Band 49, Heft 9

Background: Cystatin C (CC) has been proposed to play a role in atherosclerosis. We aimed to review the prognostic value of CC serum/plasma levels in patients with acute coronary syndromes (ACS). Methods: Fifteen observational longitudinal studies were selected by Medline. Results: Increased CC over threshold values ranging from 0.93 to 1.3 mg/L were prognostic for death (hazard ratio; HR: 2.04–3.6) and for the occurrence of any fatal and non-fatal cardiovascular events (HR: 1.7–9.6) for patients with either ACS only or coronary heart disease and prevalent ACS. Only one study showed an increased risk for future myocardial infarction (MI) in patients with marker levels higher than 1.0 mg/L. Three studies reported the risk associated with a change of one unit of CC for long-term death (HR ranging from 1.9 to 6.3) and for the composite end point of 1 year MI and death (HR 2.15). Some studies showed the additional prognostic value contributed from CC measurements to other markers and to conventional risk scores. Conclusion: Despite low to moderate evidence, there is a general agreement on the significant prognostic value of CC in ACS that might encourage further research focused on risk assessment for patients with MI.

Biochemical reactions in cells which involve oxalic acid are described. It is shown that this compound is required for the formation of uracil and orotic acid. The former is a component of RNA which is common to all cells in the human metabolism. On the basis of the biochemical reactions described a possible treatment to relieve the effects of calcium oxalate renal calculi whose origin is related to the metabolic concentration of oxalic acid is proposed.

The Athletes Biological Passport (ABP) has received both criticisms and support during this year. In a recent issue of The Lancet , Michael Wozny considered that the use of the ABP makes it more difficult to take banned substances and that it was successfully used against the Italian elite cyclist Franco Pellizotti. After that, Italy's anti-doping tribunal considered that there was not enough evidence to prove manipulation of his own blood profile in Pellizotti's case. However, the UCI appealed to the Court of Arbitration for Sport (CAS) that sanctioned Pellizotti with a suspension of 2 years. Since its implementation, some problems have emerged. From 2010 to date, a large number of reports regarding the stability of the blood variables used to determine the ABP have been published, showing mixed results. This study considers that there is a risk of misinterpreting the physiological variations of the hematological parameters determined by the anti-doping authorities in the ABP. The analytical variability due to exercise training and competitions and/or to different metabolic energy demands, hypoxia treatments, etc. could lead to an increase in false-positives when using the ABP with the dramatic consequences that they might cause in major sports events like the forthcoming London Olympic Games. Moreover, the ABP characteristics, procedures, thresholds, or individual determination of reference ranges, abnormal out-comes, strikes, “how the profile differs from what is expected in clean athletes” should be clearly stated and explained in a new public technical document to avoid misunderstandings and to promote transparency.

The Athlete's Biological Passport (ABP) is an evaluation of hematological parameters, hemoglobin (Hb), reticulocytes (Ret), and their combination in the OFF-score. Recently, the Court of Arbitration for Sport accepted it as a suitable indirect method for detecting blood doping. There are various topics which are not defined and scientifically completely explained in ABP, limiting its effectiveness as evidence and as suspect of blood manipulation. The data source the ABP used for designing a profile is unclear. The variance used for cyclists is not correct. The covariables which should be calculated together with the measures of Hb and Ret are not always considered in the statistical program. The pre-analytical warnings for correct and valid collection, transport, and storage of the specimens are not assured. Quality control of the instruments is not completely assured. Analytical variability is not appropriately considered in the program. The seasonal changes of the hematological parameters, due to training and competitions, are not calculated. Statistical analysis, based on a Bayesian-like program, not available to the scientific community, does not follow the classical decision-making approach of medicine and science. The ABP needs of additional evidences and of scientific debate.

The introduction of the Athlete Biological Passport is a landmark in the fight against doping. Contrary to traditional toxicological anti-doping analysis, its aim is to detect the biological consequences of doping through the measurement of different specific biomarkers which generate individual profiles for each athlete. This new approach allows anti-doping organizations to convict those athletes showing abnormal profiles for the use (not the presence!) of even undetectable forbidden substances or methods. Additionally, these data can be used for other purposes: to conduct targeted conventional anti-doping tests on those athletes showing unusual results and to evaluate the prevalence of doping within a population of athletes. By now only the hematological biomarkers of blood doping have been validated, but in the future additional modules will be introduced to detect other forms of doping.

Since the completion of the human genome sequence, the study of common genetic polymorphisms in complex human diseases has become a main activity of human genetics. Employing genome-wide association studies, hundreds of modest genetic risk factors have been identified. In infectious diseases the identification of common risk factors has been varied and as in other common diseases it seems likely that important genetic risk factors remain to be discovered. Nevertheless, the identification of disease-specific genetic risk factors revealed an unexpected overlap in susceptibility genes of diverse inflammatory and infectious diseases. Analysis of the multi-disease susceptibility genes has allowed the definition of shared key pathways of inflammatory dysregulation and suggested unexpected infectious etiologies for other “non-infectious” common diseases.

This paper is the ninth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase; Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase; Part 6. Reference procedure for the measurement of catalytic concentration of γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 °C; Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase. The procedure described here is derived from the previously described 30 °C IFCC reference method. Differences are tabulated and commented on in Appendix 1.

Background: Automated DNA sequencing produces large amounts of data that need to be analyzed by appropriate software. Personalization of software can be a difficult and time-consuming task, especially if a large number of mutations have to be analyzed. Methods: The Applied BioSystems SeqScape software, based on the KB basecaller algorithm, is a versatile tool that can be used for mutational analysis and for data quality assessment of sequences belonging to any gene of interest. Using this software we analyzed over 1400 sequences of CFTR exons and adjacent intronic zones, representing over 500,000 bases. Results: We present an up to date specific template and a linked set of instructions for automated labeling of all point mutations and polymorphisms of the CFTR gene, whose mutations cause cystic fibrosis (the most common genetic disease among Caucasian individuals). We also describe our refined software settings for mutational analysis, in order to keep to a minimum the need of manual validation. Conclusions: The use of our template greatly simplifies the mutational analysis of the CFTR gene, reducing human intervention. In our opinion, it might not only be useful to researchers that already perform CFTR mutational analysis by sequencing methods but it should also improve the approach in those laboratories that already use ABI PRISM instrumentation for a limited mutational analysis of the CFTR gene. Similar mutational templates can also be used for other disease causing genes, thus improving molecular genetics protocols.

Background: High resolution melting (HRM) analysis of PCR amplicons was recently introduced as a closed-tube, rapid, and inexpensive method of genotyping. This study evaluated this system as an option for detecting the three most common mutations in the HFE gene (C282Y, H63D, S65C), accounting for the main form of hereditary haemochromatosis. Methods: Ninety samples, previously screened by direct sequencing, and 27 controls were used. The analysis were performed on the Rotor Gene Q, using the commercial HRM mix containing the Eva Green dye (Qiagen). Specific primers allowed the amplification of the regions of interest in the HFE gene. Following amplification, a HRM analysis was conducted to detect DNA variants. The thermal denaturation profiles of the samples were compared with those of the controls. Results: One hundred percent of heterozygous and homozygous samples were readily identified. Heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves, but significant differences were also present in the melting curves of the homozygous carries compared with those of the wild-type subjects. Conclusions: HRM analysis is an appealing technology for HFE gene screening. It is a robust technique that can be widely adopted in diagnostic laboratories to facilitate gene mutation screening.

Background: Immunoassays are biochemical tests applied to measure even very small amounts of substance using the highly specific binding between an antibody and its antigen. They have a wide range of applications. The measurement however, might be associated with substantial uncertainty; this can have significant consequences for any diagnosis, or clinical decision. An international comparability study was thus performed to assess the sources of uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA. Methods: In contrast to the original publication for this international comparability study, we reanalyse the data using Bayesian inference. This provides a statistically coherent approach to estimate ELISA concentrations and their associated uncertainties. Results: The Bayesian uncertainties of individual ELISAs and laboratory estimates are considerably larger than previously reported uncertainties. The average concentrations estimated here differ from the ones estimated by each study participant. In general, this leads to different conclusions about the study. In particular, the inter- and intra-laboratory consistency is increased, and repeatability problems occur for fewer laboratories. Conclusions: Decisions which are based on plausible ranges of measurements (such as credible intervals), are generally superior to those solely based on point estimates (such as the mean). Reliable uncertainties are thus vital, and not only in metrology. In this paper, a general method is developed to derive concentration estimates and valid uncertainties for ELISAs. Guidance on applying this Bayesian method is provided and the importance of reliable uncertainties associated with ELISAs is underlined. The applicability and virtues of the presented method are demonstrated in the context of an international comparability study.

Background: Preeclampsia is characterized by hypertension and proteinuria that begins in the second half of pregnancy. Endothelial dysfunction and trophoblastic hypoperfusion seen in preeclampsia suggested to be part of an increased maternal inflammatory response to pregnancy. In this study, we aimed to evaluate some inflammatory markers in pre-eclamptic and normotensive pregnants. Methods: The study included 36 cases with mild preeclamp-sia, 36 cases with severe preeclampsia and 33 cases of normotensive pregnant. High sensitive C-reactive protein (hsCRP) and serum amyloid A (SAA) were measured by enzyme-linked immunosorbent assays, serum procalcitonin was measured by enzyme-linked fluorescent immunassay. Mean arterial pressure (MAP) was used as an indicator of the severity of the disease. Results: In severe preeclampsia group hsCRP, serum amyloid A and procalcitonin levels were significantly higher than mild preeclamptic and normotensive groups. SAA and hsCRP levels were higher in mild preeclamptic group when compared with normotensive pregnant but no significant difference was found in procalcitonin between these groups. There were significant correlations betweeen hsCRP, SAA, procalcitonin and MAP. Conclusions: The results confirm that inflammatory reactions are closely associated with preeclampsia.

Background: In this study the pre-analytical effects of sample storage on frequently used routine clinical chemistry assays were evaluated by comparing four different lithium heparin plasma separation tubes to a reference collection procedure. Methods: Blood was collected from 20 healthy volunteers using plasma separation tubes from four different manufacturers together with manually separated plasma as reference. In total, 15 clinical chemistry parameters were determined at 0 h, 24 h, and 72 h. Samples were stored at 4°C. Statistical differences were evaluated using a generalized estimating equation regression model. Results: Significant differences could be demonstrated for almost every parameter when comparing the separation tubes to the reference collection system. The estimated maximum allowable storage time in the primary tube was considerably reduced using separation tubes, e.g., for glucose the maximum storage time was reduced from >72 h to 7–15 h, and for potassium from 60 h to 10–13 h, respectively. Conclusions: These data indicate that sample storage in the primary tube using plasma separation tubes is associated with clinically relevant changes for certain parameters. Therefore, storing samples for retesting should be avoided when using plasma separation tubes, in particular for parameters susceptible to interference by erythrocyte or platelet contamination.

Background: Pre-acquisition system assessment of clinical laboratory analysers and/or methods are generally repeated independently in each individual organisation planning their introduction. In the course of replacing our 10-year-old Sysmex ® CA-1500 for Sysmex ® CS-2100 i coagulometers, we designed and tested a model based on CLSI protocols in which one laboratory performs an extensive validation, allowing others to rely on concise verification. Methods: Validation of the Sysmex ® CS-2100 i was performed largely according to CLSI Guideline H57-A and included EP-5, 7, 9 and 10 in the evaluation of 10 assays encompassing all measurement principles available. EP-15 was used for end-user verification. Practicability and results of validation and verification were compared. Results: Analytical performance of the CS-2100 i was as claimed by the manufacturer and complied with our own criteria. System verification results were compatible with those of the validation. Verification was time- and cost-effective. Conclusions: We have approved the Sysmex ® CS-2100 i analyser for introduction in our laboratory. For colleague laboratories in our region introducing this analyser, a system verification is proposed to be sufficient when referring to our data. It is our intention to use the validation vs. end-user verification model for future method introduction, and when harmonising between our different laboratory locations.

Background: Demonstration of substantially increased urinary excretion of porphobilinogen is the cornerstone of diagnosing acute porphyria crisis. Because porphobilinogen testing is not implemented on clinical chemistry analysers, respective analyses are available in rather few clinical laboratories. The aim of this study was to critically describe and to evaluate a semi-quantitative rapid test for urinary porphobilinogen determination which is commercially available and recommended by the American Porphyria Foundation. Methods: Urinary samples from patients with acute intermittent porphyria and control samples were analysed and the semi-quantitative results were compared with the results obtained by a manual quantitative spectrophotometric method. Results: In all 32 samples studied, acceptable agreement between the results of the rapid test and the quantitative test was observed. Handling of the test was found to be convenient. Conclusions: The assay was found to be reliable and has the potential to increase the availability of porphobilinogen testing in the field.

Background: Although results from blood gas analyzers are frequently used in clinical work surprisingly few and small studies have examined reference intervals for arterial blood gases and acid-base status. We have established reference values based on a large group of healthy people with a wide age distribution. Methods: A group of medical students (n=182) aged 20–32 years old and a group of health professionals aged 21–76 years were used in this study. Arterial samples were analyzed on the blood gas analyzer ABL from Radiometer TM . Age and gender dependency was examined for all analytes and reference intervals were calculated non-parametrically. Results: Females had significantly higher pH and lower PaCO 2 (partial pressure of carbon dioxide in an arterial sample), base excess (BE, standard, extra cellular fluid), plasma standard and actual HCO 3 , when compared to males (p<0.01). However, the differences were minor and common reference intervals were therefore also determined, generally at the same level as previously published. The lactate values were similar among the genders but with a high upper limit of 2.5 mmol/L. The non-smoker group of females and males had similar PaO 2 values (partial pressure of oxygen in an arterial sample). However, an age dependent effect was found and PaO 2 decreased by 0.29 kPa per decade (confidence interval of slope –0.11 to –0.47 kPa). Electrolytes and anion gap results depicted smaller differences from previous published reference intervals for sodium (136–141 mmol/L) and anion gap (10–16 mmol/L, with potassium included or 6–12 mmol/L without potassium). Conclusions: Reference intervals for analytes on modern blood gas analyzers were established on a large group of healthy people. Gender and age dependency is generally without clinical importance, except for a lower PaCO 2 in women and a decreasing PaO 2 with higher age.

Background: Several articles describing within-subject biological variation of fasting glucose and HbA 1c in healthy populations have been published, but information about biological variation of glucose and HbA 1c in patients with type 1 diabetes is scarce. It is reasonable to assume that type 1 diabetics differ from their healthy counterparts in this matter. The aim of our study was to estimate the biological variation of glucose and HbA 1c in healthy subjects and in patients with type 1 diabetes. Methods: Fifteen healthy individuals and 15 type 1 diabetes patients were included. Biological variations were calculated based on blood samples collected weekly for 10 consecutive weeks from the healthy and the eligible of the type 1 diabetes patients. Results: The within-subject variations of glucose were approximately 5% in healthy individuals and 30% in diabetes patients, and for HbA 1c they were 1.2% in healthy individuals and 1.7% in diabetes patients. Conclusions: In conclusion, we found a high within-subject biological variation of glucose in diabetes patients as expected compared to healthy individuals (30% vs. 5%). The short-term (2 months) within-subject biological variation of HbA 1c did not differ significantly between well regulated type 1 diabetes patients and healthy individuals (1.7% vs. 1.2%).

Background: Magnitude and major causes of oxidative stress may be different between sexes, although limitedly addressed in clinical studies with controversial results. The present study aimed to determine whether any gender-related difference exists concerning oxidative stress in a population of 332 subjects of both sexes, in a wide age range, with and without cigarette smoking habit. Methods: The Oxidative-INDEX was calculated after evaluation of serum hydroperoxides (ROMs) and total antioxidant capacity (OXY) by means of commercial kits (d-ROMs and Oxy-adsorbent Tests, Diacron, Italy) subtracting the OXY standardized variable from the ROMs standardized variable. Results: The Oxidative-INDEX resulted higher in women with respect to men (p<0.001), in smokers (p<0.01) than in non-smokers, and correlated with cigarette number (p<0.01), age (p<0.001), and post-menopausal status (p<0.001). The multivariate analysis identified age, high blood pressure, and smoking habit as factors independently associated with the Oxidative-INDEX in men, whereas cigarette smoking and age represented the independent risk factors for an elevated oxidative stress status in women. Conclusions: Gender-based differences in oxidative stress levels may provide a biochemical basis for the epidemiologic differences in the disease susceptibility between sexes, and suggest different strategies for risk assessment, diagnosis, and treatment specifically targeted to men and women.

Background: Soluble ST2 (sST2) plasma concentrations are significantly higher in healthy men than in healthy women. The reason for the sex-specific difference of sST2 plasma concentrations is not established. The aim of this study was to evaluate the association of sST2 with sex-hormones in healthy males and females separately. Methods: We recruited 528 consecutive blood donors and measured plasma concentrations of sST2 and several sex-hormones (i.e., total testosterone, estradiol, sex hormone-binding globulin, follicle-stimulating hormone, and luteinizing hormone). Of the 528 blood donors, 338 were male and 190 were female. For data analysis, we further divided the group of females into the subgroups of pre- and postmenopausal women using the age of 50 years as a proxy for menopause. Results: In non-parametric Spearman's correlation analyses, we found a weak association between sST2 and total testosterone (r s +0.126, p=0.021) and also between sST2 and estradiol (r s +0.117, p=0.032) in males. In females <50 years of age (n=158) and ≥50 years of age (n=32), respectively, we did not detect any significant association between sST2 and sex-hormones. As a result of multiple linear regression analyses (calculated with log sST2 as dependent variable and log of age and all sex-hormones as explanatory variables), there was no independent association between sST2 and any of the sex-hormones neither in males nor in females. Conclusions: In the present study cohort we did not find an independent association of sST2 with sex-hormones in healthy males and females. Therefore, the reason for the sex-specific difference of sST2 plasma concentrations still remains unclear.

Background: Human immunodeficiency virus type-1 (HIV-1) RNA viral load is a surrogate marker that is routinely used to determine indications for, and monitor the effectiveness of HIV-1 treatment. We developed three reagents for potential use in routine quality control of HIV-1 RNA quantitative assays. In this report, we compare the stability of these re-agents in storage and compare their performance in three different HIV-1 RNA quantitative assays. Methods: The candidate reagents were derived from readily available pre-existing reagents and examined for stability at different storage temperatures. They were compared in three commercially available HIV-1 RNA quantitative assays: the Cobas TaqMan HIV-1 Test (Cobas TaqMan), the RealTime HIV-1 Assay (Abbott RealTime), and the NucliSens EasyQ HIV-1 Assay v1.1 (NucliSens EasyQ). Results: The candidate reagent derived from an HIV culture supernatant (candidate CS) was the most stable of the three candidates and showed good reproducibility. Candidate CS yielded the highest HIV-1 titer of the three candidates in the Cobas TaqMan assay and the lowest HIV-1 titer and stability of the three candidates in the NucliSens EasyQ system. Conclusions: The candidate CS is the most appropriate of the three candidate reagents for quantitative testing of HIV-1 RNA. This working reagent should be useful for use in routine calibration for quality control in centers with limited financial resources. The Cobas TaqMan assay tended to yield higher viral load results than the other assays when used with our three candidate reagents.

Background: In a previous study, a relation between decreased serum angiotensin-converting enzyme (ACE) activity and physiological parameters was observed in patients with community-acquired pneumonia. The present study aims to further assess the prognostic value of serum ACE activity for outcome of community-acquired pneumonia. Methods: This was a prospective observational study including two cohorts of patients with community-acquired pneumonia (2004–2006; n=157 and 2007–2010; n=138). Serum ACE activity was measured at time of hospital admission. Based on reference values in healthy persons, patients were divided into subgroups of serum ACE activity: normal, low and extremely low. Physiological parameters, clinical outcomes and etiology were compared between the subgroups. Results: A total of 265 patients were enrolled in this study. Mean age was 60±19 years. In patients with low serum ACE activity (<20 U/L, n=53), compared to patients with normal serum ACE activity (≥20 U/L, n=212), C-reactive protein (CRP) was significantly increased, systolic blood pressure was significantly lower and there was a trend for higher heart rate and leukocyte counts. Furthermore, Streptococcus pneumoniae was significantly more identified in patients with low serum ACE activity. Serum ACE activity <24 U/L was independently associated with bacteremia (adjusted OR 3.93 [95% CI 1.57–9.87]). Low serum ACE activity was not prognostic for length of hospital stay nor mortality. Conclusions: This study did not show prognostic value for serum ACE activity regarding clinical outcome in patients with community-acquired pneumonia. Serum ACE activity <24 U/L at time of hospitalization appeared an independent indicator for the presence of bacteremia. Further research should elucidate the role of ACE in systemic infection and sepsis during pneumonia.

Background: A low cost colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A virus was developed. The assay was evaluated in this study. Methods: Six hundred and twenty-six patients were enrolled. All patients contributed two pharyngeal swabs, one used for colloidal gold immunochromatographic rapid assay for influenza A virus immediately after the collection of specimen and one used for real-time reverse transcriptase polymerase chain reaction (RT-PCR) test or virus culture at the Centers for Disease Control and Prevention (CDC) influenza network laboratory. Result: In reference to viral culture, GICA influenza A test demonstrated a sensitivity of 64%, a specificity of 95% and an overall accuracy of 93%. Consistency between the GICA test and virus culture assay is moderate, with Kappa being 0.46. In reference to RT-PCR, GICA test demonstrated considerable high sensitivity (74%) and specificity (86%), with Kappa value being 0.61 and overall accuracy of 81%. There was no significant difference between GICA test and virus culture/RT-PCR on the detected positive rates of influenza A cases. Conclusions: GICA is a reliable, rapid, convenient and inexpensive test for the screening and diagnosis of influenza A disease. Given its lower cost than other rapid tests, the GICA test has the great potential in the management of influenza A disease in resource-poor countries.

Background: Increased plasma amino-terminal-cleavage-fragment of NP (NT-proBNP) is an established indicator for heart failure. Moreover, obese adults had low circulating NT-proBNP suggesting an obesity-related dysregulation (natriuretic handicap). Secretion and/or clearance of NT-proBNP were discussed to be impaired in obesity. As only older adults were investigated so far, it remains unclear when during the evolution of obesity the state of a natriuretic handicap develops, and whether NT-proBNP may still serve as a relevant cardiac marker in obese juveniles. Methods: We analysed NT-proBNP in juvenile (n=274, 10–18 years) and middle-aged (n=277, 18–50 years) normal weight (n=213) and obese (n=338) probands together with complex anthropometry, carotis sonography, clinical, and laboratory parameters. Results: NT-proBNP showed a significant gender and age interaction. Adult females had significant higher NT-proBNP than adult males, and higher levels than juvenile females. Adult males had lower levels than juvenile males. Only a weak age and weight interaction was seen with obese juveniles which showed higher NT-proBNP than obese adults. Moreover, normal weight probands had higher NT-proBNP than overweight and obese. In a multiple regression including all probands, gender, creatinine and uric acid were the best predictors for NT-proBNP. In adults, female gender is the strongest driver for increased NT-proBNP. Conclusions: These results argue against an essential influence of obesity to B-type cardiac natriuretic hormone system regulation in the absence of heart failure, and suggest NT-proBNP as a useful cardiac marker irrespective of age and obesity.

Background: Vaspin was a recently identified adipokine, playing a protective role in many metabolic diseases. The present study aimed to investigate the association between vaspin plasma level and stable angina pectoris (SAP) and unstable angina pectoris (UAP). Methods: A total of 88 patients with angiographically-proved coronary artery disease (CAD) (SAP 47, UAP 41) and 103 control subjects without cardiovascular diseases were enrolled in this study. Circulating vaspin, mRNA expression of vaspin in peripheral blood mononuclear cells (PBMC), clinical parameters, lipid profile and high-sensitivity C-reactive protein (hsCRP) were assayed. The severity of CAD was also assessed according to the number of vessels diseased. Results: There are significant differences in circulating vaspin levels and mRNA levels of PBMC between SAP and UAP groups (SAP 0.91±0.95 ng/mL and UAP 0.43±0.38 ng/mL, p<0.01 in circulating vaspin level; SAP 1.19±0.85 and UAP 0.82±0.56, p<0.05 in mRNA level of PBMC). An inverse correlation between the number of diseased vessels and plasma vaspin concentration was observed (r =– 0.350, p<0.01) in the CAD group. Construction of receiver operating characteristic curves confirmed that vaspin plasma concentrations significantly differentiated CAD patients (area under the curve=0.684, p<0.001), as well as UAP (area under the curve=0.640, p<0.05). Conclusion: Decreased vaspin plasma levels and mRNA levels in PBMC were observed in patients with UAP. Low vaspin concentrations correlate with CAD severity. The findings suggested that vaspin could serve as a novel biomarker of CAD as well as UAP.

Background: Abnormalities in homocysteine metabolism have been suggested as risk factors for stroke. The aim of this prospective study was to examine whether total plasma homocysteine concentration (tHcy) and its main genetic determinant, methylene tetrahydrofolate reductase ( MTHFR ) polymorphisms, were associated with first ischemic or hemorrhagic stroke. Methods: This was a nested case-referent study of 321 ischemic and 60 hemorrhagic stroke cases, defined by WHO MONICA criteria and each matched with two event-free referents for sex, age, cohort, recruitment date and geographical area. All subjects were from the population-based Northern Sweden Health and Disease Study cohorts. Odds ratios were determined by conditional logistic regression. Results: The mean follow-up time was 4.2 years. Both tHcy and MTHFR were independent predictors of hemorrhagic stroke in multivariate models including body mass index, hypertension and, for MTHFR , tHcy [OR for the highest vs. lowest tHcy quartile 8.13 (95% CI 1.83–36.1), p trend =0.002; OR for MTHFR 677TT vs. 677CC genotype 3.62 (95% CI 0.77–17.0), p trend =0.040]. Haplotype analyses confirmed that the MTHFR 677T–1298A haplotype was positively associated with hemorrhagic stroke [OR 1.81 (95% CI 1.09–3.00), p=0.022], whereas the MTHFR 677C–1298C haplotype was not significantly related to either hemorrhagic or ischemic stroke. Neither tHcy nor the MTHFR polymorphisms were significant predictors of ischemic stroke. Conclusion: Both elevated plasma homocysteine levels and the MTHFR 677T allele are indicators of increased risk of hemorrhagic stroke in the northern Swedish population.