Volume 47, Issue 4

Reduced insulin sensitivity plays a role in the early pathogenesis of type 2 diabetes, and defects in insulin secretion by pancreatic β-cells are instrumental in hyperglycemic progression. There is strong evidence that genetic factors play an important role in both of these components. Several of the single nucleotide polymorphisms (SNPs) of genes associated with an increased risk of type 2 diabetes are hypothesized to influence β-cell function. The aim of the present study was to describe the function of the latter genes, to analyze the implications of the SNP positions within or near these genes, and to evaluate the suggested primary role of pancreatic β-cells in the etiology of type 2 diabetes. Clin Chem Lab Med 2009;47:383–6.

Background : Recently, the role of the FTO (fat mass and obesity associated) gene in obesity development was described in Western European, but not in Oceanic, cohorts. The objective of this study was to test the hypothesis that the FTO single nucleotide polymorphism (SNP) is associated with body mass index (BMI) in the Slavic population and to analyze if there could be sex-specific effects of the SNP on BMI, waist-to-hip ratio (WHR), and lipid parameters. Methods : We analyzed three large population-based samples comprising the post-MONICA study (1191 males, 1368 females) and the 3PMFs study (908 females). Results : FTO rs17817449 SNP was related to BMI in males (p=0.014). In the females from both the post-MONICA and the 3PMFs study, FTO had no effect on BMI. Sub-analysis of females from the 3PMFs study demonstrated that FTO had an effect on BMI in postmenopausal females (p=0.035) but not in premenopausal females (follicle-stimulating hormone <40 U/L was used as marker of premenopausal status). WHR and lipid parameters were not associated with FTO in any of the analyzed groups. Conclusions : These results suggest that the effect of FTO SNP rs17817449 may be, in some populations at least, restricted to males and postmenopausal females. Clin Chem Lab Med 2009;47:387–90.

Background : The estrogen receptor α gene ( ESR1 ) seems to be a potential candidate in altering risk for preeclampsia due to important estrogenic biological effects. Despite its previously reported association with severe preeclampsia in combined genotype in one selected ethnic group, further studies in other populations are required. The objective of this study was to investigate the relationship of the gene polymorphisms of the estrogen receptor α with preeclampsia in a southwest Chinese population. Methods : We genotyped two polymorphisms in the ESR1 gene ( Pvu II and Xba I) by polymerase chain reaction-restriction fragment length polymorphism analysis in a Chinese population. Results : In the case-control study which included 204 preeclampsia case subjects and 236 normal control subjects, the frequencies of allele T for the Pvu II site and allele G for the Xba I site in the patients (41.67% and 23.53%, respectively) were similar to those observed in controls (43.01% and 19.70%, respectively). Furthermore, the combined genotype analysis of ESR1 Pvu II and Xba I polymorphisms showed no increased risk of either severe or total preeclampsia in our study populations. However, it showed that the CC homozygotes and CT heterozygotes in control pregnant women had higher systolic blood pressure levels than TT homozygotes for the Pvu II site after adjustment for age and body mass index (p<0.05). Conclusions : Our work does not provide evidence in favor of ESR1 Pvu II and Xba I being associated with preeclampsia but with increased blood pressure levels in normal pregnant women, in a southwest Chinese population. Clin Chem Lab Med 2009;47:391–7.

Background : Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), produced by endotoxin-activated Kupffer cells, play a key role in the pathogenesis of alcoholic liver cirrhosis (ALC). Alleles TNFA –238A, IL1B –31T and variant IL1RN *2 of repeat polymorphism in the gene encoding the IL-1 receptor antagonist increase production of TNF-α and IL-1β, respectively. Alleles CD14 –159T, TLR4 c.896G and TLR4 c.1196T modify activation of Kupffer cells by endotoxin. We confirmed the published associations between these common variants and genetic predisposition to ALC by means of a large case-control association study conducted on two Central European populations. Methods : The study population comprised a Czech sample of 198 ALC patients and 370 controls (MONICA project), and a German sample of 173 ALC patients and 331 controls (KORA-Augsburg), and 109 heavy drinkers without liver disease. Results : Single locus analysis revealed no significant difference between patients and controls in all tested loci. Diplotype [ IL1RN *2/*2; IL1B –31T+] was associated with increased risk of ALC in the pilot study, but not in the validation samples. Conclusions : Although cytokine mediated immune reactions play a role in the pathogenesis of ALC, hereditary susceptibility caused by variants in the corresponding genes is low in Central European populations. Clin Chem Lab Med 2009;47:398–404.

Background : Tuberculosis (TB) remains a major health problem affecting millions of people worldwide. One-third of the world's population is infected with Mycobacterium tuberculosis , the etiologic agent of TB. A simple and rapid method to diagnose TB is urgently needed to be developed. The procedure of systematic evolution of ligands by exponential enrichment (SELEX) is a method in which single-stranded oligonucleotides (called aptamers) are selected from a wide variety of sequences, based on their interaction with a target molecule. Aptamers have been used in numerous investigations as therapeutic or diagnostic tools. Methods : In this study, we apply a SELEX method to develop aptamers against MPT64 protein from M. tuberculosis . On this basis, a sandwich assay scheme with the complex of aptamer-MPT64 was designed and tested the feasibility of detecting M. tuberculosis by detecting MPT64 protein levels in the culture filtrates of 77 samples including M. tuberculosis and other Mycobacterium species. Results : There was a highly significant difference (p<0.01) between group A (non-TB Mycobacterium , bacille Calmette-Guérin) and group B ( M. tuberculosis , M. bovis ), when they were diagnosed with the sandwich assay scheme based on aptamer-protein complex to detect MPT64 protein levels in the culture filtrates of samples. When the cut-off point was at the optical density value of 0.58 (95%=0.764–0.946; Z=6.130, p=0.0001), the sandwich assay scheme based on aptamer-protein complex had a high sensitivity (negative ration, 24/27, 86.3%) and specificity (positive ration, 46/52, 88.5%). Conclusions : Aptamer of MPT64 as a new detection tool, to a certain extent, is feasible to diagnose Mycobacterium tuberculosis . Clin Chem Lab Med 2009;47:405–11.

Background : Hepatocellular carcinoma (HCC) is one of the most frequent malignant neoplasms worldwide. Genetic polymorphism has been reported as a predictive factor related to a higher risk for HCC. Because the stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, have been reported to play important roles in tumor cell proliferation, angiogenesis, and metastasis of HCC, the aim of this study was to estimate the relationship between SDF-1 and CXCR4 gene variants to HCC risk and clinicopathological status. Methods : Polymerase chain reaction-restriction fragment length polymorphism was used to measure SDF-1 (rs1801157) and CXCR4 (rs2228014) gene polymorphisms in 311 healthy controls and 102 patients with HCC. Results : Compared to controls, individuals with at least one A allele had a higher risk of 1.57-fold (95% CI: 1.00–2.47) to induce HCC and had a risk of 2.81-fold (95% CI: 1.04–7.58) to develop a status of stage III or stage IV disease, after being adjusted for other confounders. However, there was no significant association between CXCR4 gene polymorphism and either HCC risk or pathological status. Additionally, both gene polymorphisms were not associated with the serum expression of liver-related clinical pathological markers. Conclusions : SDF-1–3 ′A gene polymorphism could be considered as a factor related to an increased susceptibility to the risk and pathological development of HCC. Clin Chem Lab Med 2009;47:412–8.

Background : The study of lineage markers by real-time quantitative polymerase chain reaction (RT-qPCR) at diagnosis enables differentiation between acute myeloblastic leukemia, B- or T-lineage acute lymphoblastic leukemia, without cell sorting. Our objective was to assess the relationship between protein expression and the amount of lineage marker mRNA in acute leukemia samples and to determine whether four lineage markers could be used to differentiate between normal and acute leukemia bone marrow (BM) without cell sorting. Methods : Quantification of the mRNA of CD19, CD79a, CD3e, and myeloperoxidase was performed by RT-qPCR on 130 acute leukemia BM samples at diagnosis and on 20 BM samples from healthy donors, without cell sorting. Immunophenotyping of leukemia samples was performed after manual gating around the blastic population. Results : Reference values for the four lineage markers were established by RT-qPCR for normal BM. The mRNA expression levels of these four lineage markers allowed the distinction between normal samples and 100% of acute leukemia samples. Conclusions : With 92% congruence for protein expression and amount of mRNA in acute leukemias, these four lineage markers, essential for diagnosis and subclassification of acute leukemias by flow cytometry, also represent excellent candidate genes when using RT-qPCR technology as a diagnostic tool for molecular cancer class prediction. Clin Chem Lab Med 2009;47:419–26.

Background : The eukaryotic release factor 3 (eRF3) has been shown to affect both tubulin and actin cytoskeleton, suggesting a role in cytoskeleton assembly, mitotic spindle formation and chromosome segregation. Also, direct interactions between eRF3 and subunits of the cytosolic chaperonin CCT have been described. Moreover, both eRF3a and CCT subunits have been described to be up-regulated in cancer tissues. Our aim was to evaluate the hypothesis that eRF3 expression levels are correlated with the expression of genes encoding proteins involved in the tubulin folding pathways. Methods : Relative expression levels of eRF1 , eRF3a/GSPT1 , PFDN4 , CCT2 , CCT4 , and TBCA genes in tumour samples relative to their adjacent normal tissues were investigated using real time-polymerase chain reaction in 20 gastric cancer patients. Results : The expression levels of eRF3a/GSPT1 were not correlated with the expression levels of the other genes studied. However, significant correlations were detected between the other genes, both within intestinal and diffuse type tumours. Conclusions : eRF3a/GSPT1 expression at the mRNA level is independent from both cell translation rates and from the expression of the genes involved in tubulin-folding pathways. The differences in the patterns of expression of the genes studied support the hypothesis of genetically independent pathways in the origin of intestinal and diffuse type gastric tumours. Clin Chem Lab Med 2009;47:427–31.

Background : Serum paraoxonase (PON1) exerts antiatherogenic effects. Novel PON1 enzymatic tests have been recently developed: 5-thiobutyl butyrolactone (TBBL) estimates PON1 lactonase activity, whereas 7-O-diethylphosphoryl-3-cyano-4-methyl-7-hydroxycoumarin (DEPCyMC) is considered a surrogate marker of PON1 concentration. The TBBL to DEPCyMC ratio provides the normalized lactonase activity (NLA), which may reflect the degree of PON1 lactonase catalytic stimulation. The aim of this study was to evaluate for the first time TBBLase and DEPCyMCase activity in patients with coronary artery disease (CAD). Methods : An angiography-based case-control study was conducted, including 300 sex- and age-matched subjects [100 CAD-free, 100 CAD without myocardial infarction (MI) and 100 CAD with MI]. Results : A low DEPCyMCase activity (lowest vs. highest tertile: OR 2.96, 95% CI 1.18–7.43) and a high NLA (highest vs. lowest tertile: OR 3.25, 95% CI 1.28–8.26) were both associated with CAD, independent of classical atherosclerosis risk factors, lipid-lowering therapy and PON1 genotype. Total TBBLase activity was, however, not different in CAD compared to CAD-free subjects. Conclusions : Novel PON1 activity assays may be associated with CAD. In this study, CAD patients had low DEPCyMCase activity, a possible marker of low PON1 concentration, but showed a high stimulation of PON1 lactonase activity. Clin Chem Lab Med 2009;47:432–40.

Background : The aim of this study was to evaluate the erythrocyte carbonic anhydrase (CA) activity in patients with hyperventilation syndrome. Methods : A total of 71 patients with hyperventilation syndrome and 60 controls were recruited in this study for measurement of the erythrocyte CA activity. CA activity was analyzed from erythrocyte using CA esterase activity analysis. Results : The erythrocyte CA activity was significantly elevated in the patients with hyperventilation syndrome compared to controls (31.07±1.37 U/g Hb vs. 24.67±0.99 U/g Hb, p=0.003). The standardized β value and significant R 2 value of total CA activity for prediction of hyperventilation was 0.155 (p=0.016) and 0.024, respectively. Moreover, a significant negative correlation was found between total CA activity and partial pressure of carbon dioxide (PaCO 2 ) (r=−0.185, p=0.03, n=131). Furthermore, the adjusted odds ratios for patients with hyperventilation were 11.6 (95% CI: 4.8–27.9) and 51.0 (95% CI: 5.8–445.2) for individuals with either PaCO 2 ≤28.1 mm Hg or CA activity ≥29.71 U/g hemoglobin (Hb), and for individuals with both PaCO 2 ≤28.1 mm Hg and CA activity ≥29.71 U/g Hb, respectively, compared to individuals with both PaCO 2 >28.1 mm Hg and CA activity <29.71 U/g Hb. Conclusions : Erythrocyte CA activity was significantly elevated in patients with hyperventilation. Detection of erythrocyte CA activity may provide a potential explanatory parameter for the prediction of hyperventilation syndrome. Clin Chem Lab Med 2009;47:441–5.

Background : An important role in the acceleration of vascular disease has been previously suggested for advanced glycation end products. N ε -(carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined non-enzymatic glycation and glycoxidation reactions. CML reacts with the receptor of advanced glycation end products inducing impairment of endothelium dependent relaxation and is a marker of oxidative stress. Methods : A total of 40 patients with acute myocardial infarction (17 patients with ST-elevation myocardial infarction, 23 patients with non-ST-elevation myocardial infarction) and 40 patients with stable coronary artery disease were included consecutively in this study. During coronary angiography, peripheral venous blood sample was taken for measuring CML. Results : Serum levels of CML were significantly increased in patients with acute myocardial infarction [17.9±10.7 vs. 6.6±3.1 arbitrary units (AU)/mg protein, p<0.001]. A cut-off value of CML>9.5 AU/mg protein was associated with an odds ratio of acute myocardial infarction of 39.7 [95% confidence interval (CI): 11.1–142, p<0.001], a sensitivity of 0.85 (95% CI: 0.70–0.94) and a specificity of 0.88 (95% CI: 0.73–0.96). Conclusions : CML levels are significantly elevated in patients presenting with acute myocardial infarction. These results suggest the involvement of endothelial dysfunction (through receptor interaction) and oxidative stress in acute myocardial infarction. Clin Chem Lab Med 2009;47:446–51.

Background : Recently, attention has been focused on the effect of lipoprotein(a) [Lp(a)] on tumors because of its possible role in development of tumor angiogenesis. The aim of this study was to investigate Lp(a) serum levels in patients with lung cancer and its association with the stages of disease. Methods : Fasting venous blood samples were collected from 418 untreated male patients with stages I–IV lung carcinoma and were analyzed for Lp(a). The results were compared with the data from 65 healthy male controls. Results : Lp(a) levels were elevated (median 157 mg/L, range 16–1497 mg/L) in patients with lung carcinoma compared to control subjects (median 110 mg/L, range 35–706 mg/L) (p=0.004). Subgroup analysis showed that patients with stages II–IV disease had significantly higher Lp(a) concentrations than did healthy controls (p<0.05). There was an independently positive correlation between tumor stage and Lp(a) levels among patients with stages I–III (r=0.162, p=0.006). However, there was a decrease in Lp(a) in stage IV compared to stage III patients (p=0.03). Conclusions : There is a significant association between Lp(a) and the presence and stage of lung cancer. Additional investigations with a larger number of lung cancer patients are needed to confirm these findings. Clin Chem Lab Med 2009;47:452–7.

Background : Carbamazepine is a first-choice antiepileptic drug for the treatment of simple and complex partial seizures. The use of an established therapeutic range for carbamazepine concentration is limited by the presence of carbamazepine-10,11-epoxide, its active metabolite that significantly contributes to the efficacy and toxicity and is not routinely measured and accounted for. This article describes the development of a HPLC method for determination of carbamazepine and carbamazepine-10,11-epoxide in serum, and compares it with chemiluminescence immunoassay to evaluate the importance of considering the active metabolite in therapeutic strategies. Methods : The procedure involves protein precipitation, separation on a reverse-phase column and ultraviolet detection. The analytical procedure proved to be sensitive, selective, precise, accurate and linear (regression coefficients >0.999) in the range of 0.5–25.0 μg/mL and 0.1–10.0 μg/mL for quantification of carbamazepine and carbamazepine-10,11-epoxide, respectively. For the comparison between methods, serum samples of 75 patients using the medication were evaluated. Results : The Pearson correlation coefficient showed that the carbamazepine concentrations measured by HPLC are significantly higher than those obtained by immunoassay (mean difference of 1.07 μg/mL, 95% limits of agreement from –0.65 to 2.80 μg/mL). Conclusions : This difference may be decisive for the therapy. In some cases, this may affect the individual dosage adjustment and subsequent treatment. Clin Chem Lab Med 2009;47:458–63.

Background : Pregnenolone sulfate (PregS) is known as a steroid conjugate positively modulating N-methyl-D-aspartate receptors on neuronal membranes. These receptors are responsible for permeability of calcium channels and activation of neuronal function. Neuroactivating effect of PregS is also exerted via non-competitive negative modulation of GABA A receptors regulating the chloride influx. Recently, a penetrability of blood-brain barrier for PregS was found in rat, but some experiments in agreement with this finding were reported even earlier. It is known that circulating levels of PregS in human are relatively high depending primarily on age and adrenal activity. Methods : Concerning the neuromodulating effect of PregS, we recently evaluated age relationships of PregS in both sexes using polynomial regression models known to bring about the problems of multicollinearity, i.e., strong correlations among independent variables. Several criteria for the selection of suitable bias are demonstrated. Biased estimators based on the generalized principal component regression (GPCR) method avoiding multicollinearity problems are described. Results : Significant differences were found between men and women in the course of the age dependence of PregS. In women, a significant maximum was found around the 30th year followed by a rapid decline, while the maximum in men was achieved almost 10 years earlier and changes were minor up to the 60th year. The investigation of gender differences and age dependencies in PregS could be of interest given its well-known neurostimulating effect, relatively high serum concentration, and the probable partial permeability of the blood-brain barrier for the steroid conjugate. Conclusions : GPCR in combination with the MEP (mean quadric error of prediction) criterion is extremely useful and appealing for constructing biased models. It can also be used for achieving such estimates with regard to keeping the model course corresponding to the data trend, especially in polynomial type regression models. Clin Chem Lab Med 2009;47:464–70.

Background : The aim of this study was to evaluate the distribution of pentraxin 3 (PTX3) values in healthy subjects and to characterize its relationship with gender, age, body mass index (BMI), lipid profile, and blood sugar levels. Methods : A Japanese population of 1749 healthy subjects (818 men and 931 women) with a mean (SD) age of 59.6 (11.4) years (range 37–87 years) were examined. Results : Plasma PTX3 levels (PTX3 data are expressed as the geometric mean and confidence intervals) were i) significantly lower in men than in women (1.87 [1.81, 1.94] ng/mL vs. 2.12 [2.05, 2.19] ng/mL, p<0.0001), ii) significantly higher in the high age group (men, lowest quartile 1.62 [1.50, 1.74] ng/mL vs. highest quartile 2.14 [2.02, 2.27] ng/mL, p<0.001; women, lowest quartile 2.05 [1.92, 2.18] ng/mL vs. highest quartile 2.23 [2.02, 2.46] ng/mL, p<0.05), iii) inversely correlated with triglycerides (r=−0.19 in men and r=−0.18 in women, p<0.00001), and BMI (r=−0.16 in men and r=−0.24 in women, p<0.00001), and iv) lower in subjects with metabolic syndrome (MetS) than in the absence of MetS (1.82 [1.70, 1.95] ng/mL vs. 2.11 [2.06, 2.16] ng/mL, p=0.021). Conclusions : We defined the normal range of plasma PTX3 in healthy Japanese subjects, and also showed the relationship between plasma PTX3 levels and established coronary risk factors, including MetS. PTX3 could be an ideal biomarker because it is a marker relatively independent from established coronary risk factors. Clin Chem Lab Med 2009;47:471–7.

Background : Since its first publication in 2000, the BONN-Risk-Index (BRI) has been successfully used to determine the calcium oxalate (CaOx) crystallization risk from urine samples. To date, a BRI-measuring device, the “Urolizer ® ”, has been developed, operating automatically and requiring only a minimum of preparation. Two major objectives were pursued: determination of Urolizer ® precision, and determination of the influence of 24-h urine storage at moderate temperatures on BRI. Methods : 24-h urine samples from 52 CaOx stone-formers were collected. A total of 37 urine samples were used for the investigation of Urolizer ® precision by performing six independent BRI determinations in series. In total, 30 samples were taken for additional investigation of urine storability. Each sample was measured thrice: directly after collection, after 24-h storage at T=21°C, and after 24-h cooling at T=4°C. Outcomes were statistically tested for identity with regard to the immediately obtained results. Results : Repeat measurements for evaluation of Urolizer ® precision revealed statistical identity of data (p<0.05). 24-h storage of urine at both tested temperatures did not significantly affect BRI (p<0.05). Conclusions : The pilot-run Urolizer ® shows high analytical reliability. The innovative analysis device may be especially suited for urologists specializing in urolithiasis treatment. The possibility for urine storage at moderate temperatures without loss of analysis quality further demonstrates the applicability of the BRI method. Clin Chem Lab Med 2009;47:478–82.

Background : In the course of type 2 diabetes mellitus, insulin resistance has a severe impact on endothelial function leading to decreased synthesis of nitric oxide (NO). Postprandial hyperglycemia leads to the generation of reactive oxygen species, which counteracts the beneficial NO effects. NO and superoxide combine very fast in solution to form peroxynitrite, which is a potent protein-oxidizing agent. The peroxynitrite concentrations can be indirectly monitored by the detection of nitrotyrosine residues in proteins, reflecting the extent of damage caused by oxidative stress. Methods : Four commercially available nitrotyrosine-specific immunoassays were evaluated by parallel measurement of nitrotyrosine in 224 serum samples derived from 16 patients with type 2 diabetes and 12 healthy controls (13 male and 15 female, age: 33±11 years) following a standardized meal. Results : The available ELISA tests were not applicable for nitrotyrosine determination in human plasma samples due to technical issues and implausible results. However, a competitive luminescence assay was able to provide sufficient sensitivity and lead to clinically meaningful results in our test samples. Conclusions : All three ELISA methods were disqualified and conclusions previously derived from clinical experiments using these tests should be carefully reconsidered or reconfirmed. In the absence of a liquid tandem chromatography-mass spectrometry reference method, the luminescence test appears to be the method of choice for determination of nitrotyrosine in human plasma. Clin Chem Lab Med 2009;47:483–8.

Background : We demonstrated that lyophilised EQA/PT control materials, under certain circumstances, provide equal information about bias values as pools of native patient sera, and that in some cases, long-term reliable work with such materials is possible. Methods : Bias values, estimated from results of routine surveys of EQA SEKK (Czech Republic) programmes for eight basic blood serum analytes using lyophilised control materials, were compared with bias values reached in the CAP (USA) programme where pools of native and lyophilised patient sera were used. Results : Results for the components Na, K, Mg, Cl, P, urea, glucose, and uric acid were assessed. No significant differences were found between the bias values estimated by CAP using native sera, and those estimated by SEKK using lyophilised sera. Conclusions : It can be concluded that well-prepared lyophilised control materials may show the required commutability level. If so, they constitute a practical and cost-efficient alternative to native or fresh frozen sera when assessing bias. Clin Chem Lab Med 2009;47:489–90.