There are many energy metabolism studies ongoing, including those for cardiovascular diseases and type-2-diabetes. With an increase in people being diagnosed with type-2-diabetes, there should be more ways to monitor not only the blood glucose levels but also the other biomarkers associated with type-2-diabetes. The metabolism biomarkers are essential in understanding the cause of diabetes early on. These biomarkers include: glucose, non-esterified fatty acid, lactate, urea, creatinine, glycosylated haemoglobin and cholesterol. Whilst glucose measurement has a clear role in type-2-diabetes management, the potential value of non-esterified fatty acid has not been explored or highlighted yet. The aim of this project is to develop an electrochemical biosensor for the non-esterified fatty acid in human blood, as non-esterified fatty acid can cause -cell loss in type-2-diabetes. Exploration of this biomarker would be a step forward in increasing research and patient understanding of the dynamic processes involved in establishing good metabolism control. The project uses the enzymes in commercial optical methods for non-esterified fatty acid detection. Oleic acid was used as the standard non-esterified fatty acid in this work. The electrochemical techniques employed are cyclic voltammetry, linear sweep voltammetry, chronoamperometry and electrochemical impedance spectroscopy. Enzyme electrodes were fabricated using the layer-by-layer immobilization of alternating polymer and enzyme combinations on carbon, cobalt phthalocyanine and single wall carbon nanotube screen printed electrodes. A chronoamperometric non-esterified fatty acid sensor was developed with the linear detection range of 0.10 mM to 0.90 mM oleic acid and with a sensitivity of 0.6562 A/mM oleic acid. This sensor was then further fabricated to detect non-esterified fatty acid concentrations in human plasma and serum samples. Commercial UV optical methods were used as method of validation of the blood sample concentrations. This work produced a platform for further non-esterified fatty acid detection studies.

Introduction In the absence of disease on endoscopy and failure to respond to empirical therapy, guidelines recommend manometry and reflux studies; however these investigations often fail to establish the physiological basis of oesophageal symptoms or guide therapy. Advances in technology may help provide insight into oesophageal function in health and disease and in turn direct management. The aim of this thesis was to explore the impact of introducing novel techniques and methodology through High Resolution Manometry (HRM) and prolonged wireless pH monitoring (Bravo). Methods: Bravo Study 1: 110 patients who successfully completed standard catheter-based pH monitoring (C-pH) were compared with 134 patients who failed the study and progressed to 48 hour Bravo. The total reflux time (TR; total % time pH drops below 4) was used as the diagnostic marker of reflux disease. Visceral sensitivity was assessed by Symptom Index (SI) and tolerability was measured with a questionnaire. Study 2: 38 patients who continued to have symptoms of reflux despite negative results with C-pH progressed to prolonged Bravo. ‘Worst day’ and ‘Average cumulative’ 24, 48, 72 and 96 hour Bravo measurements were compared to standard 24 hour C-pH. High Resolution Manometry (HRM) 23 asymptomatic volunteers who underwent HRM were compared to 18 patients presenting with oesophageal symptoms. Measurements of swallow responses were collected by varying workload on the oesophagus (changing bolus volume, consistency and patient position). Normative values from healthy subjects were formulated and were used to investigate patients. A novel dysmotility-symptom association parameter (Dysfunction Symptom Index; D-SI) was formulated. Clinical outcome and final diagnosis were documented at 2 years. Results Bravo Study 1: 76% of patients had a pathological TR on day 1 or 2 compared to 49% of C-pH (p<0.01). There was no difference in SI (p=0.28). A questionnaire demonstrated a preference for Bravo with reduced restriction, discomfort and dysphagia. Study 2: Using ‘Average’ and ‘Worst-day’ analysis, 61% and 76% patients were diagnosed with reflux disease based on either pathological acid exposure or reflux-symptom association at 96 hours. Of 12 patients who underwent anti-reflux surgery, 10 (83%) reported a good outcome at 2 years. HRM In health contractility and coordination improved with increased workload; from upright to supine and single liquid to solid swallows. Inter-observer agreement was high and normal values were formulated. Compared to healthy subjects, meal consumption was associated with more ineffective swallows in patients (28% vs. 51%; p<0.001). No symptoms occurred with single water swallows. With the test meal 50% of patients exhibited symptoms and 75% of these had a pathological D-SI. Furthermore, compared to water alone, 67% patients had a manometric change in diagnosis during the test meal. 2 year follow-up studies suggest that these techniques may help guide management Conclusion Bravo Tolerance, satisfaction and diagnostic yield was high in those who underwent Bravo. Prolonged pH measurement also increased the diagnostic yield in patients in whom an initial catheter study was negative. HRM The introduction of novel metrics and a protocol that mimics normal eating and drinking was more likely to identify the culprit dysmotility and associate these with symptoms. In summary, these studies advance the utility of modern technology in oesophageal testing and appear to guide clinical management.

(Carboxymethyl)lysine (CML) is one of the best characterised advanced glycation-endproducts (AGEs) and is frequently used as a marker of AGE formation in foods and plasma. Nɛ-(Carboxyethyl)lysine (CEL), is also an AGE and is the homolog of CML. The focus of this research was to expand the current knowledge on dietary AGE intakes and plasma AGE status (using CML and CEL as makers of AGE formation); examining how they correlate with nutritional, biological and physical markers relating to human health. CML and CEL were found in a wide range of commonly consumed foods with cereal products and fruit and vegetables containing the highest and lowest levels, respectively for both CML and CEL. There was no significant correlation found between dietary AGE intake and plasma AGE status in healthy volunteers. Plasma AGE status was not significantly correlated with the biological and physical markers investigated in this study in either the healthy volunteers or subjects with diabetes.

Flavonoids are known to interact with a number of membrane transporters, influencing the rate of intestinal nutrient absorption. The transcriptional effects and consequences of such interactions remained to be fully elucidated. To address this we carried out gene expression microarray analysis on intestinal Caco-2 cells treated with a flavonoid-rich berry extract (0.125 % w/v for 16 h). This microarray analysis identified alterations in numerous specific nutrient pathways, three of which were selected for further study, namely: 1) glucose, 2) iron and 3) copper. We then determined the effects of berry flavonoids on the expression and function of these transport pathways using qRT-PCR, Western blotting and functional assays. We found: 1) Chronic treatment (16 h) with flavonoids decreased the expression of glucose transporter genes (GLUT2 and SGLT1) and induced an acute (15 min) inhibitory effect on enterocytic glucose uptake. 2) Chronic treatment (16 h) with berry extract modulated the expression of genes that regulate iron uptake (down-regulation of DMT1, DCYTB and HFE; up-regulation of TfR1). Iron transepithelial transport was decreased by both acute (15 min) and chronic (16 h) berry extract treatment. 3) Chronic berry extract treatment (16 h) decreased the expression of genes that coordinate copper uptake (CTR1, HAH1, ATP7A and ATP7B). However, copper uptake was increased by both acute (15 min) and chronic (16 h) berry extract treatment. Thus overall, there is a clear effect of flavonoids on the pathways of glucose, iron and copper and a possible interactive effect between these nutrients themselves. Further research into the functional and physiological relevance of our findings will aid in optimising the dietary management of conditions such as diabetes and disturbances in copper or iron homeostasis.

This thesis describes the application of compartmental modelling techniques to data from human intervention studies with the objective of studying the absorption and metabolism of minerals (iron, copper, calcium) and vitamins (folate, carotenoids, riboflavin) labelled with stable isotopes. Compartmental modelling is a mathematical tool that uses differential equations to describe a dynamic process, such as nutrient metabolism, by adjusting the parameters of the equations until the model is a good fit to the real experimental data. A single compartment model was developed that can be used to estimate the quantity of absorbed (unlabelled) iron from a test drink containing a minimum of 10mg iron. Studies on copper demonstrated that approximately 74% of the absorbed dose is sequestered by the liver on “first-pass” and that large quantities of copper (2.4mg/d) are lost via the bile. Data from a calcium intervention trial was evaluated using a multi-compartment model and indicated that a moderately high salt intake (11.2g/d) was associated with a significantly negative bone calcium balance with a high calcium (1284mg/d) compared to a low calcium (518mg/d) diet (P=0.024). Modelling the initial metabolism of an absorbed dose of 13C-labelled folic acid resulted in the finding that it is not metabolised in the mucosal cells but (probably) in the liver. The absorptive efficiency of carotenoids was investigated by isolating the triglyceride rich lipoprotein fraction in plasma and a simple model was used to estimate β-carotene and lutein bioavailability. Results from the first stable isotope labelled riboflavin study indicated that the bioavailability of riboflavin from spinach (60±30%) was not significantly different to the bioavailability of riboflavin derived from milk (67±21%). The thesis has shown how compartmental modelling, in conjunction with stable isotope labelling, can reveal new insights into human mineral and vitamin metabolism, especially in the study of nutrient bioavailability.

It has recently been suggested that the occurrence of many organic diseases in the western world could be the result of a low intake of dietary fibre. Dietary fibre, is considered as the cell wall component of plants, varies both quantitatively and qualitatively between species and with the method of fibre preparation. The role of fibre in the diet may relate to its physical properties and those will depend on the chemical composition of the cell wall. The major physical properties are water holding capacity, cation exchange capacity, and organic adsorption ability and the major chemical properties are acid detergent fibre, lignin and cellulose content. It is important to identify these properties before a fibre source can be assessed clinically. In this investigation the carrot root developmental system was used as a fibre source to measure the physical properties of fibre and to determine whether these properties could differ either between varieties or within a variety. No differences were found between varieties but there were differences within each variety; these are probably of little dietary significance. When raw carrot was eaten the only effect that correlated with the physical properties of the prepared fibre was the bile salt adsorption ability. An increase in faecal bile acid excretion was found which continued in the absence of carrot. Serum cholesterol concentration decreased and the effect continued in the absence of carrot. This suggests that vegetable dietary fibre could influence body sterol metabolism. Differences between the nutritional effects of raw carrots and the properties of prepared fibre suggest fermentation by colonic bacteria could alter the properties of dietary fibre.

Type 2 Diabetes (T2D) is an important disorder that represents one of the most serious healthcare challenges facing the world. An estimated 293 million people worldwide are expected to have the condition by the year 2020. T2D is associated with increased cardiovascular and atherosclerotic risk, which are themselves due to increased levels of inflammation present in the disease. Inflammation in T2D is associated with the production of Advanced Glycation End-Products (AGEs), a result of chronically increased blood glucose levels. Clinical studies have demonstrated that the majority of diabetics suffer from cardiovascular disease, which accounts for 80% of all diabetic deaths. The peroxisome proliferator receptors (PPARs), an important group of nuclear receptors are members of the steroid and thyroid hormone receptor family of transcription factors and are increasingly seen as targets in the management of T2D. The dietary polyunsaturated fatty acid, Conjugated Linoleic Acid (CLA) has received considerable attention due to its anti-diabetes and anti-inflammatory effects. CLA has binding affinity for both PPAR-α and -γ and may influence both the cardiovascular risk associated with T2D and also the risk of developingT2D. This study investigated the role of CLA in regulating the inflammatory response of human monocytes. Inflammation was induced using glycated-BSA (Gly-BSA), an Amadori product that consists of 95% protein with 1-5 moles of hexose (as fructosamine) per mole albumin. Gly-BSA acts in a similar way to AGEs to activate monocytes/macrophages by binding to a surface receptor called RAGE. Stimulation of RAGE by gly-BSA leads to activation of a signal transduction pathway, which results in subsequent production of inflammatory cytokines, including Tumour Necrosis Factor -α. Treatment with CLA at a concentration of 60µM significantly reduced (p<0.001, ANOVA) gly-BSA stimulated production of Tumour Necrosis Factor-α, Interleukin-lß, Prostaglandin E2 and Cyclooxygenase-2 by the human monocytic cell-line MM6. CLA was found to modulate the activity of both the nuclear transcription factor NF-KB and RAGE in these cells. Treating MM6 cells with antisense oligonucleotide to PPAR-α and also to PPAR-γ revealed that the modulatory effects of CLA were mediated through PPAR-α but not PPAR-γ. In contrast, CLA treatment did not reduce gly-BSA stimulated Tumour Necrosis Factor-α and RAGE expression in another human monocytic/macrophage cell line, differentiated THP-1 macrophages and also in human-peripheral blood monocyte derived macrophages. The increase in RAGE expression and TNF-α secretion by CLA in these macrophages appeared to be mediated through both PPAR-γ and PPAR-α mediated mechanisms. Taken together these findings indicate CLA can interfere with the inflammatory processes of monocytic cell lines; and that both down-regulation and up-regulation of inflammatory processes by CLA are cell-specific, and mediated via both PPAR-γ and PPAR-α.

The thesis reviews the previous use of biomarkers of fatty acid intake and concludes that there is a lack of supporting evidence from large randomized controlled trials (RCT) of sufficient duration for their use to be justified. The hypothesis that fatty acid biomarkers are robust indices of the intake certain fatty acids was tested by the analysis of blood samples from three large and long-term RCTs where dietary intake had been well controlled and compared with a control treatment. Erythrocyte lipid fatty acid composition was unable to detect changes in saturated fatty acid (SFA) or oleic acid intake. Plasma total lipids and phospholipids SFA were also poor indicators of SFA intake. The intake of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) could be predicted from the proportions in plasma and erythrocytes. Principal components analysis appeared to be a valid data reduction technique to measure changes in fatty acid patterns. A co-twin study design conducted in 570 female participants enrolled in the St Thomas‟ Twins Study investigated the heritability of fatty acid biomarkers (adipose tissue and plasma). For most fatty acids, environmental factors (dietary intake) were dominant, but in the case of arachidonic acid, 65% of the variance was explained by additive genetic factors. Investigations subsequently explored the effects of variations in single nucleotide polymorphisms (SNP) in the fatty acid desaturase (FADS) genes on the fatty acid composition of the biomarkers. Polymorphism in FADS1 rs174537 explained some of this variation. Carriage of the minor allele of rs174537 SNP also influenced the proportions of n-6 LC-PUFA in an RCT. Further research is suggested to identify what appeared to be a FADS1/FADS2 haplotype predicting lower levels of LC-PUFA, which might be of public health significance. In conclusion, plasma fatty acid composition can be recommended to elucidate the potential relationships between polyunsaturated, trans-unsaturated and branched chain fatty acid intake and non-communicable diseases.

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