The relayed nuclear Overhauser effect in magnetization transfer and chemical exchange saturation transfer MRI

Abstract

Magnetic resonance (MR) is a powerful technique for noninvasively probing molecular species in vivo but suffers from low signal sensitivity. Saturation transfer (ST) MRI approaches, including chemical exchange saturation transfer (CEST) and conventional magnetization transfer contrast (MTC), allow imaging of low-concentration molecular components with enhanced sensitivity using indirect detection via the abundant water proton pool. Several recent studies have shown the utility of chemical exchange relayed nuclear Overhauser effect (rNOE) contrast originating from nonexchangeable carbon-bound protons in mobile macromolecules in solution. In this review, we describe the mechanisms leading to the occurrence of rNOE-based signals in the water saturation spectrum (Z-spectrum), including those from mobile and immobile molecular sources and from molecular binding. While it is becoming clear that MTC is mainly an rNOE-based signal, we continue to use the classical MTC nomenclature to separate it from the rNOE signals of mobile macromolecules, which we will refer to as rNOEs. Some emerging applications of the use of rNOEs for probing macromolecular solution components such as proteins and carbohydrates in vivo or studying the binding of small substrates are discussed.

Keywords: Z-spectrum; chemical exchange saturation transfer; dipolar cross relaxation; magnetization transfer contrast; molecular binding; molecular imaging; nuclear Overhauser effect; signal enhancement.

Figure 1.

¹H NMR spectrum and Z-spectrum for glycogen. (A) Chemical structure and proton assignment of glycogen. (B) One-dimensional ¹H NMR spectrum of glycogen (100 mM, pH 7.4). (C) Z-spectrum for rabbit liver glycogen solution (300 mM glucose unit, pH 7.4, 20 °C.) The bottom row shows the residual signal after subtracting out the fitted direct water saturation signal from the Z-spectrum. Reproduced with permission from Zhou et al., Proc Natl Acad Sci USA 2020; 117; 3144-3149.

Figure 2.

Examples of possible magnetization transfer pathways and signal enhancement in MT MRI. The selective irradiation of mobile or immobile molecular components with a radiofrequency (RF) pulse causes signal decreases in water via mechanisms including chemical exchange (A), relayed NOE (B), MTC (C), inhomogeneous MT (D) and rNOE after binding (E). In (C), the RF irradiation was placed on ─CH, but in principle, RF on ─OH is also a substantial source of MTC effect.

Figure 3.

The effect of molecular size on MR relaxation rates. (A) NMR correlation time (τ_c) increases with molecular size. (B-D) Calculated proton cross-relaxation rate σ (B), transverse relaxation rate R₂ = 1/T₂ (C), and longitudinal relaxation rate R₁ = 1/T₁ as well as $R_1^{eff}$ (D) as a function of NMR correlation time (τ_c) from Eqs. 4-8.

Figure 4.

Simulated rNOE (A, B) and MTC (C) signals in Z-spectra for an aliphatic proton pool (H_a) in molecules with varied rotational motion (in terms of NMR rotational correlation time, τ_c). Z-spectra were simulated using a three-pool rNOE model (M3). Here the T₁ and T₂ for aliphatic protons (H_a) and exchangeable protons (H_e) were assumed to be the same and calculated using Eqs. 6-7; Cross relaxation rates (σ) between H_a and H_e were calculated using Eq. 4. T_1w and T_2w were assumed to be 2.8 s and 1.8 s, respectively. The proton-proton distance was assumed to be 2 Å. The concentration of exchangeable protons ([H_e]) within the macromolecules (A-C) was 0.1 M, the exchange rate (k_ew) of relaying sites was 1000 s⁻¹. B₁ = 1 μT, saturation duration t_sat = 3 s.

Figure 5.

Illustration of pK_a and k_ew ranges for different exchangeable proton types. ^a Amide proton (─C(=O)NH-), pK_a: ~ 18, Ref ; k_ew: 10 to 1,200 s⁻¹, Ref ^{- b} Hydroxyl proton (─OH), pK_a: 8 to 16, Ref ^-; k_ew: 500 to 10,000 s⁻¹, Ref ^c Imino proton (=NH, secondary amine): pK_a: 8 to 11, Ref ; k_ew: 2,000 to 8,000 s⁻¹, Ref ^d Thiol proton (─SH): pK_a: 5 to 11, Ref ^-; k_ew: 270 to 104,000 s⁻¹, Ref ^e Amino protons (─NH₂, primary amine): pK_a: 2 to 11, Ref ^,-; k_ew: 500 to 10,000 s⁻¹,Ref ^f Carboxyl protons (─C(=O)OH): pK_a: 2 to 5, Ref ^,; k_ew: 100,000 to 1,000,000 s⁻¹, Ref

Figure 6.

Simulated rNOE (at −3.5 ppm) and CEST (at +3.5 ppm) signals for small (τ_c = 10 ns) and large (τ_c = 10² ns) proteins and with slow (k_ew = 30 s⁻¹), intermediate (k_ew = 10³ s⁻¹) and fast (k_ew = 10⁴ s⁻¹) exchange rates at 11.7 T and 3 T. The three-pool model (M3) that includes water (H_w), aliphatic proton (H_a), and exchangeable proton (H_e) pools was used for simulating rNOE and CEST signals, with an additional MT pool (H_MT) for simulating MTC background. (A-F) Z-spectra with varied B₁ for different protein sizes and exchange rate (k_ew) at t_sat = 3s, B₀ = 11.7 T. (G) Representative Z-spectra with varied B₁ for large protein and intermediate exchange rate (k_ew) at t_sat = 3s, B₀ = 3 T. (H, I) The dependence of rNOE signals on B₁ at B₀ = 11.7 T and 3 T. For small proteins (τ_c = 10 ns), the transverse relaxation time (T_2a) for H_a was calculated to be 1/22 s, and the longitudinal cross-relaxation rate (σ_ae) was −9 s⁻¹). For large proteins (τ_c = 10² ns), T_2a=1/224 s, σ_ae= −90 s⁻¹. Longitudinal relaxation rate for H_a (ρ_a) equal to −σ_ae, ρ_e = ρ_a, T_2e = T_2a, Ω_a = −3.5 ppm, Ω_e = +3.5 ppm. Concentration of H_a ([H_a]) is 100 mM, [H_e]=[H_a], [H_MTC]= 10 M.

Figure 7.

Examples of CEST and rNOE signals appearing in Z-spectra of rat and human brain with tumor (A, B), mouse fed liver (C) and rat brain with focal ischemia (D). (A) T₂ weighted image, NOE (−3.5 ppm) map, and the representative Z-spectra for a rat brain bearing 9L gliosarcoma tumor (B₁ = 1.17 μT, t_sat = 3s). Reproduced with permission from Cai et al., NMR Biomed 2015; 28; 1-8. (B) Gadolinium contrast enhancing T₁, NOE (−3.5 ppm) map, and the representative Z-spectra for the glioblastoma patient brain (B₁ = 0.6 μT). Note that the NOE (−3.5ppm) includes residual MTC in white matter, which is therefore highlighted in the image (see text). Reproduced with permission from Zaiss et al., Neuroimage 2015; 112;180-188. (C) T₂ image, glycoNOE (−1 ppm) map, and the representative Z-spectrum for a fed mouse liver (B₁ = 0.7 μT, using an ultra-short echo CEST sequence). Reproduced with permission from Zhou et al., Proc Natl Acad Sci USA 2020; 117; 3144-3149. (D) A representative Z-spectrum for the normal tissue in a rat brain, and rNOE (−1.6 ppm) images at different time points before and after ischemia from a rat brain (B₁ = 1 μT, t_sat = 5s). Reproduced with permission from Zhang et al., Magn Reson Imaging 2016;3;1100-1106.

Figure 8.

AREX spectra (B₁ = 0.75 μT, t_sat = 12 s) obtained from BSA solutions (A) at constant temperature and different pH values and (B) at constant pH and different temperatures. Native BSA (top row) corresponds to a concentration of 0 mM of the detergent SDS, unfolded BSA (bottom row) to an SDS concentration of 140 mM. Reprinted with permission from Goerke et al. NMR Biomed 28, 906-913, (2015).

Figure 9.

IMMOBILISE data from 100 mM N-acetylaspartate (NAA) and 100 mM lactate in crosslinked BSA phantoms. Notice the high-resolution IMMOBILISE-based differences agreeing with the spectral appearance for these compounds known from MRS, namely the NAA CH₃ resonance at −2.7 ppm, CH₂ resonances between −2.2 to −2.0 ppm, and the NH resonance at 3.2 ppm (which is temperature-dependent), corresponding to the known MRS frequencies referenced to TSP at 2.0 ppm, 2.5–2.7 ppm, and 7.9 ppm, respectively. For lactate, the CH₃ and CH peaks at −3.4 ppm and −0.6 ppm (MRS: 1.3 and 4.1 ppm) respectively are visible. Reprinted with permission from Yadav et al. Sci Rep 2017; 7; 10138.