The purpose of this study was to determine whether the methylation of nicotinamide to N-methylnicotinamide could discriminate between differences in methionine nutritional status, and by implication methylation capacity, in healthy humans. As part of this thesis, a highly selective high performance liquid chromatography (HPLC) method for the determination of N-methylnicotinamide (NMN) in urine and plasma was developed and validated. Quantification was by fluorescence detection of the 1,6-naphthyridine derivatives, formed after incubation of NMN with acetophenone in alkaline conditions. Seven volunteers participated in a trial to evaluate the ability of a nicotinamide load test to discriminate between changes in the methylation status of the individual. The methylation status was measured as the time dependent changes in plasma NMN concentrations after a nicotinamide load. A basal nicotinamide load test was performed on each individual. The methylation status was then changed, by means of a methionine load, and the nicotinamide load test was repeated during the enhanced methylation state. The dynamic changes in N-methylnicotinamide levels indicated that the methionine load changed neither the plasma NMN concentrations, nor the rates of NMN formation. The conclusion of this study was that nicotinamide loading could not be used as a dynamic function test to assess biological methylation in healthy humans. / Dissertation (MSc (Chemical Pathology))--University of Pretoria, 2006. / Chemical Pathology / unrestricted

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-a production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-α production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.