The importance of new biomarkers of inflammation and angiogenesis in peripheral arterial disease

Periferna arterijska bolest klinička je manifestacija ateroskleroze sa značajnim morbiditetom i mortalitetom. Upala i angiogeneza imaju značajnu ulogu u njenom razvoju, progresiji i komplikacijama. Cilj rada bio je ispitati postoji li povezanost izmeĎu katalitičkih koncentracija i genetskog polimorfizma paraoksonaze (PON1), katalitičkih koncentracija acetil hidrolaze trombocitnog čimbenika aktivacije (PAF-AH), te koncentracija vaskularnog endotelnog čimbenika rasta (VEGF), angiopoietina 2 (Ang-2) i njegovog Tie-2 receptora, kao novih biomarkera u procesu upale i angiogeneze, sa pojavnošću i stupnjem stenotično/okluzivnih promjena perifernih arterija. Snižene katalitičke koncentracije PON1 i polimorfizmi Q192R i -108C>T pon1 gena mogli bi imati ulogu u razvoju periferne arterijske bolesti. Razlike u frekvenciji Q i R alela izmeĎu ispitanika i kontrolne skupine mogu biti jedan od uzroka sniženih katalitičkih koncentracija PON1, što može doprinosti smanjenoj zaštitnoj ulozi HDL-kolesterola i povećanom riziku od prijevremene ateroskleroze kod rizičnih skupina bolesnika. Povećane koncentracije Ang-2 i Tie-2 receptora mogle bi ukazivati na povećano vaskularno remodeliranje kao odgovor na prisutne čimbenike rizika, te bi se mogli smatrati novim biomarkerima angiogeneze koji ukazuju na prisustvo periferne arterijske bolesti. Nepostojanje povezanosti izmeĎu koncentracija istraživanih biokemijskih parametara i angiografskog sustava bodovanja, kao mjere anatomske proširenosti aterosklerotskih promjena perifernih arterija ukazuje na važnost drugih čimbenika u progresiji bolesti.Peripheral artery disease is a clinical manifestation of atherosclerosis with significant morbidity and mortality. Inflammation and angiogenesis play an important role in its development, progression and complications. The aim of this thesis was to investigate the association of the catalytic concentrations and genetic polymorphism of paraoxonase (PON1), the catalytic concentrations of platelet activating factor acetylhydrolase (PAF-AH), the concentrations of vascular endothelial growth factor (VEGF), angiopoietin 2 (Ang-2) and its receptor Tie-2, as novel biomarkers of inflammation and angiogenesis with the appearance and degree of stenotic-occlusive changes in the periphearal arteries. Lower catalytic concentrations of PON1 and polymorphisms Q192R and - 108C>T pon1 gene could play a role in the development of peripheral arterial disease. Differences in the frequency of Q and R alleles between patient and control groups may be one of the causes of the reduced catalytic concentrations of PON1, which may contribute to a reduced protective role of HDL-cholesterol and increased risk of early atherosclerosis in the risk group of patients. An increased concentration of Ang-2 and Tie-2 receptor could indicate increased vascular remodeling in response to the presence of risk factors and could be considered new biomarkers of angiogenesis which indicate the presence of peripheral arterial disease. The absence of significant correlation between the concentrations of the biochemical parameters investigated and the angiographic score as a measure of the anatomic extent of atherosclerotic lesions to the peripheral arteries, suggests that other factors are more important in the progression of the disease

Applicability of common reference intervals for serum creatinine concentrations to the Croatian population

Background: In accordance with an ongoing activity for worldwide harmonization based on traceability in laboratory methods, the goal of this study was to validate the applicability of recommended “common” reference intervals for serum creatinine concentrations using a specific enzymatic method to the Croatian population. Methods: The reference group consisted of 240 healthy subjects (120 males and 120 females), between 18 and 74 years of age (median 57 years), who were selected in accordance with the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommendations. Creatinine in serum was measured using the creatinine enzymatic assay (Olympus OSR61204) that was standardized to the isotopic dilution mass spectrometry (IDMS) method and National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 967. In addition, creatinine was measured using a kinetic Jaffe method (Olympus OSR6178) standardized to NIST SRM 909b level 2 standard. Results: Method comparison between enzymatic creatinine (x) and the Jaffe kinetic method (y) gave the following P/B equation for the entire group (n=240): y=1.00x+17.00; r=0.968. Reference intervals for serum creatinine (central 95th percentiles) obtained using the enzymatic creatinine method ranged from 54 to 107 μmol/L for males and from 50 to 93 μmol/L for females. The IFCC recommended common reference intervals for global applications are 64–104 μmol/L and 49–90 μmol/L for males and females, respectively. Conclusions: Comparability of obtained results confirmed the applicability of recently recommended “common” reference intervals to the Croatian population for all laboratories measuring serum creatinine concentrations using enzymatic methods traceable to the IDMS method and NIST SRM 967. Clin Chem Lab Med 2010;48:231–5.Peer Reviewe

KONCENTRACIJE SRČANOG VISOKOOSJETLJIVOG TROPONINA I U MOKRAĆI ZDRAVIH OSOBA – PRELIMINARNI REFERENTNI INTERVALI

Recent studies have shown the presence of troponin molecules in urine, with kidneys considered the main organs of elimination. Availability of the new generation of high-sensitivity assays has enabled detection of low concentrations of circulating cardiac troponins, but the high-sensitivity troponin assays are not designed for analysis of urine specimens. The aim of this study was to establish preliminary reference intervals for the high-sensitivity cardiac troponin I (hs-cTnI) concentrations in urine of healthy adults in Croatia. A total of 60 reference persons were selected (30 males and 30 females) and the concentrations of hs-cTnI in random urine samples were determined on the Abbott Architect i1000SR (Abbott Laboratories) analytical system with chemiluminescent immunochemical method on microparticles (CMIA, ARCHITECT STAT High Sensitive Troponin-I), accredited according to the HR EN ISO 15189:2012 standard (Medical laboratorieds – requirements for quality and competence. Geneva, International Organization for Standardization, 2012). We determined the limit of detection, total laboratory precision, expanded measurement uncertainty and preliminary estimates of the gender-specifi c 99th percentile of the upper reference limit (URL) using nonparametric analysis (methods). The male and female 99th percentile cut-off values were 39.3 and 35.2 pg/mL, respectively. The results of this pilot study suggest that troponin I is removed from the blood by the kidneys and can be determined in the urine with CMIA, ARCHITECT STAT High Sensitive Troponin-I assays. Further research is focused on detailed studies of biochemistry and determination of troponin I in the urine as a new biological marker.Cilj ovoga istraživanja bio je odrediti preliminarne referentne intervale visokoosjetljivog troponina l u mokraći zdravih osoba. Koncentracije srčanog visokoosjetljivog troponina I u slučajnim uzorcima mokraće određene su na analitičkom sustavu Abbott Architecti1000SR (Abbott Laboratories) kemiluminiscentnom imunokemijskom metodom na mikročesticama (CMIA, ARCHITECT STAT High Sensitive Troponin-I), akreditiranom prema HR EN ISO 15189. Referentni interval izrađen je u Referentnom centru Ministarstva zdravstva Republike Hrvatske za izradu referentnih vrijednosti u području opće medicinske biokemije na uzorku od 30 referentnih osoba po spolu sljedećih karakteristika: nepušač, 25-65 godina, indeks tjelesne mase <30 kg/m2, odsutnost akutne i kronične bolesti, bez noćnog rada tijekom zadnjih 30 dana. Granica kvantifi kacija određena je sukladno smjernicama CLSI EP17-A2 i uz zadovoljavanje kriterija za preciznost za visokoosjetljivi test (KV<10 %). Linearnost metode u deklariranom području potvrđena je multikalibracijskom krivuljom. Ukupna laboratorijska preciznost izračunata je sukladno smjernicama CLSI EP15-A2 i iznosi 2,23 % (KV) u normalnom području i 1,45% (KV) u patološkom području. Proširena mjerna nesigurnost (k=2) izračunata je iz ukupne laboratorijske preciznosti i mjerne nesigurnosti kalibratora i iznosi ±4,5 % za normalno područje i ±2,9 % za patološko područje; 99. percentila gornje granice referentnog intervala izračunata je neparametarskom statističkom analizom, sukladno smjernicama CLSI C28-A3. Određena 99. Percentila gornje granice referentnog intervala za visokoosjetljivi troponin I u mokraći iznosi 39,3 ng/L za muškarce i 35,2 ng/L za žene. Dobiveni preliminarni rezultati ukazuju na to da se troponin I odstranjuje iz krvi putem bubrega i da se može pouzdano mjeriti na analitičkom sustavu Abbott Architecti1000SR (Abbott Laboratories) kemiluminiscentnom imunokemijskom metodom na mikročesticama

Implementation of the N - terminal proB-type Natriuretic Peptide Test in National Guidelines for Diagnosis of Heart Failure in Croatia

Aim. In this article we aimed to analyze the implications of using new method of determination N - terminal proB - type natriuretic peptide (NT-proBNP) biomarker for rapid diagnosis in Emergency Room of acute heart failure and for prediction of adverse cardiovascular outcome. Methods. Electro-chemiluminescence immunoassay is sandwich principle test with two monoclonal NT-proBNP-specific antibodies. PreciControl Cardiac II level 1 and level 2 with values of 150 ng/L and 4930 ng/L were analyzed by Electro-chemiluminescence immunoassay on Roche Cobas e411, in triplicate for five consecutive days in purpose for calculating within laboratory precision, according to Clinical and Laboratory Standards Institute (CLSI) protocol. Results. According to CLSI protocol we calculated standard deviation and coefficient of variation for repeatability, intermediate precision and within laboratory precision from control results. Calculated coefficient of variation for the within laboratory precision for level 1 was 4,48% and for level 2 was 4,15%. Conclusion. Despite very good curative cardiology, Croatia is still among the countries with high cardiovascular risks and mortality. Through the mutual dialogue and activities between leaders of Croatian Cardiac Society, Croatian Society of Hypertension and Society of Family Physicians, the consensus for the development of the Croatian Guidelines for the Diagnosis of Heart Failure (HF) has been reached

High sensitive troponin concentration stability in dialysate of anuric patients on hemodialysis

Background. High sensitive troponin I (hsTnI) and high sensitive troponin T (hsTnT) are markers of cardiac damage. Cardiomyocyte necrosis increases its blood levels. It is known that dialysis is cardiotoxic and that results in lack of contractility of certain myocardial segments. Tis mechanism is primarily due to hypo perfusion of the myocardium during dialysis. Te dialysis itself increases cardiovascular (CV) risk in patients by many different mechanisms. It has been proven that the incidence of heart failure is much more frequent in patients on hemodialysis than in healthy population. Te aim of this pilot study was to investigate the presence of troponin T molecules and troponin I in dialysate and compare their concentrations. Materials and Methods. Te study included 5 anuric patients (4M) on hemodialysis. Te dialysate samples were sampled for each patient three times during a dialysis cycle. Te frst sample was taken afer thirty minutes, the second sample was taken in the middle of dialysis (120 minutes) and the third sample was taken thirty minutes before the end of dialysis. Te value of hsTnI was measured using a high-sensitivity test on the Immuno-enzymatic analyzer Abbott Architest i1000SR. According to CLSI EP15-A2 protocol verifcation of hsTnT chemiluminescent micro-particle immunoassay on the analytical platform Roche cobas e411 was performed. Results. Altogether 15 samples (three for each patient) were processed. hsTnT was detected in all 15 samples (13.42 ± 1.18 ng / L), while hsTnI was detected in only 8 samples (0.14 ± 0.16 ng / L). To test the difference in detectability between hsTnT and hsTnI, chi square test was used and the difference was statistically signifcant (Yates chi-square 6.708, p = 0.009). Conclusion. Te presence of troponin molecules in dialysate was determined for the frst time in scientifc literature. Tis study has confrmed that TnT is present in all dialysate samples and that its concentration is stable in dialysate. TnI concentrations were detectable in signifcantly lower concentrations

utjecaj toplinskog stresa na energetski metabolizam u krava simentalske pasmine tijekom peripartalnog razdoblja

Heat stress adversely affects the metabolism and production of dairy cows, particularly during the transition period. The aim of this study was to evaluate energy metabolism in heat stressed dairy cows during the periparturient period. Twenty-four Simmental dairy cows were included in the study. The cows were assigned into two groups according to the season: the summer group (n = 12) and autumn group (n = 12). Physiological parameters (rectal temperature, respiratory and heart rate) and serum metabolic parameters (glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), non-esterified fatty acids (NEFA) and beta-hydroxy butyrate (BHB) were measured at days -21, -7, 8, 16, 24, 32 and 40 relative to calving. There were no significant differences in glucose concentration between the investigated groups (P0,05). Koncentracija NEFA-e bila je statistički znakovito veća (P<0,05) u ljetnoj skupini 7 dana prije teljenja, dok je 16 i 32 dana nakon teljenja bila znakovito manja (P<0,05) u usporedbi s jesenskom skupinom. U ljetnoj je skupini koncentracija BHB bila manja nakon teljenja u odnosu na jesensko razdoblje, sa statističkom znakovitošću 8., 16. i 24. dan laktacije (P<0,05). Lipidni pokazatelji (ukupni kolesterol i HDL-C) također su bili znakovito manji (P<0,05) u ljetnoj skupini nakon teljenja u usporedbi s jesenskom skupinom. Rezultati pokazuju da su promjene u energetskom metabolizmu mliječnih krava pod utjecajem toplinskog stresa usmjerene na smanjenje oksidacije slobodnih masnih kiselina, kako bi se smanjila proizvodnja metaboličke topline. Ova se metabolička prilagodba pojavljuje većinom nakon teljenja jer su mliječne krave mnogo osjetljivije na toplinski stres u ranoj laktaciji budući da se tijekom proizvodnje mlijeka stvara više metaboličke topline nego u kasnoj gravidnosti tijekom suhostaja

KONCENTRACIJE SRČANOG VISOKOOSJETLJIVOG TROPONINA I U MOKRAĆI ZDRAVIH OSOBA – PRELIMINARNI REFERENTNI INTERVALI

Recent studies have shown the presence of troponin molecules in urine, with kidneys considered the main organs of elimination. Availability of the new generation of high-sensitivity assays has enabled detection of low concentrations of circulating cardiac troponins, but the high-sensitivity troponin assays are not designed for analysis of urine specimens. The aim of this study was to establish preliminary reference intervals for the high-sensitivity cardiac troponin I (hs-cTnI) concentrations in urine of healthy adults in Croatia. A total of 60 reference persons were selected (30 males and 30 females) and the concentrations of hs-cTnI in random urine samples were determined on the Abbott Architect i1000SR (Abbott Laboratories) analytical system with chemiluminescent immunochemical method on microparticles (CMIA, ARCHITECT STAT High Sensitive Troponin-I), accredited according to the HR EN ISO 15189:2012 standard (Medical laboratorieds – requirements for quality and competence. Geneva, International Organization for Standardization, 2012). We determined the limit of detection, total laboratory precision, expanded measurement uncertainty and preliminary estimates of the gender-specifi c 99th percentile of the upper reference limit (URL) using nonparametric analysis (methods). The male and female 99th percentile cut-off values were 39.3 and 35.2 pg/mL, respectively. The results of this pilot study suggest that troponin I is removed from the blood by the kidneys and can be determined in the urine with CMIA, ARCHITECT STAT High Sensitive Troponin-I assays. Further research is focused on detailed studies of biochemistry and determination of troponin I in the urine as a new biological marker.Cilj ovoga istraživanja bio je odrediti preliminarne referentne intervale visokoosjetljivog troponina l u mokraći zdravih osoba. Koncentracije srčanog visokoosjetljivog troponina I u slučajnim uzorcima mokraće određene su na analitičkom sustavu Abbott Architecti1000SR (Abbott Laboratories) kemiluminiscentnom imunokemijskom metodom na mikročesticama (CMIA, ARCHITECT STAT High Sensitive Troponin-I), akreditiranom prema HR EN ISO 15189. Referentni interval izrađen je u Referentnom centru Ministarstva zdravstva Republike Hrvatske za izradu referentnih vrijednosti u području opće medicinske biokemije na uzorku od 30 referentnih osoba po spolu sljedećih karakteristika: nepušač, 25-65 godina, indeks tjelesne mase <30 kg/m2, odsutnost akutne i kronične bolesti, bez noćnog rada tijekom zadnjih 30 dana. Granica kvantifi kacija određena je sukladno smjernicama CLSI EP17-A2 i uz zadovoljavanje kriterija za preciznost za visokoosjetljivi test (KV<10 %). Linearnost metode u deklariranom području potvrđena je multikalibracijskom krivuljom. Ukupna laboratorijska preciznost izračunata je sukladno smjernicama CLSI EP15-A2 i iznosi 2,23 % (KV) u normalnom području i 1,45% (KV) u patološkom području. Proširena mjerna nesigurnost (k=2) izračunata je iz ukupne laboratorijske preciznosti i mjerne nesigurnosti kalibratora i iznosi ±4,5 % za normalno područje i ±2,9 % za patološko područje; 99. percentila gornje granice referentnog intervala izračunata je neparametarskom statističkom analizom, sukladno smjernicama CLSI C28-A3. Određena 99. Percentila gornje granice referentnog intervala za visokoosjetljivi troponin I u mokraći iznosi 39,3 ng/L za muškarce i 35,2 ng/L za žene. Dobiveni preliminarni rezultati ukazuju na to da se troponin I odstranjuje iz krvi putem bubrega i da se može pouzdano mjeriti na analitičkom sustavu Abbott Architecti1000SR (Abbott Laboratories) kemiluminiscentnom imunokemijskom metodom na mikročesticama

Utjecaj klinoptilolita dodanog u hranu na profil minerala u krvnom serumu mliječnih krava

The objectives of this study were to determine whether or not dietary clinoptilolite (CPL) has an influence on the levels of calcium (Ca), magnesium (Mg), phosphorus (P), potassium (K) and sodium (Na) in the blood serum of dairy cows during gravidity and early lactation. The study was conducted on 78 dairy cows of Holstein-Friesian breed. The cows were randomly assigned into two groups: the CPL- fed treated group (n = 38) which received 50 g of natural powdered zeolite CPL twice a day from day 180 days before to 60 days after parturition, and the control non-treated group (n = 40). Blood samples were taken on days 180, 90, 60, 30 and 10 before parturition, on the day of calving and on days 5, 12, 19, 26, 33, 40 and 60 following parturition. There were no significant differences in the Ca concentrations between the CPL-fed and the control group. However, after parturition it was noticeable that the Ca concentration was higher in the CPL-fed group, especially on day 33 (P = 0.06). The concentration of P was significantly lower (P<0.05) in the CPL-fed group on day 0 and day 5 in comparison to the control group. A significantly higher ratio of Ca:P was calculated in the CPL- fed group vs. the control group on days 0, and 12. There were no significant differences observed in Mg, K, and Na concentrations between the cows in the CPL-fed group and the control group throughout the duration of the study. The results of the study suggest that dietary CPL influenced the blood levels of Ca and P in dairy cows, and improved the serum Ca:P ratio of dairy cows during the early postpartal period. The CPL applied did not produce any clinically visible disorders in the metabolism of the tested minerals. In addition, the blood levels of all tested minerals were within physiological ranges, which indicates that CPL did not alter their homeostasis in dairy cows.Cilj je ovoga istraživanja bio ustvrditi utjecaj klinoptilolita (KPL) dodanog u hranu na razine kalcija (Ca), magnezija (Mg), fosfora (P), kalija (K) i natrija (Na) u krvnom serumu mliječnih krava tijekom gravidnosti i rane laktacije. Istraživanje je provedeno na ukupno 78 krava holštajnsko-frizijske pasmine. Krave su nasumično podijeljene u dvije skupine: krave s dodatkom KPL-a u hranu (pokusna skupina) (n = 38) koje su primale dva puta dnevno po 50 g prirodnoga praškastog zeolita KPL-a, počevši 180. dan prije porođaja do 60 dana nakon porođaja, i na kontrolnu skupinu (n = 40). Uzorci krvi uzeti su 180., 90., 60., 30. i 10. dan prije teljenja, na dan teljenja te 5., 12., 19., 26., 33., 40. i 60. dana nakon porođaja. Nisu ustanovljene znakovite razlike u koncentraciji Ca između krava pokusne i kontrolne skupine. No nakon porođaja bilo je vidljivo da je koncentracija kalcija bila viša u pokusnoj KPL skupini, posebice 33. dan (P = 0,06). Koncentracija P bila je znakovito niža (P<0,05) u pokusnoj skupini nulti i 5. dan u odnosu na kontrolnu skupinu. Znakovito viši omjer Ca i P izračunan je u pokusnoj skupini u odnosu na kontrolnu skupinu nulti i 12. dan. Nisu ustanovljene znakovite razlike u koncentraciji Mg, K i Na u krava iz pokusne i kontrolne skupine tijekom istraživanja. Rezultati ovog istraživanja upućuju na to da je KPL dodan u hranu utjecao na razine Ca i P u krvi mliječnih krava te da je poboljšao omjer Ca i P u serumu tijekom ranog puerperija. Primijenjeni pripravak KPL-a nije prouzročio klinički vidljive poremećaje metabolizma istraživanih minerala Osim toga razine svih istraživanih minerala u krvi bile su unutar fizioloških raspona što pokazuje da KPL nije promijenio njihovu homeostazu u mliječnih krava