Understanding the Nutritional Content and Impact of the Mediterranean Diet using 1H NMR Spectroscopy

Charlotte E Rowley

Poor nutrition is a leading cause of disease and death worldwide, contributing to many chronic diseases, including T2DM (T2DM). Not only do such conditions impact the physical and mental wellbeing of the individuals affected, but also the wider economy as it places a significant financial burden on the healthcare system. As such, improving our understanding of the impact of dietary patterns is crucial to improve population health. One promising area of research is that of Precision Nutrition, understanding the biological differences between individuals, and therefore understanding differences in health outcomes in response to different dietary patterns. Precision Nutrition is an area that has only been possible since the development of modern technologies that allow for molecular analysis of samples, both from humans and from the food we consume. These technologies, including Nuclear Magnetic Resonance spectroscopy, create a detailed metabolic profile that has been used in applications ranging from discriminating the growing regions of extra virgin olive oils (EVOO) to understanding the metabolic impacts of dietary patterns. The Mediterranean dietary (MD) pattern is one of the most researched dietary interventions, associated with multiple health benefits including reduced risk of cardiovascular disease and T2DMT2DM. Based predominantly on plant foods and relying on legumes, lentils and seafood for the majority of protein, the diet is often substantially different to dietary patterns of people in high-income countries. One main component of the diet is the high intake of EVOO, which is the main fat used in the diet. This thesis examines multiple aspects of the Mediterranean diet, from food composition to the impact on human metabolic profile, as well as comparing it to other dietary interventions. The initial project examines one of the stable foods of the MD, EVOO. Originating from countries around the Mediterranean sea, olive oil is now grown around the world, including Australia. However, minimal investigations into Australian EVOO have been conducted, and while there are quality assessment parameters, there is limited structure for the assessment of nutritional profiles of EVOO, regardless of origin. The first aim of this study was the creation of an EVOO nutritional scoring matrix to allow for easy comparison between oil samples. Using NMR to assess 170 samples from around Australia, oils were assessed against the current quality standards, and then against the nutritional scoring matrix which we created. Of the 170 oils, only 86 passed the quality standards. Nutritional score varied widely between samples, with no known association between year, cultivar or region. There were, however, regional influences on the parameters of Saturated Fatty Acid and Alpha-Linoleic Acid. This work allows for the identification of EVOO of high nutritional value, beneficial for marketing of oils, as well as supporting consumer choice. In addition to EVOO, the impact of fruit consumption on the human metabolic profile was assessed. These fruits are commonly consumed as part of a MD (particularly an Australian-adapted MD). An initial pilot study of three women examined the impact of fruit consumption on urinary metabolites. Identifying strong differences between the individuals excretion of Hippurate, PAG, p-cresol and DMA, a second study was established to examine the drivers behind this. Working on the hypothesis that orocaecal transit time (OCTT) was a major factor in speed of response, the Fibre Study was established, examining urine and blood samples, as well as hydrogen breath testing over an 8-hour period. Contrary to our hypothesis, OCTT was not the driving factor behind differences in metabolite excretion, however we did identify some interesting correlations in microbial metabolites which require further investigation. The MD was then applied, along with two other dietary interventions, to a cohort of 23 participants with risk factors for developing T2DM. The three dietary patterns were the MD, low carbohydrate (LC) diet and Australian (high carbohydrate, AUS) diet, which was followed for 4 days. Participants completed all dietary patterns sequentially, though the order was randomised. During this time, continuous glucose monitoring (CGM) was employed to assess blood glucose response to the interventions. Once completed, assessments of glycaemic response were analysed, including average blood glucose levels (BGL), fasting blood glucose levels (FBGL) and time out of range (TOOR). In addition, the two-hour postprandial glucose response (PPGR) was assessed as maximum BGL, and area under the curve (AUC) for the two-hour window. From these parameters, optimal diets for blood glucose control were suggested. Of interest, there was no one diet that suited all participants, despite all having risk factors for the development of T2DM. For 11 participants, the optimal diet changed based on the parameters considered. In addition, significant differences were noted between dietary patterns for most individuals (n =20). This indicates that, for some people, different dietary patterns produce significantly different BGL’s, whereas this is less pronounced for other individuals. Lastly, we examined the impact of a MD on a population of pregnant women, and compared the metabolic profiles of these women to pregnant women who are not following a MD. This cohort of 51 women were retrospectively recruited from the ORIGINS project, during which time they had completed Food Frequency Questionnaires (FFQ), Mediterranean Diet Questionnaires (MDQ) and had provided serum and urine samples at two time points during their pregnancy. Participants with either a high MD alignment (HMDA) or low MD alignment (LMDA) were included if they maintained their dietary pattern over the course of the pregnancy (as assessed by MDQ). Between the two groups, the LMDA group (n = 25) were associated with a higher intake of processed meat, discretionary foods and snacks, while the HMDA group (n = 26) was associated with a higher intake of fruits and vegetables. Examining the biological samples of these women, those in the HMDA group excreted significantly higher levels of Hippurate, a metabolite indicative of gut microbiome diversity and function, as well as reduced levels of inflammatory glycA and glycB molecules. Given that inflammation during pregnancy is associated with a number of negative health outcomes, including pre-eclampsia, low birth weight, and gestational T2DM, finding ways to improve the inflammatory profiles of women is important to support the health of mother and child. In addition, the maternal microbiome is the seed for infant microbiome. As the microbiome composition is now associated with a number of health outcomes later in life, including risk of T2DM, cardiovascular disease, asthma and dementia, establishing a healthy microbiome population at an early age gives our future generations the best chance for optimal health outcomes. This work ties together studies of nutrition from food to human health. By understanding the composition of our food, we can deepen our understanding of the potential implications of said food on the human metabolic profile. However, it is important to remember that digestion and metabolism of food is highly individual, as demonstrated in the Fibre and SMART-2 studies. While there are dietary patterns, such as the MD, which are generally considered healthy for any population, by understanding the individual variation in dietary responses, we can tailor dietary interventions to each person, further improving health outcomes at every stage of life.

Understanding the Nutritional Content and Impact of the Mediterranean Diet using 1H NMR Spectroscopy

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