Figure 1.
Schematic overview of the adiabatic version of the refocused insensitive nuclei enhanced by polarization transfer (BINEPT) sequence.
Segmented BIR4 pulses (400 µs per segment, 35 ppm band width) and a full BIR4 180° pulse (400 µs, 35 ppm band width) were used instead of conventional 90 and 180 degree pulses. The echo time of 1/4 J was set to 34 ms, which gave optimal signals for the acquisition of PE and PC. Abbreviations: BIR4, B1 insensitive refocusing; JPH, J-coupling constant; TE1H, effective echo time for proton refocusing; TE31P, effective echo time for phosphorus refocusing.
Figure 2.
Raw RARE images and segmentation of corresponding slices.
Necrotic tissue is depicted in red. Segmentation of (a) representative MCF-7 tumor and (b) representative MDA-MB-231 tumor. (c) Correlation between line width of PCr and necrotic fraction for all MCF-7 and MDA-MB-231 tumors measured.
Table 1.
In vivo 31P relaxation times.
Figure 3.
Example of in vivo pulse-acquire (PA, top) and BINEPT (bottom) 31P MR spectra of a representative MCF-7 (left) and MDA-MB-231 (right) tumor.
Lorentzian lines as fitted by JMRUI are shown below each MR spectrum. All phosphorylated metabolites are visible in the PA spectrum, whereas the BINEPT spectrum only contains signals from phospholipid metabolites with H-P-coupling such as PE, PC, GPE, and GPC. Note the broad, uneven baseline in the 0–5 ppm region of the PA spectra, where signals from mobile membrane phospholipids are resonating [35], [45], [46]. The signal of β-NTP is formed by β-NTP only. The signal labeled α-NTP is an overlapping signal from α-NTP, α-NDP, NAD, and DPDE. The signal labeled γ-NTP is an overlapping signal from γ-NTP and β-NDP. Typically, β-NTP is the smallest peak of the three NTP signals, however, here, γ-NTP overlaps with a broad baseline signal that makes it appear smaller than β-NTP.
Figure 4.
In vivo phospholipid metabolite levels as measured by (a, c) PA and (b, d) BINEPT 31P MRS.
Average metabolite levels ± standard error are shown for (a, b) MCF-7 (n = 7) and (c, d) MDA-MB-231 (n = 6) breast tumor xenografts. Statistical results: PA vs BINEPT MCF-7: GPE p = 0.004 *, GPC p = 0.03 **; PA vs BINEPT MDA-MB-231: GPE p = 0.02 #, GPC p = 0.06 ##; MCF-7 vs MDA-MB-231: PA, for all metabolites p>0.20; MCF-7 vs MDA-MB-231: BINEPT, for all metabolites p>0.21.
Figure 5.
31P High-Resolution MR spectrum of the same MCF-7 tumor as shown in Fig. 2.
The full spectral range is displayed in the spectrum on the bottom. Signals of NTPs are visible in the 0 to -15 ppm region. An expanded region of 0 to 8 ppm is displayed on the top, as well as the region with the PPA reference signal at 13 ppm. The signals of PE, PC, GPE, and GPC are clearly separated.
Figure 6.
Quantification of PE, PC, GPE, and GPC levels from 31P HR-MRS of (a) MCF-7 and (b) MDA-MB-231 breast tumor extracts.
Average metabolite levels ± standard errors are shown and were expressed as concentration in μmol per gram of tumor tissue. Statistical results: MCF-7 vs MDA-MB-231: PE p = 0.22 +, PC p = 0.77 x, GPE p = 0.31 *, GPC p = 0.16 #.
Figure 7.
Comparison of phospholipid metabolite ratios between in vivo PA, in vivo BINEPT, and tumor extract HR 31P MRS measurements of the same (a) MCF-7 and (b) MDA-MB-231 tumors.
Values are given as average ± standard error. The PE/GPE ratios are not shown for in vivo PA measurements because the GPE resonance could not be reliably detected in several PA spectra due to low SNR. Statistical results: PA vs BINEPT: MCF-7 PC/GPC p = 0.02 *, all other ratios in MCF-7 and MDA-MB-231 p>0.12; PA vs tumor extract: MCF-7 PE/PC p = 0.04 #, PC/GPC p = 0.05 ##, all other ratios in MCF-7 and MDA-MB-231 p>0.21; BINEPT vs tumor extract: MCF-7 PE/PC p = 0.05 +, all other ratios in MCF-7 and MDA-MB-231 p>0.24.