Sheffield Hyperpolarised Imaging of Metabolism and MR Engineering Research
Methods: Hyperpolarised MR spectroscopy and imaging
DNP operates on the principle that unpaired electrons are readily polarised in moderate magnetic fields at low temperature, figure 1.
Free radicals are a source of unpaired electrons.
Electron polarisation can be transferred to nearby 13C nuclei by microwave irradiation at low temperature.
Samples preparation involves doping the substrate to be polarised, e.g. pyruvate, with a free radical, e.g. trityl radical, OX63.
Substrate is dissolved in a mixture of solvents to form a glass when frozen to help electrons contact the 13C nuclei to be polarised.
Pyruvic acid (pyruvate) occupies an important position within the metabolic process, has a long T1 and self glasses when frozen making it a vey good sample for DNP studies.
Using the correct irradiation frequency electron polarisation is transferred to nearby nuclei and the then the bulk sample by different mechanisms, figure 2.
Substrate to be polarised e.g. pyruvic acid, is inserted into a HyperSense DNP polariser operating at 1.2K, see figure 3
Microwave irradiation is performed at the ESR freq ~94GHz (100mW)
This produces a Solid state polarisation of ~ 40%
After polarisation reaches ~90% of its maximum, the sample is returned to the liquid state by rapidly dissolution using superheated HEPES buffer solution.
Liquid state polarisation reaches ~ 5-40% (substrate dependent)
Dissolved hyperpolarised substrate passes to an automated injection system.
The 13C signal is acquired over time, e.g. every 1 s for each metabolite. For example in figure 4 the time course for injected 13C1-pyruvate and its metabolic product – lactate is shown.
The time course data is integrated and fitted to a metabolic model to provide a rate constant, see figure 4.
Figure 1: Theoretical polarisation versus temperature for electrons, 13C nuclei and 1H nuclei
Figure 2: a) Theoretical polarisation versus temperature for electrons, 13C nuclei and 1H nuclei b) sample preparation showing free electron source doped into sample and polarisation mechanisms