ASYMMETRIC DIMETHYLARGININE - COMPARSION OF CHROMATOGRAPHY AND IMMUNOMETRIC METHODS Objective:Asymmetric dimethylarginine (ADMA) is often discussed in connection with hyperhomocysteinemia and its toxic effect on vessel wall. ADMA concentration is usually measured by HPLC (High Performance Liquid Chromatography) after previous derivatisation. Recently, ELISA (Enzym Linked Immuno Assay) methods for ADMA determination were introduced and ELISA kits are commercially available. Method and Result:The aim of the study was to compare HPLC and ELISA methods for ADMA determination. For HPLC determination we used equipments from Thermo separation product (Florida, USA). After solid-phase extraction on polymer cation-exchange column and the following derivatisation with o-phthaldialdehyde the samples were separated using C18 column (mobile phase 8.7% acetonitril, 50 mmol/l phosphate buffer, pH 6.5) and a fluorescence detector. NG-monomethyl-L-arginine was used as an internal standard. ADMA® ELISA kit, based on a competitive principle, was obtained from DLD Diagnostika, Hamburg, Germany. ADMA was measured in EDTA plasma of 40 healthy blood donors and 40 hemodialysis patients with hyperhomocysteinemia. Conclusion: In spite of different principles both methods showed a very good correlation (r = 0.944, p<0.0001). ELISA...

Uma das principais causas da disfunção erétil (DE) pode ser relacionada com o déficit de óxido nítrico (NO) no corpo humano. O principal componente para a produção do NO é o aminoácido L-arginina que é utilizado pelas enzimas óxido nítrico sintase neuronal (nNOS), endotelial (eNOS) e induzida (iNOS) para sua produção. A dimetilarginina assimétrica (ADMA) atua como inibidor endógeno dos três subtipos de NOS citadas acima e é metabolizada pelas enzimas dimetilarginina dimetilaminohidrolase 1 e 2 (DDAH1 e DDAH2). Diversos estudos têm relacionado a alteração na expressão ou atividade das enzimas DDAH bem como alterações em seus genes, com distúrbios onde a sinalização de NO é prejudicada. Os objetivos deste estudo foram investigar a associação de variantes genéticas dos genes DDAH1 (rs1554597 e rs18582) e DDAH2 (rs805304 e 805305) com a predisposição à disfunção erétil (DE), scores de função erétil e concentrações plasmáticas de nitrito e ADMA. Também verificar se estes marcadores bioquímicos estão relacionados aos scores de função erétil. Foram selecionados 130 pacientes com DE clínica e 98 participantes controles saudáveis sem DE. A função erétil dos voluntários foi avaliada através do questionário Índice Internacional de Função Erétil (IIEF). Os genótipos dos rs1554597, rs805304 e rs805305 foram obtidos através da técnica de reação em cadeia da polimerase (PCR) seguida de digestão enzimática, e do rs18582 apenas por técnica de PCR alelo específica. No grupo Pacientes, foram encontradas associações do gene DDAH1 com as concentrações plasmáticas de ADMA: o rs1554597 teve os genótipos TT e TC associados positivamente (TT: ? 0,13 e P = 0,008; TC: ? 0,09 e P = 0,016;) e o genótipo CC associado negativamente (? -0,22 e P <0,001); já o rs18582 teve o genótipo GG associado positivamente (? 0,22 e P <0,001) e o genótipo AA associado negativamente (? -0,16 e P = 0,001); o haplótipo TG foi associado positivamente (? 0,12 e P = 0,016) e o haplótipo CA negativamente (? -0,18 e P = 0,002). Com relação ao nitrito, associações dos haplótipos do gene DDAH2 foram encontradas, o haplótipo CC foi associado negativamente (? -0,03 e P = 0,045) e o haplótipo AG foi associado positivamente (? 0,03 e P = 0,045).O rs18582 teve o genótipo GG associado positivamente com as concentrações plasmáticas de nitrito, no modelo aditivo (? 0,15 e P = 0,009) e no modelo dominante (? 0,08 e P = 0,009), e os genótipos GA ou AA associados negativamente com as concentrações plasmáticas de nitrito, apenas no modelo dominante (? -0,08 e P = 0,009). Não foi encontrada nenhuma outra associação significativa no estudo / One of the main causes for erectile dysfunction (ED) is related to nitric oxide (NO) deficiency in human body. The main substrate for NO synthesis is the amino acid L-arginine, which is processed by NO synthases (NOS) from three subtypes for its production: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). Asymmetric Dimethylarginine (ADMA) acts as an endogenous inhibitor of the three subtypes of NOS and is metabolized by enzymes dimethylarginine dimethylaminohydrolase types 1 and 2 (DDAH1 and DDAH2). Several studies associate the altered expression or activity of DDAH enzymes, as well as their genes, with diseases with hampered NO signaling. The objectives of this study were to investigate the association of genetic variants of DDAH1 (rs1554597 and rs18582) and DDAH2 (rs805304 and 805305) with vulnerability to develop ED, with altered scores of erectile function and with altered plasma concentrations of nitrite and ADMA. We also investigated whether these biochemical markers associated with erectile function scores and ED risk. We selected 130 patients with clinical ED and 98 healthy controls without ED. Erectile function was assessed through the International Index for Erectile Function (IIEF) questionnaire. Genotypes for rs1554597, rs805304 and rs805305 were obtained with polymerase chain reaction (PCR) followed by enzyme restriction (RFLP), while rs18582 was determined using Allele-Specific oligonucleotide PCR (ASO-PCR). At patients group, we found association of variants in DDAH1 gene with plasma ADMA levels: TT and TC genotypes of rs1554597 were associated with increases in ADMA (TT: ? 0.13 e P = 0.008; TC: ? 0.09 e P = 0.016;), while CC genotype was associated with decreases in ADMA (? 0.22 e P <0.001); regarding rs18582, GG genotype associated with increases in ADMA (? 0.22 e P <0.001), while AA genotype associated negatively (? -0.16 e P = 0.001); besides, haplotype TG was also associated with ADMA increases (? 0.12 e P = 0.016), while CA haplotype associated negatively with ADMA levels (? -0.18 e P = 0.002). Regarding nitrite, associations of the haplotypes of the DDAH2 gene were found, the haplotype CC was negatively associated (? -0,03 and P = 0,045) and the haplotype AG was positively associated (? 0,03 and P = 0,045) .O rs18582 had the GG genotype positively associated with plasma nitrite concentrations in the additive model (? 0.15 and P = 0.009) and in the dominant model (? 0.08 and P = 0.009), and negatively associated genotypes GA or AA with plasma nitrite concentrations, only in the dominant model (? -0.08 and P = 0.009). We found no further significant associations in our study

A disfunção erétil (DE) é uma doença relacionada com a sinalização deficiente de óxido nítrico (NO). O NO é produzido a partir da L-arginina pelas enzimas óxido nítrico sintase neuronal (nNOS), endotelial (eNOS) e induzida (iNOS). A dimetilarginina assimétrica (ADMA) é um inibidor endógeno dos três subtipos existentes de NOS, e é metabolizada principalmente pelas enzimas dimetilarginina dimetilaminohidrolase 1 e 2 (DDAH1 e DDAH2). Vários estudos têm associado alterações em genes, expressão ou atividade das enzimas DDAH com distúrbios em que a sinalização de NO é prejudicada. O objetivo deste estudo foi avaliar se o nível de resposta ao tratamento da DE com sildenafil pode estar associado a polimorfismos dos genes DDAH1 (rs1554597 e rs18582) e DDAH2 (rs805304 e rs805305) e, também, aos haplótipos formados por estes polimorfismos. Foram selecionados 70 pacientes com DE pós-prostatectomia (DEPP) e 70 pacientes com DE clínica (DEC). A função erétil dos voluntários foi avaliada através do questionário Índice Internacional de Função Erétil (IIEF). Para avaliação da resposta ao sildenafil, foram calculadas a diferença entre as pontuações pré e pós-tratamento (?IIEF) e a percentagem atingida da máxima resposta possível (?IIEF%) de cada paciente. Também, os pacientes de cada grupo foram divididos em bons e maus respondedores ao sildenafil de acordo com a mediana dos valores de ?IIEF%. Os genótipos dos rs1554597, rs805304 e rs805305 foram obtidos pela técnica de reação em cadeia da polimerase (PCR) seguida de digestão enzimática, e do rs18582 pela técnica de PCR alelo específica. O software PHASE 2.1 foi utilizado para estimar os haplótipos em cada grupo. Os resultados mostraram que o alelo variante A do rs18582 apresentou tendência para associação com piores respostas ao sildenafil no grupo DEC (P=0,058). No grupo DEPP, portadores dos alelos variantes A do rs805304 e G do rs805305 foram associados a melhores respostas ao sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; e score IIEF pós-tratamento, P=0,014). Não foram encontradas outras associações significativas. Estes resultados mostram que os marcadores genéticos rs805304 e rs805305 do DDAH2 podem influenciar as respostas ao sildenafil em pacientes com DE. / Erectile dysfunction (ED) is a disease related to deficient nitric oxide (NO) signaling. NO is produced from L-arginine by three isoforms of the enzyme nitric oxide synthase: neuronal (nNOS), endothelial (eNOS) and induced (iNOS). Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the three existing NOS subtypes, and is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 and 2 (DDAH1 and DDAH2) enzymes. Several studies have associated changes in genes, expression or activity of DDAH enzymes with disorders in which NO signaling is impaired. The aim of this study was to evaluate whether the response to ED treatment with sildenafil may be associated with polymorphisms of the DDAH1 (rs1554597 and rs18582) and DDAH2 genes (rs805304 and rs805305), as well as the haplotypes formed by these polymorphisms. We selected 70 patients with postprostatectomy ED (PPED) and 70 patients with clinical ED (CED). The erectile function of the volunteers was assessed using the International Index for Erectile Function (IIEF) questionnaire. To evaluate the response to sildenafil, the difference between the pre- and post-treatment scores (?IIEF) and the percentage reached from the maximum possible response (?IIEF%) of each patient were calculated. Also, patients from each group were divided into good and bad responders to sildenafil according to the median values of ?IIEF%. The genotypes of rs1554597, rs805304 and rs805305 were obtained by the polymerase chain reaction (PCR) technique followed by enzymatic digestion, and rs18582 by the allele-specific PCR technique. The PHASE 2.1 software was used to estimate the haplotypes in each group. The results showed that the variant A allele of rs18582 showed a tendency to be associated with a greater chance of worse responses to sildenafil in the DEC group (P=0,058). In the DEPP group, carriers of the variant alleles A of rs805304 and G of rs805305 were associated with better responses to sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; and post-treatment IIEF score, P=0,014). No other significant associations were found. These results show that the genetic markers rs805304 and rs805305 of DDAH2 may influence the responses to sildenafil in patients with ED.

A disfunção erétil (DE) é uma doença relacionada com a sinalização deficiente de óxido nítrico (NO). O NO é produzido a partir da L-arginina pelas enzimas óxido nítrico sintase neuronal (nNOS), endotelial (eNOS) e induzida (iNOS). A dimetilarginina assimétrica (ADMA) é um inibidor endógeno dos três subtipos existentes de NOS, e é metabolizada principalmente pelas enzimas dimetilarginina dimetilaminohidrolase 1 e 2 (DDAH1 e DDAH2). Vários estudos têm associado alterações em genes, expressão ou atividade das enzimas DDAH com distúrbios em que a sinalização de NO é prejudicada. O objetivo deste estudo foi avaliar se o nível de resposta ao tratamento da DE com sildenafil pode estar associado a polimorfismos dos genes DDAH1 (rs1554597 e rs18582) e DDAH2 (rs805304 e rs805305) e, também, aos haplótipos formados por estes polimorfismos. Foram selecionados 70 pacientes com DE pós-prostatectomia (DEPP) e 70 pacientes com DE clínica (DEC). A função erétil dos voluntários foi avaliada através do questionário Índice Internacional de Função Erétil (IIEF). Para avaliação da resposta ao sildenafil, foram calculadas a diferença entre as pontuações pré e pós-tratamento (?IIEF) e a percentagem atingida da máxima resposta possível (?IIEF%) de cada paciente. Também, os pacientes de cada grupo foram divididos em bons e maus respondedores ao sildenafil de acordo com a mediana dos valores de ?IIEF%. Os genótipos dos rs1554597, rs805304 e rs805305 foram obtidos pela técnica de reação em cadeia da polimerase (PCR) seguida de digestão enzimática, e do rs18582 pela técnica de PCR alelo específica. O software PHASE 2.1 foi utilizado para estimar os haplótipos em cada grupo. Os resultados mostraram que o alelo variante A do rs18582 apresentou tendência para associação com piores respostas ao sildenafil no grupo DEC (P=0,058). No grupo DEPP, portadores dos alelos variantes A do rs805304 e G do rs805305 foram associados a melhores respostas ao sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; e score IIEF pós-tratamento, P=0,014). Não foram encontradas outras associações significativas. Estes resultados mostram que os marcadores genéticos rs805304 e rs805305 do DDAH2 podem influenciar as respostas ao sildenafil em pacientes com DE. / Erectile dysfunction (ED) is a disease related to deficient nitric oxide (NO) signaling. NO is produced from L-arginine by three isoforms of the enzyme nitric oxide synthase: neuronal (nNOS), endothelial (eNOS) and induced (iNOS). Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the three existing NOS subtypes, and is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 and 2 (DDAH1 and DDAH2) enzymes. Several studies have associated changes in genes, expression or activity of DDAH enzymes with disorders in which NO signaling is impaired. The aim of this study was to evaluate whether the response to ED treatment with sildenafil may be associated with polymorphisms of the DDAH1 (rs1554597 and rs18582) and DDAH2 genes (rs805304 and rs805305), as well as the haplotypes formed by these polymorphisms. We selected 70 patients with postprostatectomy ED (PPED) and 70 patients with clinical ED (CED). The erectile function of the volunteers was assessed using the International Index for Erectile Function (IIEF) questionnaire. To evaluate the response to sildenafil, the difference between the pre- and post-treatment scores (?IIEF) and the percentage reached from the maximum possible response (?IIEF%) of each patient were calculated. Also, patients from each group were divided into good and bad responders to sildenafil according to the median values of ?IIEF%. The genotypes of rs1554597, rs805304 and rs805305 were obtained by the polymerase chain reaction (PCR) technique followed by enzymatic digestion, and rs18582 by the allele-specific PCR technique. The PHASE 2.1 software was used to estimate the haplotypes in each group. The results showed that the variant A allele of rs18582 showed a tendency to be associated with a greater chance of worse responses to sildenafil in the DEC group (P=0,058). In the DEPP group, carriers of the variant alleles A of rs805304 and G of rs805305 were associated with better responses to sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; and post-treatment IIEF score, P=0,014). No other significant associations were found. These results show that the genetic markers rs805304 and rs805305 of DDAH2 may influence the responses to sildenafil in patients with ED.

Metabolic syndrome is defined as a combination of obesity, elevated triglycerides, decreased high-density lipoproteins, hypertension and insulin resistance. It is at least partially caused by sedentary life style and unhealthy dietary habits and is a major risk factor for development and progression of cardiovascular disease and type 2 diabetes. Growing medical and socioeconomic impact of the metabolic syndrome warrants further active search for novel risk markers and therapeutic targets. Recent experimental and epidemiological studies have demonstrated the multiple roles of the endogenous methylarginines, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) as wells as the enzymes, which are involved in their catabolism, dimethyarginine dimethylaminohydrolases (DDAHs) and alanine:glyoxylate aminotransferase 2 (AGXT2) in the pathogenesis of metabolic syndrome and its complications. ADMA is thought to exhibit its pathological effects by inhibiting and uncoupling nitric oxide synthases (NOS), while SDMA can inhibit transport of L-arginine. DDAHs, namely DDAH1 and DDAH2, have been thought as the major enzymes metabolizing ADMA to citrulline, while being inactive towards SDMA. Experimental studies with upregulation of DDAH1 in animal models showed that lowering ADMA results in protection against endothelial dysfunction, atherosclerosis, ischemia/reperfusion injury and vascular remodeling, acceleration of angiogenesis in the settings of ischemia and improvement of insulin sensitivity. Unfortunately, all the attempts to upregulate DDAH1 using small drugs have not been successful. The data regarding the role of DDAH2 are contradictory, with some studies showing that it can metabolize ADMA under certain conditions and other studies questioning its enzymatic activity towards ADMA. AGXT2 is a mitochondrial aminotransferase, which can metabolize, among its other substrates, both ADMA and SDMA. It is a large protein with possible allosteric regulatory sites, suggesting that, in contrast to DDAH1, it could be upregulated by small molecules. The role of AGXT2 in different pathophysiological processes involving ADMA and SDMA is poorly understood. It has been recently discovered in the offspring cohort of the Framingham Heart Study participants that a composite compound, consisting of the products of metabolism of ADMA and SDMA by AGXT2 (asymmetric dimethylguanidino valeric acid (ADGV) and symmetric dimethylguanidino valeric acid (SDGV), correspondingly) is an independent biomarker of CT (computed tomography)-defined NAFLD (non-alcoholic fatty liver disease) and a predictor of future diabetes up to 12 years before disease, suggesting that AGXT2 may play a key role in development of metabolic disease and its progression. We and other have recently identified several other metabolically active substrates of AGXT2, such as a marker of cardiovascular and overall mortality homoarginine and a regulator of fatty acid oxidation and browning of adipose tissue beta-amino-isobutyric acid (BAIBA), which further supports the importance of AGXT2 in pathogenesis of cardiovascular and metabolic diseases. The data presented in the current thesis enable answering the two research aims: 1) Identification of the tissue and intracellular expression pattern of human AGXT2 and 2) Testing the hypothesis that ubiquitous transgenic overexpression of AGXT2 protects from ADMA-induced vascular damage in vivo. The first research aim provided a thorough characterization of AGXT2 expression in humans using multiple complimentary techniques and addressed the current discrepancy in the literature with previous demonstration of comparable levels of Agxt2 expression by RT-PCR and Western Blot in the kidneys and liver in mice, and previous reports on detection of predominant Agxt2 expression in the kidneys by Northern Blot and in-situ RNA-hybridization in rats. In our current study we analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. We saw the strongest expression of AGXT2 in the kidney and liver both on the mRNA and protein levels. Our immunohistochemistry stainings showed that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed the intracellular localization of AGXT2 in mitochondria. In the second research aim we investigated whether long-term upregulation of AGXT2 is safe and can protect from ADMA- mediated vascular damage in the setting of DDAH1 deficiency, which is commonly observed in cardiovascular pathologies. We generated AGXT2 transgenic (TG) mice with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of ADGV, the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. The work, included into this thesis demonstrates that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans, where the enzyme is localized in mitochondria. The expression of AGXT2 in the liver is consistent with the proposed role of AGXT2 in development and progression of NAFLD and is consistent with our previous discovery of hepatocyte nuclear factor 4 alpha (HNF4α) as the major regulator of Agxt2 expression in the mouse liver. Chronic upregulation of AGXT2 in mice lowered systemic ADMA levels without any obvious effects on viability, development, growth and fertility, suggesting potential safety of this ADMA-lowering approach. Overexpression of AGXT2 protected from ADMA-induced vascular damage in the highly clinically relevant settings of DDAH1 deficiency, suggesting that the observed vascular damage was indeed caused by ADMA itself, rather than by some ADMA-independent effects of DDAH1 deficiency. The observed protective effects of AGXT2 upregulation are especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful. The current study, therefore, provides the basis for the future screens to identify small molecules, which would upregulate AGXT2 activity.

Homocystein (tHcy) gilt als unabhängiger kardiovaskulärer Risikofaktor und korreliert eng mit einer endothelialen Dysfunktion, welche nichtinvasiv mittels der flussinduzierten Vasodilatation (FMD) messbar ist. Experimentelle Hyperhomocysteinämie ist mit einer reduzierten Bioverfügbarkeit von endothelialen Stickstoffmonoxid (NO) bei gleichzeitig erhöhten Spiegeln des kompetetiven Inhibitors der NO-Biosynthese asymmetrisches Dimethylarginin (ADMA) assoziiert. In-vivo senkt eine Östrogenbehandlung neben tHcy auch die ADMA-Spiegel und verbessert signifikant die Endothelfunktion. Hinsichtlich ihrer Wirkung als selektive Östrogenrezeptormodulatoren wird angenommen, dass Phytoöstrogene, speziell Sojaisoflavone, ähnliche Effekte hervorrufen.<br><br> Innerhalb einer europäischen, multizentrischen, doppelblinden Interventionsstudie an 89 gesunden, postmenopausalen Frauen wurde der Einfluss von Sojaisoflavonen auf den Homocysteinmetabolismus, den Blutdruck und die in-vivo Endothelfunktion untersucht. Die cross-over Studie umfasste zwei achtwöchige Interventionsperioden, die von einer gleichlangen Wash-out-Phase unterbrochen waren. Die Zuteilung zum Isoflavon- (50 mg/d) oder Plazeboregime für die erste Interventionsphase erfolgte randomisiert. Endpunkterhebungen fanden jeweils in den Wochen 0 und 8 der Interventionsperioden statt.<br><br> Die renale Ausscheidung von Genistein, Daidzein und Equol war während der Isoflavonintervention signifikant erhöht (P>0,001). Die Phyoöstrogene hatten weder einen Effekt auf die tHcy-Konzentration (P=0,286), noch auf ADMA, Erythrozytenfolat und Vitamin B-12 (P>0,05) im Plasma. Während die Summe aus Nitrat und Nitrit (NOx), welche die NO-Bioverfügbarkeit reflektiert, im Verlaufe der Plazebobehandlung abfiel, wurde ein leichter Anstieg bei der Isoflavonsupplementation beobachtet (Delta Wo8-Wo0: -2,60 [-8,75; 2,25] vs. 1,00 [-6,65; 7,85] µmol/L P<0,001), was zu einem signifikanten Behandlungseffekt führte. Weiterhin wurde eine positive Korrelation zwischen ADMA und Vitamin B-12 gefunden (R=0,252; P=0,018). Die flussinduzierte Vasodilatation (P=0,716), ein Maß für die Endothelfunktion, blieb durch die Isoflavonbehandlung unbeeinflusst, obwohl sich diese über die Zeit insgesamt verbesserte (P>0,001). Bis auf einen marginalen Anstieg des systolischen Wertes (P=0,032) im Vergleich zur Plazebobehandlung blieb der Blutdruck während der Isoflavonintervention unverändert.<br><br> Im Gegensatz zu Östrogen übten Sojaisoflavone weder einen Einfluss auf die in-vivo Endothelfunktion noch auf die traditionellen und neuen kardiovaskulären Risikofaktoren den Blutdruck, tHcy und ADMA aus. Demzufolge ist der gesundheitliche Nutzen isolierter Isoflavone hinsichtlich einer Prävention hormonmangelbedingter Erkrankungen in gesunden postmenopausalen Frauen fraglich. / Homocysteine (tHcy) is a strong and independent risk factor for cardiovascular disease. Hyperhomocysteinemia contributes to endothelial dysfunction as assessed by flow-mediated vasodilation (FMD). The mechanisms by which homocysteine generates endothelial dysfunction remain incompletely understood although a growing body of data suggests that the bioavailability of nitric oxide (NO) is reduced. The principal competitive inhibitor of endothelial NO-synthase asymmetric dimethylarginine (ADMA) may play a central role in homocysteine related dysfunction as it is derived from homocysteine metabolism. Cardiovascular risk factor modification has suggested beneficial effects of estrogen on endothelial function by lowering homocysteine and ADMA levels. We hypothesize that phytoestrogens particular isoflavones act in a similar manner.<br><br> The effects of soy isoflavones on homocysteine metabolism and endothelial function were investigated within a multi-centre, double blind, cross-over intervention trial in 89 European postmenopausal women. Subjects consumed either fruit cereal bars with or without soy isoflavones (50 mg/d) for 8 weeks each with a 8 weeks washout period in between. Endpoint measurements were during both treatment phases at baseline and weeks 8, respectively. <br><br> Urinary phytoestrogens increased significantly after isoflavone intervention (P<0.001). Isoflavone supplementation did affect neither plasma total homocysteine (P=0.286) nor ADMA, vitamin B-12 or folate (P<0.05). Isoflavones had a favorable effect on NO-metabolism assessed by analysis of NO-metabolites (NOx) nitrite and nitrate. While NOx concentration significantly decreased during placebo there was a slight increase after isoflavone supplementation leading to a significant treatment difference (delta wk8-wk0: -2.60 [-8.75; 2.25] vs. 1.00 [-6.65; 7.85] µmol/L P<0.001). There was no association between total homocysteine and ADMA whereas a positive correlation was found for ADMA and vitamin B-12 (R=0.252; P=0.018). The endothelial function model did not demonstrate any difference between either treatment regime (P=0.716), although endothelial function assessed by flow-mediated vasodilation improved in general (P<0.001). A potential adverse effect was noted, with an elevation in systolic blood pressure (P=0.032) whereas diastolic blood pressure and mean arterial pressure remained unaffected.<br><br> Soy isoflavones did not have beneficial effects on endothelial function as well as on traditional and novel cardiovascular risk factors like plasma homocysteine, blood pressure and ADMA as observed for estrogen treatment. The health benefit of isolated isoflavones in healthy postmenopausal women is questionable.

Severe Traumatic Brain Injury (TBI) and aneurysmal Subarachnoid Hemorrhage (SAH) are severe cerebral emergencies. They are common reasons for extensive morbidity and mortality in young people and adults in the western world. This thesis, based on five clinical studies in patients with severe TBI (I-IV) and SAH (V), is concentrated on examination of pathophysiological developments and of evaluation of therapeutic approaches in order to improve outcome after cerebral emergency. The treatment for severe TBI patients at Umeå University Hospital, Sweden is an intracranial pressure (ICP)-targeted therapy according to “the Lund-concept”. This therapy is based on physiological principles for cerebral volume regulation, in order to preserve a normal cerebral microcirculation and a normal ICP. The main goal is to avoid development of secondary brain injuries, thus avoiding brain oedema and worsened microcirculation. Study I is evaluating retrospectively 41 children with severe TBI, from 1993 to 2002. The boundaries of the ICP-targeted protocol were obtained in 90%. Survival rate was 93%, and favourable outcome (Glasgow Outcome Scale, score 4+5) was 80%. Study II is retrospectively analysing fluid administration and fluid balance in 93 adult patients with severe TBI, from 1998 to 2001.The ICP-targeted therapy used, have defined fluid strategies. The total fluid balance was positive day one to three, and negative day four to ten. Colloids constituted 40-60% of total fluids given/day. Severe organ failure was evident for respiratory insufficiency and observed in 29%. Mortality within 28 days was 11%. Study III is a prospective, randomised, double-blind, placebo-controlled clinical trial in 48 patients with severe TBI. In order to improve microcirculation and prevent oedema formation, prostacyclin treatment was added to the ICP-targeted therapy. Prostacyclin is endogenously produced, by the vascular endothelium, and has the ability to decrease capillary permeability and vasodilate cerebral capillaries. Prostacyclin is an inhibitor of leukocyte adhesion and platelet aggregation. There was no significant difference between prostacyclin or placebo groups in clinical outcome or in cerebral microdialysis markers such as lactatepyruvate ratio and brain glucose levels. Study IV is part of the third trial and focus on the systemic release of pro-inflammatory mediators that are rapidly activated by trauma. The systemically released pro-inflammatory mediators, interleukin-6 and CRP were significantly decreased in the prostacyclin group versus the placebo group. Study V is a prospective pilot study which analyses asymmetric dimethylarginine (ADMA) concentrations in serum from SAH patients. Acute SAH patients have cerebral vascular, systemic circulatory and inflammatory complications. ADMA is a marker in vascular diseases which is correlated to endothelial dysfunction. ADMA concentrations in serum were significantly elevated seven days after the SAH compared to admission and were still elevated at the three months follow-up. Our results show overall low mortality and high favourable outcome compared to international reports on outcome in severe TBI patients. Prostacyclin administration does not improve cerebral metabolism or outcome but significantly decreases the levels of pro-inflammatory mediators. SAH seems to induce long-lasting elevations of ADMA in serum, which indicates persistent endothelial dysfunction. Endothelial dysfunction may influence outcome after severe cerebral emergencies.

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

L’infarctus du myocarde (IDM) résulte des complications de l’athérosclérose, dont le développement serait initié par une dysfonction endothéliale, s’accompagnant d’un état de stress oxydant. Le fer interagirait dans cette pathogenèse à différents niveaux et aurait également un rôle majeur dans la survenue de la dysfonction endothéliale. L’hepcidine et l’érythroferrone (découverte auparavant sous le nom de myonectine/CTRP 15), participeraient dans la régulation de certaines étapes du métabolisme du fer. La diméthyl-arginine asymétrique (ADMA), marqueur de dysfonction endothéliale, est associée à la plupart des facteurs de risque cardiovasculaire. Son stéréo-isomère, la diméthyl-arginine symétrique (SDMA), a une élimination exclusivement rénale et est considéré comme un puissant marqueur de fonction rénale.Nous nous sommes proposés d’étudier, chez des patients en phase aiguë d’IDM, la signification des paramètres traditionnels du statut du fer, des protéines impliquées dans la régulation du fer, l’hepcidine et la myonectine, ainsi que des dérivés méthylés de la L-arginine, l’ADMA et la SDMA.Dans notre première étude prospective, nous observons que les valeurs de SDMA, et dans une moindre mesure celles d'ADMA, sont associées à la glycémie d'admission et pourraient donc exercer des actions biologiques indépendantes de la fonction rénale. Notre second travail suggère que les patients présentant de l’insuffisance cardiaque à la phase aiguë de l’IDM sont caractérisés par une anémie et un certain degré de carence en fer. Le dosage des nouveaux biomarqueurs de la régulation du métabolisme du fer, l'hepcidine et la myonectine a montré une tendance à l’augmentation de ces taux sériques chez ces patients en lien avec une augmentation de la CRP. Ainsi nos travaux laissent entrevoir les liens qui uniraient, à la phase aiguë de l’infarctus du myocarde, ces nouveaux biomarqueurs régulateurs du métabolisme du fer aux conséquences fonctionnelles des pathologies cardiovasculaires, notamment en termes d’anémie et d’insuffisance cardiaque. / Myocardial infarction (MI) is mostly caused by complications of atherosclerosis, whose the development would be initiated by a dysfunction of the vascular endothelium, characterized by an inflammatory condition and oxidative stress.In this pathogenesis, iron interacts at different levels and also has a major role in the development of endothelial dysfunction. Hepcidin and erythroferrone (discovered earlier as the myonectin/CTRP 15) participate in a major way in regulating certain stages of iron metabolism.Asymmetric dimethylarginine (ADMA), a marker of endothelial dysfunction is associated with most cardiovascular risk factors. Symmetrical dimethyl-arginine (SDMA), its stereoisomer, has an exclusively renal elimination and is considered as a powerful renal function marker.We aimed to study, in patients with acute MI, the meaning of the "traditional" status iron parameters, two proteins involved in the regulation of iron, hepcidin and myonectin, as well as the two L-arginine derivatives, ADMA and SDMA. In our first prospective study, we observe that, in patients with acute MI, the values of SDMA, and only weakly ADMA, are associated with admission blood glucose, beyond traditional dimethylarginine determinants and may therefore have biological activity beyond renal function.Our second work suggests that patients with heart failure in the acute phase of MI present more frequently anemia and a certain degree of iron deficiency. New iron metabolism regulators biomarkers, hepcidin and myonectin showed a trend toward an increase in the serum levels in these patients characterized by an increase in CRP.Thus, our work suggests the links between these new regulators of iron metabolism in acute MI with functional consequences of cardiovascular diseases, particularly in terms of anemia and heart failure.

1 Cardiovascular risks in chronic airway disease in childhood The aim of this thesis was to evaluate cardiovascular risk by using a combined diagnostic approach by measuring RHI and specific biochemical markers in patients with chronic respiratory disease, where we could assume a possible risk of CVD. A total of 119 probands were examined, including 22 patients with cystic fibrosis (CF) and 52 asthma patients. We evaluated RHI using a new plethysmographic method that has a number of advantages over the ultrasonographic methods used in other studies, including non-invasiveness, high sensitivity, low biological variability and objectivity due to automatic processing. Of the biochemical parameters, we measured 4 biomarkers in relation to endothelial dysfunction (ED): hsCRP, ADMA, E-selectin, and VCAM-1. We compared RHI and biomarkers in CF and asthma patients with healthy controls and sought mutual correlations. We did not prove a statistically significant difference in RHI between the test groups with CF children but we confirmed the decreasing trend of RHI since adolescence and significantly lower RHI values in CF adults, confirming the progressive development of atherogenesis and worsening of ED with age. Biochemical parameters showed significantly higher levels of hsCRP, sVCAM-1 and E-selectin in CF...