Volume 43, Issue 6

The Czech Republic is an iodine-deficient area. Insufficient iodine intake was reduced by enriching cooking salt with iodine in the range 20–34mgI/kg. An important indicator for tracking changes in iodine nutrition over time is accurate information about urinary iodine concentrations in the population. In this paper we describe and characterize our method used for the determination of iodine in biological material, which is based on alkaline ashing of urine specimens preceding Sandell-Kolthoff reaction using brucine as a colorimetric marker. The losses of radioiodine added during sample preparation have not exceeded 0.001%. The detection limit is 2.6μgI/L and the limit of quantification is 11.7μgI/L, with intra-assay precision of 4% and inter-assay precision of 4.9%. During the period 1994–2002, the urinary iodine concentration was determined in 29,612 samples in the Institute of Endocrinology. The mean basal urinary iodine concentrations±SD were 115±69μgI/L. Of the samples, 0.7% were in severe (<20μgI/L), 9.6% in moderate (20–49μgI/L), 40.1% in mild (50–99μgI/L), 35.6% in adequate (100–200μgI/L), and 14.0% in more than adequate (>200μgI/L) subsets of iodine nutrition. A statistically significant (p<0.00001) difference was found between mean male (127μgI/L) and female (112μgI/L) urinary iodine, and an inversely proportional trend also exists in the age-related data.

It has been suggested that iodine plays an important role in gastric cancer. Gastric cancer ranks first among the cancers in the north-eastern Anatolia region, Turkey, where iodine deficiency is common. In this study, iodine levels were determined in gastric cancer and surrounding normal tissues in 19 patients with gastric cancer. Tissue iodine levels were determined by the Foss method based on the Sandell-Kolt-hoff reaction. Tissue iodine levels were lower in gastric cancer tissue (17.8±3.4ngI/mg protein, mean±SEM) compared with surrounding normal tissue (41.7±8.0ngI/mg protein) (p<0.001). There was positive correlation between the iodine levels in gastric cancer tissue and surrounding normal tissue (r=0.845, p<0.001). There was no significant difference in iodine levels in cancer and surrounding normal tissue between male and female subjects. The iodine deficiency in our region may be one of the factors for increased gastric cancer prevalence. Our results support the hypothesis that iodine plays an important role in gastric cancer development.

Increasing urinary volume is an important tool in the prevention of calcium renal stones. However, the mechanism of how it actually works is only partially understood. This study aimed at assessing how urine dilution affects urinary calcium oxalate crystallization. A total of 16 male idiopathic calcium oxalate (CaOx) stone-formers and 12 normal male subjects were studied and 4 h urine samples were taken twice, under low (undiluted urine) and high hydration conditions (diluted urine). An equal oxalate load (1.3mmol/L) was added to both types of urine and the crystallization parameters were assessed. In both stone-formers and normal subjects, the crystallization processes were significantly (p<0.05 or less) more marked in the undiluted urine than in the diluted urine in terms of: a) total quantity of calcium oxalate dihydrate (COD) and calcium oxalate monohydrate (COM) crystals; b) total quantity of crystalline aggregates; and c) aggregation index (i.e., ratio between the area occupied by crystalline aggregates and the area occupied by all the crystals present). The comparison between stone-formers and normal subjects showed that the greatest difference was for the size of COD crystals, which were larger in the urine of the stone-formers. A further important finding was an inverse relationship between changes in urinary volume and in the aggregation index (r=–0.53, p=0.004). In conclusion, urine dilution considerably reduces crystallization phenomena induced in vitro by an oxalate load in both calcium stone-formers and normal subjects.

Background: The site of origin of idiopathic recurrent calcium urolithiasis (IRCU) – a disorder characterized by stones composed of calcium oxalate (CaOx) and/or calcium phosphate (CaPi) – is uncertain, because in urine such risk factors for stones as disturbed Ox, Ca and Pi are not regularly observed. Aims: To evaluate whether imbalance of antioxidants and oxidants might be present in IRCU patients that is then followed by abnormal urine, plasma and intracellular mineral homeostasis, and stones. Methods: Males were investigated in the laboratory under standardized conditions, and three trials were organized. Trial 1 was cross-sectional, comparing IRCU patients with (n=111) and without stones in situ (n=126), focussing on abnormalities of oxypurines and minerals in urine and plasma, and metabolic activity (MA) of the disease. Trial 2 was partly controlled (n=14 healthy subjects; n=53 IRCU patients), comparing the plasma levels of total antioxidant status (TAS) and uric acid, the major antioxidant in humans, using the subsets Low (n=26) and High (n=27) TAS among IRCU patients in terms of plasma levels of uric acid, ascorbic acid, albumin, α-tocopherol and minerals, urinary minerals, CaOx and CaPi (hydroxyapatite) supersaturation. Trial 3, comprising stone-free IRCU patients (n=8) and healthy controls (n=8), compared minerals and mineral ratios in plasma and red blood cells (RBCs). Established analytical methodologies were used throughout. Results: In trial 1, uricemia, hypoxanthinuria and proteinuria were elevated, fractional urinary clearance (FE) of uric acid was decreased in stone-bearing patients, and MA correlated positively with uricemia and urinary total protein excretion. In trial 2, TAS was significantly decreased in IRCU patients vs. healthy controls; low TAS coincided with low plasma uric acid and albumin, unchanged ascorbic acid, α-tocopherol and parathyroid hormone, but increased FE-uric acid and Pi excretion; the latter correlated negatively with TAS. In trial 3, plasma minerals were significantly decreased in IRCU patients vs. controls, and Ca/Pi, (Ca/Pi)/Mg and (Ca/Pi)/Na molar ratios increased; the latter ratio was also increased in RBCs, and correlated highly positively with the same ratio in plasma. Conclusions: In IRCU 1) renal stones in situ in combination with high fasting uricemia, high hypoxanthinuria and protein-uria, and high MA suggest that a systemic metabolic anomaly underlies stone formation; 2) antioxidant deficit is frequent, unrelated to the presence or absence of stones but apparently related to poor renal uric acid recycling, low uricemia and albuminemia, exaggerated urinary Pi excretion, and low MA; 3) the combination of low plasma TAS, disordered Ca/Pi and other mineral ratios in urine, plasma and RBCs, but unchanged urinary Ca salt supersaturation is compatible with the view that CaPi solid and Ca microlith formation start inside oxidatively damaged cells.

Recent work has shown that high-density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-chlorotyrosine. A higher nitrotyrosine content in lipoprotein is significantly associated with diminished cholesterol efflux capacity of the lipoprotein. Since accelerated atherogenesis is a key complication of diabetes mellitus, and nitrosative stress has recently been implicated in diabetic pathology, we set out to demonstrate an increase in the circulating levels of nitrated apolipoprotein A (apoA)-I in type 2 diabetic patients and its putative correlation with metabolic biomarkers. In this work we addressed this hypothesis in a case-control study with 30 type 2 diabetic patients and 30 age-matched control subjects. Nitrated apoA-I was 3280±1910 absorbance peak area/apoA-I (g/L) for diabetic patients and 2320±890 for control subjects (p<0.037). This represents a 50% increase in circulating nitrated apoA-I in diabetic patients to age-matched controls. Diabetic patients also showed increases of a similar magnitude in circulating advanced glycation endproducts measured as pentosidine fluorescence (44.16±16.26 vs. 30.84±12.86 AU; p<0.01) and in circulating lipoperoxides (46.0±18.0 vs. 37.2±18.0nmol/L; p<0.03). No significant correlation was found between nitration of apoA-I and glycosylated hemoglobin or any of the other parameters measured. If proven in subsequent functional and in vivo studies, increased nitrated apoA-I would represent another mechanism by which nitrosative stress participates in diabetic macro-angiopathy.

Type 2 diabetes mellitus (DM) is associated with significant abnormalities of lipoprotein metabolism and coronary heart disease (CHD). The most commonly recognized lipid abnormality in type 2 DM is hypertriglyceridemia, which is known to be an independent risk factor for CHD in diabetics. The –1131T→C polymorphism found in the newly identified apoli-poprotein A5 ( APOA5 ) gene has been found to be associated with elevated plasma triglyceride (TG) concentrations in different racial groups. In this study, DNA samples from 155 control subjects, 172 type 2 diabetics and 113 type 2 DM patients with CHD were analyzed to examine the influence of APOA5 1131T→C polymorphism on plasma lipids and the susceptibility to CHD in type 2 diabetics. The frequency of the APOA5 –1131C allele in the DM+CHD group was significantly higher than that of control subjects (37.2% vs. 27.7%, p=0.021). The distribution of the APOA5 –1131T→C genotypes (TT, TC and CC) was 36.3%, 53.1% and 10.6% in type 2 DM patients with CHD, and 53.6%, 37.4% and 9.0% in controls, respectively (p=0.018). The frequencies of alleles and genotypes in type 2 diabetics were not significant compared to controls. In controls, plasma TG concentrations in subjects with the TT genotype were significantly lower than in those with TC/CC (0.92, 1.28 and 1.35mmol/L for TT, TC and CC, respectively; p=0.003 by ANOVA). These data suggest that the APOA5 –1131T→C polymorphism might play a role in elevated plasma TG levels in type 2 diabetic patients in the Chinese population.

An algorithm was developed to evaluate equilibrium constants for testosterone (Te) and sex hormone-binding globulin (SHBG) or albumin from serum free testosterone (FTe) measurements performed in a panel of 30 healthy elderly men by means of a near-reference method, i.e., symmetric dialysis (affinity constants: SHBG-Te, 1.13×10 9 L/mol; albumin-Te, 4.4×10 4 L/mol). Using these estimates, a free testosterone index (FTeI) was calculated from total Te and SHBG concentrations in a further 35 elderly men. This FTeI perfectly matches with actually measured free testosterone concentrations by symmetric dialysis in this second group, with a mean ratio index/measurement of 0.998±0.016 (SEM). The efficacy of the algorithm, which represents a simple alternative to previous cumbersome methods for estimation of equilibrium constants, is thereby demonstrated.

Detection and quantitation of circulating tumor cells from solid epithelial tumors could become a valuable tool for therapy monitoring if the procedure can be standardized. In the present work we assessed the influence of preanalytical handling, storage and white blood cell isolation on analysis of a population of spiked tumor cell-line cells and intrinsically present epithelial cells in the peripheral blood of breast and lung cancer patients and the sensitivity of their detection. Sucrose density separation did not enrich epithelial cells, and even depleted them, leading to a gross underestimation of their numbers (3/13 positive, between 2.9 and 50cells/mL) in comparison to red blood cell lysis (13/13 positive, between 77,200 and 800cells/mL). Short-term storage of whole blood samples for up to 7days had little influence on the number of epithelial cells recovered. The effectiveness of magnetic bead enrichment was dependent on the number of relevant cells and the volume used for enrichment. Red blood cell lysis and fluorochrome-labeled antibody staining in a no-wash procedure with subsequent laser scanning cytometry allowed the detection of circulating epithelial cells in 92% of breast and lung cancer patients. Two examples of how this method can be applied for the longitudinal analysis in individual patients are shown, with an increase in numbers preceding relapse and a decrease paralleling tumor reduction. The proposed simple and easy method allows close monitoring, which may help in real-time analysis of the response of solid tumors, especially their systemic component, to therapy and hopefully will contribute to more individually tailored therapy.

Background: Elevated plasma levels of total homocysteine (tHcy) are associated with an increased risk of developing occlusive vascular diseases. To better illustrate the relationship between plasma tHcy concentration, oxidative stress, and inflammation in patients with coronary artery disease (CAD), we measured plasma 8-isoprostane-prostaglandin F 2 (Iso-P), plasma malondialdehyde (MDA), and several markers of inflammation. We also aimed to demonstrate the effects of vitamin supplementation on these markers. Methods: A total of 93 patients with ischemic heart disease were investigated. Of these, 34 had plasma tHcy ≤8μmol/L, while 59 had plasma tHcy ≥15.0 μmol/L. The 59 patients were randomized to open therapy with folic acid, 5mg, pyridoxine, 40mg, and cyancobalamin, 1mg once daily for 3months (n=29) or to no vitamin treatment (n=30). Blood samples were obtained from both groups before randomization and 3months later. A sample was also obtained from the remaining 34 patients. Results: Plasma Iso-P, serum amyloid A (S-AA), and plasma intercellular adhesion molecule-1 (ICAM-1) concentrations were higher in patients with high plasma tHcy levels than in patients with low to normal tHcy levels. Plasma levels of P-, L-, E-selectins, MDA, C-reactive protein (CRP), and orosomucoid did not differ between the groups. Vitamin therapy reduced plasma tHcy from 17.4 (15.3/20.1) to 9.2 (8.3/10.3)μmol/L (25th and 75th percentiles in parentheses) (p<0.0001). Plasma levels of Iso-P remained unchanged and, of all inflammatory markers, only the S-AA concentrations were slightly reduced by the vitamin treatment, from 5.3 (2.2/7.0)ng/L at baseline to 4.6 (2.1/6.9)ng/L (p<0.05) after 3months of vitamin supplementation. Conclusion: Patients with CAD and high plasma tHcy levels had elevated plasma levels of Iso-P. The increase remained unaffected by plasma tHcy-lowering therapy, suggesting that homocysteine per se does not cause increased lipid peroxidation. Levels of plasma ICAM-1 and S-AA were increased in patients with high plasma tHcy, suggesting an association between homocysteinemia and low-grade inflammation.

Background: There are several methods for calculating serum osmolality, and their accordance with measured osmolality is the subject of controversy. Methods: The concentrations of sodium, potassium, glucose, blood urea nitrogen (BUN) and osmolalities of 210 serum samples were measured. Two empirical equations were deduced for the calculation of serum osmolality by regression analysis of the data. To choose the best equation, chemical concentrations were also used to calculate osmolalities according to our formulas and 16 different equations were taken from the literature and compared with the measured osmolalities. Correlation and linear regression analyses were performed using Excel and SPSS software. Results: Multiple linear regression analysis showed that serum concentrations of sodium (β=0.778, p≤0.000), BUN (β=0.315, p≤0.000), glucose (β=0.0.089, p≤0.007) and potassium (β=0.109, p≤0.008) are strong predictors of serum osmolality. The data were also analyzed by manual linear regression to yield the equations: osmolality=1.897[Na + ]+glucose+BUN+13.5, and osmolality=1.90[Na + +K + ]+glucose+BUN+5.0. The osmotic coefficient for sodium and potassium solutes was deduced to be 0.949 from the slope of the curves of measured osmolality vs. [Na + ] and [Na + +K + ], respectively. The inclusion of a BUN value in the equation for osmolality increased the correlation coefficient by approximately 450% and decreased the SD of difference by approximately 35% (p≤0.002). Inclusion of the osmotic coefficient for sodium solutes caused an underestimation of measured osmolality and positive osmolal gap unless an appropriate coefficient, constant value and/or the potassium value were included in the equation. The agreement was not improved when molal chemical concentrations were used instead of molar values. The formula presented by Dorwart and Chalmers gave inferior results to those obtained with our formulas. Conclusions: Our data suggest use of the Worthley et al. formula Osm=2[Na + ]+glucose+BUN for rapid mental calculation and the formulas of Bhagat et al. or ours for calculation of serum osmolality by equipment linked to a computer.

C-Type natriuretic peptide (CNP) is a member of the family of natriuretic peptides with vasodilatory properties, and is produced and secreted by endothelial cells. It seems to play a central role in the paracrine vasomotor control of tone and to be important in several clinical conditions characterized by endothelial dysfunction. We evaluated the analytical performance of a commercially available radioimmunoassay for CNP after a preliminary extraction with Sep-Pak C18. Its analytical reliability was checked by determination of CNP plasma levels in healthy subjects (n=23) and in patients with different diseases, likely characterized by endothelial dysfunction, such as chronic heart failure (n=133) and cirrhosis (n=84). The extraction yield was 78±3%. Accuracy of the radiommunological determination was evaluated by dilution (45–370μL of extracted plasma) and recovery tests (>80%). Between- and within-assay variabilities were ≤10% and analytical sensitivity was 0.41±0.015pg/tube. Plasma CNP in patients with chronic heart failure and with cirrhosis was significantly raised compared to controls (p<0.0001 and p=0.001, respectively). The sensitivity, accuracy and variability levels of the method proposed for CNP assay was suitable for reliable detection of changes in CNP plasma levels in the clinical setting.

The Konelab 20XT (Thermo Electron Oy, Finland) is a clinical chemistry analyzer for colorimetric, immunoturbidimetric and ion-selective electrode measurements. The aim of our work was to evaluate the analytical performances of the Konelab 20XT according to the European Clinical Chemistry Laboratory Standards Guidelines. A total of 30 analytes including substrates, enzymes, electrolytes and specific proteins were tested. Investigation results showed low imprecision (within-run coefficient of variation was below 3.5% and between-day coefficient of variation was less than 2.5% for most analytes at all three levels studied) and acceptable accuracy of the analyzer. No significant sample- or reagent-related carry-over was found. It was demonstrated that the analytical system operates within the claimed linearity ranges. The results compared well with those obtained by instruments routinely used in our laboratory (Olympus AU2700, Behring Nephelometer II). In general, the data on interference by hemoglobin, hyperbilirubinemia and turbidity are in accordance with known facts. However, slight hemolysis was found to interfere with the alkaline phosphatase (ALP) assay and mild lipemia affected the glucose assay. The Konelab 20XT is an easy-to-use analyzer that is suitable for routine and emergency analyses in small laboratories.

A definition has been agreed for the most senior professional (consultant) in clinical chemistry and laboratory medicine. A model job description for a consultant has been determined, which is intended to act as a toolkit to assist employing authorities and professional bodies to define the role of individual consultant posts. A total of 86 competences for a consultant have been designated and expressed in the form of simple generic proficiency standards. These competences have been allocated to six broad areas: clinical [13]; scientific [15]; technical [12]; communication [12]; management and leadership [20]; professional autonomy and accountability [14]. The competences are intended to be illustrative rather than definitive and to enable the duties of any consultant post to be defined. Assessment of competence is likely to entail consideration of qualifications, registration status, continuing professional development and performance review. The project is intended as a guide to European societies of clinical chemistry and laboratory medicine. The guide should be capable of local interpretation to encourage a greater degree of commonality in the role of the consultant whilst protecting national identity. The guide should stimulate international understanding and collaboration and contribute to an overall improvement in the quality of practice.