Introduction

Dissolution Dynamic Nuclear Polarization (dDNP) permits measurement of in vivo tissue function in ways not possible through established molecular imaging methods.¹ The hyperpolarized (HP) agent is typically administered via intravenous injection, and must traverse the vascular tree before interacting with tissues of interest. Due to the highly transient nature of HP signals, the amount of HP agent delivered to capillary blood in these tissues depends in part on the rate of depolarization of the agent in blood prior to the arrival of the bolus in the capillary bed. The apparent longitudinal relaxation rate of the most widely used DNP agent, ¹³C pyruvate, has been shown to vary with albumin concentration in vitro,^2,3 however the effects of blood oxygenation on agent depolarization has not been thoroughly studied. In this work, we investigate the effects of variations in blood oxygenation on the apparent T₁ of HP agents that probe cellular metabolism and tissue perfusion.

Materials and Methods

Separate quantities of fresh heparinized bovine blood (Animal Technologies, Tyler, TX, USA) were gently bubbled with oxygen and nitrogen gas to enrich oxy- and deoxyhemoglobin content. Aliquots of oxygenated blood were used to reproduce the arterial oxygen state, and mixtures of oxygenated and deoxygenated blood were used to simulate venous blood. Each blood sample was assayed using a spectrophotometric CO-oximeter (GEM OPL; Instrumentation Laboratory, Bedford, MA, USA). All arterial samples had hemoglobin oxygen saturation (sO₂) values >95%, and venous samples had sO₂ values between 65% and 70%.

1 mL of each blood sample was placed in a 10mm Shigemi NMR tube and inserted in a 7T Bruker NMR system equipped with a 10mm broadband probe maintained at a temperature of 37 C. Shim preset values measured in a sample containing 1 mL of blood and 100 µL of D₂O were used. HP samples of [1-¹³C]pyruvate,⁴ [¹³C,¹⁵N₂]urea,⁵ and [2-¹³C]dihydroxyacetone⁶ were prepared in a HyperSense dDNP system using published methods. 100 µL of each HP dissolution was injected into a blood sample and a pulse-acquire ¹³C spectroscopy scan (TR = 2s, 10 degree excitation angle) was initiated. The primary signal in each set of dynamic HP spectra was integrated across a frequency range corresponding to the full-width at half-max (FWHM) measured in a time-averaged spectrum, and integral values were corrected for the applied excitations by multiplying the spectral integral from the $$$n$$$th repetition by $$$\sec(10^{\circ})^{n-1}$$$. Exponential decay curves of the form $$$y(t) = C + A e^{-t / T_1}$$$ were then fit to these data, and decay time constants were compared for HP agents assayed in blood with arterial and venous oxygen content using a two-sample t-test. All data processing was completed in MATLAB R2020b (The Mathworks, Natick, MA, USA) with custom-written scripts.

Results

Representative time-averaged spectra for each HP agent are shown in Figure 1. No chemical conversion of urea and dihydroxyacetone was observed, however some lactate production was seen in whole blood following administration of pyruvate. The ratio of lactate to pyruvate FWHM integrals in time-averaged spectra was <1% in all blood samples, and no difference in lactate production was observed with blood oxygenation.

Blood oxygenation had a statistically significant effect on the measured T₁ values for all three HP agents (Figure 2, Table 1), with more rapid depolarization occurring in whole blood with arterial sO_­2. Relative to the T₁ values measured in venous blood, relaxation time constants measured in arterial blood were on average decreased by 23% for [1-¹³C]pyruvate, 17% for [¹³C,¹⁵N₂]urea and 7% for [2-¹³C]dihydroxyacetone.

Discussion

Our experiments demonstrate that blood oxygenation imparts variations in depolarization rate for HP ¹³C agents. This effect was strongest for the HP signal corresponding to the carboxyl carbon of pyruvate, but was also observed for carbamide and ketone moieties. While our methods cannot identify the precise mechanism of oxygen-accelerated relaxation, these results suggest that dipole-dipole interactions between the HP ¹³C nucleus and paramagnetic O₂ are a significant factor at 7T, and that the paramagnetic effect of deoxyhemoglobin is less impactful on longitudinal relaxation than that of O₂ in whole blood.

The observation of lactate production from HP pyruvate in whole blood is notable since it could complicate quantitative analysis of HP imaging data. However, the amounts of lactate observed in these experiments are likely insignificant relative to those typically measured in metabolically active tissues.

Future work will investigate these effects in human blood at MRI field strengths more commonly used for HP imaging in patients. These variations in T₁ relaxation with blood oxygenation state could bias quantitative results of HP imaging studies in patients and animals with abnormal sO₂, for example due to impairment of respiratory function.

Acknowledgements

This work was supported in part by the National Cancer Institute and National Institute of Diabetes and Digestive and Kidney diseases of the National Institutes of Health (R01-DK105346, R01-CA211150, P30-CA016672). The content is solely the responsibility of the authors and does not necessarily represent the official views of any funding agency.