Quantum coherence spectroscopy to measure dietary fat retention in the liver

Abstract

The prevalence of fatty liver reaches alarming proportions. Fatty liver increases the risk for insulin resistance, cardiovascular disease, and nonalcoholic steatohepatitis (NASH). Although extensively studied in a preclinical setting, the lack of noninvasive methodologies hampers our understanding of which pathways promote hepatic fat accumulation in humans. Dietary fat retention is one of the pathways that may lead to fatty liver. The low (1.1%) natural abundance (NA) of carbon-13 (¹³C) allows use of ¹³C-enriched lipids for in vivo MR studies. Successful implementation of such methodology, however, is challenging due to low sensitivity of ¹³C-magnetic resonance spectroscopy (¹³C-MRS). Here, we investigated the use of 1-dimensional gradient enhanced heteronuclear single quantum coherence (ge-HSQC) spectroscopy for the in vivo detection of hepatic ¹H-[¹³C]-lipid signals after a single high-fat meal with ¹³C-labeled fatty acids in 5 lean and 6 obese subjects. Postprandial retention of orally administered ¹³C-labeled fatty acids was significant (P < 0.01). Approximately 1.5% of the tracer was retained in the liver after 6 hours, and retention was similar in both groups (P = 0.92). Thus, a substantial part of the liver fat can originate directly from storage of meal-derived fat. The ge-HSQC can be used to noninvasively reveal the contribution of dietary fat to the development of hepatic steatosis over time.

Figure 1. Spectra acquired from the phantom containing naturally abundant ¹³C Intralipid.

(A and B) The result from the conventional quantum coherence sequences. In both cases, the ¹³C chemical shift evolution during the t₁ period resulted in phase distortions in the spectrum. Addition of the ¹³C inversion pulse led to nonphase distorted lipid spectra, in which both CH₂ and CH₃ signals were observable. (C and D) Both relative and absolute signals were higher in the 1D ge-HSQC (heteronuclear single quantum coherence) as compared with the 1D ge-HMQC (heteronuclear multiple quantum coherence).

Figure 2. Spectra acquired from the Intralipid phantom with increasing t₁ in the 1D ge-HMQC (heteronuclear multiple quantum coherence) and the 1D ge-HSQC (heteronuclear single quantum coherence).

(A and B) In A, the results for the 1D ge-HMQC are shown, while in B, the outcome of the 1D ge-HSQC sequence is presented. The evolution time (t₁) was increased with 1 ms every step and started at, respectively, 7.6 and 4.2 ms. It is apparent that the signal acquired with the ge-HMQC decays rapidly with an increasing t₁.

Figure 3. Spectra acquired from the phantom with the uniformly labeled ¹³C tracer with increasing t₁ in the 1D ge-HSQC (heteronuclear single quantum coherence).

The evolution time (t₁) was increased with 1 ms every step and started at 4.2 ms. Due to the ¹³C-¹³C couplings in the tracer, the signal now decays rapidly with increasing t₁.

Figure 4. Results from the in vivo experiments.

(A and B) Typical example of the total lipid spectrum, acquired with STEAM (stimulated echo acquistion mode) ¹H-MRS, and the concomitant ¹³C-edited lipid spectrum, acquired with the ge-HSQC (heteronuclear single quantum coherence) sequence. As no decoupling was applied, two lipid CH₂ peaks are visible, which are separated by approximately 127 Hz. (C) The total IHL (intrahepatic lipid) pool (g/kg ww) for both lean (n = 5) and obese (n = 6) subjects at 1,5, 3.0, 4.5, and 6.0 hours after a high-fat meal. Total IHL did not change after the meal (repeated-measures ANOVA, P = 0.35). (D) The absolute concentration of ¹³C lipids (g/kg ww) in the liver is plotted in time, showing retention of ¹³C-labeled fatty acids after the meal (repeated-measures ANOVA, P < 0.01).

Figure 5. Pulse sequences used in this study.

(A) The 1D ge-HMQC (heteronuclear multiple quantum coherence) sequence is shown. The HMQC block was placed after PRESS (point resolved spectroscopy) localization. Two additional ¹³C inversion pulses were used to refocus the ¹³C chemical shift evolution. Minimum evolution time (t₁) was 7.6 ms. In contrast to the conventional ge-HMQC sequence, 5 gradients were used for coherence selection. Gradients were applied in a ratio of 1:–1:–1:1:1. (B) The 1D ge-HSQC (heteronuclear single quantum coherence) sequence, based on STEAM (stimulated echo acquistion mode) localization. The inversion pulse during the t₁, which was used on the ¹H channel previously, was now applied on the ¹³C channel to again refocus ¹³C chemical shift evolution. Minimum t₁ was 4.2 ms, and gradients were applied in a 2:–2:1 ratio. In both sequences, 1/2J_CH was chosen as 3.95 (J = 127 Hz for CH₂ lipids). No decoupling was applied.