidimeters and commercial reagent kits for the evaluation of serum proteins. However, with growing emphasis on cost containment, laboratories are forced to seek additional operational efficiencies by capitalizing on the use of existing analyzers whenever possible. In the present paper we describe the development of immunoturbidimetric assays for routine analysis of 14 human serum proteins (α 1 -antitrypsin, α 2 -macroglobulin, albumin, apolipoproteins AI and B, complement components 3 and 4, haptoglobin, immunoglobulins A, G, and M, orosomucoid, prealbumin, and transferrin) on the Hitachi 912™, a general chemistry analyzer. With this system, we obtained excellent precision at levels corresponding to low, normal, and high physiologic concentrations of each protein (within-run imprecision CVs ≤3.4%, total imprecision CVs ≤4.1%). Linearity for each method was within 5% of the expected value throughout the calibration range, and method comparisons with either the Roche turbidimetric or Dade Behring nephelometric assays were in good agreement (r >0.97). We observed no significant interference from bilirubin (up to 718 μmol/l), hemoglobin (up to 8 g/l), triglyceride (up to 14.7 mmol/l) or rheumatoid factor (up to 4140 IU/ml). Calibration for the 14 protein assays was stable for at least 7 days and onboard refrigerated reagents were stable for at least 3 months. The instrument's automated sample re-run feature minimized sample handling and helped to conserve specimens. In conclusion, the newly developed assays on the Hitachi 912™ offer high throughput (>250 tests per hour) without the associated cost of a dedicated instrument for protein assays.

Soluble transferrin receptor (sTfR) is reported to be a reliable marker for the diagnosis of iron deficiency, especially when iron metabolism is influenced by inflammatory disorders such as infection, chronic inflammation and cancer-related anemia. In the present multicenter study the analytical performance of a recently introduced, latex-enhanced immunoturbidimetric assay for the determination of soluble transferrin receptor (Tina quant ® [a] sTfR, Roche Diagnostics) on different fully mechanized analyzers such as Hitachi 917 and 911, and Cobas Integra 400 and 700 was evaluated. Within-run and between-run imprecision showed good results (CV<5% and <7%, respectively). The assay was found to be linear over a wide measuring range (0.4–35 mg/l). Endogenous substances did not interfere with the test results. Comparison of serum sTfR concentrations with those of heparinized plasma revealed good correlation (r>0.976). Method comparison with an existing fully mechanized method as well as with ELISA tests for sTfR showed very good correlation (r>0.987). Because of the lack of international standardization the results differed from each other up to 2.5-fold. The 95% of serum levels in healthy individuals ranged from 1.9 to 4.4 mg/l (n=427). However, the reference ranges should be reported in a sex-dependent manner, as 2.2–5.0 mg/l for men (n=211) and as 1.9–4.4 mg/l for premenopausal (n=216) and postmenopausal (n=45) women. The Tina quant ® [a] sTfR assay enables the precise, accurate, rapid and convenient determination of sTfR concentrations for routine clinical chemistry purposes.