Volume 46, Issue 4

Free fatty acids (FFAs) serve as physiologically important energy substrates and their release from the adipose tissue by lipolysis is regulated according to the energy demands of the body. FFAs are increased in obese patients and contribute to type 2 diabetes, hepatic steatosis and several cardiovascular diseases. In patients with heart failure and acute coronary syndromes, elevated FFA levels are a consequence of an increased lipolysis due to a surge in catecholamines and natriuretic peptides. FFAs contribute to myocardial dysfunction and are proarrhythmic, and their oxidation requires more oxygen than does glycolysis. Therapeutic approaches have already emerged that aim to reduce the uptake and/or oxidation of fatty acids in the myocardium. The routine use of FFAs as a diagnostic tool is limited by their high variability, this being strongly influenced by nutrition and the effects of several hormones. In addition, it remains to be clarified whether fasting or postprandial values or dynamic measurements such as changes in FFA concentrations induced by stress are better parameters for evaluating cardiovascular risk. In this review, we present an overview of the metabolism and role of FFAs as a cardiovascular risk factor and discuss the potential of FFAs in the diagnosis and treatment of cardiovascular diseases. Clin Chem Lab Med 2008;46:429–34.

Laboratory diagnostics has undergone relevant changes in organization and complexity, providing new opportunities and risks. Many laboratory tests are now available on compact, easy to operate and reliable instruments, making it possible for facilities other than the traditional clinical laboratories (e.g., pharmacies) to conduct on-site or mobile testing. Ideally, although there is nothing wrong with a rational decentralization, on-site laboratory testing must still be considered a knowledge service, and a core set of competency-quality-consultancy prerequisites should be fulfilled to ensure that results of tests performed outside the clinical laboratories can reach the highest degree of efficiency and clinical efficacy. Pharmacies were historically involved in clinical chemistry. At the European level, the existing pharmacy syllabus gives to students and postgraduates the physiopathological knowledge a good basis for such an evolution. Clin Chem Lab Med 2008;46:435–6.

Authors are usually invited to follow the instructions to authors in scientific journals, because this section is likely to contain unique requirements, such as the references style requested. Authors not only must accurately check the instructions, but are also expected to verify references against the original documents before submitting the article, consuming a considerable amount of time. This aspect may contribute to the large number of references errors in scientific manuscripts, some of which would be prevented by introducing a more homogeneous and uniformed style. A variety of international, peer-reviewed journals currently requires a rather different style, especially for the numbers of authors to be cited, the placing of the year of publication, the style of the journal title. A broad implementation of reasonable standards of practice and consensus guidelines for the format of manuscript references should be promoted for an advantageous process for both the journals and the authors. Clin Chem Lab Med 2008;46:437–8.

Background : Several factors participate in the pathogenesis of Alzheimer's disease (AD), including free radicals, which when out of balance with their antioxidant capacity contribute to the oxidative stress process and neuronal death. The glutathione S-transferase (GST) polymorphisms are associated with the organism detoxification capacity and can help with the identification of sub-groups that present susceptibility to the development of AD. The aim of this study was to analyze the association of GSTs , including GSTP1 , GSTT1 and GSTM1 and apolipoprotein E ( apoE ) with AD and the distribution of these polymorphisms in the first-degree relatives of patients. Methods : For this, 41 patients with AD, 24 elderly without cognitive deficits (control group), 109 relatives of patients with AD and 41 relatives of controls were studied. A sample of peripheral blood was drawn for leukocyte DNA extraction. The genetic polymorphisms were analyzed by PCR-RFLP. Results : There was a significantly higher frequency of the ɛ4 allele in the patients (0.21) and in their relatives (0.25) when compared to controls (0.04; p=0.01) and their relatives (0.03; p<0.0001). The V allele of the GSTP1 polymorphism was higher in patients compared to controls (0.35 and 0.19, respectively; p=0.04). In contrast, the presence of the GSTT1 polymorphism prevailed in controls (79%) and their relatives. Conclusions : The V allele may be a risk factor for AD, mainly in the presence of the apoE ɛ4 allele, while the presence of GSTT1 may indicate protection against the disease. Clin Chem Lab Med 2008;46:439–45.

Background : Angiotensin II is produced primarily by angiotensin I-converting enzyme (ACE) within atherosclerotic lesions and ACE level in plaques correlates with the severity of vessel wall damage. Therefore, we investigated the possible association of ACE gene insertion/deletion (I/D) polymorphism and the severity of atherosclerosis, estimated on the basis of the number of coronary stenoses and critical arterial occlusions observed during coronary angiography. Methods : The study cohort included 172 patients with angiographically confirmed premature coronary artery disease. The ACE gene I/D polymorphism was genotyped using a PCR method. Results : The frequencies of DD genotype, D allele carrier-state ( DD+ID genotypes) and the D allele increased with the number of stenoses in coronary vessels. D allele carriers ( DD+ID genotypes) were more frequent in the subgroup of patients with stenoses in at least four coronary vessels than in other patients including subjects with one-, two- and three-vessel disease (97.4% vs. 74.4%, OR=13.05, 95% CI: 1.81–100.00, χ 2 =9.84, p=0.0017). Furthermore, the D allele was significantly more frequent in patients with critical arterial occlusions (>90%) than in subjects without critical stenoses (61.1% vs. 49.3%, χ 2 =9.84, p=0.023). Conclusions : The ACE I/D polymorphism influences individual differences in severity of coronary artery disease and the D allele promotes generation of numerous and critical atherosclerotic lesions. Clin Chem Lab Med 2008;46:446–52.

Background : Variations of the apolipoprotein A5 ( APOA5 ) gene are strongly associated with hypertriglyceridemia. Vitamin E is transported in triglyceride (TG)-rich lipoproteins and therefore could also be modulated by apoAV. Patients with type 2 diabetes have a tendency towards high TG values and increased oxidative stress. Methods : We examined the impact of genetic APOA5 variation ( – 1131 T → C ) on vitamin E and oxidative status in 169 non-smoker type 2 diabetic patients. Plasma samples were analyzed for lipids, lipoproteins, vitamin E, oxidized low-density lipoprotein (oxLDL), lipoperoxides, autoantibodies against oxLDL and diene formation of LDL. Results : Vitamin E concentrations were higher in TC carriers compared with TT carriers (45.48±8.20  μmol/L vs. 40.32±10.47 μmol/L; p=0.02). The prevalence of the TC genotype was 2.6-fold higher among individuals with high vitamin E concentrations (p=0.02). The APOA5 polymorphism did not determine any differences in oxidative status. Fasting TG concentration was a significant 21% higher in carriers of the TC genotype (p=0.04) due to higher TG concentrations in very-low-density lipoprotein (VLDL) and high-density lipoprotein. Conclusions : The APOA5– 1131 T → C polymorphism is associated with both higher vitamin E concentrations and higher VLDL-TGs in diabetic patients. Clin Chem Lab Med 2008;46:453–7.

Background : In this study, we evaluated the analytical reliability and clinical sensitivity of two tests (indirect immunofluorescence and ELISA) for anti-DNA antibodies which use cell membrane DNA (cmDNA) as antigen (Wil2 NS lymphoblastoid B cell line). Methods : We tested 97 sera of patients with systemic lupus erythematosus (SLE) and 140 control sera from healthy subjects or from patients with other systemic autoimmune diseases or infectious diseases. The results obtained with the two anti-cmDNA kits were compared with those obtained with three other methods: indirect immunofluorescence on Crithidia luciliae , ELISA anti-double-stranded DNA (anti-dsDNA) and ELISA anti-nucleosome. Results : The diagnostic sensitivity was 85% for immunofluorescence cmDNA and 90% for ELISA cmDNA, i.e., much higher than that of Crithidia (51%) and ELISA dsDNA (71%) methods, and the same as that of the ELISA nucleosome test (85%). The specificity of the cmDNA tests proved lower (92% for immunofluorescence and 87% for ELISA) than that found by anti-dsDNA Crithidia (99%), ELISA (99%) and anti-nucleosome tests (100%). Conclusions : The results of our study indicate that cmDNA tests may be used as screening tests in patients with suspected SLE. The anti-nucleosome test offers the best diagnostic accuracy overall and can play an important role in the serological diagnosis of SLE. Clin Chem Lab Med 2008;46:458–62.

Background : There is a need for reference materials (RMs) in the field of genetic testing for verification of test results obtained in patients and probands. For the frequent genetic variation G20210A in the prothrombin gene, it has been shown that purified plasmids containing the gene fragment harbouring the mutation constitute good candidate RMs. Methods : Plasmid-type RMs were characterised for homogeneity, stability, sequence identity and fitness for purpose. Their certification required the use of different real-time PCR methods for genotyping and quantification of the plasmid copy number. Results : Homogeneity, stability and fitness for the purpose of the plasmids could be demonstrated. The long-term stability (up to 24 months) of the materials was confirmed by highly sensitive and specific quantitative real-time PCR methods. Conclusions : New types of certified RMs (CRMs) for genetic testing of the human prothrombin gene G20210A sequence variant are available. Their fitness for purpose was demonstrated and no evidence was found that they would not work with other methods as long as these are targeting the whole or parts of the prothrombin gene fragment inserted into the plasmids. The described CRMs support the efforts of the international community in development, validation and harmonisation of tests for molecular genetic testing. Clin Chem Lab Med 2008;46:463–9.

Background : The aim of this study was to detect the expression of apoptosis factor caspase-3 in transferred HepG 2 cells and provide feasible evaluation of the treatment for primary liver cancer with gene methods. Methods : The pcDNA4C- LIGHT cDNA was extracted from Escherichia coli JM-109; then, the pcDNA4C- LIGHT cDNA was transferred into the HepG 2 cells by a cationic liposome mediated method. Meanwhile, the blank group was established as the control group and the HepG 2 cells were collected after transfection at 12 h, 24 h, 48 h, 3 days and 5 days. The expression of caspase-3 was identified in the supernatants by ELISA. A standard curve was generated for the set of samples assayed. Statistical significance was analyzed by SPSS. Results : The quantity of caspase-3 protein was the greatest at 48 h and the least on day 5. The secretion of caspase-3 did not increase in the control group. The coefficient of correlation was equal to 0.9986 and had evident significance. Conclusions : The pcDNA4C- LIGHT was effectively transfected in human HepG 2 cells mediated by liposome. The expression of caspase-3 increased in the transfected group. This study provides necessary theoretic support for the treatment of liver cancer with gene methods. Clin Chem Lab Med 2008;46:470–4.

Background : Polymerase chain reaction (PCR) methods play an essential role in providing data relating to diagnosis, monitoring and treatment of hepatitis C virus (HCV) infection. The real-time reverse transcription PCR (RT-PCR) assay is an established and promising tool in terms of quantifying HCV RNA for clinical application. This study aimed to evaluate the performance characteristics of a real-time RT-PCR-based test in a clinical setting. Methods : Validation and reproducibility tests were performed using a standard panel. Sera from 197 chronic HCV patients were analyzed by the real-time RT-PCR assay and the results were compared with the Versant bDNA3.0 assay (bDNA3.0). Results : The real-time RT-PCR assay showed an acceptable linear response (r 2 =0.989–0.995) in the serial dilutions regarding genotypes 1b, 2a, 2b and 1b+2a. HCV viral loads were quantifiable in all 197 patients (100%) by the real-time RT-PCR assay and in 194 (98.5%) by the bDNA3.0. HCV RNA quantification values measured by the real-time RT-PCR and bDNA3.0 assays were positively correlated (Pearson's correlation coefficient r=0.734, p<0.001). The real-time RT-PCR assay values were on average 0.13 logs higher than the bDNA3.0 results. The correlation coefficients with genotypes 1b, 2a, 2b and mixed were 0.737, 0.711, 0.791 and 0.766, respectively (p<0.01). Conclusions : The real-time RT-PCR assay showed comparable performance with bDNA3.0 regarding quantification of HCV viral loads with genotype 1 and 2 HCV infections. Clin Chem Lab Med 2008;46:475–80.

Background : Apolipoprotein E polymorphisms have important effects on plasma lipid levels and in the genetic susceptibility to development of cardiovascular diseases. Thus, the purpose of this study was to investigate the association of apolipoprotein E polymorphisms with coronary artery disease and with plasma lipid levels in a group of Mexican Mestizo patients. Methods : Apolipoprotein E polymorphisms were determined in 156 Mexican patients with coronary artery disease and 200 non-related healthy controls using the restriction fragment length polymorphism technique. The correlation of these polymorphisms with lipid profile (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides) in the patient group was determined. Results : A similar distribution of allele and genotype frequencies in coronary artery disease patients and healthy controls was found. Higher serum levels of high-density lipoprotein cholesterol and lower levels of low-density lipoprotein cholesterol, triglycerides and glucose were found in patients with the APOE*2/3 genotype when compared to patients with the APOE*3/4 and APOE*3/3 genotypes, although these differences were not significant. Conclusions : Our data suggest that genetic variation at the APOE is not a genetic factor related to the genetic susceptibility to coronary artery disease in Mexican individuals, but the role of this polymorphism in determining the lipid profile cannot be excluded. Clin Chem Lab Med 2008;46:481–5.

Background : The establishment of new methods to detect human genetic variations, such as single nucleotide polymorphisms, is of importance in hereditary disease diagnosis and pharmacogenomics. Several single nucleotide polymorphism genotyping technologies have been presented in recent years. However, techniques which would allow accurate, fast and cheap allelic determination and multiple single nucleotide polymorphism detection in parallel are still in great need. Methods : Here, we present a new genotyping technique based on gap ligase chain reaction and a fluorescent polystyrene microsphere measurement platform. We chose the human polymorphism rs3730386 as our candidate to establish this method. Probes for gap ligase chain reaction were designed to recognize the target alleles sensitively and specifically and to produce templates for amplifying the correspondent target fragments used in the following hybridization. The genotypes were finally determined by a hybridization process based on the Luminex fluorescent polystyrene microspheres measurement platform. Results : This method was successfully applied for the detection of selected single nucleotide polymorphisms with high sensitivity and specificity. The genotypes were validated by DNA sequencing. Conclusions : The new genotyping method benefited from the high sensitivity and specificity of gap ligase chain reaction and the detection platform of Luminex. The method allows multiplex analysis of single nucleotide polymorphism, because 100 types of microspheres are available from Luminex. Clin Chem Lab Med 2008;46:486–9.

Background : High-density lipoprotein (HDL) subfractions are among the new emerging risk factors for atherosclerosis. In particular, HDL 2b has been shown to be linked to cardiovascular risk. This study uses a novel microfluidics-based method to establish HDL 2b clinical utility using samples from the Prospective Cardiovascular Muenster (PROCAM) Study. Methods : Method performance was established by measuring accuracy, precision, linearity and inter-site precision. Serum samples from 503 individuals collected in the context of the PROCAM study were analyzed by electrophoresis on a microfluidics system. Of these, 251 were male survivors of myocardial infarction (cases), while 252 individuals were matched healthy controls. HDL cholesterol, HDL 2b concentration and HDL 2b percentage were analyzed. Results : This novel method showed satisfactory assay performance with an inter-site coefficient of variance of <10% for HDL 2b percentage. Parallel patient testing on 52 samples between two sites resulted in a correlation coefficient of r=0.95. Significant differences were observed in the HDL 2b subfraction between cases and controls independent of other risk factors. Including HDL 2b percentage in logistic regression reduced the number of false positives from 64 to 39 and the number of false negative cases from 48 to 45, in the context of this study. Conclusions : The novel method showed satisfactory assay performance in addition to drastically reduced analysis times and improved ease of use as compared to other methods. Clinical utility of HDL 2b was demonstrated supporting the findings of previous studies. Clin Chem Lab Med 2008;46:490–8.

Background : Obesity and metabolic syndrome increase the risk of cardiovascular morbidity and mortality. Oxidative stress seems to be involved in the pathophysiology of diabetes and cardiovascular complications of metabolic syndrome. The aim of our study was to evaluate the level of oxidative stress and inflammation in obese adults with and without metabolic syndrome. Methods : Oxidative stress and inflammation markers (total amount of free radicals, malondialdehyde, allantoin, α 1 -antiproteinase, oxidized/reduced glutathione ratio, high-sensitive C-reactive protein, fibrinogen), total antioxidant capacity and lipid standardized α-tocopherol were determined in obese subjects fulfilling at least three criteria of metabolic syndrome according to the National Cholesterol Education Program-Adult Treatment Panel III guidelines (n=20 patients), in obese subjects without metabolic syndrome (n=20 patients) and in 48 healthy controls. Results : Oxidative stress and inflammation markers were significantly elevated in the obese subjects, especially in those exhibiting metabolic syndrome. According to multidimensional statistical analysis, oxidative stress was independently related to triacylglyceride concentration, abdominal fat, low high-density lipoprotein cholesterol and low lipid standardized α-tocopherol in the patients with metabolic syndrome. Conclusions : High levels of free radicals together with low antioxidant capacity detected in obese adults indicate elevated oxidative stress, which is – together with systemic inflammation – further potentiated in the case of obese patients with metabolic syndrome. This imbalance in oxidative/antioxidative status and subclinical inflammatory state leads to higher risk of atherosclerotic and diabetic complications. Clin Chem Lab Med 2008;46:499–505.

Background : Adenosine deaminase (ADA) catalyzes the irreversible hydrolytic deamination of adenosine to inosine. The purpose of this study was to determine the plasma activities of total adenosine deaminase (ADA T ), and its isoenzymes, ADA 1 and ADA 2 , and ADA 1 /ADA 2 ratio of male and female ischemic stroke patients. Methods : We determined activities of plasma ADA T , ADA 1 , ADA 2 and ADA 1 /ADA 2 ratio in 30 patients (15 men and 15 women) with acute ischemic stroke within 12 h of the onset of the attack, as well as in 30 control subjects (15 men and 15 women) of comparable age. Results : There were significant differences between the ADA 1 activity and ADA 1 /ADA 2 ratio in male and female stroke patients (p<0.05). Compared with male stroke subjects, females had higher ADA 1 activity and ADA 1 /ADA 2 ratios. There were no significant differences between activities of ADA T and ADA 2 in men and women of the stroke and control groups. In addition, the Canadian Neurological Scale in men was significantly higher than that of women in the stroke group (p<0.05). Conclusions : Our results suggest that the primary mechanism in men with ischemic stroke might involve the reduction of ADA 1 activity. The reduction is probably an adaptation mechanism for induced increase in adenosine availability and protection of brain to ischemic injury. Clin Chem Lab Med 2008;46:506–9.

Background : Osteoprotegerin (OPG) and receptor activator of nuclear factor κ-B ligand (RANKL) are critical regulators of bone remodeling and RANKL/RANK signaling could also play an important role in the remodeling process of several tissues, such as myocardium. Therefore, we investigated whether the serum concentrations of OPG and RANKL correlate with the serum levels of metalloproteinase-1 (MMP-1), MMP-9 and tissue inhibitors of MMP-1 (TIMP-1), which are known regulators of myocardial healing in acute myocardial infarction (AMI) patients. Methods : We analyzed blood samples from 51 consecutively hospitalized men with AMI, 12 men with established ischemic heart failure (New York Heart Association category II, NYHA-II) and 12 healthy men age-matched to the NYHA-II patients. Serum levels of MMP-1, MMP-9, TIMP-1, OPG and RANKL were quantified using commercially available ELISA kits. AMI patients were sampled 4 days and 6 months after MI. Results : Our data revealed increased serum levels of OPG, RANKL, MMP-1 and TIMP-1 levels and significant correlations between increased RANKL levels and MMP-1 and TIMP-1 serum levels 6 months after MI. In addition, the ratio OPG/RANKL was very low 6 months after MI, suggesting that the nuclear factor κ-B signaling is possibly more active 6 months post-MI than it is on day 4 post-MI. Conclusions : Our data suggest that OPG, RANKL, MMP-1 and TIMP-1 serum levels can be potential mediators of myocardial healing after MI. However, further large studies are needed to confirm the utility of OPG and RANKL as markers of healing after ST elevation in MI. Clin Chem Lab Med 2008;46:510–6.

Background : Preeclampsia is associated with accumulation of collagen and proteoglycans in the umbilical cord tissues as a result of increased biosynthesis and decreased degradation of these components. Matrix metalloproteinases (MMPs) are enzymes engaged in the degradation of collagen and the protein core structures of proteoglycans, including those which bind peptide growth factors. Methods : We used Western immunoblots, immunoenzymatic assay (ELISA) and zymography techniques for the detection of gelatinases and their inhibitors. Results : We found that both umbilical cord blood plasma and serum of controls and preeclamptic newborns contained MMP-2 and MMP-9, as well tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2. The umbilical cord plasma of preeclamptic subjects contained large amounts of MMP-9 in a form of complexes with other plasma components, and zymographic analysis demonstrated increased gelatinolytic activity at a position corresponding to MMP-9, compared to control samples. By contrast, MMP-2, TIMP-1 and TIMP-2 data showed no significant differences between preeclamptic and control samples. Conclusions : The high activity of MMP-9 in preeclamptic plasma suggests its participation in the proteolytic release of peptide growth factors from their complexes with other matrix components, with subsequent stimulation of cell division and matrix biosynthesis. We suggest this might represent one of the mechanisms for matrix remodeling in the umbilical cord of preeclamptic newborns. Clin Chem Lab Med 2008;46:517–22.

Background : Alkaline phosphatase (ALP) is a biomarker for hepatobiliary and skeletal diseases. It is also raised in sepsis. In atherosclerotic plaques, ALP is expressed. Similar to C-reactive protein (CRP), it may be another marker of systemic inflammation. Therefore, we investigated their association in a Hong Kong Chinese population. Methods : Plasma ALP and CRP were measured in 205 subjects (110 men, 95 women; age 55.2±11.6 years) in the Hong Kong Cardiovascular Risk Factor Prevalence Study-2 cohort. Results : The blood levels of ALP and CRP were significantly correlated (r=0.30, p<0.001), which was due to a significant correlation in women (r=0.43, p<0.001). In a multivariate model, CRP level was related to ALP (β=0.18, p=0.008). After adjusting for confounding factors and other liver enzymes, the relationship between ALP and CRP remained significant in women (β=0.28, p=0.019), but in men, ALP was not an independent determinant of CRP levels. Conclusions : ALP may be another marker of systemic inflammation, especially in women. Whether it provides clinical information additional to CRP requires further study. Clin Chem Lab Med 2008;46:523–7.

Background : In normal ageing, as well as in diabetes mellitus, blood platelets are exposed to increasing amounts of reactive oxygen species. Because occurrence of carbonyl groups is the first step in oxidative damage of proteins, we questioned the formation of carbonylated proteins (i) in vitro: platelets exposed to oxidant hydrogen peroxide (H 2 O 2 ) and agonist thrombin (in the presence/absence of antioxidants), and (ii) in vivo: during ageing progression, and in type 2 diabetes. Methods : Platelets were isolated from the blood of experimental animals (rats and hamsters) and humans (healthy, young and elderly), as well as from type 2 diabetics (matures and elderly). 2,4-Dinitrophenyl derivatization of the amino acid side chains was used to quantify protein carbonyls (spectrophotometry) and to immunodetect carbonylated protein bands (Western blotting). Results : In animal models and humans, H 2 O 2 produced dose-dependent increases in carbonylation of platelet proteins (vs. basal condition). Thrombin activation stimulated protein carbonyl formation in a process quenched by antioxidant catalase, suggesting that carbonylation was induced by the oxidative stress generated by activated platelets. Progression of ageing caused increased carbonylation of platelet proteins (vs. young age); enriched in carbonylated proteins, platelets of elderly subjects were less sensitive to H 2 O 2 . Type 2 diabetes additionally enhanced carbonylation of human platelet proteins (vs. the levels at young and elderly healthy subjects). In all experiments, protein carbonyl concentrations were correlated with changes in intensity of 2,4-dinitrophenyl-hydrazine-reactive protein bands on immunoblots. Conclusions : The results suggest that exogenous oxidative stress, thrombin activation, progression of ageing and type 2 diabetes lead to protein carbonyls formation in platelets, and this modification can be attenuated by antioxidant enzymes. Clin Chem Lab Med 2008;46:528–36.

Background : Urinary enzymes, including the brush border enzyme alanine aminopeptidase (AAP), are reliable biomarkers of nephrotoxicity and represent early indicators of tubular damage. The aim of the present study was to develop and validate an automated urinary AAP activity assay. Methods and results : For this purpose, we adapted a photometric test using the substrate L-alanine-4-nitroanilide to a dedicated clinical chemistry analyser. Due to the unavailability of calibrators with established AAP activity, we assessed the AAP activity of a commercially available control material using the photometric test. This material was subsequently employed as a calibrator for the automated assay. Quality assurance was accomplished with independent control materials. A fast gel filtration step of urine samples using Sephadex G50 was demonstrated to be necessary before AAP analysis to avoid underestimation of enzyme activity due to ammonia and amino acids. We compared the automated assay with a manual photometric assay and obtained excellent correlation between both methods (r 2 =0.946, n=50). Conclusions : We conclude that the newly developed automated assay represents a user-friendly and analytical tool to determine urinary AAP activity in a medium- to high-throughput laboratory. Potential applications include clinical diagnostic work and toxicological studies. Clin Chem Lab Med 2008;46:537–40.

Background : Serum non-high-density lipoprotein cholesterol (HDL-C), an easily identifiable atherogenic index, has attracted attention within a clinical meaning different from that of other lipid indexes. The link between body weight gain and the occurrence of cardiovascular diseases may be mediated through non-HDL-C. However, there have been few reports examining the independent associations between weight gain and non-HDL-C, over a period of at least 1 year, especially in females. Methods : We examined data on 200 asymptomatic Japanese females (mean 49.1 years) with an involuntary weight gain of at least ≥0.1 kg/m 2 as a body mass index (BMI) 1 year after a baseline check-up. At baseline and after the 1-year period, we measured BMI, blood pressure (BP) and blood metabolic variables, including non-HDL-C. Results : The mean BMI levels rose from 22.9 to 23.5 kg/m 2 during this period. Non-HDL-C levels had a significant increase (from 3.87 to 3.93 mmol/L), and a partial correlation test, adjusted for age and all measured metabolic variables, revealed that BMI change was significantly and independently correlated to non-HDL-C levels (r=0.25, p<0.0001), along with systolic BP. The subgroup with an age of <50 years had a clear correlation between BMI change and non-HDL-C levels (r=0.34, p=0.001), contrary to those of ≥50 years. Conclusions : A short-term involuntary weight gain was significantly and independently correlated to an increase in non-HDL-C levels among asymptomatic Japanese females, particularly in relatively young subjects. In achieving a more favorable lipid profile of non-HDL-C, even a modest weight gain may need attention. Clin Chem Lab Med 2008;46:541–4.

Background : The levels of angiogenic cytokines are important in the definition of baseline characteristics of cancers and to predict patient prognosis; however, the reference intervals for angiogenic cytokines have received only limited attention. Herein, we obtained reference intervals for angiogenic cytokines, including vascular endothelial growth factor (VEGF), hepatocyte growth factor, basic fibroblast growth factor, interleukin-6, vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2. Methods : We measured these materials in serum, plasma and urine of 131 controls, according to the guidelines of the Clinical and Laboratory Standards Institute. We analyzed the association between angiogenic cytokine levels and other laboratory parameters and explored whether polymorphisms in angiogenic cytokine genes affect the levels of cytokines. Results/Conclusions : These data allowed us to construct reference intervals for angiogenic cytokines, and we also observed that concentrations of angiogenic cytokines generally do not vary with genotype or haplotype. An exception was serum VEGF concentration; this level was marginally affected by the –1154 VEGF polymorphism. Clin Chem Lab Med 2008;46:545–50.

Background : The objective of this study was to describe the natural kinetics of serum soluble transferrin receptor (S-TfR), ferritin and reticulocyte indices in preterm neonates, and to find out whether these analytes relate to hematocrit (Hct) level in determining the need for red cell (RBC) transfusions. Methods : During a 2-year period, 100 preterm neonates were recruited in a tertiary level neonatal intensive care unit. Inclusion criteria were gestational age ≤34 weeks or birth weight <2000 g. Biochemical markers of iron deficiency and hematological indices were serially analyzed from birth. This report focuses on the first 16 weeks after birth. Results : The trends of the studied analytes were presented with reference ranges. RBC transfusions did not have a significant effect on reticulocyte hemoglobin content (CHr) or reticulocyte count. Reticulocytes were lowest after the first week and S-TfR at 9 weeks of age. CHr and fraction of immature reticulocytes were highest at birth and decreased thereafter. CHr and reticulocyte count were significantly different in two groups determined by Hct level (Hct < or ≥0.30). This difference was not observed in S-TfR or ferritin concentrations. Conclusions : In addition to reflecting the activity of erythropoiesis, S-TfR seems to reflect iron balance in preterm neonates. By using CHr and reticulocyte, it is possible to obtain more information about iron balance in relation to erythropoiesis, and it might be useful to combine this information with Hct before making a decision about a transfusion. Clin Chem Lab Med 2008;46:551–7.

Background : The chemical inertness of hydroxyethyl starch (HES) might cause interferences of the colloid with a variety of laboratory tests. We aimed to evaluate potential influences of HES 130/0.4, the newest HES type, on several common hematology and clinical chemistry parameters. Methods and results : A convenient sample of 25 patients scheduled for rheological therapy with 500 mL 6% HES 130/0.4 was evaluated. Blood samples were drawn before and after colloid application. Comparing pre- and post-infusion values of a battery of laboratory tests (i.e., hematology and hemostasis parameters, electrolytes, enzymes, kidney and metabolic parameters, lipids, etc.) in time course, a median difference greater than the reference change value for a specific parameter was considered clinically relevant. Among all parameters tested, only serum amylase activity displayed a clinically relevant difference between pre- and post-infusion values (median increase of 85% due to HES administration). By applying in vitro experiments, we demonstrated that serum amylase values obtained in the samples diluted in a 1:1 ratio with HES 130/0.4 and in samples diluted in a 1:1 ratio with 0.9% NaCl displayed a negligible median difference of 3%. Conclusions : The in vivo effect of HES 130/0.4 administration on serum amylase activity observed in our study was pharmacological (real) in nature. With the exception of the influence of HES 130/0.4 on amylase activity, the effects of HES 130/0.4 on other parameters tested in this study can be interpreted as having no clinical relevance. Clin Chem Lab Med 2008;46:558–62.

Accurate serum creatinine measurements in glomerular filtration rate estimation (eGFR) using equations are critical to ongoing global public health efforts to improve the diagnosis and treatment of chronic kidney disease. There is now an ongoing activity to promote worldwide standardization of methods to determine creatinine together with the introduction of a revised eGFR equation appropriate for use with standardized creatinine methods. Standardization of calibration, i.e., implementation of calibration traceability to high-order reference measurement procedures and reference materials, does not, however, correct for analytical interferences of field methods (non-specificity bias). To account for the sensitivity of alkaline picrate-based methods to non-creatinine chromogens, some manufacturers have adjusted the calibration to minimize the pseudo-creatinine contribution of plasma proteins, producing results more closely aligned to the reference method (isotope dilution-mass spectrometry), but this strategy makes an assumption that the non-creatinine chromogen interference is a constant among samples, which is an oversimplification. Thus, analytical non-specificity for substances found in individual patient samples can affect the accuracy of eGFR computed from serum creatinine values for any alkaline picrate method, including the so-called “compensated” Jaffé methods. The use of assays that are more specific for serum creatinine determination, such as those based on enzymatic reactions, may provide more reliable eGFR values. Supporting the choice of more specific assays by clinical laboratories represents one of the main tasks of our profession in order to achieve the ultimate clinical goal, which is to routinely report an accurate eGFR in all the pertinent clinical situations. Clin Chem Lab Med 2008;46:567–72.