No Inhibitory Effect of Heparinized Blood on Real-Time PCR Analysis of Thrombophilic Mutations.

We compared the efficiency of real-time PCR analysis of FII (c.*97G>A, G20210A) and FV Leiden (c.1601G>A) thrombophilic mutations in the samples obtained from venous blood treated with various anti coagulant agents (EDTA, heparin, and sodium fluoride with potassium oxalate), or from clotted venous blood; one hundred samples of wild-type subjects were tested. Genomic DNA extracts and whole blood specimens modified by 90 °C heating were analysed by real-time PCR analysis; cycle threshold values were subsequently evaluated. Real-time PCR analysis for the FII gene assay performed in DNA extracts from EDTA blood samples revealed a median Ct value of 19.3. Similar Ct values were apparent in the DNA extracts obtained from the heparinized blood and sodium fluoride with potassium oxalatetreated samples: 18.5 and 18.9, respectively. Significantly higher Ct values were found in extracts from clotted blood with medians of 20.6 (tubes with inert separation gel) and 20.5 (tubes without the gel, both P < 0.001). The data on the FV real-time PCR analysis were very comparable to the FII assay. In the modified whole blood, the samples treated with heparin salts showed significantly lower Ct values (P < 0.001) in both assays when compared with the samples with EDTA, sodium fluoride with potassium oxalate, and with the samples with clotted blood. Our results indicate that real-time PCR analyses of thrombophilic mutations were not negatively influenced by the presence of heparin salts in collection tubes. Blood samples with various anticoagulants might be exchangeable for each other when DNA analysis of thrombophilic mutations is required.

Variation of selected genotoxic and epigenetic markers due to therapeutic exposure to PAHs and ultraviolet radiation.

BACKGROUND: Goeckerman therapy (GT) of psoriasis involves dermal application of crude coal tar containing polycyclic aromatic hydrocarbons (PAHs) and exposure to ultraviolet radiation (UVR). Little is known about GT influence on DNA epigenetics. OBJECTIVE: The study aim was to discover epigenetic mechanisms altered by the exposure related to the GT of psoriasis. METHODS: Observed group of patients with plaque psoriasis (n = 23) was treated by GT with 3 % CCT. Before and after GT, we analyzed the levels of benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts (BPDE-DNA), p53 protein in serum, 5-methylcytosine (5-mC, global DNA methylation), and methylation in selected CpG sites of p53 gene. RESULTS: We found a significant increase in the levels of BPDE-DNA (p < 0.01) and serum levels of p53 protein (p < 0.01) after GT, and an insignificant decrease in the percentage of 5-mC in peripheral blood DNA. Methylation of p53 CpG sites was affected neither by psoriasis nor by GT. The study confirmed good effectiveness of GT (significantly reduced psoriasis area and severity index; p < 0.001). CONCLUSION: Our findings indicate that there is a significantly increased genotoxic hazard related to the exposure of PAHs and UV radiation after GT of psoriasis. However, global DNA methylation and p53 gene methylation evade the effect of GT, as they remained unchanged (Tab. 4, Fig. 3, Ref. 50).

Deregulation of Selected MicroRNAs in Sinonasal Squamous Cell Carcinoma: Searching for Potential Prognostic Biomarkers.

Sinonasal carcinomas are head and neck tumours arising from the nasal cavity and paranasal sinuses characterized by unfavourable outcome, difficult treatment, diagnosis and prognosis. MicroRNAs are key molecules in the regulation of development and progression of cancer and their expression profiles could be used as prognostic biomarkers, to predict the patients' survival and response to treatment. In this study, we used quantitative real­time PCR with TaqMan® Advanced miRNA Assays to investigate the relative expression values of selected micro- RNAs in a unique set of formalin-fixed paraffin-embedded tissue samples obtained from 46 patients with sinonasal squamous cell carcinoma. Our results showed statistically significant up-regulation of three mature microRNAs: miR-9-5p (fold change: 6.80), miR-9-3p (fold change: 3.07) and let-7d (fold change: 3.93) in sinonasal carcinoma patients. Kaplan-Meier survival analysis and logrank test identified association between higher expression of miR-9-5p and longer survival of the patients (P = 0.0264). Lower expression of let-7d was detected in the patients with impaired survival, and higher expression of miR-137 was linked to shorter survival of the patients. We also identified several correlations between expression of the studied microRNAs and recorded clinicopathological data. Higher expression of miR-137 and lower expression of let-7d correlated with local recurrence (P = 0.045 and P = 0.025); lower expression of miR-9-5p and higher expression of miR-155-5p correlated with regional recurrence (P = 0.045 and P = 0.036). Higher expression of miR-9-3p correlated with occupational risk (P = 0.031), presence of vascular invasion (P = 0.013) and perineural invasion (P = 0.031). Higher expression of miR-155-5p was present in the samples originating from maxillary sinus (P = 0.011), cN1-3 classified tumours (P = 0.009) and G2-3 classified tumours (P = 0.017). In conclusion, our study supports the hypothesis of future prospect to use expression of miRNAs as prognostic biomarkers of squamous cell sinonasal carcinoma. In particular, miR-9-5p and miR-9-3p seem to be important members of the sinonasal cancer pathogenesis.

Serum Levels of Aryl Hydrocarbon Receptor, Cytochromes P450 1A1 and 1B1 in Patients with Exacerbated Psoriasis Vulgaris.

The aryl hydrocarbon receptor (AhR) is highly expressed in psoriasis skin lesions. The aim of this study was to investigate serum concentrations of AhR, cytochromes P450 (CYP) 1A1 and 1B1 in patients with exacerbated psoriasis vulgaris treated with combined therapy of ultraviolet radiation (UVR) and crude coal tar. The analyses were performed by using enzyme-linked immunosorbent assays. Before the treatment, the patients had significantly higher serum levels of AhR and CYP1A1 than healthy controls. AhR median noticeably decreased after the therapy; nevertheless, it remained significantly higher compared to the controls. CYP1A1 levels measured before and after the therapy did not differ significantly. Serum CYP1A1 positively correlated with AhR values before and after the treatment. The serum values of CYP1B1 were very low and we did not see any differences between the study group and the control group. The study demonstrated that serum levels of AhR and CYP1A1 could indicate their immunopathological and metabolic roles in exacerbated psoriasis.

Importance of Tumour Suppressor Gene Methylation in Sinonasal Carcinomas.

Epigenetic changes are considered to be a frequent event during tumour development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in samples of sinonasal carcinoma by comparison with normal sinonasal tissue. To search for epigenetic events we used methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to compare the methylation status of 64 tissue samples of sinonasal carcinomas with 19 control samples. We also compared the human papilloma virus (HPV) status with DNA methylation. Using a 20% cut-off for methylation, we observed significantly higher methylation in RASSF1, CDH13, ESR1 and TP73 genes in the sinonasal cancer group compared with the control group. HPV positivity was found in 15/64 (23.4 %) of all samples in the carcinoma group and in no sample in the control group. No correlation was found between DNA methylation and HPV status. In conclusion, our study showed that there are significant differences in promoter methylation in the RASSF1, ESR 1, TP73 and CDH13 genes between sinonasal carcinoma and normal sinonasal tissue, suggesting the importance of epigenetic changes in these genes in carcinogenesis of the sinonasal area. These findings could be used as prognostic factors and may have implications for future individualised therapies based on epigenetic changes.

Adherence to oral bisphosphonates: 30 more minutes in dosing instructions matter.

OBJECTIVES: Low adherence to treatment with bisphosphonates significantly impedes its effectiveness. The objectives were: (1) to compare adherence to oral weekly and monthly bisphosphonates with emphasis on dosing instructions; and (2) to study associations between adherence and beliefs about the bisphosphonate treatment among women ≥ 55 years. METHODS: A multicenter survey was performed in secondary-care patients with osteoporosis. Osteoporosis Specific Morisky Medication Adherence Scale (OS-MMAS), questions on compliance with five dosing instructions and Beliefs about Medicines Questionnaire (BMQ) Specific were used. RESULTS: As many as 363 questionnaires (response rate 95%) were analyzed. Respondents (mean age 69 years) were treated with weekly bisphosphonates (37%) or monthly ibandronate (63%). Based on OS-MMAS, 67% of respondents showed high adherence with no differences between the subgroups. Only 44% of respondents were compliant with all dosing instructions. Compliance with dosing instructions concerning time interval (fasting and staying upright) was 71% in weekly and 52% in monthly subgroups, respectively (p < 0.001). Compliance with dosing instructions correlated positively with education (p = 0.009). The mean BMQ necessity score of 18.4 was greater than the mean BMQ concerns score of 13.3. OS-MMAS score correlated with necessity (p = 0.010). Persistence derived from OS-MMAS correlated with both necessity (p = 0.014) and concerns (p = 0.041). CONCLUSION: Despite relatively high adherence to the treatment, most patients do not follow dosing instructions. Reduced bioavailability, particularly of monthly ibandronate, can be expected in clinical practice. Adherence-related outcomes are associated with beliefs about the oral treatment with bisphosphonates.

Preparing Triple-Compound Heterozygous Control Material for Molecular Diagnostics of TPMT Allelic Variants.

The aim of the study is to present a novel approach for preparing triple-compound heterozygous reference material (TCH-RM) for thiopurine S-methyltransferase (TPMT) genotyping by using the gene synthesis technology. The polynucleotide chain we prepared consisted of three wild-type and three mutant segments corresponding to the TPMT 238G>C, 460G>A, and 719A>G polymorphic sites. TCH-RM characteristics were assessed via four methods: reverse hybridization, real-time PCR with hydrolysis probes, real-time PCR followed by subsequent melting temperature analysis, and DNA sequencing. Consequently, we investigated the TPMT genotype of 371 patients suffering from autoimmune diseases requiring immunosuppressive therapy with thiopurine drugs, mostly inflammatory bowel disease. All methods confirmed the triple heterozygous character and commutability of TCH-RM. In evaluating its stability we obtained very comparable data before and after six months of storage at -80 °C. The determined genotypes were as follows: 352 wild-type subjects (94.8%), 17 TPMT*3A heterozygotes (460G>A and 719A>G, 4.6%), one patient heterozygous for the TPMT*2 allele (238G>C, 0.3%), and one TPMT*3C heterozygote (719A>G, 0.3%). The frequencies of TPMT*1, *3A, *3C, and *2 in the patients were 97.5%, 2.3%, 0.1%, and 0.1 %, respectively. Assembling segments of synthetic DNA into long polynucleotide chains is a universal way of obtaining compound heterozygous material for performing any simultaneous analysis of polymorphic sites in the human genome. The batches are manufactured with a perfect concentration match of wildtype and mutant fragments, and can be made in large quantities for most diagnostic techniques.

Promoter methylation of GATA4, WIF1, NTRK1 and other selected tumour suppressor genes in ovarian cancer.

Ovarian cancer is the leading cause of death from gynaecologic tumours, but the molecular and especially epigenetic events underlying the transformation are poorly understood. Various methylation changes have been identified and show promise as potential cancer biomarkers. The aim of this study was to investigate promoter methylation of selected tumour suppressor genes in ovarian cancer by comparison with normal ovarian tissue. To search for epigenetic events we used methylation-specific multiplex ligation-dependent probe amplification to compare the methylation status of 44 tissue samples of ovarian cancer with 30 control samples. Using a 20% cut-off for methylation, we observed significantly higher methylation in genes NTKR1, GATA4 and WIF1 in the ovarian cancer group compared with the control group. These findings could potentially be used in screening of ovarian cancer, and may have implications for future chemotherapy based on epigenetic changes.

Hypermethylation of RAD51L3 and XRCC2 genes to predict late toxicity in chemoradiotherapy-treated cervical cancer patients.

Cervical cancer affects women worldwide, especially in developing countries. Approximately 500,000 cases of this disease are diagnosed per year. The method of choice in the treatment of advanced cervical cancers (in accordance with the International Federation of Gynecology and Obstetrics staging system (FIGO) starting from stage IIB) is combined radiotherapy with concomitant chemotherapy. This treatment provides good tumour control, but it carries a risk of late complications in the irradiated area in 10-15 % of cases. Methylation is one of the methods of epigenetic control, which has an important role in gene expression. Aberrant methylation of normal CpG islands in promoters of tumour suppressor genes such as RB, p53 or DNA reparation genes ATM, BRCA1,2, and RAD51 gene family causes silencing of their function and cell cycle deregulation, which is one of the efficient ways of neoplastic transformation. The significantly decreased expression of molecules involved in DNA response may cause facilitated radiosensitivity in predisposed individuals. We looked for the relationship between hypermethylation of 18 DNA reparation genes and late toxicity occurrence in cervical cancer patients treated by chemoradiotherapy using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The cut-off value for the hypermethylation was set at 10 %. We confirmed significant association between promoter hypermethylation in the XRCC2 gene and occurrence of late grade III-IV toxicity in cervical cancer patients (P = 0.0357). This finding could be useful in the late toxicity prediction in radiotherapy-treated patients.

Selection of endogenous control and identification of significant microRNA deregulations in cervical cancer.

Introduction: Cervical cancer causes approximately 350,000 deaths each year. The availability of sensitive and specific diagnostic tests to detect cervical cancer in its early stages is essential to improve survival rates. Methods: In this study, we compared two strategies for selecting endogenous controls: miRNA profiling by small-RNA sequencing and a commercially available microfluidic card with 30 recommended endogenous controls preloaded by the manufacturer. We used the RefFinder algorithm and coefficient of variation to select endogenous controls. We selected the combination of miR-181a-5p and miR-423-3p as the most optimal normalizer. In the second part of this study, we determined the differential expression (between tumor/non-tumor groups) of microRNA in cervical cancer FFPE tissue samples. We determined the comprehensive miRNA expression profile using small-RNA sequencing technology and verified the results by real-time PCR. We determined the relative expression of selected miRNAs using the 2-ΔΔCt method. Results: We detected statistically significant upregulation of miR-320a-3p, miR-7704, and downregulation of miR-26a-5p in the tumor group compared to the control group. The combination of these miRNAs may have the potential to be utilized as a diagnostic panel for cervical cancer. Using ROC curve analysis, the proposed panel showed 93.33% specificity and 96.97% sensitivity with AUC = 0.985. Conclusions: We proposed a combination of miR-181a-5p and miR-423-3p as optimal endogenous control and detected potentially significant miRNAs (miR-320a-3p, miR-7704, miR-26a-5p). After further validation of our results, these miRNAs could be used in a diagnostic panel for cervical cancer.

Methylation analysis of tumour suppressor genes in ovarian cancer using MS-MLPA.

Epigenetic changes are considered to be a frequent event during tumour development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in ovarian cancer by comparison with normal ovarian tissue. To search for epigenetic events we used methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to compare the methylation status of 69 tissue samples of ovarian cancer with 40 control samples. Using a 15% cut-off for methylation, we observed significantly higher methylation in genes MGMT, PAX5, CDH13, WT1, THBS1, GATA5 in the ovarian cancer group, while in the ESR1 gene we observed significantly higher methylation in the control group compared with the ovarian cancer group. These findings could potentially be used in screening of ovarian cancer and may have implications for future chemotherapy based on epigenetic changes.

[What is the prognostic importance of molecular genetic factors in endometrial carcinoma?]. / Jaký je prognostický význam molekulárne genetických faktoru u karcinomu endometria?

BACKGROUND: Evaluation of the importance of molecular genetic factors in endometrial carcinoma based on our review of available literature, and in the case of K- ras mutation based on our own data. The aim of the original part of our study was to compare the presence of K- ras mutation in early stages of endometroid carcinoma with normal endometrium and evaluate the role of the mutation in endometrial carcinogenesis. MATERIAL AND METHODS: Molecular bio-logical analysis was performed to detect K- ras mutation in samples of endometrial tissue obtained from women treated in the past at the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove. The detection was made from DNA isolated from paraffin embedded sections using K- ras -StripAssay, ViennaLab Diagnostics GmbH. RESULTS: K- ras mutation was found in 7 out of 30 specimens of endometroid carcinoma in stage I (23%) and in 3 of 20 specimens of normal endometrium in the control group (15%). K- ras mutations were more frequent in IA stage and grade 1 of endometroid carcinoma. CONCLUSION: The importance of molecular genetic factors in endometrial carcinoma differs depending on the type of carcinoma. In more common type 1 endometroid cancer, published data are not as clear as in type 2 carcinoma, in which prevalence of alteration of p53 reaches 90%. Results of our study performed on local population of women support the theory about the possible role of K- ras mutation as an early event in the process of endometrial carcinogenesis in type 1 tumors.

Whole blood samples for faster real-time PCR analysis of thrombophilic mutations in SARS-CoV-2 virus positive patients.

High incidence of thrombosis and venous thromboembolism was reported in patients with COVID-19. In this study, we focused on analysis of thrombophilic mutations performed without a standard DNA extraction step. In one hundred of COVID-19 positive outpatients, real-time PCR for Leiden mutation in the FV gene and G20210A mutation in the FII gene was carried out from DNA extracts and modified whole blood samples, and their cycle threshold (Ct) values were evaluated. In the extracts, healthy homozygotes (wt/wt), heterozygotes (M/wt), and homozygous carriers of Leiden mutation (M/M) provided median Ct values of 18.5, 19.4/22.0, and 20.9. In the whole blood, Ct values were 25.3 (wt/wt), 24.8/27.2 (M/wt), and 26.9 (M/M). Median Ct values for G20210A in the extracts were 19.6 for homozygotes (wt/wt), and 19.7/20.4 for heterozygous carriers. The whole blood samples provided Ct values of 23.9 in healthy homozygotes and 26.3/27.2 in heterozygotes for G20210A mutation. No homozygous subjects for G20210A and no double heterozygotes (for Leiden and G20210A mutations) were found. Despite significant differences in the Ct values, genotyping showed complete result concordance of the DNA extracts and the whole blood samples. The integrity and amplificability of DNA molecules in the whole blood samples during 28 days of deep freezing, interrupted by four cycles of thawing, did not significantly change. In conclusion, we demonstrated a new protocol for the detection of the thrombophilic mutations via real time PCR on the modified whole blood of COVID-19 positive patients. The blood modification was reliable, easy, cheap, and saving costs and turnaround time of the whole laboratory process.

The role of atorvastatin in bone metabolism in male albino Wistar rats.

OBJECTIVE: Statins have been widely used for the treatment of hypercholesterolemia, and recent studies have shown that these drugs also affect bone metabolism. The aim of this experiment was to follow the effect of atorvastatin on bone metabolism in male albino Wistar rats. METHODS: Our study was carried out on 16 rats (240 +/- 10g) which were randomly divided into 2 groups of 8 animals. The control group (CO) was given aqua pro injectione (0.2 mL/100 g BW; gavage) and the experimental group atorvastatin suspension (AT; 0.3 mg in 0.2 mL aqua pro inj./100 g BW; gavage) daily for 8 weeks. We examined serum markers of bone turnover using ELISA - C-terminal crosslinking telopeptide of type I collagen (CTX-I), total osteocalcin (total OC), procollagen type I N propeptide (PINP) and bone alkaline phosphatase (bone ALP). We investigated bone morphogenetic protein-2 (BMP-2) in the proximal tibia using Western blot analysis. Additionally, we measured bone mineral density (BMD). The femurs were used for a three-point bending test and compression test of the femoral neck. RESULTS: After 8 weeks of atorvastatin administration, a significant decrease was found in serum level of bone ALP to 30% vs. CO (p = 0.005). PINP, CTX-I and OC did not change significantly. The expression of BMP-2 was increased. There were no significant differences in BMD measurements, three-point bending test or compression test of the femoral neck. CONCLUSIONS: Our results suggest that atorvastatin has a positive effect on bone metabolism in rats by maintenance of BMD and the biomechanical characteristics of bone. Atorvastatin influenced bone metabolism by decreasing bone ALP, and probably in consequence increasing expression of BMP-2 in rats.

C-reactive protein, chemerin, fetuin-A and osteopontin as predictors of cardiovascular risks in persons with psoriasis vulgaris.

The study aimed to contribute to understanding the role of CRP, chemerin, fetuin-A and osteopontin and to assess their suitability as biomarkers of early stages of cardiovascular diseases in psoriasis vulgaris. Serum levels measured in 28 patients and 22 controls. Patients: increased levels of CRP (p<0.001), chemerin (p<0.05), osteopontin (p<0.05) and decreased levels of fetuin-A (p<0.05), significant relationships between CRP and fetuin-A (rho=0.530, p<0.01), CRP and chemerin (rho=0.543, p<0.01), CRP and age (rho=0.590, p<0.001), osteopontin and fetuin-A (r=-0.415, p<0.05), chemerin and PASI score (rho=-0.424, p<0.05). We confirmed specific roles of the biomarkers in psoriasis. CRP, fetuin-A and osteopontin could be considered appropriate markers for the detection of early stages of cardiovascular diseases.

Alendronate lowers cholesterol synthesis in the central nervous system of rats - a preliminary study.

Nitrogen-containing bisphosphonates were found to inhibit farnesyl diphosphate synthase - an essential enzyme in the cholesterol biosynthesis pathway, but their effect on cholesterol synthesis per se in the central nervous system (CNS) remains unknown. The aim of the present study was to examine possible influence of a representative agent alendronate on cholesterol synthesis rates in selected parts of rat CNS and on plasma cholesterol level. Two groups of rats were orally administered either alendronate (3 mg/kg b.w.) or vehicle for 9 days. At the end of experiment, brain (basal ganglia, frontal cortex and hippocampus) and spinal cord were isolated and cholesterol synthesis was determined using the technique of deuterium incorporation from deuterated water. In the alendronate group significant reductions of cholesterol synthesis rates were detected in frontal cortex, hippocampus and spinal cord (p<0.001). However, the experimental treatment did not produce a significant alteration in the levels of plasma cholesterol. In conclusion, this study brings the first experimental evidence of the inhibition of cholesterol biosynthesis with alendronate in central nervous system.

Time-course of tissue factor plasma level in patients with acute coronary syndrome.

Enhanced expression of tissue factor (TF) may result in thrombosis contributing to acute clinical consequences of coronary artery disease. Several studies demonstrated elevated plasma levels of TF in patients with acute coronary syndrome (ACS). The aim of our study was to compare the concentrations of TF in coronary sinus (CS), proximal part of the left coronary artery (LCA) and peripheral vein (PV) of patients with ACS and stable coronary artery disease (SCAD). Time course of the TF plasma levels in PV was followed on day 1 and day 7 after index event of ACS presentation and was compared to day 0 values. No heparin was given prior to the blood sampling. Twenty-nine patients in the ACS group (age 63.6+/-10.8 years, 20 males, 9 females) and 24 patients with SCAD (age 62.3+/-8.1 years, 21 males, 3 females) were examined. TF plasma level was significantly higher in patients with ACS than in those with SCAD (239.0+/-99.3 ng/ml vs. 164.3+/-114.2 ng/ml; p=0.016). There was no difference in TF plasma levels in PV, CS and LCA (239.0+/-99.3 ng/ml vs. 253.7+/-131.5 ng/ml vs. 250.6+/-116.4 ng/ml, respectively). TF plasma levels tended to decrease only non-significantly on the day 7 (224.4+/-109.8 ng/ml). Significant linear correlation between TF and high sensitivity CRP (hs-CRP) levels on day 0 was found. In conclusion, TF plasma levels are elevated in patients with ACS not only locally in CS but also in systematic circulation. Our data support the relationship between TF production and proinflammatory mediators.

[Multi-marker approach in the diagnostics of cardiac diseases by protein biochip technology]. / Multianalytový prístup k diagnostice srdecních chorob technologií proteinových biocipu.

BACKGROUND: Multi-marker approach is recommended for rapid diagnostics and risk stratification of acute coronary syndrome. We tested the analytical performance of protein biochip technology for determination cardiac markers. METHODS: Analysis of cardiac markers: CK-MB mass, cTnl, myoglobin, glycogen phosphorylase BB (GPBB), heart type of fatty acid binding protein (h-FABP) and carbonic anhydrase III (CAIII) was performed by system Evidence Investigator (Randox). Analytical parameters of Cardiac array were tested. The Evidence Investigator results were compared with Elecsys 2010 (Roche) CK-MB mass and myoglobin methods. Markers of myocardial injury were determined in 28 blood donors, 28 patients with acute myocardial infarction diagnosis and 21 patients after chemotherapy containing anthracyclines (monitoring of cardiotoxicity). RESULTS: The Passing-Bablok regression shows statistically significant differences in results. The reasons for these differences are poor standardization of methods and discrepancies between calibrations. New substances h-FABP and GPBB are promising early markers of acute myocardial infarction and diagnostic sensitivity of h-FABP would be better than myoglobin test. These markers can be useful for monitoring of cardiotoxicity of anthracyclines. CONCLUSIONS: In future, the use of biochip technology in cardiology diagnostic represents an important challenge but it is a necessary standardization of immunochemical methods.

[Drugs influencing bone metabolism in diabetic patients]. / Léky ovlivnující kostní metabolizmus diabetiku.

A significant improvement in the knowledge of physiology, pathophysiology and pathobiochemistry of bone metabolism has enhanced the understanding not only of regulatory mechanisms of bone remodelation in adulthood but also of the dysfunction of such regulatory processes with other diseases. In the pathophysiology of metabolic bone disease in diabetic patients, attention is currently being focused on the method in which the differentiation of mesenchymal stem cells in osteoblasts, or adipocytes is regulated. The key role of PPAR-gamma, its activation or activity inhibition and thus also the directing of the differentiation in fat cells or osteoblasts is especially important in diabetic patients. Thiazolidinediones, in particular of the rosiglitazone type, have a positive impact on increased tissue sensitivity to insulin built on the activation of PPAR-gamma. Therefore, they can provoke the image of an aging bone in case of prolonged administration. Nevertheless, many factors contribute to the causes of increased fragility of bones in diabetic patients, both directly and indirectly--AGE, changes in insulin and IGF-I concentrations, renal dysfunctions, chronic inflammatory processes and impaired immunity. The treatment of osteoporosis in diabetic patients does not principally differ from the treatment of postmenopausal osteoporosis.

UHPLC-HRMS study of anti-Alzheimer's drug candidates: metabolism of 7-MEOTA-tryptophan hybrids hampers their passage into brain.

Being among the top five causes of death in the developed world, Alzheimer's disease represents a major socio-economic issue. We administered a single intramuscular dose of two new hybrid anti-Alzheimer's compounds, with 7-methoxytacrine (7-MEOTA; acetylcholinesterase inhibitor) and tryptophan (inhibitor of amyloid accumulation) in their structure, to rats. Using validated ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) methods, we uncovered their inability to enter the site of action - the brain. We discuss four possible explanations: i) physico-chemical properties, ii) lack of active/facilitated transport, iii) effective efflux and/or iv) extensive metabolism. High-resolution mass spectrometric analyses proved that the compounds are easily hydrolysed at amide bond between tryptophan and the linker both in vitro and in vivo. Contrary to the parent compounds these metabolites - analogues of 7-MEOTA - can enter the brain in significant amounts.