A rapid method for direct detection of metabolic conversion and magnetization exchange with application to hyperpolarized substrates

Abstract

In this work, we present a new MR spectroscopy approach for directly observing nuclear spins that undergo exchange, metabolic conversion, or, generally, any frequency shift during a mixing time. Unlike conventional approaches to observe these processes, such as exchange spectroscopy (EXSY), this rapid approach requires only a single encoding step and thus is readily applicable to hyperpolarized MR in which the magnetization is not replenished after T(1) decay and RF excitations. This method is based on stimulated-echoes and uses phase-sensitive detection in conjunction with precisely chosen echo times in order to separate spins generated during the mixing time from those present prior to mixing. We are calling the method Metabolic Activity Decomposition Stimulated-echo Acquisition Mode or MAD-STEAM. We have validated this approach as well as applied it in vivo to normal mice and a transgenic prostate cancer mouse model for observing pyruvate-lactate conversion, which has been shown to be elevated in numerous tumor types. In this application, it provides an improved measure of cellular metabolism by separating [1-(13)C]-lactate produced in tissue by metabolic conversion from [1-(13)C]-lactate that has flowed into the tissue or is in the blood. Generally, MAD-STEAM can be applied to any system in which spins undergo a frequency shift.

Figure 1

Illustration of the phase-sensitive signal encoding in MAD-STEAM. (a) Stimulated-echo pulse sequence. (b,c) Signal encoding with no net frequency shift during the mixing time (TM), where the received signal has no phase shift (ie Δϕ = 0 from Eq. 8) (d) Signal encoding with a frequency shift during the mixing time, where the received signal a phase shift. In MAD-STEAM and this illustration example, TE is chosen such that Δϕ = π/2.

Figure 2

Illustration of the relationship between 2D exchange spectroscopy (EXSY) and MAD-STEAM. MAD-STEAM is a single step in an EXSY experiment, and their spectra are related by the relationship in Eq. 13. The MAD-STEAM TE is chosen such that the complex exponential weighting gives a phase difference of ±π/2 between the two peaks of interest.

Figure 3

MAD-STEAM pulse sequence. The Encoding (90°-90°) section stores the magnetization along M_Z with a sinusoidal encoding (M_Z,enc). The encoding was refocused and data acquired at multiple mixing times (TM). These used a progressive flip angle, α[n], (43) to account for the depletion of stored magnetization by previous excitation pulses. An adiabatic double spin-echo was used following the excitation pulse and refocusing gradient to allow for symmetric sampling of the echo.

Figure 4

Validation of the MAD-STEAM method observing pyruvate (pyr) hydration following pre-saturation of the pyruvate-hydrate (pyr-hyd) resonance. (A) Pyruvic acid dissolved into an aqueous solution creates exchange between pyruvate and pyruvate-hydrate, which are in equilibrium. (Teal indicates enriched ¹³C.) (B) TE = 13.0 ms experiment showing urea (phase reference) and the conversion from pyruvate to pyruvate-hydrate. (C) Various TE experiments showing the phase of the generated pyruvate-hydrate. In all of different TE experiments shown, the experimental data (x’s) phase matches well with the expected curves (dashed lines), which are based on the predicted phase shift, Δϕ, and the fitted conversion rate. Diffferences between the data and expected curves are likely due to imperfect pyruvate-hydrate saturation.

Figure 5

Comparison of pyruvate hydration kinetics using (A) Spin-echo MR spectroscopy with pre-saturation of pyruvate-hydrate. (B) Spin-echo MR spectroscopy without presaturation (Note: the kinetics cannot be esimated from this experiment). (C) MAD-STEAM with TE = 13.0 ms and pre-saturation of pyruvate-hydrate. (D) MAD-STEAM with TE = 13.0 ms and no pre-saturation. The two-site exchange fits (dashed lines) were very similar between both spin-echo and MAD-STEAM saturation recovery, as well as between MAD-STEAM with and without pre-saturation. The experimental data is denoted by x’s.

Figure 6

Ex vivo LDH enzyme assay validation using TE = 14.0 ms to observe the conversion from pyruvate to lactate. (A) Pyruvate to lactate conversion is mediated by the LDH enzyme and NAD cofactors. (B) Spectra at first and last time points. (C) Peak amplitude time courses: data (x’s) and fitted two-site exchange model with exponential decay (dashed lines). Lactate was partially suppressed prior to the experiment by pre-saturation pulses.

Figure 7

Normal mouse results from a 10 mm slice through the abdomen. The pulse sequence started 20 sec after the start of injection to allow for perfusion of the bolus. (A) Slab location on a coronal image. (B) Spectra at first and last time points. (C) Peak amplitude time courses: data (x’s) and fitted two-site exchange model with exponential decay (dashed lines).

Figure 8

Prostate tumor mouse model results from a 20 mm slab across a tumor. The MAD-STEAM pulse sequence was started 25 sec after the start of injection to allow for perfusion of the bolus. (A) Peak amplitude time courses without pre-saturation: data (x’s) and fits (dashed lines). (B) Slab location on a coronal image. (C) Lactate time courses following pre-saturation of lactate. These experiments used TE = 14.0 ms (left) and 15.3 ms (right) to modulate the phase of lactate generated from pyruvate (Δϕ_pyr→lac) similarly to Fig. 4. All curves were normalized to the peak pyruvate, and the relative amounts of lactate generated were similar across all three experiments.