AbstractDespite the biological importance of essential metals, the roles they play in maintaining health and the regulation and dysregulation of their trafficking - from diet to tissues, tissues to tissues and excretion – in diseases such as dementias, cancer and diabetes - are poorly understood. Studies of in vivo metal trafficking have been limited hitherto to invasive, spatiotemporally restricted destructive analytical techniques such as laser-ablation-inductively-coupled-plasma-mass-spectrometry (LA-ICP-MS). Fluorescent probes can study intracellular metal pools in vitro but their limited tissue penetration permits only superficial imaging in vivo. A new, complementary technique - PET imaging using radiometals (e.g. manganese-51/52, zinc-62/63 and copper-62/64) - is emerging that will allow non-invasive study of acute metal trafficking repeatedly across the whole body of living subjects. I applied two approaches to study zinc in models of health and disease in vivo: 1) radioactive forms of the trace metal itself and 2) radiolabelled ligands designed to become intracellularly trapped following zinc binding.
In the first part of this project, I evaluated an overlooked zinc radioisotope, 62Zn (β+, 8.2%, t1/2 = 9.3 hours), that has previously been considered a poor choice to study zinc trafficking in vivo due to its unfavourable decay to 62Cu (β+, 97 %, t1/2 = 10 min). I have shown for the first time, in healthy animals and a mouse model of breast cancer, that 62Zn localises to zinc-rich organs such as the pancreas, has stark differences in biodistribution compared to 64Cu, and can therefore be used to longitudinally and non-invasively image zinc trafficking across the whole body. This provides an investigative and potentially clinical diagnostic tool that can be used to study zinc trafficking for up to 2 days, exceeding the 2 hours of imaging that can be achieved with 63Zn (t1/2 = 38.5 minutes).
62Zn was applied to a mouse model with a loss of function mutation in ZnT8, a critical zinc transporter exclusively expressed in pancreatic islet cells. Zinc dynamics and biodistribution mapped by 62Zn showed no difference between homozygous mutant and wild-type mice over 1 hour, whereas mapping of tissue distribution of zinc and manganese at the microscopic level by LA-ICP-MS showed significant differences in the metal content of the exocrine and endocrine pancreas. This project demonstrates the need for combining complementary multi-scale techniques together to build a comprehensive picture of metal homeostasis in health and disease.
To investigate the potential for radiolabelled metal sensors to probe endogenous pools of metals in vivo, I synthesised and characterised a fluorine-18 labelled zinc sensor, [18F]AQA-F, based on a promising fluorescent probe. In vivo studies revealed rapid pharmacokinetics with dual excretion via hepatobiliary and renal routes. In stark contrast to 62Zn, [18F]AQA-F did not localise to zinc-rich organs such as the pancreas and so the potential for this approach is yet to be realised or confirmed.
This thesis has presented two approaches that set the foundation for the non-invasive study of zinc trafficking in vivo and warrant further investigation in disease models where the homeostasis of zinc is disrupted.
|Date of Award||1 May 2022|
|Supervisor||Philip Blower (Supervisor)|