Abstract
This thesis focuses on the study of molecular interactions of metabolites in biofluids by Nuclear Magnetic Resonance (NMR). NMR is uniquely suited for measurement of complex mixtures as biofluids as it provides qualitative and quantitative information in a non-destructive manner at molecular/atomic detail.
In Chapter 1 is discussed the role of biofluids and show how NMR is useful to study molecular interactions in whole crude biofluids. We also discuss the biological role of human serum albumin (HSA), the most abundant protein and present in almost all human's biofluids. Moreover, an introduction to the NMR is presented with a specific focus on chemical shift and bulk magnetic susceptibility (BMS).
Chapter 2 give theoretical explanation and demonstrate experimental use of signal frequency shift caused by protein-induced BMS to determine total protein content in blood plasma. The BMS method has an error estimated to be = 5 %, which is comparable to the ~4 % error of the standard clinical colorimetric method.
Chapter 3 is about the impact of fatty acids (FAs) on metabolites-HSA interactions in blood plasma. Also, ultrafiltration, the method employed for separation of metabolites from blood-plasma proteins, was evaluated. And chemical shift change due to sample dilution was used to identify metabolites interacting in fast exchange rate and their respective dissociation constant.
In Chapter 4 specific FAs - myristic acid and stearic acid - were used and the metabolite lactate was strongly release from HSA. Due to poor FA solubility, effect of dilution on free metabolite concentration was also followed upon increasing FA-HSA ratio.
Chapter 5 deals with the formation of complexes between metabolites, metal ions and HSA. This chapter focuses on composition of the prostatic fluid that is affected as part of malignant growth and transformation. Here chemical shift and transverse relaxation were measured and calculated for different composition.
