Insulin-like growth factor-I (IGF-I) is the only known potentially modifiable risk factor for prostate cancer. Intake of dietary protein, especially from dairy products, might also be associated with risk and with circulating IGF-I, but it is not clear if amino acids play a role in these relationships. Moreover, investigations of circulating concentrations of metabolites might reveal novel risk factors for prostate cancer. This thesis investigates plasma concentrations of amino acids and other metabolites in relation to protein intake, IGF-I, and prostate cancer risk using data from the European Prospective Investigation into Cancer and Nutrition (EPIC). To characterise plasma metabolite profile in men consuming markedly different amounts and types of animal products (meat-eaters, fish-eaters, vegetarians and vegans), cross-sectional analyses of 392 men in the EPIC-Oxford sub-cohort were conducted. Of 21 amino acids, six varied significantly by diet group, and the metabolite profile of vegans was different from those of other diet groups owing to lower concentrations of several glycerophospholipids and sphingolipids. In a case-control study nested within EPIC, with a mean follow-up time of seven years, the relationship of plasma metabolites with risk of prostate cancer overall, by time to diagnosis, by tumour characteristics, and with risk of prostate cancer death, was investigated. Data from 1,077 matched sets suggested that seven metabolites, from various classes, were associated with risk of prostate cancer overall (p < 0.05). After correction for multiple testing, 12 glycerophospholipids were inversely associated with risk of advanced prostate cancer (the strongest OR_1SD = 0.54; 95%CI: 0.40-0.72). In multivariate analyses, including data from 1,593 matched sets, principal component analysis (PCA) and treelet transform (TT) were used to identify patterns in metabolite profile, five of which were associated with risk of more aggressive tumour sub-types (high grade, advanced and aggressive disease) and/or prostate cancer death. There was a ≈ 50% lower risk of advanced and high grade prostate cancer in men with metabolite profiles characterised by high glycerophospholipids and sphingolipids (for advanced OR_{TT, top vs bottom third} = 0:48; 95%CI: 0:31-0:74), with similar results for high grade and PCA). To investigate if associations between protein intake and circulating IGF-I may be mediated by plasma amino acid concentrations, cross-sectional analyses of amino acid concentrations with protein intake and IGF-I concentrations were carried out in 1,697 and 1,142 control participants, respectively, from the nested case-control study. Dairy protein intake was positively associated with concentrations of branched-chain amino acids and several other essential amino acids, while plant protein intake was strongly associated only with histidine. Serum IGF-I was positively associated with arginine and inversely with ornithine and certain amino acid ratios. In conclusion, men with different dietary habits with respect to the consumption of protein types have different amino acid and metabolite profiles, and metabolite concentrations may be associated with risk of more high-risk prostate cancer sub-types (high grade, advanced and aggressive disease) and prostate cancer death. Further large-scale studies are needed to determine if metabolites play a role in aetiology or are markers of sub-clinical prostate cancer.

Synopsis My research work is focused on: (i) development of novel Fast NMR methods in solution state and their application to metabolomics and small molecules. (ii) NMR based metabolic study of human IVF to assess embryo viability for implantation. The major components of the embryo growth media were identified for evaluating the embryo quality. Described below are the projects carried out towards the dissertation of my PhD. Chapter 1 describes NMR methods which are the foundation stones for new Fast NMR methods developed. Typical 1D and 2D NMR experiments used in metabolomics and statistical methods for analysis are described. A few applications of metabolomics are also covered in the chapter. Chapter 2 describes a new Fast NMR method based on polarization sharing and parallel acquisition using the dual receiver system. The method developed helps in acquiring simultaneously three 2D NMR spectra: 2D [13C-1H] HETCOR, 2D [1H-1H] TOCSY and 2D [13C-1H] HSQC-TOCSY in a single data set. This method achieves a time saving of about two fold. All the experiments are acquired on molecules with natural abundance of 13C. The method was used to assign the side chain atoms (1H and 13C) of two important peptides. i) 12 amino acid residue peptide, which is a part of central linker domain of Human Insulin like Growth Factor Binding Protein-2 known to play a vital role in the IGF system and ii) a 18 amino acid residue peptide which acts as an antimicrobial agent. Chapter 3 describes extension of the Fast NMR method described in chapter 2. The method is combined with G-matrix Fourier Transform NMR spectroscopy. In this method we have acquire simultaneously two 2D NMR experiments and one reduced dimensional 3D experiment. The three experiments are 2D [13C-1H] HETCOR, 2D [1H-1H] TOCSY and GFT (3,2)D [13C-1H] HSQC-TOCSY, which provide complementary information for rapid assignments. GFT (3,2)D [13C-1H] HSQC-TOCSY gives 3D correlations in a 2D manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete assignment of 21 unlabeled metabolite mixtures corresponding to the Innovative Sequential medium (ISM1) used for culturing human embryos for IVF. Further, a 13C multiplicity edition block is added to the method to simplify the resonances assignment in GFT (3,2)D [13C-1H] HSQC-TOCSY. Taken together, experiments provide time gain of order of magnitudes compared to conventional data acquisition. Chapter 4 of the thesis describes a metabolomics study of Human in-vitro fertilization to assess viable embryos of implantation potential using NMR as non-invasive tool. NMR study included the analysis of 127 embryo culture media (Innovative Sequential Media-1) and 29 controls (culture media without embryo) of both day-2 and day-3 transferred. The embryos were divided into 3 categories 1) implanted (successful) 2) transferred not-implanted (unsuccessful) 3) not transferred based on morphological studies. All NMR experiments were acquired with CPMG (T2 filter) incorporated in 1D 1H presaturation pulse scheme. The study was based on estimation of lactate, pyruvate and alanine levels in the embryo culture media (ISM1). The study reveals higher uptake of pyruvate and high pyruvate/alanine ratios in case of implanted embryos compared to one which failed to implant. Present study provides pyruvate/alanine ratio as a biomarker to select the embryos with high implantation potential. The method combined with morphology based assessment or with other biomarkers can be serve as a powerful tool to assess the embryo quality. Chapter 5 describes a novel NMR method for rapid characterization of translation diffusion of molecules in solution either in mixture or pure form. Unlike acquisition of several 2D [13C-1H] HSQC experiments with varying gradients to get diffusion measurement, a single 2D [13C-1H] HSQC is sufficient to measure the diffusion coefficients which is in the linewidths of peaks. The method uses the idea of accordion NMR spectroscopy, wherein gradients are linearly co-incremented with 13C chemical shift evolution period during t1. The methodology speeds up the acquisition by replacing series of 2D [13C-1H] HSQC with single 2D constant time [13C-1H] HSQC. The method was used to monitor the diffusion of metabolites in a time-resolved manner during polymerization of SDS-PAGE gel. Using this method, it was possible to detect the presence of oligomers of diphenylalanine (FF) during its self assembly to form nanotubular structures.