Lectures L9.1 Session 9. Metabolic Diseases L9.2
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Lectures L9.1 Session 9. Metabolic Diseases L9.2
Session 9. Metabolic Diseases Lectures L9.2 L9.1 Mechanisms underlying pathological manifestations of genetic hyperhomocysteinemia in cystathionine β-synthase deficiency Congenital disorders of glycosylation (CDG) Maciej Adamowicz The Children's Memorial Health Institute, Department of Biochemistry and Experimental Medicine, Warsaw, Poland e-mail: Maciej Adamowicz <[email protected]> Congenital Disorders of Glycosylation (CDG) comprise permanently expanding group of inherited diseases caused by defects of multiple glycosylation pathways which disturb structure of many glycoconjugates classes. The first patients were described by Jaak Jaeken in 1980, in 1984 simple test was implemented to identify new cases. In 1995, fifteen years later, enzymatic defect was revealed as phosphomannomutase (PMM2) by van Schaftingen group and in 1999 the first classification was proposed. Defects in N-glycosylation pathway are detected most often due to the broad application of the simple screening method, isofocusing (IEF) of serum transferrin. Transferrin normally carries two complex type N-glycans terminated by negatively charged sialic acid. The loss of whole glycan on transferrin molecule or truncation of terminal sugars with sialic acid on glycan result in abnormal, diverse patterns in IEF. The type of pattern suggests location of defect in multistep N-glycosylation pathway and that was the basis of the first CDG classification. New patients identified were classified as CDG of type I or II, for defects in the assembly of dolichol-linked oligosaccharides (LLO) or for defects of subsequent processing steps of glycans bound to protein. Once the gen was found, the newly identified CDG subtype was lined up alphabeticaly as CDG Ia-Iq and CDG-IIa-h. These were mainly defects in the basic N-glycosylation machinery, in glycosyltransferases, glycosidases, sugar transporters but also in the biosynthesis of nucleotide-sugar. With time this nomenclature became inadequate because a novel defects were identified such as: combined N- and O- glycosylation defects due to disturbed vesicular transport (e.g. conserved oligomeric Golgi complex, V-ATPase a2 subunit ), O-mannosylation defects, defects of dolichol biosynthesis and glycolipids glycosylation. Therefore in 2008 the new nomenclature was proposed based on official gen symbol followed by CDG. At present CDG group encompass more than sixty inborn errors of metabolism of which majority are defects of glycoconjugates biosynthesis and small part concern proteins taking part in vesicular transport. Glycoconjugates are ubiquitous molecules in an organism, its function is various and play an important role for many complex processes such growth, differentiation, organ development, signal transduction, immunologic defense and many others. This knowledge well explains extremely broad and diverse clinical spectrum of individual CDG subtypes, from fatal, multisystem disease to milder disease involving only one organ or system. The study of patients with impaired glycosylation have provided already and may provide in the future the new insight into the basic mechanisms of glycosylation. References 1. Hennet T (2012) Biochim Biophys Acta doi:10.1016/j.bbagen.2012.02.001. 2. Jaeken J et al. (2009) Biochim Biophys Acta 1792: 825-826. Hieronim Jakubowski Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland; University of Life Sciences, Department of Biochemistry & Biotechnology, Poznań, Poland; UMDNJ-New Jersey Medical School, Department of Microbiology & Molecular Genetics, Newark, NJ, USA e-mail: Hieronim Jakubowski <[email protected]> Severe hyperhomocysteinemia due to cystathionine β-synthase (CBS) deficiency is associated with mental retardation, abnormalities in the skeletal system (osteoporosis, Marfanoid appearance), the skin (fine hair, brittle skin), the lung (pulmonary embolism), the eye (ectopia lentis), and the vascular system (atherothrombosis). Vascular complications are the major cause of morbidity and mortality in untreated patients. Mechanisms underlying these pathologies are unclear. Studies of homocysteine (Hcy) metabolism in cultured human fibroblasts from CBS deficient patients have led to a hypothesis that metabolic conversion of Hcy to Hcy-thiolactone and the reactivity of Hcy-thiolactone towards protein lysine residues provides biochemical bases that account for the pathological consequences of hyperhomocysteinemia (Jakubowski, JBC 1997). Hcy-thiolactone is a product of an enzymatic error-editing reaction in protein biosynthesis when Hcy is erroneously selected in place of methionine by methionyl-tRNA synthetase. The Hcy editing reaction and the biosynthesis of Hcy-thiolactone are universal, occur in all organisms examined from bacteria to human (Jakubowski, Wiley Interdiscip Rev RNA 2012). N-Hcy-protein is formed in a chemical reaction — called protein N-homocysteinylation - of Hcy-thiolactone with the ε-amino group of a protein lysine residue (Jakubowski, FASEB J 1999). We found that protein N-homocysteinylation is greatly enhanced in human CBS deficiency (Jakubowski et al., FASEB J 2008; Perła-Kajan & Jakubowski, FASEB J 2010) and in Cbs-null mouse models (Jakubowski et al., FASEB J 2009). Specifically, human CBS-deficient patients have elevated levels of plasma N-Hcy-protein, including pro-thrombotic N-Hcyfibrinogen, which can account for thrombosis, observed in these patients. Plasma N-Hcy-protein levels are negatively correlated with Hcy-thiolactonase activity of serum paraoxonase 1 (PON1) (r=-0.43, p=0.01), i.e. the higher the Hcythiolactonase activity the lower N-Hcy protein levels. These findings provide evidence that the Hcy-thiolactonase activity of PON1 is a determinant of plasma N-Hcy-protein levels and suggest that Hcy-thiolactonase/PON1 protects proteins against N-homocysteinylation in humans. In a Tg-I278T Cbs-/- mouse model, bone N-Hcy-collagen is significantly elevated compared with wild type littermates (61.0±41.2 vs. 18.3±5.7 pmol/mg bone, P=0.042. At the same time, collagen pyridinoline cross-links are significantly decreased in Tg-I278T Cbs-/- mice compared with Tg-I278T Cbs+/+ animals (20.8±3.5 vs. 15.2±2.7 pmol/mg bone, P=0.022) (Rusek & Jakubowski, unpublished). These finding suggest that N-homocysteinylation of collagen lysine residues prevents their cross-linking, which could account for connective tissue abnormalities observed in CBS-deficient patients. 47th Congress of the Polish Biochemical Society, 2012 Session 9. Metabolic Diseases215 L9.3 L9.4 Twenty years experience with enzyme replacement therapy for metabolic diseases Substrate reduction therapies for lysosomal storage diseases Anna Tylki-Szymańska Grzegorz Węgrzyn The Children’s Memorial Health Institute, Warsaw, Poland University of Gdańsk, Department of Molecular Biology, Gdańsk, Poland e-mail: Anna Tylki-Szymańska <[email protected]> e-mail: Grzegorz Węgrzyn <[email protected]> Lysosomal storage diseases (LSDs) are a class of more than 40 diseases in some of them the deficiency of a single lysosomal enzyme activity causes the progressive accumulation of undigested macromolecules in the lysosomes in cells of most tissues. Accumulation of lysosomal storage causes progressive lysosomal dysfunction, leading to increased manifestations of disease. The phenotypes vary from moderate to severe physical impairment and, in the worst case, can lead to death at an early age. LSDs represent “rare” or “ultra rare” diseases with an aggregate frequency of 1/7 000 live births. In the past, no specific therapy was available for the affected persons, and management consisted solely of supportive care and treatment of complications. Almost 50 years ago experiments conducted by Neufeld and all revealed that the metabolic defect of cultured fibroblasts from mucopolysaccharidosis patients can be compensated by the addition of corrective factors which are secreted by cells not having the same defect. In further studies it could be demonstrated that the corrective factors are enzymes that are taken up from outside the cell into the lysosomal compartment by receptor-mediated endocytosis via the mannose 6-phosphate receptor. The first disease treated with enzyme replacement therapy (ERT) was Gaucher disease, the treatment is commercially available from 1991. Since enzyme replacement therapy has been successfully introduced for patients with Gaucher disease, this principle of treatment has been taken into consideration for other lysosomal storage disorders as well. Clinical trials could demonstrate the clinical benefit of this therapeutic principle in Fabry disease, mucopolysaccharidoses type I, II and VI and in Pompe disease. However, the usefulness of ERT is limited due to the fact that a given enzyme preparation does not have beneficial effects on all aspects of a disorder in the same degree. Additionally, clinical studies have shown that many symptoms of a lysosomal storage disorder even after long-term treatment are no more reversible. However, ERT is not curative nevertheless treated patients achieve a much better state of health than in the absence of such therapy, and ERT is life saving/prolonging for several of these LSDs. The success of ERT for Gaucher disease represents a major advance in basic and clinical sciences, as well as in bringing major health improvements to affected people. Lysosomal storage diseases (LSD) are a group of inherited metabolic disorders caused by dysfunction of one of lysosomal proteins. Such defects lead to accumulation of various compounds in lysosomes, which in turn causes dysfunctions of the cells, tissues and organs. Bone marrow transplantation and enzyme replacement therapy have been developed as first attempts to treat LSD. Despite unquestionable successes in treatment of somatic symptoms of several LSD, these therapeutic procedures are not effective when central nervous system (CNS) is affected, since administered enzyme cannot cross the blood-brain-barrier (BBB). Therefore, alternative approaches are required to treat patients suffering from neuronopathic forms of LSD. One of them is substrate reduction therapy (SRT), based on the use of small molecules, which can cross BBB and slow down synthesis of compounds that cannot be degraded due to specific metabolic defect. Molecular mechanisms of SRT range from inhibition of activity of particular enzyme involved in a biosynthetic pathway to reduction of efficiency of expression of genes coding for enzymes necessary to the substrate synthesis. Current state of SRT for LSD will be presented, and achievements and specific problems will be discussed. The First Polish-German Biochemical Societies Joint Meeting, 2012 216 Session 9. Metabolic Diseases Oral presentations O9.2 O9.1 Activity regulation of GlcN-6-P synthase as an alternative way for diabetes treatment The comparison of direct LDL cholesterol measurement and calculated LDL cholesterol level in relation to matrix effects Aleksandra Miszkiel, Marek Wojciechowski, Sławomir Milewski Department of Pharmaceutical Technology and Biochemistry, Chemical Faculty, Gdańsk University of Technology, Gdańsk, Poland e-mail: Aleksandra Miszkiel <[email protected]> K. Mamica, K. Sztefko Diabetes mellitus is nowadays considered as one of the most common and the fastest growing chronic diseases in e-mail: katarzyna mamica <[email protected]> the world. According to The Centers for Disease Control and Prevention, the problem has already reached epidemic Background: Hypercholesterolemia is a metabolic dis- proportions in all developed countries. It is estimated that order characterized by high concentration of low-density in 2010, over 285 million people suffered from diabetes. lipoprotein cholesterol (LDL-C) in plasma. Reducing con- Diabetes is a group of metabolic diseases caused by inhercentration of LDL-C is the primary aim of therapy which ited and/or acquired deficiency in production of insulin by improves lipids profile. Reliable measurement of LDL-C is the pancreas or by the ineffectiveness of the insulin provery important for correct diagnosis of lipid disturbances duced. This deficiency causes an increase in the concentrain both adults and children. The calculation of LDL-C tion of blood glucose, which can provoke damages of the from Friedewald formula has many limitation and accuracy body’s systems, in particular the blood vessels and nerves. of direct measurement of LDL-C in pediatric population There are 3 most main types of diabetes. Type 1 results is largely unknown. from autoimmune destruction of insulin-producing beta Aim of the study: The aim of study was to compare the cells of the pancreas which leads to a complete deficiency direct measurement of LDL-C (D-LDL-C) and calculated of the insulin hormone. Type 2, accounting for 90–95% of LDL-C (C-LDL-C) levels in pediatric population in rela- all diabetes, consists of insulin resistance followed by failtion to different concentration of serum triglycerides (TG), ure in insulin production. The third type is the gestational urea, creatinine and glucose. diabetes: pregnant, so far non-diabetic women, have a high Material and Methods: Serum samples were collected blood glucose level during pregnancy [1]. from 162 children (mean age 9 months±11 month, 92 Although type 1 and 2 diabetes are still incurable, all forms boys, 70 girls) admitted to Children’s University Hospital have been successfully treatable since insulin became availin Krakow. LDL-C was measured by direct method using able. Moreover, type 2 diabetes may be controlled with Vitros analyzer and Fridewald formula was used for calcu- proper lifestyle and medications. Available drugs are effeclation of LDL-C. tive but not selective and thus, newer approaches are desResult: The good correlation between D-LDL-C and C- perately needed. LDL-C was observed for whole group of children, regard- In both type 1 and type 2 diabetes, insulin resistance can less the level of TG, urea and glucose (r=0.83). However, result from hyperglycemia itself. As for the mechanism of no acceptable correlation was observed for children with target tissues sensing and responding to hyperglycemia, it triglycerides levels above >3.0 mmol/l (r=0.77) and cre- has been suggested that glucose flux through the hexosaatinine levels above 100.0 umol/l (r=0.33). Both methods mine biosynthetic pathway might be responsible for some (for D-LDL-C measurement and C-LDL-C calculation) of the adverse regulatory effects of excessive glucose. For were less affected by pathological glucose (r=0.81) and urea example, glucosamine has been shown to be much more (r=0.80) levels. potent than glucose in inducing insulin resistance in culConclusion: In pediatric population, interpretation of tured adipocytes, and inhibitors of hexosamine synthesis LDL-C level obtained by direct method should take into block the ability of glucose to cause insulin resistance [2]. account the kidney disease. Glucosamine-6-phosphate synthase is responsible for catalysis of the first and rate-limiting step in hexosamine metabolism. Thus, understanding the mechanism of the enzyme activity regulation can be crucial for determining a new class of anti-diabetes drugs. The eukaryotic enzyme has a physiological inhibitor, UDP-GlcNAc and can be regulated by phosphorylation/ dephosphorylation mediated by protein kinase A and protein phosphatase. Computational approaches, i.e. molecular modeling tools have been used to succor traditional methods [3, 4]. College of Medicine, Jagiellonian University, Clinical Biochemistry Department, Kraków, Poland References 1. Williams RH et al. (2012) Textbook of endocrinology. 12th edition. 2. Hebert LF et al. (1996) J Clinic Invest 98: 930-936. 3. Raczynska J et al. (2007) J Mol Biol 372: 672-688. 4. Miszkiel A et al. (2011) J Mol Modeling 17: 3103-3115. 47th Congress of the Polish Biochemical Society, 2012 Session 9. Metabolic Diseases217 O9.3 O9.4 Sepsis and marker endothelial damage A pragmatic methodology as a measure of the effectiveness in metabolic diseases research Uladzimir Spas, Viktar Predka Grodno State Medical University, Anesthesiology and Intensive Care Department, Grodno, Bielarus e-mail: Viktor Predko <[email protected]> Sepsis is a leading cause of death in critically ill patients. Sepsis and septic shock are common causes for admission to intensive care units. The morbidity and mortality remain unacceptably high despite the advanced treatments, use of modern antibiotics and resuscitation therapies. Severe sepsis accounts for 20% of all admissions to intensive care units (ICUs) and is the leading cause of death in non-cardiac ICUs, yet comprehensive clinical practice guidelines had not existed. After numerous unsuccessful trials of antiinflammatory agents in patients with sepsis, investigators doubted that mortality could be decreased. Advances in unraveling the pathophysiology and genetic basis for the host response to sepsis have changed the prevailing understanding of the syndrome, and several therapies have demonstrated surprising efficacy. Sepsis is accompanied by profound metabolic disturbances and endothelial damage. Extracorporeal blood purification treatment methods have been used in the treatment of human sepsis in a variety of settings. One of the effective methods of treatment sepsis is hemosorption.Homocystein (Hcy) is one of endothelium toxic amino acid. Numerous epidemiological studies have reported that even moderate hyperhomocysteinemia is a risk factor, associated with cardiovascular diseases, neuropsy-chiatric disorders, complications of pregnancy. The aim of the studies was to measure the concentration of plasma Hcy (HPLC methods) under healthy people and septic patients.Plasma concentrations of homocysteine were measured using a HPLC technique with fluorescence detection of the derivatives obtained with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate. Blood was drawn into chilled EDTA-evacuated tubes. After centrifugation at 4 degrees C without delay, plasma samples were kept frozen at -20 degrees C until analysis. In plasma samples from 28 healthy subjects, concentration of homocysteine was 5.5 (4.4; 6.1) (Me, (25;75)) mumol/l. Homocystein level in blod plasma under sepsis patients was 10.3 (7.2; 14.2) mumol/l. Hemosorption sessions with biospecific antiproteinase hemosorbent “Ovosorb” has been performed 39 patients for hyperhomocysteinemia correction. 4–7 sessions each lasting 60 minutes were performed. During treatments the hemodynamics were stable.A significant reduction was observed in levels of Hcy 5.6 (4.3; 7.2) mumol/l, (P=0.05). The time at the ICU was 7.1±3.1 days. Our findings suggest that hyperhomocysteinemia could associated with sepsis. Hemosorption sessions with biospecific hemosorbent “Ovosorb” has allowed to reduce homocystein level to the healthy donors’ level. Application of hemosorption also reduces treatment time and mortality. Łukasz Kaczyński, Bogdan Solnica Department of Diagnostics, Jagiellonian University Medical College, Kraków, Poland e-mail: Łukasz Kaczyński <[email protected]> Background: Acquisition of scientific data became the gold standard in making decisions concerning the selection procedure in the course of the treatment the patient. The primary source of scientific evidence for health technology assessment (HTA) are randomized controlled trials (RCTs), but possibility of transfer of the results and conclusions from RCTs to the level of routine practice is problematic. In this situation an important role begin to play highly reliable studies that provide data on effectiveness – pragmatic clinical trials (PRCTs) (Patsopoulos, 2011, Dialogues Clin Neurosci 13: 217-224). Aim: The aim of this study was to gather and systematize the current information about pragmatic randomized trials in metabolic diseases. Methods: A systematic review in Medline through Pubmed using the following queries: “(pragmatic OR practical OR naturalistic OR real world) AND metabolic diseases” was performed. Results: Using this search strategy 3750 hits were obtained, of which a preliminary evaluation included nearly 20 publications. The included research (e.g. DEWL (Krebs et al., 2012, Diabetologia 55: 905-914), DEPICTED (Robling et al., 2012, BMJ 344: e2359) or J-STOP-MetS 2 (Munakata et al., 2008, Vasc Health Risk Manag 4: 415-420) demonstrated that the best reflection of the conditions of routine practice (generalizability) in metabolic PRCTs can be obtained mostly through the development of broader inclusion criteria, minimizing the exclusion criteria or broadening the scope of patients evaluation. Found studies will be also evaluated using tools which can solve the problem of determining whether the results of a randomized controlled trial may be really regarded as pragmatic: PRECIS or CONSORT criteria. Conclusion: Properly assessed PRCTs data in conjunction with information about the efficacy from RCTs will serve as a whole to determine effectiveness of investigated drugs or methods in metabolic diseases trials. The First Polish-German Biochemical Societies Joint Meeting, 2012 218 Session 9. Metabolic Diseases Posters P9.2 P9.1 Influence of incretinomimetics on adhesion molecules ICAM-1 and VCAM-1 expressed on artery endothelial cells Insulin increases glomerular filtration barrier permeability through dimerization of protein kinase G type Iα subunits Agnieszka Chyra, Agnieszka Kłych, Tomasz Francuz, Wojciech Garczorz Agnieszka Piwkowska1, Dorota Rogacka1, Irena Audzeyenka1, Małgorzata Kasztan2, Stefan Angielski1, Maciej Jankowski1,2 Medical University of Silesia, Department of Biochemistry in Katowice, Poland 1Mossakowski Medical Research Centre Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdańsk, Poland; 2Medical University of Gdańsk, Department of Therapy Monitoring and Pharmacogenetics, Gdańsk, Poland The world statistics demonstrate the epidemic nature of diabetes. This disorder carries an increased risk of heart attack, stroke, and complications related to cardiovascular dysfunction. Furthermore, diabetes is associated with significant changes to the structure and function of cardiac and vascular tissue. Recent studies indicate that in addition to the ability to control glucose level, incretinomimetics - a novel therapy used in diabetes, have beneficial effect on the cardiovascular system. These novel group of drugs can affect on endothelial cells through GLP-1 receptor leading to diminished endothelial cells dysfunction. However, the underlying related mechanisms are not known yet. The aim of study was to assess the influence of incretinomimetics (natural GLP-1 and synthetic exendin-4) on endothelial cells function. The cronary artery endothelial cells function was determined by measurement expression of the adhesion molecules (ICAM-1 and VCAM-1) under dysfunction conditions induced by TNF-α (2,5 or 10 ng/ml) and glycated albumin (100, 500, 1000 mg/l). Both TNF-α (proinflammatory cytokine) and glycated proteins contribute to progression of atherosclerosis among diabetics. In such conditions the expression of tested adhesion molecules was measured by ELISA. The activation of NFκB was determined using cells transduced with lentivirus containing a reporter gene, which undergoes expression in effect of NFκB activation. Results: Both GLP-1 (10 nM and 100 nM) and exendin-4 (1 nM and 10 nM) have a potent anti-inflammatory properties mediated through diminished expression of adhesion molecules ICAM-1 and VACM-1. Moreover, the possible effect is related to their influence on NFκB signaling pathway. Conclusions: A novel group of drugs used in diabetes therapy, incretinomimetics, reveals not only the ability to modulate the glycemia but also an anti-inflammatory action. This properties may be important in prevention of atherosclerosis, that is one of the main causes of death in western countries. e-mail: Irena Audzeyenka <[email protected]> The increase in the permeability of the glomerular barrier filtration to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor for vascular complications [1]. However, the underlying mechanisms are incompletely understood. We demonstrated recently that superoxide anion generation increases dimerization of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction [2]. Here we investigated whether insulin is involved in PKGI-dependent hyperpermeability of the glomerular filtration barrier. We assessed changes in insulin-induced glomerular permeability by measuring the transmembrane albumin flux in cultured rat podocytes. Expression of PKGIα and upstream proteins was confirmed in the podocytes using Western blotting and immunofluorescence. In this study, we confirmed that insulin (300 nM) enhanced superoxide anion generation, which increased from 3.84±0.19 to 5.55±0.18 nmol/mg protein in podocytes. When podocytes were exposed to exogenous insulin in non-reducing conditions, we observed a 2.2-fold increase in the formation of interprotein disulfide bonds in PKGIα. In addition, the amount of dimerized PKGIα/actin increased from 0.17±0.02 to 0.38±0.02. Podocyte exposure to insulin also caused an increase in permeability to albumin; the transmembrane flux for albumin increased from 45.3±2.1 to 81.0±10.3 mg/ml. This effect was abolished in the presence of either an NAD(P) H oxidase inhibitor (apocynin, 100 µM; 58.8±6.9 mg/ml) or a PKG inhibitor (Rp-8-cGMPS; 100 µM; 37.8±5.9mg/ ml). Moreover, using siRNA directed against NOX4, we were able to achieve a substantial knockdown of NOX4 (to about 40% of control levels) as assessed by immunoblot analysis. Insulin-evoked activation of NAD(P)H oxidase and increase transmembrane flux for albumin were reduced after NOX4 knockdown. Thus, we propose that an insulininduced redox imbalance, through activation NOX4 subunit of NAD(P)H oxidase, may affect the dimerization of PKGIa in podocytes and affect filtration barrier permeability. Taken together, these data demonstrate that insulin increases glomerular barrier albumin permeability via a PKGI-dependent mechanism involving NAD(P)H-dependent generation of superoxide anion. These findings reveal a role for insulin in the pathophysiology of diabetic glomerular nephropathy. e-mail: Agnieszka Chyra <[email protected]> Reference 1. Patrakka J et al. (2009) Nat Rev Nephrol 5: 463-468. 2. Piwkowska et al. (2012) J Cell Physiol 227: 1004-1016. Acknowledgments This work was supported by grants from the Foundation for Polish Science (POMOST/2011-4/6) and from the National Science Centre (Grants No. N N401 063337). 47th Congress of the Polish Biochemical Society, 2012 Session 9. Metabolic Diseases219 P9.3 P9.4 Genetic predisposition to inflammatory bowel disease Effects of incretin agonists on eNOS expression and nitric oxide synthesis in human endothelial cells Bartłomiej Ferra1, Magdalena Góra-Gębka2, Roman Kotłowski1, Lucyna Holec-Gąsior1, Dorota Drapała1 1Department of Microbiology, Faculty of Chemistry, Gdańsk University of Technology, Gdańsk, Poland; 2Department of Pediatrics, Gastroenterology, Hepatology and Nutrition, Medical University of Gdańsk, Gdańsk, Poland e-mail: Bartłomiej Ferra <[email protected]> Wojciech Garczorz1, Tomasz Francuz1, Agnieszka Chyra1, Agnieszka Kłych1, Krystyna Jagoda2 1Medical University of Silesia, Department of Biochemistry in Katowice, Poland; 2Medical University of Silesia, Department of Hematology and Bone Marrow Transplantation in Katowice, Poland e-mail: Wojciech Garczorz <[email protected]> Inflammatory bowel disease (IBD) is a chronic, incurable inflammatory disease of the digestive system. The two main disease entities included in the IBD are ulcerative colitis and Crohn's disease. According to epidemiological studies there are more and more new cases every year. In especially among the youngest patients with symptoms of malnutrition and growth inhibition to land up in hospital with cancer suspected. The purpose of this study was to determine the genetic predisposition of the disease in the population of children. On the literature study basis there were genes slc11a1, irgm and atg16l1 selected and analyzed. The proteins encoded by these genes probably play a key role in the process of phagocytosis and autophagocytosis. They also interact with other components of the immune system and their dysfunction can lead to inflammation. The study was conducted on a selected group of 77 patients with confirmed IBD and 79 control patients. We used PCR-RFLP technique to detect point mutations, deletions and insertions within the promoter sequence, untranslated region (UTR) and ORF sequences. The results obtained for gene slc11a1 show that the D543N point mutation, 823C/T and 1729+55del4 deletion in the gene do not predispose to the risk of IBD among the studied population of children. Based on the preliminary results obtained for gene irgm can be concluded that 20.1 kb deletion from the promoter region of the gene and point mutation 281C/A also do not predispose to the risk of disease. However, the disease may be associated with point mutation T300A in the gene atg16l1, which encodes a protein essential for the phagophore formation in autophagocytosis process of pathogenic cells. Cardiovascular diseases are predominant cause of death in type 2 diabetes. Numerous vascular dysfunctions may be caused as a complication of metabolic disorders. Tumor necrosis factor (TNFα) is a cytokine involved in systemic inflammation. Evidence suggests that TNFα plays a pivotal role in the injury of macro- and microvascular circulation both in vivo and in vitro. TNFα-mediated signaling initiates and accelerates atherogenesis, vascular remodelling, oxidative stress and impaired NO bioavailability, resulting in vascular dysfunction. TNFα level is elevated in diabetic patients. An incretin hormone, glucagon-like peptide 1 (GLP-1) is a gut-derived hormone that stimulates insulin and suppresses glucagon secretion, inhibits gastric emptying, and reduces appetite and food intake. GLP-1 receptor is expressed in pancreatic islet α and β cells and in peripheral tissues, including heart, kidney, lung, gastrointestinal tract and vasculature. The pleiotropic actions of GLP-1 and synthetic agonist — exenatide on the control of blood glucose are of interest in the use of these agents for the treatment of type 2 diabetes. Some experimental and clinical data have suggested a considerable protective role of GLP-1 analogs in the cardiovascular system. Human vascular endothelial cells from the coronary artery (CAEC), aorta (AOEC) and iliac artery (IAEC) were used in this experiment. Nitric oxide synthase gene expression was assessed by Real-Time PCR. Protein quantity was assessed by western blot and ELISA. Nitric oxide production was assessed by flow cytometry using DAF-FM/DA. Each cell line was stimulated with TNFα, GLP-1 and exenatide. The project aims to assess the influence of incretin receptor agonists during stimulation of TNFα on: endothelial nitric oxide synthase (eNOS) mRNA expression, eNOS protein expression and nitric oxide synthesis. There were assessed potential differences in the influence of tested substances on endothelial cells from different vascular locations. The highest basic eNOS protein synthesis and NO production was detected in CAEC cells, lower in AOEC cells and the lowest in IAEC cells. TNFα significantly decreased the expression of eNOS at mRNA and protein level. GLP-1 and exenatide did not change eNOS mRNA expression. Both GLP-1 and exenatide increased eNOS protein level only in IAEC cells. Incretin agonists decreased NO production compared to TNFα stimulated cells. We suppose that this effect is caused by anti-inflammatory action of incretins. The First Polish-German Biochemical Societies Joint Meeting, 2012 220 Session 9. Metabolic Diseases P9.5 P9.6 Lipid signalling in NiemannPick disease type A and B Thrombophilias and recurrent pregnancy loss Constantin Bode, Markus H. Gräler Tatiyana Grinevich, Searhei Lialikau Molecular Cancer Research Centre, Charité – University Medical School, Berlin, Germany Grodno State Medical University, Grodno, Belarus e-mail: Markus Gräler <[email protected]> Niemann-Pick disease (NPD) type A and B originates from loss-of-function-mutations in the gene encoding for acid sphingomyelinase (ASM). The predominant molecular phenotype is a pathological increase of sphingomyelin (SM), the substrate of ASM. Similar to other members of the family of lysosomal storage diseases, no causative treatment is available due to the lack of appropriate pharmacological targets. In order to find new ways to combat this disease, we analyzed ASM deficient mice with regard to their lipid profile beyond SM using liquid chromatography coupled to triple-quadrupole mass spectrometry. Importantly not only SM was elevated in most tested tissues, but also sphingosine (Sph), the precursor of the lipid signalling molecule sphingosine 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC), which is also active as a low affinity ligand for S1P receptors. Based on this observation we hypothesized that lipid signalling may have a relevant impact on the pathology of NPD-A/B. Since S1P signalling is required to induce emigration of lymphocytes from lymph nodes and thymus, lymphocyte populations were analyzed in blood and lymphoid compartments of ASM deficient mice. While the amount of mature CD4 and CD8 single-positive thymocytes were increased and respective double-positive immature thymocyte numbers were decreased in ASM deficient mice compared to heterozygous littermates, T cell counts in the blood were normal, but B cell numbers were increased. This phenotype was different from S1P accumulation in lymphoid organs, which prevents lymphocytes from exiting lymph nodes and thymus via lymph and blood, respectively. Application of the S1Plyase inhibitor 4-deoxypyridoxine (DOP) induced massive accumulation of S1P and Sph in lymphoid organs of heterozygous mice, but not ASM-deficient mice. A similar phenotype was observed in sphingosine kinase 2 (SphK2) deficient mice, which suffered from defective distribution of S1P from blood into peripheral tissues. Tracing intravenously injected synthetic C17-S1P over time however revealed that ASM deficient mice did not show this defect, suggesting the involvement of a different metabolic pathway that was not S1P-lyase-dependent. The identification of this additional metabolic pathway and the analysis of abnormal lipid signalling leading to the observed differential lymphocyte counts in heterozygous and ASM deficient mice potentially results in the identification of novel target molecules that could pave the way for a future causative treatment of this devastating disease. e-mail: TATSIANA GRINEVICH <[email protected]> Thrombophilia has been associated with adverse pregnancy outcome. The term thrombophilia is an umbrella term for a diverse group of blood clotting disorders of the haemostatic mechanisms. Thrombophilias may be hereditary or acquired or sometimes mixed (as a result of exogenous factors for example with estrogen use in combined oral contraceptives or hormone replacement therapy) superimposed on a genetic predisposition. Inherited thrombophilia includes a single point mutation on the Factor V gene (factor V Leiden), prothrombin (PT) G20210A gene mutation, deficiencies in protein C and protein S as well as antithrombin (AT) deficiency. The most entrenched acquired thrombophilia is the antiphospholipid syndrome (APS). The routine thrombophilia screening includes testing for antiphospholipid antibodies, lupus anticoagulant, and anticardiolipin antibodies. Patients with recurrent miscarriages should be tested for genetic markers of thrombophilia. In addition to the traditional laboratory testing, modified thromboelastometry analyses were used to detect possible disturbances in coagulation. Thromboelastometry was performed with ROTEM Gamma (Pentapharm, Deutschland). Thromboelastometry is an easy, rapid and reproducible test that offers the opportunity to investigate all phases of coagulation and fibrinolysis in whole blood. The purpose of this study was to determine if an additional group of women with coagulopathy could be found with the use of thromboelastometry, that are not yet identified with routine thrombophilia screening and that are at risk of adverse pregnancy outcome. Study group consisted of 60 women with recurrent pregnancy loss. In 6 women thrombophilia was diagnosed by routine testing the antiphospholipid syndrome (APS). Thromboelastometry identified only 30% of women with an abnormal thrombophilia screening. However, in 33% of the women with normal thrombophilia screening an abnormal thromboelastometry was found. Conclusion Thromboelastrometry does detect a group of women with abnormal coagulation that are not detected by routine thrombophilia screening. Additional studies are warranted to determine if thromboelastometry identifies a subset of women with a prothrombotic tendency that are at an increased risk of recurrent adverse pregnancy outcome. 47th Congress of the Polish Biochemical Society, 2012 Session 9. Metabolic Diseases221 P9.7 P9.8 Evaluation of antioxidant properties of selected natural antioxidants towards albumin Influence of incretin mimetics on matrix metalloproteinases expression in TNF-α or glycated albuminstimulated vascular endothelial cells Ewa Grzebyk, Ewa Zurawska-Plaksej, Agnieszka Piwowar Chair and Department of Pharmaceutical Biochemistry, University of Medicine in Wroclaw, Wrocław, Poland e-mail: Ewa Grzebyk <[email protected]> Currently many civilization diseases are connected with intensified oxidative stress (OS), derived from different origin. OS can act on the molecular and cellular level, causing many negative structural and functional changes. The oxidative modifications of proteins, lipids and nucleic acids are very important. OS, in consequence, leads to functional disturbances of cells, tissues and organs, and may disturb the activity of many enzymes and hormones, intensifying disorders and aging of organism. Many attention is paid on the possibility of reduction and/or prevention of oxidative modifications induced by OS. The aim of our investigation was the answer to the question: if natural antioxidants commonly used as dietary supplements, revealed inhibitory effects in direction to oxidative modification of proteins. On the basis of current literature three substances were chosen: vitamin C, quercetin and green tea as the most popular in usage. As in vitro model the bovine serum albumin (BSA), in concentration reflecting physiological concentration of human serum albumin in the blood, was applied. As oxidized agent the chloramine T was chosen. The concentration of advanced oxidation protein products (AOPPs), thiol (-SH), carbonyl (-CO) and amino (-NH2) groups as well as fluorescence spectrum of tryptophan were measured as markers of oxidative modification of proteins. Our research showed that all examined substances significantly suppressed the formation of AOPP (p less than 0.05). The oxidative modifications reflected by concentration of SH groups was the most strongly nhibitedrcetin (p less than 0.05), and the least by vitamin C (this action was statistically significant (p less than 0.05) only during the 30 minutes on). The inhibition of the protein oxdain elced by concentration of CO groups, was the highest for vitamin C (p less than 0.05), and the weakest for quercetin (no statistical significance). We did not reveal any significant inhibitory effect of examined supplements elce y oncentration of NH2 groups. Comparison of the fluorescence spectra of tryptophan revealed that the most ability to inhibit of proteins oxidation is shown subsequently for: vitamin C, green tea, and quercetin (which acts definitely the weakest). These results show that examined suplements inhibit the oxidation of proteins, but not with the same strength and on the same way. The vitamin C has the best protective action towards protein carbonyls, quercetin — towards thiols, and green tea is the most universal (it protects the CO and SH groups and prevents from excessive formation of AOPP). Agnieszka Kłych, Agnieszka Chyra, Tomasz Francuz, Wojciech Garczorz Medical University of Silesia, Department of Biochemistry in Katowice, Poland e-mail: Agnieszka Kłych <[email protected]> Nowadays, diabetes mellitus is a major global problem. It is closely related with the progression of hypertension and dyslipidaemia, that lead to accelerated development of coronary heart disease and generalized vascular atherosclerosis. This complications are mainly caused by smooth muscle cells and endothelial cells dysfunction. TNF-α and glycated proteins contribute to progression of atherosclerotic changes among diabetics. Incretin mimetics are new drugs used in diabetes therapy. These compounds acts through GLP-1R stimulating postprandial insulin secretion and increasing glucose uptake by peripheral tissues. Recent studies indicate that in addition to the ability to control glucose level, they have beneficial effect on the cardiovascular system. The related mechanisms are not known yet. It is believed that among other functions, these drugs can affect the function of vascular endothelial cells. In atherosclerosis these cells exhibit increased production of matrix metalloproteinases (MMPs). MMPs are involved in all stages of the atherosclerosis process, from the initial lesion to plaque rupture. The project assumed that GLP-1 receptor agonists alter the expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in vascular endothelial cells. Aim of the study: Assess the influence of GLP-1 receptor agonists (vatious concentrations of natural GLP-1 and synthestic exendin-4) on mRNA expression and concentration of matrix metalloproteinases protein: MMP-1, MMP-9 and their inhibitors: TIMP-1, TIMP-2 in TNF-α or glycated albumin- stimulated human iliac artery endothelial cells (IAEC). Results: The results of presented study have shown significant changes in basic and TNF-α or glycated albumin stimulated expression of tested molecules. Tested incretin mimetics decreased MMP-1 and MMP-9 expression stimulated by proinflammatory factors. On the other hand, they slightly induced expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2 ). This may result in inhibition of TNF-α or glycated albumin stimulated metalloprotease activity, and thereby inhibiting the degradation of ECM. Conclusion: Tested incretin mimetics exert protective effects on vascular endothelial cells by antagonizing negative action of TNF-α and glycated albumin and reducing extracellular matrix degradation. This may play important role in atherosclerosis prevention among patients with diabetes. The First Polish-German Biochemical Societies Joint Meeting, 2012 222 Session 9. Metabolic Diseases P9.9 P9.10 Dynamics of changes in the levels of biogenic amines and neuroactive amino acids in the rat striatum after a single injection of lead acetate Perfringolysin O, a bacterial toxin as a probe detecting cholesterol deposits in cells Ivan V. Liakh, Evgeniy M. Doroshenko, Vitaliy Y. Smirnov, Vladimir M. Sheibak Nencki Institute of Experimental Biology PAS, Department of Cell Biology, Warsaw, Poland Grodno State Medical University, Grodno, Belarus e-mail: Ivan Liakh <[email protected]> Among the numerous studies on the toxic effects of lead much of the works is devoted to its effects on the nervous system of mammals. It is known that lead intoxication provokes changes levels of the main neurotransmitters in the brain (Leret et al., 2002), including changes levels of monoamines (Antonio et al., 2000). To study the effects of a single injection of lead on the level of biogenic amines and neuroactive amino acids in the rat striatum, we performed an experiment in which animals one time intraperitoneally were injected lead acetate in dose of 150 mg/kg. Animals were divided into three groups. The first group animals were decapitated after twenty-four hours, the second group - after three and a third group — 10 days after introduction of lead. Thereafter, in the striatum by HPLC were determined levels of biogenic amines and neuroactive amino acids such as aspartate, glutamate, glycine, taurine, GABA, and determined ratio index of excitatory amino acids to the inhibitory amino acids (EAA/ IAA). The most significant changes in the striatum were marked one day after the introduction of lead: an increase in the concentration of dihydroxyphenylalanine (DOPA) and reduced levels of dioxyphenylacetik acid (DOPAC) and homovanillic acid (HVA), this resulted in the increase index of DA turnover (DA/DOPAC) and index (DA/HVA), which may indicate inhibition of dopaminergic system. Three days after of lead injection was observed normalization of all parameters except level of dihydroxyphenylalanine (DOPA). On the tenth day inhibition of dopamine turnover by the index DA/HVA was observed again, like a reducing the concentration of HVA. Levels of neuroactive amino acids in the striatum didn’t change at 1 and 3 day. On the tenth day there was decrease aspartate concentration and decrease index of EAA/IAA, which may indicate the prevalence of inhibitory processes. Conclusions. Lead exposure in the rat striatum have the time dependent nature and manifests in the depression of the dopaminergic system in the first day, that disappears on the third day and returns to the tenth day with enhancement of inhibitory processes. Ewelina Marszałek-Sadowska, Gabriela Traczyk, Magdalena Kulma, Katarzyna Kwiatkowaska, Andrzej Sobota e-mail: Ewelina Marszałek-Sadowska <[email protected]> Niemann-Pick disease type C (NPC) is based on defects in cholesterol transport system in lysosome membrane. As a result, a massive deposition of cholesterol in these cellular organelles is observed. Until now, diagnosis of NPC disease is based on the determination of the presence of intracellular cholesterol by filipin staining. The drug is a polyene antibiotic, which specifically binds to free cholesterol, forming a fluorescent complex. Unfortunately, the excitation of the filipin/cholesterol complex is within the UV range where light scattering and fast photobleaching of the fluorophore occur. To avoid these difficulties in detection of cholesterol in cells we used bacterial toxin, perfringolysin O (PFO) which selectively interacts with the lipid. We prepared a construct containing PFO gene fragment, lacking the signal sequence, coupled with GST protein, which allows convenient purification and detection of recombinant protein. We optimized the process of GST-PFO expression in E. coli and its purification by affinity chromatography. Perfringolysin O selectively recognizes cholesterol among a variety lipids, as we demonstrated by Lipid-Protein Overlay Assay. The data were also confirmed by Surface Plasmon Resonance technique showing that PFO binds more strongly to liposomes containing cholesterol. There was no PFO interactions with liposomes containing no cholesterol. We applied immunofluorescent microscopy to analyze cholesterol using GST-PFO and anti-GST-FITC in cells derived from patients with NPC. We found abundant deposits of the lipid in lysosome compartment. The specificity of PFO binding to cholesterol in these cells was confirmed by colocalization of PFO with filipin staining. The structures stained by PFO were identified as lysosome on the basis of their colocalization with LAMP1, lysosomal marker protein. No colocalization of PFO staining with protein markers of Golgi apparatus, mitochondrion or endoplasmic reticulum was seen. Our results show that perfringolysin O can be used as a probe for diagnostic purposes to detect the Niemann-Pick disease type C instead of the test based on filipin staining widely applied in the world. 47th Congress of the Polish Biochemical Society, 2012 Session 9. Metabolic Diseases223 P9.11 P9.12 Biochemical, genetic and clinical investigations in Polish Fabry disease female heterozygotes Hepatoprotective action of glutamine and glutamine-containing dipeptides during subchronic alcohol intoxication in rats M. Musielak1, A. Ługowska1, A. Tylki-Szymańska2, I. Małecka1, A. Wiśniewska1, B. Czartoryska1 Alyaxej H. Shlyahtun, Pavel S. Pronko 1Institute of Psychiatry and Neurology, Department of Genetics, Warsaw, Poland; 2The Children’s Memorial Health Institute, Department of Metabolic Diseases, Warsaw, Poland e-mail: malgorzata musielak <[email protected]> Background: Fabry disease is a rare lipid storage disorder caused by a deficiency of lysosomal α-galactosidase A (GALA, EC 3.2.1.22) activity. The enzyme is responsible for the hydrolysis of terminal α-galactosyl residues from glycolipids and glycoproteins, and is coded for by an X linked gene, GLA. The deficient GALA activity leads to accumulation of neutral glycosphingolipids, mainly globotriaosylceramide Gb3, in various tissues, leading to angiokeratoma, neuronopathic pain, progressive renal dysfunction, cardiomyopathy, and stroke. Aim: The description of biochemical, genetic, and clinical studies in female carriers of Fabry disease, and the possible genotype-phenotype correlation. Patients and Methods: Eight female carriers from six families of Polish origin were studied. Among them 5 were symptomatic whereas the others were initially asymptomatic. The activity of GALA was measured in blood leukocytes and plasma using 4-methyl-umbelliferyl-α-D-galactoside by means of fluorescence spectrophotometer. Mutations in the GLA gene were identified in gDNA samples by sequencing procedure. Results: GALA activity ranged from 6.0 to 15.7 nmol/ mg/h in leukocytes and from 2.1 to 13.5 nmol/ml/h in plasma, in non-Fabry reference samples from females. For non-Fabry reference samples from males, the values were similar: from 2.9 to 18.9 nmol/mg/h in leukocytes and from 2.8 to 11.7 nmol/ml/h in plasma. Female carriers of a mutation in the GLA gene expressed lowered GALA activity, although in some cases still in the reference range: 1.1 to 5.7 nmol/mg/h in leukocytes and 0.8 to 4.5 nmol/ml/h in plasma. We found private mutations in the GLA gene of the females: 1 missense (p.N34S), 2 nonsense (p.Y132X, p.R301X) and 3 small deletions. All female carriers investigated in this study stammed from families where probands suffered from the renal type of Fabry disease. Thus, it can be speculated that mutations identified by us lead to the “renal’ phenotype. Symptomatic females reported pronounced pain in hands and feet, episodes of fever and later were found to have progressive renal insufficiency and also hypertrophic cardiomyopathy in one case. Conclusions: We conclude that female carriers develop similar clinical phenotypes to male probands in their families but at more advanced age. GALA activity is not a reliable mean of heterozygotes identification and it should be always followed by GLA gene sequencing analysis. Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Grodno, Belarus e-mail: Alexej Shlyahtun <[email protected]> Purpose: Alcoholic liver disease is the most common complication of chronic alcohol intoxication. One of the promising directions in the development hepatoprotectors is to find products based on amino acids and their derivatives. Previously we shown that glutamine can reduce the toxic effects of alcohol due to the antioxidant properties [1] and the acceleration of ethanol excretion from the body [2]. Synthetic glutamine derivatives — L-alanyl-L-glutamine and glycyl-L-glutamine — are most stable, soluble and have greater bioavailability than glutamine. Methods: Adult male Wistar rats were used in all experiments. The subchronic alcohol intoxication was induced as described earlier [1]. Glutamine (146 mg/kg), alanylglutamine (216 mg/kg), glycyl-glutamine (203 mg/kg) or an equivolume amount of saline was injected i.p., twice after 14 and 16 hours following ethanol withdrawal. Animals were sacrificed after 18 hours following ethanol withdrawal. The protein content in liver tissue was determined by the Lowry method, the free thiol groups and the levels of malondialdehyde as described everywhere [3]. Measurement of transaminase activities in blood plasma were carried out according to the instructions of the kits. Amino acids contents were measured by RP-HPLC/FD. Results: In the liver, alcohol intoxication was accompanied by a decrease in the level of free thiols. There was a significant increase in the levels aspartate, glutamate, glutamine, and decreased levels of methionine, threonine, citrulline, ornithine and taurine. All the investigated compounds showed the ability to rapidly improve the content of free thiols and to normalize the levels of aspartate, glutamate, taurine and methionine in the rat liver. This probably occurs as a result of complex factors, first and foremost, a rapid increase in the NAD/NADH ratio and supplying the necessary metabolic substrates. Conclusions: The results obtained show the ability of glutamine and its dipeptides to normalize the levels of free thiols content and free amino acid in a liver tissue. This fact raises the possibility that these compounds might possess some therapeutic benefits in treatment for alcoholic liver disease. References 1. Pronko PS et al. (2009) J ECNP 19: 644. 2. Shlyahtun AH et al. (2011) Vesnik of State University of Grodno 112: 134138. 3. Pronko PS et al. (2011) Alcohol and Alcohol 46 (Suppl 1): 44-48. The First Polish-German Biochemical Societies Joint Meeting, 2012 224 Session 9. Metabolic Diseases P9.13 P9.14 The influence of serine and glycine on homocysteine level in rat’s blood plasma Oxidative modifications of serum albumin in diabetic patients with ischemic heart disease Anna Volkovich, Aleksandr Naumov Grodno state Medical University, Grodno, Belarus e-mail: Anna Volkovich <[email protected]> Recently, much attention is paid to sulfur-containing amino acids — an important diagnostic and prognostic indicators in various pathologies. A special place here takes homocysteine (Hcy). It’s proved a dependency between the increased levels of Hcy and the development of a number of pathologies: • cardiovascular diseases (arteriosclerosis, stroke) [2]; • neurodegenerative diseases (Alzheimer's disease, schizophrenia, cognitive impairment) [1]; • pregnancy complications [3]; • congenital fetal malformations (neural tube defect) [3]. Currently a number of drugs are used in biology and medicine to decrease the level of this amino acid. However due to their low efficiency is a very important task to find the ways to normalize the level of homocysteine and other sulfur containing compounds. Since homocysteine and methionine metabolism incorporates a quite large number of amino acids, the possibility of hyperhomocysteinemia correction by their exogenous administration was assessed. In our work we considered the possibility of homocysteine levels decrease by the introduction of serine (Ser) amino acid, involved in homocysteine metabolism on transsulfuration stage. As a comparison amino acid, we used glycine (Gly) – neutral amino acid, presumably not involved in homocysteine metabolism. The study was performed on white laboratory rats of heterogeneous strain. The animals of experimental groups were modeled as hyperhomocysteinemia group, and then we introduced exogenous serine and glycine in a dosage of 100 mg/kg. As a result, it was observed that the concentration of Ser and Gly in plasma statistically significantly increased with respect to the results of control group after the administration of exogenous amino acids. At the same time serine significantly reduced the level of Hcy to control values, which presumably is associated with the activation of transsulfuration process for homocysteine. It restores the correlation between the individual members of Hcy metabolism cycle. This allows us to consider that serine as a possible factor in the correction of hyperhomocysteinemia. Gly introduction did not develop such effect & the development of hyperhomocysteinemia was not affected. References 1. Clarke R et al. (1998) Arch Neurol 55: 1449-55. 3. Lipton P (1999) Physiol Rev 79: 1431-568. 3. Navumau AV et al. (2006) Acta Biochem Pol 53: 195. Ewa Zurawska-Plaksej1, Ewa Grzebyk1, Aneta Stachurska2, Andrzej Mysiak2, Agnieszka Piwowar1 1Department of Pharmaceutical Biochemistry, Poland; 2Department and Clinic of Cardiology, Wroclaw Medical University, Wrocław, Poland e-mail: Ewa Zurawska-Plaksej <[email protected]> Diabetes mellitus (DM) is a complex metabolic disease, characterized not only with prolonged hyperglycemia, but also with intensified oxidative stress and coexistence of many clinical disorders, such as heart diseases. Oxidative stress (OS) is considered as an important factor in pathomechanism both in DM and ischemic heart disease (IHD). The aim of this study was to evaluate differences in levels of biochemical markers reflecting oxidative modifications of HSA (as degree of OS intensification) in diabetic patients accompanied by IHD, with and without myocardial infarction (MI). Fifty six cardiologic patients (therein 35 with MI) were enrolled for this study. DM occurred in 29 of these patients. The control group consisted of 19 healthy volunteers. Blood samples were taken on admission to hospital and plasma levels of ischemia modified albumin (IMA), advanced oxidation protein products (AOPP) and sulfhydryl groups (SH) were measured. Patients were divided into 4 groups: A1 (DM with MI), A2 (DM without MI), B1 (NDM with MI) and B2 (NDM without MI). To estimate association of OS markers with DM and IHD in these 4 groups statistical analysis was carried. Values of measured markers in these groups varied from 0.378 to 0.473 ABSU for IMA, 0.136–0.188 mM for AOPP and 0.421–0.493 mM for SH. These values differed significantly from control group, with the exception of IMA in B2 group. We also found some correlations between these markers and certain groups: SH correlated with IMA in groups without MI (A2 and B2) and with AOPP in groups with MI (A1 and B1), both independently from presence of DM. Multivariate analysis of variance have shown SH as the most differentiating parameter between examined groups. IMA, considered as helpful marker in myocardial ischemia, did not have discriminative power between A1 and A2 groups. Both, diabetes and ischemic heart disease predispose to intensification of oxidative modification of HSA, what hinders interpretation of results of OS-associated markers measurements in diabetic patients with ischemic heart diseases. Our study has shown the greatest usefulness of SH in patients with diabetes complicated with IHD, whereas IMA should be interpreting with carefulness in these patients. 47th Congress of the Polish Biochemical Society, 2012 Session 9. Metabolic Diseases225 P9.15 Mutations in the gene for betaglucocerebrosidase are more frequent in patients with early onset Parkinson disease than in control individuals A. Ługowska1, A. E. Wiśniewska1, J. Sławek3, P. Janik2, A. Potulska-Chromik2, D. Kosiorowski4, A. Friedman4, M. Kuźma-Kozakiewicz2, D. Hoffman-Zacharska5, Z. Jamrozik2 1Institute of Psychiatry and Neurology, Department of Genetics, Warsaw, Poland; 2Department of Neurology, Poland; 3Department of Neurological-Psychiatric Nursing, Gdańsk, Poland; 4Faculty of Health Science, Department of Neurology, Medical University of Warsaw, Warsaw, Poland; 5Institute of Mother and Child, Department of Medical Genetics, Warsaw, Poland e-mail: Agnieszka Ługowska <[email protected]> Objective: To evaluate the presence of two most common mutations in the GBA gene in Polish patients with early onset Parkinson’s disease. Background: Mutations in the gene coding for lysosomal beta-glucocerebrosidase (GBA) were recently proposed as a probable risk factor for Parkinson’s Disease (PD) and Levy Body Dementia (DLB). The incidence of mutations in the GBA gene in PD patients is variable and related to population studied, methods of DNA testing (sequencing versus evaluating most common mutations only) and control groups. Methods: To identify mutations in the GBA gene, genomic DNA was extracted from the white blood cells by standard techniques. A screening for mutations, p.L444P and p.N370S was performed in a group of 115 PD patients with early onset (45 year-old) and in a group of 56 patients with PD onset between 45 and 50 year-old. PCR-RFLP methods were used as described earlier. Results were compared to incidence in other European poplations and PD patients published earlier. Results: In the group of 115 PD patients with early onset we found 5 heterozygotes for the examined mutations in the GBA gene (4- p.L444P and 1- p.N370S). In the group of 56 patients with later onset of disease none of the examined mutations was identified. Conclusions: Our data suggest that incidence of p.L444P and p.N370S mutations in GBA gene is strongly related to the age of PD onset. This 2.2 % incidence in our group is higher as compared to general European populations and similar to PD patients reported earlier. Acknowledgements Supported by: Polish Ministry of Science and Higher Education, Grant # N N402 4404 33. The First Polish-German Biochemical Societies Joint Meeting, 2012