Enzyme activity and AGE formation in a model of AST glycoxidation by D-fructose in vitro

Neenzimska glikacija je proces kojim se reducirajući šećeri kovalentnim vezama povezuju s amino skupinama proteina, bez posredovanja enzima. Taj proces dijelom je i oksidativan pa se često naziva glikooksidacija. Konačni produkti glikacije (AGE) nastaju polimerizacijom mnogih intermedijarnih produkata. U ovom radu, praćen je u in vitro uvjetima, mjerenjem katalitičke aktivnosti enzima i fluorescencije nastalih ukupnih AGE produkata i pentozidina, utjecaj D-fruktoze kao reaktivnog šećera na aspartat aminotransferazu (EC 2.6.1.1.) kao modelni protein. Promjena katalitičke aktivnosti enzima pokazatelj je promjenjene funkcije proteina glikacijom dok je intenzitet fluorescencije ukupnih AGE produkata i pentozidina samo djelomičan pokazatelj glikoksidacijskih promjena. Katalitička aktivnost AST izmjerena IFCC preporučenom metodom u inkubacijskoj smjesi koja je sadržavala 50 mmol L-1 fruktoze, smanjena je na 42% (p<0.05) petog dana, te na 11% (p<0.05) dvadeset i prvog dana. Statistički su značajne razlike u katalitičkoj aktivnosti enzima inkubiranog sa i bez fruktoze već nakon petog dana.Stvaranje ukupnih AGE produkata i pentozidina statistički je značajno nakon pet, odnosno 15 dana inkubacije enzima s fruktozom. Katalitička aktivnost enzima jasno ukazuje na funkcionalne promjene uzrokovane glikiranjem, dok je evaluacija stvaranja ukupnih AGE produkata, a pogotovo pentozidina, mjerenjem fluorescencije manje pouzdan pokazatelj promjena.Non-enzymatic glycation as the reaction chain between reducing sugars and free amino groups of proteins has been shown to correlate with physiological ageing and severity of diabetes. The process involves oxidative steps (glycoxidation). In this paper, the effect of D-fructose as a reactive sugar on aspartate aminotransferase (AST) as a model protein was monitored by measurements of the enzyme activity and formation of fluorescent advanced glycation end products (AGEs). Change in the AST activity was considered as a measure of the overall protein damage caused by glycation, and total AGEs and pentosidine represent, at least partly, the formation of glycoxidation products. Catalytic activity of AST in an incubation mixture containing D-fructose (50 mmol L-1), decreased compared to control values to 42% (p < 0.05) and to 11% (p < 0.05) on 5th and on 21st day of incubation, respectively. In the presence of fructose, total fluorescent AGEs concentrations were significant higher since 5th day of incubation (110 %, p < 0.05) and the fluorescent pentosidine concentrations from 15th day of incubation (117 %, p < 0.05) compared to control values, respectively. Catalytic activity of AST clearly and quantitatively demonstrated functional changes in the enzyme molecule caused by structural modifications initiated by fructose, while the evaluation of AGE formation and especially that of pentosidine by fluorescence measurement was less reliable

Oxidative Damage to Nucleic Acids and Benzo(a)pyrene-7,8-diol-9,10-epoxide-DNA Adducts and Chromosomal Aberration in Children with Psoriasis Repeatedly Exposed to Crude Coal Tar Ointment and UV Radiation

The paper presents a prospective cohort study. Observed group was formed of children with plaque psoriasis (n = 19) treated by Goeckerman therapy (GT). The study describes adverse (side) effects associated with application of GT (combined exposure of 3% crude coal tar ointment and UV radiation). After GT we found significantly increased markers of oxidative stress (8-hydroxy-2 -deoxyguanosine, 8-hydroxyguanosine, and 8-hydroxyguanine), significantly increased levels of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) DNA adducts (BPDE-DNA), and significantly increased levels of total number of chromosomal aberrations in peripheral lymphocytes. We found significant relationship between (1) time of UV exposure and total number of aberrated cells and (2) daily topical application of 3% crude coal tar ointment (% of body surface) and level of BPDE-DNA adducts. The findings indicated increased hazard of oxidative stress and genotoxic effects related to the treatment. However, it must be noted that the oxidized guanine species and BPDE-DNA adducts also reflect individual variations in metabolic enzyme activity (different extent of bioactivation of benzo[a]pyrene to BPDE) and overall efficiency of DNA/RNA repair system. The study confirmed good effectiveness of the GT (significantly decreased PASI score)

Katalitička aktivnost i stvaranje AGE-produkata u modelu glikoksidacije AST D-fruktozom in vitro

Non-enzymatic glycation as the reaction chain between reducing sugars and free amino groups of proteins has been shown to correlate with physiological ageing and severity of diabetes. The process involves oxidative steps (glycoxidation). In this paper, the effect of D-fructose as a reactive sugar on aspartate aminotransferase (AST) as a model protein was monitored by measurements of the enzyme activity and formation of fluorescent advanced glycation end products (AGEs). Change in the AST activity was considered as a measure of the overall protein damage caused by glycation, and total AGEs and pentosidine represent, at least partly, the formation of glycoxidation products. Catalytic activity of AST in an incubation mixture containing D-fructose (50 mmol L-1), decreased compared to control values to 42% (p < 0.05) and to 11% (p < 0.05) on 5th and on 21st day of incubation, respectively. In the presence of fructose, total fluorescent AGEs concentrations were significant higher since 5th day of incubation (110 %, p < 0.05) and the fluorescent pentosidine concentrations from 15th day of incubation (117 %, p < 0.05) compared to control values, respectively. Catalytic activity of AST clearly and quantitatively demonstrated functional changes in the enzyme molecule caused by structural modifications initiated by fructose, while the evaluation of AGE formation and especially that of pentosidine by fluorescence measurement was less reliable.Neenzimska glikacija je proces kojim se reducirajući šećeri kovalentnim vezama povezuju s amino skupinama proteina, bez posredovanja enzima. Taj proces dijelom je i oksidativan pa se često naziva glikooksidacija. Konačni produkti glikacije (AGE) nastaju polimerizacijom mnogih intermedijarnih produkata. U ovom radu, praćen je u in vitro uvjetima, mjerenjem katalitičke aktivnosti enzima i fluorescencije nastalih ukupnih AGE produkata i pentozidina, utjecaj D-fruktoze kao reaktivnog šećera na aspartat aminotransferazu (EC 2.6.1.1.) kao modelni protein. Promjena katalitičke aktivnosti enzima pokazatelj je promjenjene funkcije proteina glikacijom dok je intenzitet fluorescencije ukupnih AGE produkata i pentozidina samo djelomičan pokazatelj glikoksidacijskih promjena. Katalitička aktivnost AST izmjerena IFCC preporučenom metodom u inkubacijskoj smjesi koja je sadržavala 50 mmol L-1 fruktoze, smanjena je na 42% (p<0.05) petog dana, te na 11% (p<0.05) dvadeset i prvog dana. Statistički su značajne razlike u katalitičkoj aktivnosti enzima inkubiranog sa i bez fruktoze već nakon petog dana.Stvaranje ukupnih AGE produkata i pentozidina statistički je značajno nakon pet, odnosno 15 dana inkubacije enzima s fruktozom. Katalitička aktivnost enzima jasno ukazuje na funkcionalne promjene uzrokovane glikiranjem, dok je evaluacija stvaranja ukupnih AGE produkata, a pogotovo pentozidina, mjerenjem fluorescencije manje pouzdan pokazatelj promjena

Is a Highly Linear Relationship Between the Dose of Quercetin and the Pharmacological Effect Possible? — A Comment on Liu, et al. Evaluation of Antioxidant and Immunity Activities of Quercetin in Isoproterenol-Treated Rats. Molecules 2012, 17, 4281–4291

We wish to offer some comments on the article by H. Liu et al. entitled “Evaluation of antioxidant and immunity activities of quercetin in isoproterenol-treated rats”, published in Molecules in 2012 [1]. [...

Patterns of non-adherence to supplementation with calcium and vitamin D in persistent postmenopausal women are similar at start and one year later: a qualitative longitudinal study

Background: Osteoporosis is a chronic disease and adherence can fluctuate over time. Therefore, longer observation is necessary to investigate the stability of patients’ adherence. The study aim was to compare the overall adherence (OA) with supplementation with the fixed combination of calcium and vitamin D (Ca/D) in postmenopausal women at baseline and after 1 year, and to evaluate the fluctuation of the OA in individual months. Furthermore, we studied whether adherence is influenced by signing of informed consent and routine medical check-up.Methods: This was a longitudinal, observational study. The data were obtained from the Osteocenter of University Hospital in Hradec Kralove, Czech Republic. Adherence was measured using electronic bottles type Medication Events Monitoring System (MEMS). The study was carried out in two 3-month periods; the baseline in 2013 (signing of informed consent while medical check-up) and the follow-up (medical check-up) in 2014. The adherence and adherence-related outcomes were studied in patients who had initiated osteoporosis treatment and were persistent.Results: 21 (49%) out of 43 patients who avoided drug dispenser and were persistent both at baseline and at follow-up, completed the study and were included. Median age was 76. Evaluating the whole 3-month periods, the OA did not differ significantly at baseline and at follow-up, the OA was 71% and 68% respectively. However, the adherence in month 1 at baseline was significantly higher than the adherence in month 2 at baseline (p < 0.001) and also than the adherence in month 1 at follow-up (p = 0.010). Analysing the study period without month 1, a stable adherence was observed in 48% of patients. About 33% of doses were omitted at baseline and 34% at follow-up. As many as 71% of the patients took drug holidays at baseline, and 76% at follow-up. Conclusion: The OA was insufficient, around 70% both at baseline and at follow-up. One half of the patients showed a stable adherence. The patterns of non-adherence were very similar at follow-up. Signing of the informed consent seems to act as bias more than regular medical check-up

Multicenter evaluation of the hemolysis index in automated clinical chemistry systems

Background: In vitro hemolysis, the prevailing cause of preanalytical error in routine laboratory diagnostics, might influence the reliability of several tests, affect the quality of the total testing process and jeopardize patient safety. Although laboratory instrumentation is now routinely equipped with systems capable of automatically testing and eventually correcting for hemolysis interference, to our knowledge there are no reports that have compared the efficiency of different analytical platforms for identifying and classifying specimens with hemolysis. Methods: Serum from a healthy volunteer was spiked with varying amounts of hemolyzed blood from the same volunteer, providing a serum free hemoglobin concentration ranging from 0.0 g/L to 2.0 g/L as measured by the reference cyanmethemoglobin assay. The spiked serum samples were shipped to seven separate laboratories and the hemolysis index (HI) was tested in triplicate on the following analytical platforms: Roche Modular System P (n=4) and Integra 400 Plus (n=1), Siemens Dimension RxL (n=3), ADVIA 2400 (n=1) and ADVIA 1800 (n=1), Olympus AU 680 (n=1) and Coulter DXC 800 (n=1). Results: Satisfactory agreement of HI results was observed among the various analytical platforms, despite a trend toward overestimation by the ADVIA 2400 and 1800. After normalizing results according to the instrument-specific alert value, discrepancies were considerably reduced. All instruments except for the Dimension RxL gave values normalized to the instrument-specific alert value, &#60;1.0 for the sample with 0.048 g/L free hemoglobin, and &#62;1.0 for the sample with 0.075 g/L free hemoglobin. The results of the four Modular System P tests were also highly reproducible among the different facilities. When evaluating instruments that provided quantitative HI results, the mean intra-assay coefficient of variation (CV) calculated for the triplicate determinations was always between 0.1% and 2.7%. Conclusions: The results of this multicenter evaluation confirm that efficiency of different analytical platforms to correctly identify and classify unsuitable samples is satisfactory. However, more effort should be placed on the standardization of reporting HI. All the instruments that we tested provide either quantitative or qualitative results that are essentially comparable, but which should always be compared with the instrument-specific alert values to harmonize their efficiency. Clin Chem Lab Med 2009;47:934–9.Peer Reviewe

Vascular Endothelial Growth Factor Is Associated with the Morphologic and Functional Parameters in Patients with Hypertrophic Cardiomyopathy

Background. Hypertrophic cardiomyopathy (HCM) is mostly autosomal dominant disease of the myocardium, which is characterized by myocardial hypertrophy. Vascular endothelial growth factor (VEGF) is involved in myocyte function, growth, and survival. The aim of study was to analyze the clinical significance of VEGF in structural and functional changes in patient with HCM. Methods. In a group of 21 patients with nonobstructive HCM, we assessed serum VEGF and analyzed its association with morphological and functional parameters. Compared to healthy controls, serum VEGF was increased: 199 (IQR: 120.4–260.8) ng/L versus 20 (IQR: 14.8–37.7) ng/L, P<0.001. VEGF levels were associated with left atrium diameter (r=0.51, P=0.01), left ventricle ejection fraction (r=-0.56, P=0.01), fractional shortening (r=-0.54, P=0.02), left ventricular mass (r=0.61, P=0.03), LV mass index (r=0.46, P=0.04), vena cava inferior diameter (r=0.65, P=0.01), and peak gradient of tricuspid regurgitation (r=0.46, P=0.03). Conclusions. Increased VEGF level is associated with structural and functional parameters in patients with HCM and serves as a potential tool for diagnostic process of these patients

How General Practitioners and Their Patients Adhere to Osteoporosis Management: A Follow-Up Survey among Czech General Practitioners

Introduction: General practitioners (GPs) are key participants in osteoporosis (OP) management. The aim was to evaluate their adherence to lege artis management of the disease, potential barriers, and to discuss differences observed in comparison with the baseline survey carried out in 2007; the focus was on secondary prevention.Methods: On behalf of two professional associations, 2-round postal survey among randomly selected GPs (&gt;1/4 of all Czech GPs) was performed in 2014. The questionnaire covered areas concerning GP's role in the fight against OP, knowledge about OP, management of OP-related fractures, barriers to the management of OP, system- and patient-related in particular, and availability and use of information sources.Results: The overall questionnaire return rate was 37% (551 respondents); mean age of the respondents was 53 year (37% men). The GP's role in the treatment of OP was rated as essential in 28 and 37% of men and women, respectively (P = 0.012). The guideline for diagnosis and treatment of OP for GPs was considered accessible by 92% of respondents. As much as 60% of the respondents were adherent to the guideline, i.e., used it repeatedly. The knowledge of several risk factors was very good, however, recommended daily intake of calcium was stated correctly by only 41% of respondents, and daily intake of vitamin D by only 40%. Three quarters reported active steps after a fracture: referral to a specialist, life-style recommendations, prescription of calcium/vitamin D supplements. Half of the respondents focus on fall prevention. System-related barriers, such as lack of possibility to prescribe selected drugs (61%) and financial limits set by health insurance company (44%) were most frequently reported. Patient-related barriers were also common, patient's non-adherence (reported by 29%) and patient's reluctance to go to a specialist (18%).Conclusion: GPs adhered to OP management more than in 2007. Knowledge of risk factors and involvement in post-fracture care was relatively high. Compared to baseline survey, patient-related barriers, patient non-adherence in particular, were more common. Prescribing conditions are still an important issue. Among GPs, education should be focused on calcium and vitamin D intake, doses, sources, and supplements