Volume 44, Issue 3

A constant extracellular Ca 2+ concentration is required for numerous physiological functions at tissue and cellular levels. This suggests that minor changes in Ca 2+ will be corrected by appropriate homeostatic systems. The system regulating Ca 2+ homeostasis involves several organs and hormones. The former are mainly the kidneys, skeleton, intestine and the parathyroid glands. The latter comprise, amongst others, the parathyroid hormone, vitamin D and calcitonin. Progress has recently been made in the identification and characterisation of Ca 2+ transport proteins CaT1 and ECaC and this has provided new insights into the molecular mechanisms of Ca 2+ transport in cells. The G-protein coupled calcium-sensing receptor, responsible for the exquisite ability of the parathyroid gland to respond to small changes in serum Ca 2+ concentration was discovered about a decade ago. Research has focussed on the molecular mechanisms determining the serum levels of 1,25(OH) 2 D 3 , and on the transcriptional activity of the vitamin D receptor. The aim of recent work has been to elucidate the mechanisms and the intracellular signalling pathways by which parathyroid hormone, vitamin D and calcitonin affect Ca 2+ homeostasis. This article summarises recent advances in the understanding and the molecular basis of physiological Ca 2+ homeostasis.

Background: Because apolipoprotein E (apoE) and lipopoprotein lipase (LPL) polymorphisms interact with each other and with other factors to affect lipid metabolism, we sought to determine their separate and combined effects in association with ischemic vascular disease. Methods: We performed a case-control study of 816 subjects: 246 acute ischemic stroke patients, 234 acute myocardial infarction patients, and 336 controls. APOE exon 4 and LPL S447X genotypes were determined. Results: APOE ɛ2 and ɛ4 homozygotes were increased in stroke (4.5% vs. 1.0%, p=0.008), while in myocardial infarction the ɛ4 allele was increased (12.6% vs. 9.5%, p=0.006) but ɛ2 was decreased (3.7% vs. 12.1%, p=0.000006). For subjects with either APOE ɛ2 or ɛ4 alleles, LPL X alleles were increased in vascular disease (OR=2.2, p=0.01). LPL X alleles displayed opposite tendencies toward association with disease when subjects were divided by sex, smoking, or APOE genotype. Meta-analysis and regression analysis of previous studies supported the sex and smoking dichotomies. Conclusion: This is the first report of an association of vascular disease with an interaction of APOE exon 4 and LPL S447X genotypes. Therefore, APOE genotypes and LPL S447X interactions with apoE, sex, and smoking may affect the risk of myocardial infarction and ischemic stroke.

Background : Polymorphisms of the angiotensin II type 1 receptor ( AGTR1 ) gene are associated with essential hypertension and cardiovascular disease, but the correlation between AGTR1 A1166C polymorphism and carotid intima-media thickness (IMT) remains unclear. We sought to demonstrate correlation between AGTR1 gene polymorphism and carotid atherosclerosis in a Chinese population. Method : A total of 150 patients diagnosed with essential hypertension were included in this study. The AGTR1 A1166C polymorphism was detected by restriction analysis of the polymerase chain reaction product with Dde l digestion. Carotid IMT was measured by B-mode ultrasonography. Results : The AC genotype frequency and the C1166 allele frequency of the AGTR1 gene in essential hypertensive patients were significantly higher than in controls (22.00% vs. 6.00% for AC , p<0.01; 18.67% vs. 8.00% for the C allele, p<0.05). Hypertensive subjects with the AC genotype had increased carotid artery IMT and IMT/D (common carotid artery diameter) ratio compared with the AA genotype (IMT 1.14±0.39 vs. 0.88±0.16, p<0.05; IMT/D 14.08±2.88 vs. 10.51±1.94, p<0.01). Conclusion : These results suggest that the AGTR1 A1166C polymorphism is associated with essential hypertension and carotid atherosclerosis in a Chinese population.

Background : Pharmacogenetic testing for drug-metabolizing enzymes is not yet widely used in clinical practice. Methods : In an attempt to facilitate the application of this procedure, we have compared two real-time PCR-based methods, the TaqMan ® and the LightCycler™ for the pharmacogenetic evaluation of CYP2C9*2/*3 polymorphisms. Results and Conclusion : Both procedures are suitable for pharmacogenetic studies. The TaqMan procedure was less expensive in terms of cost per sample, but the TaqMan apparatus is more expensive than the LightCycler apparatus.

Background : Cystatin C (CST3), a strong inhibitor of cysteine proteinases, is freely filtered by the kidney glomerulus and is reabsorbed by the tubules, where it is almost totally catabolized, with the remainder then eliminated in urine. In tubular diseases, it seems sensible to postulate that CST3 degradation would be reduced and consequently an increase in its urinary elimination would be observed. Methods : We report here the development of an automatic quantitative assay to measure CST3 concentrations in urine using a Behring N-Latex Cystatin C kit on a BNII laser nephelometer. We tested its clinical relevance on several kidney disease patients. Results : This assay is sensitive (limit of detection 0.008mg/L) and precise (within- and between-day CVs <4%). Reference values for freshly collected urine samples range from 0.03 to 0.18mg/L. Mean urine CST3 concentrations obtained from 52 patients with kidney tubular disease (4.31±3.85mg/L) were significantly higher than those for 60 controls (0.096±0.044mg/L; p<0.0001) and 47 glomerular disease patients (0.106±0.133mg/L; p<0.0001). Conclusion : Increased urinary CST3 concentrations allow the accurate detection of tubular dysfunction among pure and mixed nephropathies. Because of its ability to be processed on automated clinical chemistry analyzers, this assay could easily be used as an adjunct to the standard panel used to screen kidney pathologies, even in emergency situations.

Background : Growing evidence in the literature shows the clinical importance of the calprotectin assay in faeces, especially for the differential diagnosis and monitoring of patients with inflammatory bowel diseases. Methods : We developed a time-resolved fluorimetric immunoassay for calprotectin to extend the limited measuring range of the commercially available ELISA method currently used in our laboratory. Together with the introduction of a non-enzymatic label, this new method offers the advantages of better precision and higher sensitivity. Results : The new assay shows a dynamic measuring range extended by a factor four, which reduces the number of samples with concentrations outside the measuring range from 30% to only 4%. The value of the assay was confirmed in various patient groups suffering from active and inactive gastrointestinal diseases. We suggest that the frequent coincidence of a high calprotectin concentration with intestinal blood loss is not the consequence of mere blood loss, but can be ascribed to neutrophil infiltration and subsequent shedding into the intestinal lumen as a result of intestinal inflammation or malignancy. Conclusion : We expect that in the near future faecal calprotectin will be used as a non-invasive routine diagnostic marker and an effective laboratory parameter to monitor patients with inflammatory bowel disease.

Background : In this study antiretroviral drugs of the classes protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were quantified for the first time directly in patient plasma samples by means of an automated and validated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) method using the Symbiosis Pharma ® system (Spark Holland) for XLC coupled to an API 2000 for MS/MS analysis. Methods : The PI drugs amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, and atazanavir, and the NNRTI drugs nevirapine and efavirenz in real patient samples were analysed in a 25-μL sample volume, which was only diluted with 200μL of H 2 O (containing 500ng/mL of the internal standard reserpine) to minimise the matrix concentration and to add the internal standard. No additional tedious and time-consuming sample preparation steps such as protein precipitation, centrifugation, and pipetting were per-formed for sample clean-up. Results : The high-throughput method developed allowed the simultaneous analysis of two samples (first analysis 6.6min, subsequent analyses 3.3min between injections) and has been validated in terms of the limit of detection (LOD, 2–70ng/mL), lower limit of quantification (LLOQ, 78–156ng/mL), linearity (R 2 , 0.9971–0.9989), linear concentration range (from LLOQ to 10,000ng/mL), intra- and inter-day precision (<13.5% at LLOQ, <7.5% at high concentrations), proficiency testing accuracy (78–127%), laboratory internal accuracy (86–113%), recovery (60–110%), and drug stability (freeze-thaw, short-term temperature, long-term and post-preparative) and inter-subject variability. Conclusion : Although direct analysis of diluted plasma was performed, post-column experiments showed efficient matrix minimisation by the XLC-MS/MS technique, which is perfectly appropriate for routine therapeutic drug monitoring of HIV/AIDS patient samples.

Background : Persons with Down syndrome have increased vulnerability to oxidative stress caused by overexpression of superoxide dismutase, an antioxidant enzyme coded on chromosome 21. Increased oxidative stress may lead to oxidative damage of important macromolecules. We monitored this damage by measuring levels of different biomarkers of oxidative stress (protein carbonyls and 4-hydroxy-2-nonenal), as well as plasma antioxidant capacity, in children with Down syndrome. A total of 20 children with Down syndrome and 18 healthy individuals were recruited for this purpose. Methods : Plasma protein carbonyls were measured using an ELISA technique, 4-hydroxy-2-nonenal was monitored by HPLC and the antioxidant capacity was evaluated using a ferric reducing ability of plasma (FRAP) assay. Results : We found that children with Down syndrome had significantly elevated levels of protein carbonyls compared to healthy controls (p<0.01). Levels of 4-hydroxy-2-nonenal and antioxidant capacity were similar in both groups. Conclusion : Our results on oxidative damage to proteins confirm the assumption of increased oxidative stress in individuals with Down syndrome.

Background : Preanalytical factors are the main source of variation in clinical chemistry testing and among the major determinants of preanalytical variability, sample hemolysis can exert a strong influence on result reliability. Hemolytic samples are a rather common and unfavorable occurrence in laboratory practice, as they are often considered unsuitable for routine testing due to biological and analytical interference. However, definitive indications on the analytical and clinical management of hemolyzed specimens are currently lacking. Therefore, the present investigation evaluated the influence of in vitro blood cell lysis on routine clinical chemistry testing. Methods : Nine aliquots, prepared by serial dilutions of homologous hemolyzed samples collected from 12 different subjects and containing a final concentration of serum hemoglobin ranging from 0 to 20.6g/L, were tested for the most common clinical chemistry analytes. Lysis was achieved by subjecting whole blood to an overnight freeze-thaw cycle. Results : Hemolysis interference appeared to be approximately linearly dependent on the final concentration of blood-cell lysate in the specimen. This generated a consistent trend towards overestimation of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, creatine kinase (CK), iron, lactate dehydrogenase (LDH), lipase, magnesium, phosphorus, potassium and urea, whereas mean values of albumin, alkaline phosphatase (ALP), chloride, γ-glutamyltransferase (GGT), glucose and sodium were substantially decreased. Clinically meaningful variations of AST, chloride, LDH, potassium and sodium were observed in specimens displaying mild or almost undetectable hemolysis by visual inspection (serum hemoglobin <0.6g/L). The rather heterogeneous and unpredictable response to hemolysis observed for several parameters prevented the adoption of reliable statistic corrective measures for results on the basis of the degree of hemolysis. Conclusion : If hemolysis and blood cell lysis result from an in vitro cause, we suggest that the most convenient corrective solution might be quantification of free hemoglobin, alerting the clinicians and sample recollection.

Background : Obesity and increased waist-to-hip ratio, emphasizing the importance of truncal obesity, have been found to correlate positively with increased cardiovascular disease risk and mortality. Owing to the inflammatory nature of atherosclerosis, the aim of our study was to find possible correlations between body mass index and waist-to-hip ratio, and the inflammatory markers C-reactive protein (CRP) and neopterin in healthy lean and overweight adults. Methods : A total of 49 healthy adults (mean age 42.4±1.8years, 32 females and 17 males) were classified according to their body mass index (BMI) and waist-to-hip ratio values. CRP and neopterin levels were measured. Results : CRP levels were found to be significantly higher in the group with BMI≥25kg/m 2 compared to the group with BMI<25kg/m 2 (p=0.014). Subjects with increased waist-to-hip ratio displayed significantly higher serum CRP and neopterin levels (p=0.014 and p=0.033, respectively) compared with the group in which the waist-to-hip ratio was <0.9. A strong positive correlation was found between CRP and BMI in the whole group (r=0.658, p=0.0001). Conclusions : Grouping overweight subjects according to their waist-to-hip ratio, which is an indicator of truncal obesity, seems to be convenient in studying the inflammatory process in relation to the elevation of adipose tissue. Elevated CRP and neopterin levels may be useful in the assessment of cardiovascular risk in overweight as well as obese subjects.

Background : There is consolidated evidence that physical activity exerts beneficial effects on several chronic conditions and longevity, on the basis of its proposed biological effects, especially on lipid profiles. However, debate continues regarding the intensity of activity required for good health, as vigorous physical activity might overwhelm advantageous changes. In addition, little is known so far on the effect of a vigorous and regular aerobic training regimen on emerging markers of cardiovascular risk, such as lipoprotein(a), total/high-density lipoprotein cholesterol ratio and the atherogenic index of plasma. Methods : To further investigate this topic, an extensive lipid profile, in accordance with the most recent guidelines issued by the American Heart Association (AHA)/American College of Cardiology (ACC) and the National Cholesterol Education Program (NCEP), was evaluated in 60 healthy male sedentary controls and in a wide population of professional endurance athletes, including 40 male professional cross-country skiers and 102 male professional road cyclists. Results : Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides, TC low-density lipoprotein cholesterol (LDL-C) ratio and the atherogenic index of plasma were significantly lower in both categories of professional athletes, whereas the mean HDL-C concentration was significantly higher. The concentration of lipoprotein(a) did not differ significantly between the groups. When compared to current NCEP or AHA/ACC goals, the percentage of patients with undesirable values was statistically different for all parameters tested, apart from lipoprotein(a). According to multiple stepwise logistic regression analysis, lower TC/HDL-C ratio in professional skiers and lower TC/HDL-C ratio and TC in professional cyclists were significantly associated with increased aerobic physical activity. Conclusions : Results of this case-control study confirm that elevated aerobic energy expenditure might be associated with a highly favorable stabilization of most traditional and emerging cardiovascular risk predictors. Therefore, a substantial increase in aerobic physical activity within the population might be recommended to reverse adverse lipid abnormalities, especially in subjects with a higher cardiovascular risk.

Background : In modern medicine many laboratory measurements are used as a surrogate for clinical outcome. There is therefore a need for models to quantify this approach. The target plot presented here is based on the difference plot. The difference between two measurements is plotted against the initial measurement, of which the first measurement is the index measurement (M index ) and the second the outcome measurement (M outcome ). A vertical line separates normal vs. increased M index concentrations according to a selected target concentration, and a horizontal line describes the situation of no change. A target line (M target ) with a slope equal to −1 and indicating the selected target concentration is drawn through the crossing point. The six outcome areas thus refer to the combined effect of M index and M outcome . A target plot taking the biological and analytical variation in consideration is considered. Materials and model : The target plot addresses sources of outcome categories, and calculates the percentage in each of these categories according to a defined target value. Hemoglobin A 1c (HbA 1c ) in diabetes in patients from Vejle County, Denmark was taken as an example. Results : Different strategies for target setting for HbA 1c were investigated: the Danish Diabetes Association (DDA) target of 5.84% HbA 1c , the target from the American Diabetes Association (ADA) of 7.0% HbA 1c , and 6.62% HbA 1c , which is the 99.9 centile of the Danish national reference interval. All three strategies can be validated from the target plot for differences in surrogate clinical outcome in spite of identical patient material. Use of the ADA target (the highest nominal value) reveals the lowest percentage of index values above the target, and of those the highest percentage outcome values of the surrogate measure reached the target. Conclusion : The target plot allows detailed stratification of outcome based on surrogate parameters and assessment of risk of disease based on the selected target. The need to standardize outcome targets in diabetes to allow comparison of quality of treatment is obvious.

Background : The metrological traceability of values for the catalytic concentration of several enzymes assigned to a calibration material has been assured by following the recently published International Standard ISO 18153. Methods : A traceable value with a measurement uncertainty was assigned for the catalytic concentration of alanine aminotransferase, creatine kinase, γ-glutamyltransferase and lactate dehydrogenase in two materials from different sources. These are all measurable quantities, with the primary reference measurement procedure described by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and a primary calibrator giving metrological traceability to the SI unit of measurement. The metrologically traceable calibration was validated by measuring human serum samples using the primary reference measurement procedure and a routine commercial measurement procedure calibrated with the traceable materials. Results : Results showed that the primary reference procedure, selected manufacturers' procedures and the end-user's routine procedure for each enzyme have the same analytical specificity. Four of eight commercial calibrators tested were commutable, whereas the others had a very small difference in absolute terms, indicating that these materials would be useful for calibration. Conclusion : The implementation of a reference system for enzyme measurements was demonstrated that assures the traceability of patient results to SI units.

We evaluated the analytical performances of the new Sebia kit for quantification of hemoglobin fractions (HbA, HbF and HbA 2 ) and structural hemoglobin variants on the Capillarys ® system. This automated capillary zone electrophoresis method uses an alkaline buffer with silica capillaries and spectrophotometric detection. Specimen stability was evaluated during 1 month. The reproducibility of migration and the imprecision of quantification were also investigated. Comparison with the Beckman P/ACE ® system was performed on 202 samples. A total of 131 subjects without any hematological abnormality were analyzed to establish the HbA 2 reference ranges based on our local population. Quantification of the Hb fractions and variants exhibited excellent stability for 4weeks of storage at 4°C, with CVs <0.3%. The imprecision of the migration normalized to that of HbA 2 for all hemoglobins tested (fractions and variants) was low, with a CV of <2.5%. At physiological and pathological levels, total imprecision ranged from 1.9% to 4.6% for HbA 2 , from 0.6% to 9.7% for HbF, and from 0.6% to 1% for HbS. Statistical analysis revealed a small proportional negative bias for HbA 2 (−8.6%). Small systematic bias (−0.2%) and proportional bias (−28%) were observed for HbF. No statistically significant difference was found for HbS. The reference range for HbA 2 was 2.1–3.2%. The Capillarys ® system is a fully automated and accurate system that gives high-resolution performance and displays appropriate characteristics for use as a routine method for the diagnosis of thalassemias and hemoglobinopathies.

The proposed recommendation for measuring and reporting chloride in undiluted plasma or blood by ion-selective electrodes (ISEs) will provide results that are identical to chloride concentrations measured by coulometry for standardized normal plasma or blood samples. It is applicable to all current ISEs dedicated to chloride measurement in undiluted samples that meet the requirements. However, in samples with reduced water concentration, results by coulometry are lower than by ion-selective electrode due to volume displacement. The quantity measured by this standardized ISE procedure is called the ionized chloride concentration. It may be clinically more relevant than the chloride concentration as determined by coulometry, photometry or by ISE after dilution of the sample.