Quantitation of enteroendocrine hormones as metabolic disease biomarkers in human biological matrices by liquid chromatography tandem mass spectrometry
Polypeptides are widely present in biology and many of them act as biomarkers, both for disease and system regulation. Many gut hormone peptides are secreted by enteroendocrine cells (EECs) and some, known as incretins, are released after a meal to control blood glucose and stimulate neuronal satiety. These are being studied as novel targets for treatment of metabolic diseases such as obesity. Traditionally quantitative analysis of these hormones is performed by enzyme-linked immunosorbent assays (ELISAs). The development of these immunological techniques can take many years and is not always cost effective, so new biomarker assay approaches are being explored in research. Liquid Chromatography Mass Spectrometry (LC-MS/MS) methods are becoming an alternative approach and analytical technique combines the physical separation abilities of liquid chromatography with specific mass analysis of mass spectrometry. The methods described in this thesis are traditional bioanalytical extractions used to isolate small molecule analytes from biological matrices. Assay development can be a challenging process, especially when the target biomarker is expected to be in the low (picomolar) ranges. To improve sensitivity of the methods a number of different extraction procedures were investigated and the optimal conditions for each peptide target were identified. Additionally the mass spectrometry hardware systems were assessed and modified, working to find a balance between sensitive detection limits and reducing sample analysis time to maintain high throughput methods for clinical application. Where applicable, the optimised LC-MS/MS methods were validated by commercial immunoassays and the strong correlation in concentrations confirmed the accuracy of the developed assay. An additional advantage of mass spectrometry is the ability to monitor multiple analytes in a single sample, so additional data on all possible isoforms of the target analyte are available, as opposed to the ‘total’ response usually measured by antibody capture techniques. This advantage is clearly important in the instance of prohormone intermediate species (as discovered in Chapter 3) and enzymatically cleaved and inactive forms peptides (Chapter 4 and Chapter 5). Plasma is one of the most accessible biological matrices, and the majority of the methods were found to be suitable for analysis of circulatory concentrations of peptide hormones. With support from clinicians, the final methods were applied to plasma samples from participants who had had specific meal tests, to gauge the postprandial response of insulin and C-peptide (Chapter 3 and Chapter 4), motilin and gastric inhibitory peptide (Chapter 4), and cholecystokinin (Chapter 5) following nutrient stimulation. As intestinal tissue is a more complex and invasive matrix to collect, organoid models have been developed for *in vitro* studies of specific cell activity. An extraction method for organoid secretion supernatants was optimised (in collaboration with Emily Miedzybrodzka), to be able to quantify the target analytes in direct response to stimuli. Analysis of organoid cultures was utilised to understand the secretory response of motilin (Chapter 4), cholecystokinin (Chapter 5) and glucagon-like peptide 2 (Chapter 6), respectively. To summarise, this thesis describes the development of analytical methods for metabolic gut hormone peptides, for clinical applications. The manuscript is separated into chapters based on specific gut hormone analytes, as multiple high throughput methods were developed on UPLC liquid chromatography system and triple quadrupole mass spectrometer. Each chapter in this thesis describes the method development, validation and application of the optimised methods to biological samples, with the results providing some insightful and novel discoveries to aid in metabolic disease biomarker quantitation.
bioanalysis, enteroendocrine, mass spectrometry, metabolic diseases, peptides
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