Tracing TET1 expression in prostate cancer: discovery of malignant cells with a distinct oncogenic signature.

BACKGROUND: Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is involved in DNA demethylation and transcriptional regulation, plays a key role in the maintenance of stem cell pluripotency, and is dysregulated in malignant cells. The identification of cancer stem cells (CSCs) driving tumor growth and metastasis is the primary objective of biomarker discovery in aggressive prostate cancer (PCa). In this context, we analyzed TET1 expression in PCa. METHODS: A large-scale immunohistochemical analysis of TET1 was performed in normal prostate (NOR) and PCa using conventional slides (50 PCa specimens) and tissue microarrays (669 NOR and 1371 PCa tissue cores from 371 PCa specimens). Western blotting, RT-qPCR, and 450 K methylation array analyses were performed on PCa cell lines. Genome-wide correlation, gene regulatory network, and functional genomics studies were performed using publicly available data sources and bioinformatics tools. RESULTS: In NOR, TET1 was exclusively expressed in normal cytokeratin 903 (CK903)-positive basal cells. In PCa, TET1 was frequently detected in alpha-methylacyl-CoA racemase (AMACR)-positive tumor cell clusters and was detectable at all tumor stages and Gleason scores. Pearson's correlation analyses of PCa revealed 626 TET1-coactivated genes (r > 0.5) primarily encoding chromatin remodeling and mitotic factors. Moreover, signaling pathways regulating antiviral processes (62 zinc finger, ZNF, antiviral proteins) and the pluripotency of stem cells were activated. A significant proportion of detected genes exhibited TET1-correlated promoter hypomethylation. There were 161 genes encoding transcription factors (TFs), of which 133 were ZNF-TFs with promoter binding sites in TET1 and in the vast majority of TET1-coactivated genes. CONCLUSIONS: TET1-expressing cells are an integral part of PCa and may represent CSCs with oncogenic potential.

Novel insights into peptide amidation and amidating activity in the human circulation.

C-terminal α-amidation is the final and essential step in the biosynthesis of several peptide hormones. Peptidylglycine α-amidating monooxygenase (PAM) is the only known enzyme to catalyse this reaction. PAM amidating activity (AMA) is known to be present in human circulation, but its physiological role and significance as a clinical biomarker remains unclear. We developed a PAM-specific amidation assay that utilizes the naturally occurring substrate Adrenomedullin-Gly (ADM-Gly, 1-53). Using our amidation assay we quantified serum amidating activities in a large population-based cohort of more than 4900 individuals. A correlation of serum amidating activity with several clinical parameters including high blood pressure was observed. Increasing PAM-AMA was an independent predictor of hard outcomes related to hemodynamic stress such as cardiovascular mortality, atrial fibrillation and heart failure during long-term follow-up (8.8 ± 2.5 years). Moreover, results from an animal study in rats utilizing recombinant human PAM provide novel insights into the physiological role of circulating PAM and show its potential significance in circulating peptide amidation.

Prognostic role of high TET1 expression in patients with solid tumors: A meta-analysis.

BACKGROUND: Recently, increased expression of TET1 has been shown to inhibit tumor development in many studies. Therefore, a meta-analysis was conducted to assess the prognostic role of TET1 in solid tumors. METHODS: PubMed, Embase, and the Web of Science (last updated on June 13, 2019) were searched and 16 eligible studies involving 3100 patients were eventually taken forward into the meta-analysis. RESULTS: Pooled results indicated that higher TET1 expression in cancer tissues was associated with improved overall survival (OS) [hazard ratio (HR) = 0.736, 95% confidence interval (95% CI) = 0.542-0.998, P = .049]. In the subgroup analysis, higher TET1 expression in respiratory tumors (HR = 0.778, 95% CI = 0.639-0.946, P = .012) and breast cancer in Asian patients (HR = 0.326, 95% CI = 0.199-0.533, P < .001) were significantly associated with better OS. In addition, the association between high TET1 expression and prolonged OS was also statistically significant in the following subgroups; data source from samples (HR = 0.561, 95% CI = 0.384-0.819, P = .003), reported in text (HR = 0.539, 95% CI = 0.312-0.931, P = .027), TET1 protein (HR = 0.635, 95% CI = 0.409-0.984, P = .042), Asians (HR = 0.563, 95% CI = 0.376-0.844, P = .005). CONCLUSION: This meta-analysis displays that high expression levels of TET1 in tissues is significantly associated with better survival in patients with solid tumors. This finding can be used as evidence to the tone that TET1 may be a useful target for the treatment of patients with solid tumors in the future.

Variant analysis of HPD genes from two families showing elevated tyrosine upon newborn screening by tandem mass spectrometry (MS/MS).

Background Alterations in the structure and activity of 4-hydroxyphenylpyruvate dioxygenase (HPD) are causally related to two different metabolic disorders: recessively inherited tyrosinemia type III and dominantly inherited hawkinsinuria. The aim of this study was to provide a new perspective for the clinical understanding of the pathogenesis of tyrosinemia type III or hawkinsinuria. Case presentation A full-term newborn baby born after a safe pregnancy and childbirth with a birth weight of 3200 g and another full-term baby born after a safe pregnancy and childbirth with a birth weight of 2800 g are reported and analysed. DNA extraction, next-generation sequencing, bioinformatics analysis, Sanger sequencing and biochemical analysis were performed. One patient with a heterozygous HPD gene (NM_002150.2) c.460G > A mutation and one patient with a heterozygous HPD gene (NM_002150.2) c.248delG mutation showing elevated tyrosine levels upon newborn screening by tandem mass spectrometry (MS/MS) are reported. Conclusions The HPD gene may not be a strictly autosomal recessive pathogenic gene, which provides a new perspective for the clinical understanding of the pathogenesis of tyrosinemia type III or hawkinsinuria.

Elevated Expression of A-Raf and FA2H in Hepatocellular Carcinoma is Associated with Lipid Metabolism Dysregulation and Cancer Progression.

BACKGROUND: Identification of events leading to hepatocellular carcinoma (HCC) progression is essential for understanding its pathophysiology. The aims of this study are to identify and characterize differentially expressed proteins in serum of HCC-bearing rats and the corresponding controls during cancer initiation, progression and tumorigenesis. METHODS: Chemical carcinogens, N-Nitrosodiethylamine and 2-aminoacetylfluorine are administered to induce HCC to male Wistar rats. The 2D-Electrophoresis and PD-Quest analyses are performed to identify several differentially expressed proteins in serum of HCC-bearing animals. These proteins are further characterized by MALDI-TOF-MS/MS analyses. Using pathwaylinker a HCC-specific network is analyzed among the MALDITOF- MS/MS characterized proteins and their interactors. RESULTS: Carcinogen administration caused inflammation leading to liver injury and HCC development. Liver inflammation was confirmed by increase in the levels of TNF-α and IL-6 in carcinogen treated rats. We report significant increase in expression of two differentially expressed proteins, namely, A-Raf and Fatty Acid 2- Hydroxylase (FA2H), at early stage of HCC initiation, during its progression and at tumor stage. Real-time PCR analysis of mRNA for these proteins confirmed up-regulation of their transcripts. Further, we validated our experimental data with sera of clinically confirmed liver cancer patients. CONCLUSION: The study suggests that FA2H and A-Raf play a major role in the progression of HCC.

Reduced mRNA and Protein Expression Levels of Tet Methylcytosine Dioxygenase 3 in Endothelial Progenitor Cells of Patients of Type 2 Diabetes With Peripheral Artery Disease.

Endothelial progenitor cells (EPCs) with immunological properties repair microvasculature to prevent the complications in patients with diabetes. Epigenetic changes such as DNA methylation alter the functions of cells. Tet methylcytosine dioxygenases (TETs) are enzymes responsible for the demethylation of cytosine on genomic DNA in cells. We hypothesized that EPCs of diabetic patients with peripheral artery disease (D-PAD) might have altered expression levels of TETs. Subjects who were non-diabetic (ND, n = 22), with diabetes only (D, n = 29) and with D-PAD (n = 22) were recruited for the collection of EPCs, which were isolated and subjected to analysis. The mRNA and protein expression levels of TET1, TET2, and TET3 were determined using real-time PCR and immunoblot, respectively. The TET1 mRNA expression level in ND group was lower than that in the D and D-PAD groups. The TET3 mRNA level in the ND group was higher than that in the D group, which was higher than that in the D-PAD group. The TET1 protein level in the D-PAD group, but not the D group, was higher than that in the ND group. The TET2 protein level in the D-PAD group, but not the D group, was lower than that in the ND group. The TET3 protein level in the ND group was higher than that in the D group, which was higher than that in the D-PAD group, which is the lowest among the three groups. The changes of TETs protein levels were due to the alterations of their transcripts. These probably lead to epigenetic changes, which may be responsible for the reductions of EPCs numbers and functions in patients with the D-PAD. The expression pattern of TET3 mRNA and TET3 protein in EPCs may be a biomarker of angiopathy in diabetic patients.

Assessment of a combination of Serum Proteins as potential biomarkers to clinically predict Schizophrenia.

Schizophrenia (SZ) is a devastating psychiatric disorder. Validation of potential serum biomarkers during first-episode psychosis (FEP) is especially helpful to understand the onset and prognosis of this disorder. To address this question, we examined multiple blood biomarkers and assessed the efficacy to diagnose SZ. The expression levels of Neuregulin1 (NRG1), ErbB4, brain-derived neurotrophic factor (BDNF), DNA methyltransferases 1 (DNMT1) and ten-eleven translocation 1 (TET1) proteins in peripheral blood of 53 FEP patients and 57 healthy controls were determined by enzyme-linked immunosorbent assay (ELISA). Multivariable logistic regression including biomarker concentration as covariates was used to predict SZ. Differentiating performance of these five serum protein levels was analyzed by Receiver Operating Characteristic (ROC) curve analysis. We found that patients with SZ present a higher concentration of DNMT1, and TET1 in peripheral blood, but a lower concentration of NRG1, ErbB4 and BDNF than controls. Multivariable logistic regression showed that ErbB4, BDNF and TET1 were independent predictors of SZ, and when combined, provided high diagnostic accuracy for SZ. Together, our findings highlight that altered expression of NRG1, ErbB4, BDNF, DNMT1 and TET1 are involved in schizophrenia development and they may serve as potential biomarkers for the diagnosis of the schizophrenia. Therefore, our study provides evidence that combination of ErbB4, BDNF and TET1 biomarkers could greatly improve the diagnostic performance.

DNA Hydroxymethylation Levels Are Altered in Blood Cells From Down Syndrome Persons Enrolled in the MARK-AGE Project.

Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the aging process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work, we investigated the levels of 5-hydroxymethylcytosine and of the Ten-eleven translocation dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in aging. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease in 5-hydroxymethylcytosine, TET1, and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS.

A rare case of sterol-C4-methyl oxidase deficiency in a young Italian male: Biochemical and molecular characterization.

Inborn defects of cholesterol biosynthesis are metabolic disorders presenting with multi-organ and tissue anomalies. An autosomal recessive defect involving the demethylating enzyme C4-methyl sterol (SC4MOL) has been reported in only 4 patients so far. In infancy, all patients were affected by microcephaly, bilateral congenital cataracts, growth delay, psoriasiform dermatitis, immune dysfunction, and intellectual disability. Herein, we describe a new case of SC4MOL deficiency in which a 19-year-old Italian male was affected by bilateral congenital cataracts, growth delay and learning disabilities, behavioral disorders and small stature, but not microcephaly. Our patient had abundant scalp dandruff, without other skin manifestations. Analysis of the blood sterol profile showed accumulation of C4-monomethyl and C4-dimethyl sterols suggesting a deficiency of the SC4MOL enzyme. Sequencing of the MSMO1 gene (also known as the "SC4MOL" gene) confirmed mutations in each allele (c.731A>G, p.Y244C, which is already known, and c.605G>A, p.G202E, which is a novel variant). His father carried c.731A>G mutation, whereas his mother carried c.605G>A. Thus, the combination of multiple skills and methodologies, in particular, blood sterol profiling and genetic analysis, led to the diagnosis of a new case of a very rare defect of cholesterol biosynthesis. Consequently, we suggest that these two analyses should be performed as soon as possible in all undiagnosed patients affected by bilateral cataracts and developmental delay.

The Effect of Selenium Supplementation on Glucose Homeostasis and the Expression of Genes Related to Glucose Metabolism.

The aim of the study was to evaluate the effect of selenium supplementation on the expression of genes associated with glucose metabolism in humans, in order to explain the unclear relationship between selenium and the risk of diabetes. For gene expression analysis we used archival samples of cDNA from 76 non-diabetic subjects supplemented with selenium in the previous study. The supplementation period was six weeks and the daily dose of selenium was 200 µg (as selenium yeast). Blood for mRNA isolation was collected at four time points: before supplementation, after two and four weeks of supplementation, and after four weeks of washout. The analysis included 15 genes encoding selected proteins involved in insulin signaling and glucose metabolism. In addition, HbA1c and fasting plasma glucose were measured at three and four time points, respectively. Selenium supplementation was associated with a significantly decreased level of HbA1c but not fasting plasma glucose (FPG) and significant down-regulation of seven genes: INSR, ADIPOR1, LDHA, PDHA, PDHB, MYC, and HIF1AN. These results suggest that selenium may affect glycemic control at different levels of regulation, linked to insulin signaling, glycolysis, and pyruvate metabolism. Further research is needed to investigate mechanisms of such transcriptional regulation and its potential implication in direct metabolic effects.

Effects of electromagnetic fields exposure on plasma hormonal and inflammatory pathway biomarkers in male workers of a power plant.

PURPOSE: The potential health risks of electromagnetic fields (EMFs) have currently raised considerable public concerns. The aim of this study was to evaluate the effects of EMF exposure on levels of plasma hormonal and inflammatory pathway biomarkers in male workers of an electric power plant. METHODS: Seventy-seven male workers with high occupational EMF exposure and 77 male controls with low exposure, matched by age, were selected from a cross-sectional study. Moreover, high EMF exposure group was with walkie-talkies usage and exposed to power frequency EMF at the work places for a longer duration than control group. A questionnaire was applied to obtain relevant information, including sociodemographic characteristics, lifestyle factors, and EMF exposures. Plasma levels of testosterone, estradiol, melatonin, NF-κB, heat-shock protein (HSP) 70, HSP27, and TET1 were determined by an enzyme-linked immunosorbent assay. RESULTS: EMF exposure group had statistically significantly lower levels of testosterone (ß = -0.3 nmol/L, P = 0.015), testosterone/estradiol (T/E2) ratio (ß = -15.6, P = 0.037), and NF-κB (ß = -20.8 ng/L, P = 0.045) than control group. Moreover, joint effects between occupational EMF exposure and employment duration, mobile phone fees, years of mobile phone usage, and electric fees on levels of testosterone and T/E2 ratio were observed. Nevertheless, no statistically significant associations of EMF exposures with plasma estradiol, melatonin, HSP70, HSP27, and TET1 were found. CONCLUSIONS: The findings showed that chronic exposure to EMF could decrease male plasma testosterone and T/E2 ratio, and it might possibly affect reproductive functions in males. No significant associations of EMF exposure with inflammatory pathway biomarkers were found.

[Prognostic significance of indices of endogenous intoxication and of a monooxygenase system on the stages of surgical treatment in patients, suffering abdominal sepsis].

Some indices of endogenous intoxication and lymphocytic monooxygenase activity in the blood of patients, suffering abdominal sepsis (AS), were investigated, their prognostic significance was determined. In 28 patients the signs of AS were revealed, in 14 of a severe sepsis, in 11--of a septic shock. For peritoneal AS there were operated 37 patients, and for a pancreatogenic one--15. Relaparotomy "on demand" was performed in 12, and "the programmed" one--in 15 patients. A 30-days lethality in AS was 10.7%, in severe shock--28.6%, and in a septic one--63.6%. While AS occurrence a rising of metabolic activity of the monooxygenase system were registered in certain terms, and significant inhibition of this index--while severe state of the patients in a refractory shock occurrence. A safety correlational connections of indices in the blood and the lymphocytic monooxygenase system activity were determined in patients, who died.

Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates.

BACKGROUND: Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. METHODS: Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. RESULTS: The pooled human AB serum contained 0.38 µg/ml anti-Neu5Gc IgG and 0.085 µg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. CONCLUSIONS: Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.

A serotonin-induced N-glycan switch regulates platelet aggregation.

Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation.

Retrospective evidence for clinical validity of expanded genetic model in warfarin dose optimization in a South Indian population.

AIM: To optimize warfarin dose in patients at risk for thrombotic events, we have recently developed a pharmacogenomic algorithm, which explained 44.9% of the variability in warfarin dose requirements using age, gender, BMI, vitamin K intake, CYP2C9 (*2 and *3) and VKORC1 (*3, *4 and -1639 G>A) as predictors. The aim of the current study is to develop an expanded genetic model that can explain greater percentage of warfarin variability and that has clinical validity. PATIENTS & METHODS: CYP2C9*8, CYP4F2 V433M, GGCX G8016A and thyroid status were added to an expanded genetic model (n = 243). RESULTS: The expanded genetic model explained 61% of the variability in warfarin dose requirements, has a prediction accuracy of ±11 mg/week and can differentiate warfarin sensitive and warfarin resistant groups efficiently (areas under receiver operating characteristic curves: 0.93 and 0.998, respectively; p < 0.0001). Higher percentage of International Normalized Ratios in therapeutic range (52.68 ± 4.21 vs 43.80 ± 2.27; p = 0.04) and prolonged time in therapeutic range (61.74 ± 3.18 vs 47.75 ± 5.77; p = 0.03) were observed in subjects with a prediction accuracy of <1 mg/day compared with subjects with prediction accuracy >1 mg/day. In the warfarin-resistant group, primary hypothyroidism was found to induce more resistance while in the warfarin-sensitive group, hyperthyroidism was found to increase sensitivity. CONCLUSION: The expanded genetic model explains greater variability in warfarin dose requirements and it prolongs time in therapeutic range and minimizes out-of-range International Normalized Ratios. Thyroid status also influences warfarin dose adjustments.

A common X-linked inborn error of carnitine biosynthesis may be a risk factor for nondysmorphic autism.

We recently reported a deletion of exon 2 of the trimethyllysine hydroxylase epsilon (TMLHE) gene in a proband with autism. TMLHE maps to the X chromosome and encodes the first enzyme in carnitine biosynthesis, 6-N-trimethyllysine dioxygenase. Deletion of exon 2 of TMLHE causes enzyme deficiency, resulting in increased substrate concentration (6-N-trimethyllysine) and decreased product levels (3-hydroxy-6-N-trimethyllysine and γ-butyrobetaine) in plasma and urine. TMLHE deficiency is common in control males (24 in 8,787 or 1 in 366) and was not significantly increased in frequency in probands from simplex autism families (9 in 2,904 or 1 in 323). However, it was 2.82-fold more frequent in probands from male-male multiplex autism families compared with controls (7 in 909 or 1 in 130; P = 0.023). Additionally, six of seven autistic male siblings of probands in male-male multiplex families had the deletion, suggesting that TMLHE deficiency is a risk factor for autism (metaanalysis Z-score = 2.90 and P = 0.0037), although with low penetrance (2-4%). These data suggest that dysregulation of carnitine metabolism may be important in nondysmorphic autism; that abnormalities of carnitine intake, loss, transport, or synthesis may be important in a larger fraction of nondysmorphic autism cases; and that the carnitine pathway may provide a novel target for therapy or prevention of autism.

[Dynamics of antipirin test at patients with alcoholic liver disease on the background of metadoksin drug receiving].

AIM: To study the effect of the drug metadoxin on drug-metabolizing liver function in patients with liver lesions alcoholic etiology (ALD). MATERIALS AND METHODS: 36 patients with ALD, of which 16 patients were diagnosed with hepatitis, while 20 - with the liver cirrhosis. All the patients underwent biochemical blood analysis and the study of drug-metabolizing liver function according to the pharmacokinetics of antipyrine in saliva before and after treatment with metadoxin. Metadoxin was administered at a dose of 500 mg once a day for 28 days. Concentrations of antipyrine in saliva samples were determined by HPLC. RESULTS. It was shown that of 36 patients examined in the 28-patients with ALD (group 2) there was a significant decrease in activity of liver enzymes according to the test with antipyrine (T1/2 = 28.7 +/- 3.4, CL = 17,9 +/- 5.2; p < 0,01 vs normal), whereas in 8 patients (group 1) was noted the typical for alcohol inductive influence on the activity of liver monooxygenases (T1/2 = 7.8 +/- 1.5, CL = 39.1 +/- 6.8; p < 0,05 vs normal). As a result of the 28-day therapy with metadoxin was a normalization of the pharmacokinetic parameters of AP in Gr. 1 (12.6 +/- 1.8; p < 0.05; NS vs normal) and a significant improvement of it in patients of Gr. 2 (17.9 +/- 5.2, vs N, p <0.05). Biochemical markers of ALD (AST/ALT, GGT, ALP) also demonstrated a positive dynamics in patients of both study groups. Correlation analysis of changes in CL and GGT (r1) and changes in AST/ALT and T1/2 (r2) showed a fairly high degree of correlation between these parameters (r1 = 0.58, r2 = 0.65). CONCLUSION: The results showed marked improvement of drug-metabolizing liver function according to the test with antipyrine in patients with ALD after treatment with metadoxin.

Failure of pharmacogenetic-based dosing algorithms to identify older patients requiring low daily doses of warfarin.

INTRODUCTION: Warfarin doses vary greatly among patients and warfarin administration is accompanied by risk of bleeding. Genes responsible for its metabolism (CYP2C9) and effect on clotting (VKORC1) have been identified. It has been suggested that genotyping for variants in these genes can improve warfarin dosing and decrease bleeding complications. We evaluated performance of pharmacogenetic-based warfarin dosing estimation algorithms in old and very old patients. METHODS: Cross-sectional study of stable patients older than 65 years receiving warfarin with therapeutic International Normalized Ratio (INR) anticoagulation. Medical and laboratory data were reviewed and genotyping for CYP2C9 and VKORC1 performed. Warfarin dose estimates with and without genotype information were compared to clinically established therapeutic doses. RESULTS: Sixty-nine patients (32 men, 37 women; 41 nursing home residents; 28 senior care community residents) aged 81.4 ± 8.3 (mean ± S.D) years; ethnicity: Caucasian in 53, Asian in 10, Hispanic in 4, and African American in 2, received 3.3 ± 1.7 mg/d (range 0.7-9) warfarin achieving target INRs of 2.5 ± 0.2. Pharmacogenetic-based dose estimates (in combination with age, weight, height, smoking history, ethnicity/race, history of liver disease, selected co-medications such as amiodarone and enzyme inducers, baseline INR, clinical indication, and target INR), explained 50% of variability (P < .0001) compared with 12% without genetic data (P = .003). However, doses were overestimated in 15 of 16 patients requiring less than 2 mg/d (2.6 ± 0.9 mg/d compared with observed 1.5 ± 0.3 mg/d, P = .0001). Renal disease was a potential variable contributing to low warfarin requirements. DISCUSSION: The role of pharmacogenetic testing in the management of warfarin administration in patients is undergoing evaluation. Currently available pharmacogenetic- based warfarin dose estimation algorithms reduce variability in estimates for groups of older patients but consistently overestimate doses for older patients requiring the lowest doses of warfarin. CONCLUSIONS: Pharmacogenetic data add to our understanding of variability in warfarin dosing requirements but do not accurately identify older patients requiring the lowest warfarin doses. Therefore, the most prudent approach to warfarin therapy in older patients should include low initial doses in the absence of genotype variants associated with very low warfarin sensitivity and careful monitoring of INR responses.

Interactions of peptide amidation and copper: novel biomarkers and mechanisms of neural dysfunction.

Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for peptidylglycine alpha-amidating monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM(+/-) mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM(+/-) mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency.

Vitamin K epoxide reductase complex subunit 1 gene polymorphism is associated with atherothrombotic complication after drug-eluting stent implantation: 2-Center prospective cohort study.

BACKGROUND: Single nucleotide polymorphisms of vitamin K epoxide reductase complex subunit 1 (VKORC1) was reported to have association with arterial vascular disease. We investigated whether single nucleotide polymorphism of VKORC1 +2255 is associated with clinical outcomes among patients who underwent drug-eluting stent (DES) implantation. METHODS: We prospectively collected genomic DNA in patients who underwent DES deployment from September 2003 to December 2006 and compared clinical outcomes according to their VKORC1 genotype at the locus + 2255 (rs 2359612). The primary end point was composite of atherothrombotic events (cardiac death, myocardial infarction, and nonhemorrhagic stroke). RESULTS: Mean follow-up duration was 631 +/- 251 days. Genotyping was completed in 764 cases (TT genotype [n = 640, 83.8%] vs non-TT [CC or CT] genotype [n = 124, 16.2%]). Non-TT group showed more composite events than TT group (7.3% vs 3.0%, P = .032). In the Cox regression analysis, non-TT genotype of VKORC gene was a significant predictor of atherothrombotic events (hazard ratio 2.56, 95% confidence interval 1.14-5.78). In the event-free survival analysis, non-TT group also showed significantly poorer cardiovascular events-free survival rate than TT group (P = .02). CONCLUSIONS: VKORC1 genotype is associated with cardiovascular events in patients with DES implantation, suggesting the role of coagulation system.