Frequency of galactose-1-phosphate uridyl transferase gene mutations in healthy population of Croatia

Galactosemia is a human disease caused by deficient activity of each one of the three enzymes involved in galactose metabolism, galactokinase (GALK), galactose-1-phosphate uridyl transferase (GALT) and UDP-galactose-4-epimerase (GALE). Absence or deficiency of GALT activity results in classical galactosemia. This disorder exhibits allelic heterogeneity in different populations and ethnic groups. The aim of this study was to search for galactosemia mutations Q188R, N314D, and K285N in healthy population of Croatia. DNA samples from 221 subjects were analyzed by the polymerase chain reaction, followed by digestion with restriction endonucleases (PCR-RFLP procedure). Allele frequencies for Q188R, N314D, and K285N were found to be 0.2 %, 7.5 % and 0 %, respectively, and correlate well with those published for most other healthy Caucasian populations

Toxicological Assessment of P-9801091 Plant Mixture Extract after Chronic Administration in CBA/HZg Mice – A Biochemical and Histological Study

Acute, subchronic and chronic effects of the P-9801091 plant mixture extract at a dose of 20 mg/kg body mass were assessed in serum of healthy CBA/HZg mice at 24 hours, 7 days, 3 months and 6 months of treatment (experimental group), and compared with the values obtained in the control group of untreated healthy CBA/HZg mice. The P-9801091 plant mixture extract is an antihyperglycemic preparation containing Myrtilli folium (Vaccinium myrtillus L.), Taraxaci radix (Taraxacum officinale Web.), Cichorii radix (Cichorium intybus L.), Juniperi fructus (Juniperus communis L.), Centaurii herba (Centaurium umbellatum Gilib.), Phaseoli fructus sine semine (Phaseolus vulgaris L.), Millefolii herba (Achillea millefolium L.), Mori folium (Morus nigra L.), Valerianae radix (Valeriana officinalis L.) and Urticae herba et radix (Urtica dioica L). Toxic effect of the P-9801091 plant mixture extract was assessed by the following biochemical parameters: urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and cholesterol. Also, histopathological examination of the kidneys, liver, spleen, pancreas, testes and lungs was performed. Results of biochemical testing performed at specified time points generally showed no statistically significant differences from control values, with the only exception of the catalytic concentration of AST in the experimental group measured on day 7, which was significantly increased as compared with the control group (p<0.05). Pathohistological examination including characteristic organ and tissue structure, and parenchyma relationship to the adjacent blood vessels and connective tissue in the examined organs revealed no major pathologic changes

Frequency of Galactose-1-phosphate Uridyl Transferase Gene Mutations in Healthy Population of Croatia

Galactosemia is a human disease caused by deficient activity of each one of the three enzymes involved in galactose metabolism, galactokinase (GALK), galactose-1-phosphate uridyl transferase (GALT) and UDP-galactose-4-epimerase (GALE). Absence or deficiency of GALT activity results in classical galactosemia. This disorder exhibits allelic heterogeneity in different populations and ethnic groups. The aim of this study was to search for galactosemia mutations Q188R, N314D, and K285N in healthy population of Croatia. DNA samples from 221 subjects were analyzed by the polymerase chain reaction, followed by digestion with restriction endonucleases (PCR-RFLP procedure). Allele frequencies for Q188R, N314D, and K285N were found to be 0.2 %, 7.5 % and 0 %, respectively, and correlate well with those published for most other healthy Caucasian populations

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

Concentration of malondialdehyde in a NOD mice treated with acarbose

Cilj: Malondialdehid (MDA) je jedan od toksičnih produkata lipidne peroksidacije, a koristi se u evaluaciji oksidativnog stresa tijekom šećerne bolesti. Cilj ovog rada bio je ispitati učinak akarboze (inhibitora α-glukozidaza) na koncentraciju glukoze u serumu i MDA u homogenatu jetre NOD (engl. non-obese diabetic) miševa. Materijali i metode: U NOD miševa šećerna bolest inducirana je i.v. aplikacijom aloksan-monohidrata (75 mg/kg t. mase). NOD miševi podijeljeni su u 4 skupine (n=6): Kontrolni, zdravi NOD miševi (K), Kontrolni, zdravi NOD miševi tretirani 7 dana akarbozom (K/A), dijabetični NOD miševi (D) te dijabetični NOD miševi tretirani 7 dana akarbozom (D/A). Koncentracija glukoze u krvi izmjerena je glukoza oksidaza-peroksidaza metodom, a koncentracija MDA u homogenatu jetre određena je upotrebom metode s tiobarbiturnom kiselinom. Rezultati: Nakon sedmodnevnog tretmana akarbozom zabilježeno je statistički značajno smanjenje koncentracije glukoze u krvi u skupini D/A u odnosu na skupinu D (p<0.05), a isto tako je uočen i statistički značajan pad koncentracije MDA (p<0.05). Zaključak: Ovi rezultati potvrđuju pozitivan antioksidacijski učinak akarboze što se može objasniti njezinim antihiperglikemijskim djelovanjem.Aim. Malondialdehyde (MDA) is one of the toxic product of the lipid peroxida-tion, and that plays an important role in the evaluation of oxidative stress in the diabetes mellitus. The aim of this study was to study the effect of acarbo-se (α-glucosidase inhibitor) on serum glucose concentration and MDA in liver homogenate of NOD (non-obese diabetic) mice. Material and methods. Diabetes was induced in mice by i.v. administration of alloxan-monohydrate (75 mg/kg b.w.). The mice were divided into 4 groups (N=6): control, healthy mice (C), control healthy mice on 7-day treatment with acarbose (C/A), NOD mice (D), and NOD mice on 7-day acarbose treatment (D/A). The levels of blood glucose and of MDA in liver homogenate were determined by glucose oxidase-peroxidase method and a method with thiobarbituric acid, respectively. Results. Significant reduction in blood glucose levels was observed after 7-day administration of acarbose in D/A group compared to D group (p<0.05), together with a significant fall in MDA concentration (p<0.05). Conclusion. This results confirmed thefavorable antioxidative effect of acarbose which may be explained by its antihyperglycemic action

Genetic Frequencies of Paraoxonase 1 Gene Polymorphisms in Croatian Population

Paraoxonase 1 (PON1), a HDL-associated enzyme, is believed to contribute to the protective effects of HDL by decreasing the generation of lipid peroxidation products during the process of LDL oxidation. It has been reported that polymorphisms in promoter and coding regions of the pon1 gene could affect PON1 activity. The aim of this study was to determine the frequencies of pon1 polymorphisms Q192R, L55M and –108C>T and their influence on PON1 activity in healthy population of Croatia. The following genotype frequencies were determined: 60 % QQ, 34 % QR, 6 % RR for Q192R; 44 % LL, 43 % LM, 13 % MM for L55M; 30 % CC, 48 % CT, 22 % TT for –108C>T polymorphism. Genotypes RR, LL and CC were found to be associated with higher PON1 activity. The most frequent genotypes in healthy Croatian population were QQ, CT and equally present LL and LM

Paraoksonaza/arilesteraza u serumu ispitanika s dijabetesom tipa II

The aim of this study was to determine whether the paraoxonase (PON1) status, i.e. PON1 activities and phenotypes (AA, AB and BB), and its relationship with lipid status are different in patients with type II diabetes as compared to healthy population. Diabetic group comprised 175 patients with type II diabetes mellitus (94 men and 81 women) who came to their regular control examination and took the oral glucose tolerance test. Patients with type II diabetes mellitus diagnosis for 12 years on average were on peroral antidiabetics, or insulin or diet, and 3 patients had no therapy prescribed yet. Control group comprised 114 apparently healthy individuals (28 men and 86 women) who were not on any medication. The paraoxonase activity was measured with 2.0 mmol L-1 paraoxon in the absence and in the presence of 1.0 mol L-1 NaCl, and with 2.0 mmol L-1 phenylacetate. Both activities were measured spectrophotometrically at 37 oC in 0.1 mol L-1 Tris-HCl buffer, pH = 8.0, containing 2.0 mmol L-1 CaCl2. Sera of diabetic and control subjects were assigned to the paraoxonase phenotypes on the basis of the basal paraoxonase activity distribution. We assigned 45% sera of male and 49% sera of female diabetic patients, and 64% sera of both genders of the control group to the AA low activity phenotype. There were no differences in paraoxonase activities between the gender- and phenotype-matched diabetic and control groups. Enzyme activity against the phenylacetate was higher and phenotype-dependent only in diabetic patients. In contrast to AA phenotype individuals, total cholesterol and LDL-cholesterol in the female diabetic group and triglyceride concentration in the male diabetic group assigned to pooled AB and BB phenotypes were higher than in the corresponding controls. It follows from PON1 phenotype distribution that less antiatherogenic paraoxonase B allele is more frequent in type II diabetes mellitus than in the healthy population. Their lipid status is more atherogenic, which could indicate a risk of premature atherosclerosis.Cilj rada je usporediti katalitičku aktivnost paraoksonaze (PON1) te učestalost fenotipova AA, AB i BB paraoksonaze i njihovu povezanost s lipidnim statusom u serumu ispitanika s dijabetesom tipa II i kontrolnoj skupini. U skupini ispitanika s dijabetesom tipa II bilo je 175 osoba (81 žena i 94 muškaraca), s prosječnim trajanjem bolesti od 12 godina, koji su bili na peroralnoj terapiji antidijabeticima ili inzulinom ili na dijeti, dok trojici pacijenata još nije predložena terapija. Aktivnost paraoksonaze mjerena je s paraoksonom (O,O-dietil-O-p-nitrofenilfosfat). Koncentracije reagensa u reakcijskoj smjesi za određivanje bazalne aktivnosti paraoksonaze bile su: 2.0 mmol L-1 paraokson i 2.0 mmol L-1 CaCl2 u 0.1 mol L-1 Tris-HCl puferu, pH=8.0. Reakcijska smjesa za određivanje NaCl-stimulirane aktivnosti paraoksonaze sadržavala je još 1.0 mol L-1 NaCl. Arilesterazna aktivnost enzima mjerena je s fenilacetatom. Reakcijska smjesa je sadržavala 2.0 mmol L-1 fenilacetata i 2.0 mmol L-1 CaCl2 u 0.1 mol L-1 Tris-HCl puferu, pH=8.0. Broj ispitanika s AA fenotipom odnosno skupno AB i BB fenotipom paraoksonaze određen je iz raspodjelne krivulje bazalnih aktivnosti (bez prisutnosti 1.0 mol L-1 NaCl) paraoksonaze u serumu. U serumima 45% žena i 49% muškaraca skupine ispitanika s dijabetesom tipa II te u 64% seruma oba spola u skupini zdravih ispitanika potvrđen je AA homozigotni fenotip paraoksonaze. Katalitičke aktivnosti enzima prema paraoksonu nisu se značajno razlikovale ovisno o spolu i fenotipu izmedu dijabetične i kontrolne skupine, dok su aktivnosti enzima prema fenilacetatu bile veće i ovisne o fenotipu samo u dijabetičnoj skupini ispitanika. Značajno veće koncentracije ukupnog kolesterola i LDL-kolesterola izmjerene su u serumima žena te veće koncentracije triglicerida u serumima muškaraca s dijabetesom tipa II koji su razvrstani u zajedničku skupinu AB+BB fenotipova što bi ukazivalo da su AB i BB fenotipovi uglavnom povezani s lipidnim statusom većeg rizika za razvoj ateroskleroze u ispitanika s dijabetesom tipa II

Paraoksonaza/arilesteraza u serumu ispitanika s dijabetesom tipa II

The aim of this study was to determine whether the paraoxonase (PON1) status, i.e. PON1 activities and phenotypes (AA, AB and BB), and its relationship with lipid status are different in patients with type II diabetes as compared to healthy population. Diabetic group comprised 175 patients with type II diabetes mellitus (94 men and 81 women) who came to their regular control examination and took the oral glucose tolerance test. Patients with type II diabetes mellitus diagnosis for 12 years on average were on peroral antidiabetics, or insulin or diet, and 3 patients had no therapy prescribed yet. Control group comprised 114 apparently healthy individuals (28 men and 86 women) who were not on any medication. The paraoxonase activity was measured with 2.0 mmol L-1 paraoxon in the absence and in the presence of 1.0 mol L-1 NaCl, and with 2.0 mmol L-1 phenylacetate. Both activities were measured spectrophotometrically at 37 oC in 0.1 mol L-1 Tris-HCl buffer, pH = 8.0, containing 2.0 mmol L-1 CaCl2. Sera of diabetic and control subjects were assigned to the paraoxonase phenotypes on the basis of the basal paraoxonase activity distribution. We assigned 45% sera of male and 49% sera of female diabetic patients, and 64% sera of both genders of the control group to the AA low activity phenotype. There were no differences in paraoxonase activities between the gender- and phenotype-matched diabetic and control groups. Enzyme activity against the phenylacetate was higher and phenotype-dependent only in diabetic patients. In contrast to AA phenotype individuals, total cholesterol and LDL-cholesterol in the female diabetic group and triglyceride concentration in the male diabetic group assigned to pooled AB and BB phenotypes were higher than in the corresponding controls. It follows from PON1 phenotype distribution that less antiatherogenic paraoxonase B allele is more frequent in type II diabetes mellitus than in the healthy population. Their lipid status is more atherogenic, which could indicate a risk of premature atherosclerosis.Cilj rada je usporediti katalitičku aktivnost paraoksonaze (PON1) te učestalost fenotipova AA, AB i BB paraoksonaze i njihovu povezanost s lipidnim statusom u serumu ispitanika s dijabetesom tipa II i kontrolnoj skupini. U skupini ispitanika s dijabetesom tipa II bilo je 175 osoba (81 žena i 94 muškaraca), s prosječnim trajanjem bolesti od 12 godina, koji su bili na peroralnoj terapiji antidijabeticima ili inzulinom ili na dijeti, dok trojici pacijenata još nije predložena terapija. Aktivnost paraoksonaze mjerena je s paraoksonom (O,O-dietil-O-p-nitrofenilfosfat). Koncentracije reagensa u reakcijskoj smjesi za određivanje bazalne aktivnosti paraoksonaze bile su: 2.0 mmol L-1 paraokson i 2.0 mmol L-1 CaCl2 u 0.1 mol L-1 Tris-HCl puferu, pH=8.0. Reakcijska smjesa za određivanje NaCl-stimulirane aktivnosti paraoksonaze sadržavala je još 1.0 mol L-1 NaCl. Arilesterazna aktivnost enzima mjerena je s fenilacetatom. Reakcijska smjesa je sadržavala 2.0 mmol L-1 fenilacetata i 2.0 mmol L-1 CaCl2 u 0.1 mol L-1 Tris-HCl puferu, pH=8.0. Broj ispitanika s AA fenotipom odnosno skupno AB i BB fenotipom paraoksonaze određen je iz raspodjelne krivulje bazalnih aktivnosti (bez prisutnosti 1.0 mol L-1 NaCl) paraoksonaze u serumu. U serumima 45% žena i 49% muškaraca skupine ispitanika s dijabetesom tipa II te u 64% seruma oba spola u skupini zdravih ispitanika potvrđen je AA homozigotni fenotip paraoksonaze. Katalitičke aktivnosti enzima prema paraoksonu nisu se značajno razlikovale ovisno o spolu i fenotipu izmedu dijabetične i kontrolne skupine, dok su aktivnosti enzima prema fenilacetatu bile veće i ovisne o fenotipu samo u dijabetičnoj skupini ispitanika. Značajno veće koncentracije ukupnog kolesterola i LDL-kolesterola izmjerene su u serumima žena te veće koncentracije triglicerida u serumima muškaraca s dijabetesom tipa II koji su razvrstani u zajedničku skupinu AB+BB fenotipova što bi ukazivalo da su AB i BB fenotipovi uglavnom povezani s lipidnim statusom većeg rizika za razvoj ateroskleroze u ispitanika s dijabetesom tipa II

Antioksidacijska i protuupalna aktivnost paraoksonaze i lipidni pokazatelji u svinja nakon splenektomije i autotransplantacije slezene.

The objective of this study was to investigate serum PON1 activity and lipid and lipoprotein status after total splenectomy and autologous spleen transplantation in pigs, in order to assess the effect of surgical stress and trauma on PON1 activity and lipid metabolism. Nineteen piglets used in the experiment were randomly divided into three groups: sham-operation with spleens intact (n = 6), total splenectomy (n = 6), and splenic autotransplantation (n = 7) with small fragments of the spleen autotransplanted into the greater omentum. The blood samples were taken just before surgery and on the 1st, 5th, 12th, 26th and 40th days postoperatively. PON1 activity, total cholesterol and HDL-C were assayed in the sera. PON1 activity was significantly decreased postoperatively in splenectomized pigs (on days 5, 12 and 40) and in autotransplantated pigs (on days 5, 12, 26 and 40) while there were no PON1 changes in the sham-operated. In sham-operated pigs, total cholesterol was significantly decreased only on the 12th day postoperatively, while HDL-C was significantly decreased postoperatively on the 1st, 5th, 12th, 26th and 40th days with the minimum value on the 12th day. In splenectomized pigs total cholesterol was significantly decreased on the 5th, 12th, 26th and 40th days postoperatively, while HDL-C was decreased as well on the 1st, 5th, 12th and 40th days and both were the lowest on the 12th day. In pigs with autologous splenic transplants, total cholesterol was significantly decreased on the 12th, 26th and 40th days, while there was no difference in HDL-C. The results demonstrated decreased PON1 activity and alterations in the concentrations of total cholesterol and HDL-C after surgery due to surgical trauma and post-surgical inflammatory response.Cilj je ovoga rada bio istražiti aktivnost PON1 te lipidni status u serumu svinja nakon splenektomije i autotransplantacije slezene u namjeri da se procijeni učinak stresa i traume na aktivnost PON1 i metabolizam lipida u tijeku kirurškoga zahvata. U istraživanje je bilo uključeno 19 praščića koji su nasumično bili raspodijeljeni u tri skupine: (1) laparoskopija s intaktnom slezenom (n = 6), (2) splenektomija (n = 6) i autotransplantacija slezene (n = 7). Uzorci krvi uzimani su neposredno prije kirurškoga zahvata, te 1., 5., 12., 26. i 40. dan nakon operacije. U serumu je određena aktivnost PON1, te koncentracija ukupnoga kolesterola i HDL-kolesterola. Aktivnost PON1 bila je značajno snižena postoperativno u splenektomiranih (5., 12. i 40. dan) i autotransplantiranih životinja (5., 12., 26. i 40. dan), dok u skupini u kojoj je učinjena laparoskopija nije bilo značajnih razlika u aktivnosti PON1 nakon operacije. U skupini podvrgnutoj laparoskopiji, ukupan kolesterol bio je značajno manji samo 12. dan nakon operacije, dok je HDL-C bio manji 1., 5., 12., 26. i 40. dan postoperativno s najmanjom vrijednošću 12. dan. U splenektomirane prasadi, ukupan kolesterol bio je značajno manji 5., 12., 26. i 40. dan postoperativno, a HDL-C je bio manji 5., 12. i 40. dan nakon operacije. U prasadi kojoj je autotransplantirana slezena, ukupni je kolesterol bio značajno manji 12., 26. i 40. dan nakon operacije, dok se koncentracija HDL-C nije značajno razlikovala prije i nakon operacije. Rezultati pokazuju smanjenu aktivnost PON1 i promjene koncentracija ukupnog kolesterola i HDL-C uzrokovane kirurškom traumom i upalnim odgovorom nakon operacije