Rodriguez R. Molecular features of folate-dependent metabolism in the human placenta are normal, in hyperhomocysteinemia and depending on the polymorphism of the methylenetetrahydrofolate reductase gene in in vivo, ex vivo and in silico studies.

The dissertation is devoted to the analysis of molecular features of folate-dependent metabolism (FOCM) in the human placenta and the influence of elevated concentrations of homocysteine and C677T mutation in the MTHFR gene on metabolic fluxes. Hypehomocysteinemia (elevated level of Hcy) often accompanies pregnancy disorders related to placental dysfunctions. Homocysteine is the toxic intermediate of folate-dependent one-carbon metabolism, but exact mechanisms of its adverse effects are still debated. One-carbon metabolism supplies substrates and cofactors for methylation reactions, polyamine synthesis, purine ring and thymidylate synthesis thus aking difficult to assess homocysteine impact on a system level. In such cases one can exploit mathematic models of metabolism. Among them stoichiometric models offer the best balance between required data input and predictive power. For system analysis, a stoichiometric model of FOCM was created, taking into account tissue-specific gene expression. For the latter mRNA expression analysis of folate-dependent metabolism genes was conducted in 1 st trimester and term placenta. Four cases were simulated with the model: normal metabolite fluxes and wild-type enzymes; elevated homocysteine concentration with activation of direct SAHH reaction (direct input of homocysteine by transport reaction has not yield data that align with experiments); decreased activity of MTHFR due to C677T genotype; combination of both adverse factors. Simulation of elevated homocysteine concentrations in the system alters metabolic fluxes through most FOCM reactions and, in particular, shifts metabolic fluxes toward remethylation by reducing folate cycle reactions and purine base synthesis. Modeling of the C677T genotype of the MTHFR gene leads to a slight decrease in metabolic fluxes to the methionine cycle reaction, and an increased reactions of purine synthesis. The prediction of the model was tested experimentally on placental explant culture either in normal conditions and with added 20 µM Hcy, with in-house developed assays for S-adenosylmethione (SAM) and S-adenosylhomocysteine content, as well as for purine bases. Content of universal methylation substrate SAM and its product SAH metabolites was measured in explants by high performance liquid chromatography / mass spectrometry (HPLC/MS). Purine synthesis de novo was assayed by adding to the explant culture heavy ( 13 C) glycine and monitoring its appearance in purine ring by HPLC/MS. Experimental evidence supported model predictions about activation of methionine cycle and slowdown of purine synthesis due to competition for the limited folate cofactors.