Competitive Displacement Restores the Hyperpolarized 15N NMR Signal in Blood Plasma

Abstract

Hyperpolarized (HP) NMR can improve the sensitivity of conventional NMR experiments by several orders of magnitude, thereby making it feasible to detect the signal of low sensitivity nuclei such as ¹³C and ¹⁵N nuclei in vivo. Hyperpolarized substrates are usually administered by direct injection into the bloodstream, and interaction with serum albumin can cause rapid decay of the hyperpolarized signal due to the shortening of the spin-lattice (T₁) relaxation time. Here we report that the ¹⁵N T₁ of ¹⁵N labeled, partially deuterated tris(2-pyridylmethyl)amine decreases dramatically upon binding to albumin to such an extent that no HP-¹⁵ signal could be detected. We also demonstrate that the signal could be restored using a competitive displacer, iophenoxic acid, which binds stronger to albumin than tris(2-pyridylmethyl)amine. The methodology presented here eliminates the undesirable effect of albumin binding and should widen the range of hyperpolarized probes for in vivo studies.

Chart 1. Structure of ¹⁵N Labeled Tris(2-pyridylmethyl)amine Zinc Sensors, Salicylic Acid, and Iophenoxic Acid

Figure 1

Effect of increasing amount of competitive displacer 3 on the ¹⁵N NMR spectra of HP-(1b) (1.72 mM) in human plasma (HSA concentration ∼0.45 mM) at 9.4 T. The 3/1b mole ratio increases from 0 (no displacer) to 2. (A) Dynamic ¹⁵N NMR spectra recorded every 5 s with a 5° flip angle at 9.4 T showing the decay of ¹⁵N magnetization. (B) The first spectrum of each array showing the relative ¹⁵N signal intensities. Maximum signal intensity was observed at 1:1 mol ratio. (C) Plot of ¹⁵N signal intensity vs the mole ratio of 3 to 1b.

Figure 2

Effect of competitive displacer 3 on the ¹⁵N NMR spectra of HP-(1b) (1.72 mM) in the presence of half equivalent Zn²⁺ (0.85 mM) in human plasma (0.45 mM). (A) First spectrum in the absence and presence of equimolar 3 (1.72 mM). (B) Plot of the respective ¹⁵N signal intensities. (C) HP-1b can detect endogenous Zn²⁺ in blood plasma even in the absence of 3.