Evaluation of Hepcidin and Atherosclerosis in Dialysis Patients.

BACKGROUND: Chronic kidney disease (CKD) is a condition that involves 10% - 15% of population worldwide, which increases the risk of cardio-vascular diseases (CVD). Chronic kidney disease is one of the main reasons for illness and mortality in the world. Chronic kidney disease is a serious health problem caused by involvement of a large number of patients with kidney injury, especially in industrial countries. Among the main reasons for this are population living longer and the number of diseases in elderly persons, such as diabetes mellitus type 2, hypertension, and cardio-vascular diseases. METHODS: We evaluated 63 patients on chronic dialysis at the Dialysis Centre at University "Aleksandrovska" Hospital; the average age was 49.9 ± 7.8. Their results were compared to 63 age matched controls. Blood samplings were taken before dialysis procedure. In the included groups, we measured CBC, serum iron (by Ferrozine method), ferritin, soluble transferrin receptors and hsCRP (by nephelometric method), hepcidin (by ELISA method), and homocysteine (by CLIA method). IMT was measured by using electronic calipers and evaluated by automated software programs. RESULTS: We established elevated serum hepcidin levels in CKD patients (205.1 ± 29.9 µg/L) compared to the control group (20.8 ± 3.1 µg/L), p < 0.001. Serum homocysteine and hsCRP concentrations were elevated in CKD cases (48.7 ± 6.8 µmol/L; 29.7 ± 4.1 mg/L) compared to controls (7.9 ± 1.8 µmol/L; 1.1 ± 0.4 mg/L), p < 0.005. In patients with CKD we found a strong positive correlation between serum hepcidin and homocysteine concentrations, r = 0.879, p < 0.001. In patients with impaired kidney function soluble transferrin receptors correlated negatively to hepcidin: r = -0.799, p < 0.001. In dialysis, the transferrin concentration correlated highly positive to hepcidin: r = 0.691, p < 0.001. IMT in CKD patients correlated positively to hepcidin and homocysteine levels: r = 0.788 and r = 0.841, respectively, p < 0.005. CONCLUSIONS: Chronic kidney disease is connected to cardio-vascular disease risk factors. CKD might be an independent CVD risk factor. In early kidney injury stages, increased morbidity is found from CVD. The risk of fatal and non-fatal cardio-vascular incidents is connected to kidney injury. For clinical practice, early evaluation of hepcidin and atherosclerosis in chronic kidney disease patients is very important.

Plasma Selenium Levels in Chronic Cocaine Abuse.

BACKGROUND: Cocaine abuse is a significant health problem worldwide. We aim to evaluate the role of selenium in oxidative stress in patients with cocaine related cardio- and cerebrovascular diseases. Selenium is of fundamental importance to human health. It is an essential component of several major metabolic pathways. METHODS: We included 26 patients with chronic cocaine abuse separated into two groups according to duration of intake--from six months up to one year and more than a year, with and without only vascular incidents (TIA, stroke, myocardial infarction), or presence of hypertension. All included groups were analyzed for laboratory parameters: CBC, CRP, AST, ALT, γ-GT, serum creatinine, urea, K, Na, and Ca. Main risk factors were evaluated: total cholesterol, LDL and HDL-cholesterol, triglycerides, glucose, and selenium. RESULTS: Our result shows that in the first group only 20% have cardiovascular problems. In the group with cocaine abuse more than a year, number of vascular incidents has increased (58.3%). Patients from the group cocaine abuse up to 1 year showed no changes in lipid profile, but cocaine abuse more than a year showed interesting changes in the lipid profile. We found a high positive correlation between the two cocaine groups for plasma selenium concentrations. This means that after the first six months oxidative stress has occured. It increases with duration of cocaine abuse. CRP correlated positively between these two groups, showing an endothelial function disorder. CONCLUSIONS: Development of oxidative stress increased with cocaine abuse, which leads to lower plasma selenium levels in patients with different duration of intake--less and more than a year. It is possible to supplement selenium during the early period of cocaine dependence to prevent hard vascular accidents, which are common after more than one year of cocaine abuse.

Is There a Link Between Changes in Levels of Hepcidin and Stroke?

BACKGROUND: Iron is an essential element for the living body. It is well known that iron homeostasis disorders are important in two ways--its deficiency and its overload lead to several pathologies. METHODS: We measured 17 patients with iron deficiency anemia (IDA); 19 with anemia of chronic diseases (ACD); 15 with ischemic stroke (IS). The results were compared to a previously selected control group. For evaluation of iron metabolism status, we measured serum iron levels, ferritin, and soluble transferrin receptors. For inflammation, serum interleukin-6 and hsCRP were measured. Serum hepcidin quantification was performed using a previously validated immunosorbent method. Ferritin was measured by an ECLIA method; serum iron on AAS; hsCRP using a nephelometric analysis. RESULTS: We found statistically significant elevated serum hepcidin levels in patients with ACD and IS compared to the control group (p < 0.001). Patients with IDA had statistically significant lower hepcidin levels compared to the control group (p < 0.001). Serum ferritin levels in the IS group was higher compared to the control and other groups (p < 0.001). The lowest ferritin concentrations were established in the IDA group compared to the control (p < 0.001). We found a strong correlation between serum hepcidin and ferritin levels in the IS group (r = 0.583; p < 0.001). CONCLUSIONS: Quantification of serum hepcidin levels might be used as a link for prediction of acute ischemic stroke and future therapeutic influences.

ADMA--a Possible Marker for Early Therapeutic Outcome in Acute Stroke.

BACKGROUND: Ischemic acute stroke is a leading cause for mortality and invalidity in recent years. Clinical trials show the role of ADMA as endogenous inhibitor of nitric oxide synthase and its connection to acute stroke. An early decrease of serum ADMA levels might help in recovery of the blood stream in patients with acute stroke and non-affective changes in the brain. METHODS: We compared 18 patients with acute stroke before and after thrombolysis and 21 patients (without history of cardiovascular disease, diabetes, kidney and liver injury, non-smokers) before and after thrombolysis. All results were compared to 30 healthy persons. Patients were evaluated by NIHSS, with ultrasound, CT and laboratory methods (traditional risk factors and CRP, D-dimmer, homocysteine, and ADMA). RESULTS: Classic vascular factors showed statistical significance in patients with acute stroke. Serum hsCRP levels and D-dimer in our study showed no connection to acute stroke. We found a statistically significant correlation in patients with stroke between ADMA and homocysteine levels (r = 0.469, p < 0.001). We found no significant difference between basic serum ADMA concentrations in groups with stroke who underwent different treatment (r = -0.449, p < 0.001). In patients with thrombolysis ADMA concentrations are reduced earlier, which also shows that earlier clinical recovery leads to no brain damaging effects. CONCLUSIONS: Through its pathophysiological changes, ADMA might be a predictor for acute stroke (along with already known risk factors) and can be a marker for the outcome of stroke, depending on the treatment.

Serum hepcidin levels in Bulgarian population.

BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Hepcidin quantification in human serum provides new insights for the pathogenesis of disorders of iron homeostasis. This study describes an ELISA immunoassay for hepcidin quantification in human serum and reference ranges for Bulgarian population. METHODS: We used a sandwich ELISA method from USCN Life Science inc. that consists of ready to use, pre-coated 96-well strip plate with 2 antihepcidin-25 monoclonal antibodies. A recombinant Hepcidin in 16 µg/L concentration is used as a standard. We correlated ELISA results of hepcidin-25 measurements in healthy population to ferritin, hemoglobin concentration in reticulocytes, transferrin, and iron levels. RESULTS: The sandwich ELISA was highly specific for hepcidin-25. We found that serum hepcidin levels for Bulgarian population are 3.052 µg/L - 37.750 µg/L, which is quite similar to that established by WCX-TOF MS from the Laboratory of Genetic, Endocrine and Metabolic Diseases; Dept. of Laboratory Medicine, Radbound University Medical Centre; Nijmegen, The Netherlands. Ferritin levels and hemoglobin concentration in reticulocytes correlated significantly to serum hepcidin levels (0.3 < r < 0.5, p < 0.010). Transferrin levels showed negative and no significant correlation to hepcidin in serum (r = -0.111). CONCLUSIONS: The use of two monoclonal antibodies in a sandwich ELISA format provides a reliable, reproducible, and not very expensive method for measuring serum concentrations of the bioactive form of hepcidin in Bulgarian laboratory practice.

The EC4 European syllabus for post-graduate training in clinical chemistry and laboratory medicine: version 4--2012.

Laboratory medicine's practitioners across the European community include medical, scientific and pharmacy trained specialists whose contributions to health and healthcare is in the application of diagnostic tests for screening and early detection of disease, differential diagnosis, monitoring, management and treatment of patients, and their prognostic assessment. In submitting a revised common syllabus for post-graduate education and training across the 27 member states an expectation is set for harmonised, high quality, safe practice. In this regard an extended 'Core knowledge, skills and competencies' division embracing all laboratory medicine disciplines is described. For the first time the syllabus identifies the competencies required to meet clinical leadership demands for defining, directing and assuring the efficiency and effectiveness of laboratory services as well as expectations in translating knowledge and skills into ability to practice. In a 'Specialist knowledge' division, the expectations from the individual disciplines of Clinical Chemistry/Immunology, Haematology/Blood Transfusion, Microbiology/ Virology, Genetics and In Vitro Fertilisation are described. Beyond providing a common platform of knowledge, skills and competency, the syllabus supports the aims of the European Commission in providing safeguards to increasing professional mobility across European borders at a time when demand for highly qualified professionals is increasing and the labour force is declining. It continues to act as a guide for the formulation of national programmes supplemented by the needs of individual country priorities.

Correlation between rotation thrombelastometry ROTEM analysis and standard haemostatic parameters during liver transplantation.

BACKGROUND: The determination of the correlations between simultaneously performed rotation thrombelastometry ROTEM analysis and standard haemostatic analysis during liver transplantations is indispensable for performing an adequate perioperative haemostatic monitoring. METHODS: Perioperative haemostatic monitoring was performed to 30 patients undergoing orthotopic liver transplantation (13 male (42%) and 17 female (58%), age: (mean +/- SD; 21 +/- 17 years). Standard coagulation parameters (PT, APTT, FIB) were assessed chronometrically on STA-Compact Analyzer (Diagnostica Stago - La Roche), rotation thrombelastometry analyses - on ROTEM analyzer (Petapharm GmbH) and platelets (PLT) - on Cell Dyn 3700 (Abbott Diagnostica), MAPSS technology. RESULTS: A protocol was successfully developed for the implementation of perioperative haemostatic control during orthotopic liver transplantations, performing parallel thrombelastometric and standard haemostatic analyses. Significant correlation was established between PT(INR) and EXTEM_CFT ( r = 0.834; p < 0.001) and between APTT and INTEM_CFT (r = 0.707; p < 0.001) in the preoperative period (R1). The correlation was reduced to insignificant during the intraoperative periods (R2-R5) and two hours postoperatively (R6). Significant correlation was determined between PLT/INTEM and between FIB/MCF_FIBTEM during all perioperative periods (R1 -R6). CONCLUSIONS: The correlations found in the present study suggest to perform the haemoststic liver transplantation monitoring through a parallel systematic analysis of both standard and rotation thrombelastometry parameters and confirm the ROTEM method as preferable and highly informative.

The European Register of Specialists in Clinical Chemistry and Laboratory Medicine: guide to the Register, version 3-2010.

In 1997, the European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) set up a Register for European Specialists in Clinical Chemistry and Laboratory Medicine. The operation of the Register is undertaken by a Register Commission (EC4RC). During the last 12 years, more than 2200 specialists in Clinical Chemistry and Laboratory Medicine have joined the Register. In 2007, EC4 merged with the Forum of European Societies of Clinical Chemistry and Laboratory Medicine (FESCC) to form the European Federation of Clinical Chemistry and Laboratory Medicine (EFCC). Two previous Guides to the Register have been published, one in 1997 and another in 2003. The third version of the Guide is presented in this article and is based on the experience gained and development of the profession since the last revision. Registration is valid for 5 years and the procedure and criteria for re-registration are presented as an Appendix at the end of the article.

The European Register of Specialists in Clinical Chemistry and Laboratory Medicine: Code of Conduct, Version 2--2008.

In 1997, the European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) set up a Register for European Specialists in Clinical Chemistry and Laboratory Medicine. The operation of the Register is undertaken by a Register Commission (EC4RC). During the last 10 years, more than 2000 specialists in Clinical Chemistry and Laboratory Medicine have joined the Register. In 2007, EC4 merged with the Federation of European Societies of Clinical Chemistry and Laboratory Medicine (FESCC) to form the European Federation of Clinical Chemistry and Laboratory Medicine (EFCC). A Code of Conduct was adopted in 2003 and a revised and updated version, taking account particularly of the guidelines of the Conseil Européen des Professions Libérales (CEPLIS) of which EFCC is a member, is presented in this article. The revised version was approved by the EC4 Register Commission and by the EFCC Executive Board in Paris on 6 November, 2008.

Ascorbic acid--important for iron metabolism.

Ascorbic acid is actively involved in the control of iron metabolism. It has long been known to enhance absorption of iron from test meals. At first this effect was ascribed to luminal reduction and solubilsation of iron. Later, molecular cloning of mammalian duodenal brush-border reductase activity and studies in animals and man strongly supported ascorbate as the intracellular electron donor for duodenal ferri-reductase activity and provided molecular mechanism for an intracellular role of ascorbate in intestinal iron absorption. Factors that alter duodenal ascorbate levels (dietary intake of ascorbate, dehydroascorbate, or oxidants) may therefore alter the rate of absorption. Ascorbate could play dual role in human cells; it could react as pro-oxidant and as antioxidant. The balance of these contradictory effects depends on ascorbate concentration. Pro-oxidant reactions predominate at low concentrations; at higher concentrations vitamin C reacts as antioxidant. The increase of plasma ascorbate in human iron deficiency, especially in females in active age, could explain gender-related biological variation of plasma levels of vitamin C. The possible participation of ferric reductase activity Dcytb in transferrin cycle in liver and in neutrophil host defense implies new aspects of the role of vitamin C in the regulation of iron homeostasis.

Duodenal ascorbate and ferric reductase in human iron deficiency.

BACKGROUND: The first step in iron absorption requires the reduction of ferric iron to ferrous iron, a change that is catalyzed by duodenal ferric reductase. Iron deficiency is associated with high iron absorption, high ferric reductase activity, and high duodenal ascorbate concentrations in experimental animals, but it is not known whether a relation between reductase and ascorbate is evident in humans. OBJECTIVE: The objective of the study was to assess the relation between ferric reductase activity in human duodenal biopsy specimens and ascorbate concentrations in iron-replete and iron-deficient subjects. DESIGN: Patients and control subjects were overnight-fasted adults presenting sequentially for upper gastrointestinal endoscopic investigation. Ferric reductase activity in duodenal biopsy specimens was assayed by using nitroblue tetrazolium. Ascorbate was assayed in duodenal biopsy specimens and plasma. RESULTS: Iron-deficient patients had significantly higher reductase activity (n = 6-9; P < 0.05) and duodenal (n = 20; P < 0.001) and plasma (n = 6; P < 0.001) ascorbate concentrations than did control subjects. Incubation of biopsy specimens with dehydroascorbate (to boost cellular ascorbate) increased reductase activity in the tissues that initially had normal activity (n = 9; P < 0.01) but inhibited reductase activity in the tissues that already had high reductase activity (n = 13; P < 0.001). CONCLUSIONS: Iron deficiency in humans is associated with increased duodenal ascorbate concentrations. This finding suggests that increased reductase activity is partly due to an increase in this substrate for duodenal cytochrome b reductase 1.

Duodenal ascorbate levels are changed in mice with altered iron metabolism.

Ascorbate has long been thought to play an important role in intestinal iron absorption. The recent identification of a possible ascorbate-dependent duodenal ferric reductase suggests a role for intracellular ascorbate in the control of iron absorption. We set out to determine whether duodenal ascorbate concentrations are altered by treatments known to alter the rate of iron absorption and whether ascorbate levels affect duodenal reductase activity. Duodenal ascorbate was extracted and assayed by HPLC and/or a chemical assay. Ferric reductase was assayed in vitro with ferric nitrilotriacetate or nitroblue tetrazolium as substrates. Duodenal ascorbate concentrations were increased by iron deficiency, genetic hypotransferrinemia, and hypoxia. Parenteral iron overload increased iron stores but did not affect duodenal ascorbate concentrations. Hemolytic anemia induced in mice by phenylhydrazine injection also did not affect duodenal ascorbate concentrations. In vitro studies with incubated duodenum showed that decreased tissue ascorbate was associated with decreased mucosal ferric reductase activity, whereas incubation with dehydroascorbate prevented both the decrease in ascorbate concentration and reductase activity. Mouse duodenum ascorbate concentrations changed in response to treatments that altered iron absorption rates; in particular, ascorbate levels generally increased when iron absorption was increased by iron deficiency, hypoxia, or genetic hypotransferrinemia. We conclude that changes in ascorbate levels are associated with changes in ferric reductase activity. These findings are consistent with the proposal that duodenal ascorbate plays a role in intestinal iron absorption.