Quality assurance of PASADENA hyperpolarization for 13C biomolecules

Object  Define MR quality assurance procedures for maximal PASADENA hyperpolarization of a biological ¹³C molecular imaging reagent. Materials and methods  An automated PASADENA polarizer and a parahydrogen generator were installed. ¹³C enriched hydroxyethyl acrylate, 1-¹³C, 2,3,3-d₃ (HEA), was converted to hyperpolarized hydroxyethyl propionate, 1-¹³C, 2,3,3-d₃ (HEP) and fumaric acid, 1-¹³C, 2,3-d₂ (FUM) to hyperpolarized succinic acid, 1-¹³C, 2,3-d₂ (SUC), by reaction with parahydrogen and norbornadiene rhodium catalyst. Incremental optimization of successive steps in PASADENA was implemented. MR spectra and in vivo images of hyperpolarized ¹³C imaging agents were acquired at 1.5 and 4.7 T. Results  Application of quality assurance (QA) criteria resulted in incremental optimization of the individual steps in PASADENA implementation. Optimal hyperpolarization of HEP of P = 20% was achieved by calibration of the NMR unit of the polarizer (B ₀ field strength ± 0.002 mT). Mean hyperpolarization of SUC, P = [15.3 ± 1.9]% (N = 16) in D ₂O, and P = [12.8 ± 3.1]% (N = 12) in H ₂O, was achieved every 5–8 min (range 13–20%). An in vivo ¹³C succinate image of a rat was produced. Conclusion  PASADENA spin hyperpolarization of SUC to 15.3% in average was demonstrated (37,400 fold signal enhancement at 4.7 T). The biological fate of ¹³C succinate, a normally occurring cellular intermediate, might be monitored with enhanced sensitivity.     

Ultra-fast three dimensional imaging of hyperpolarized 13C in vivo

Objective: PASADENA, a chemical method of enhancing nuclear spin polarization has demonstrated ¹³C polarizations of order unity for the nascent products of molecular addition by parahydrogen. The extreme brevity of signal enhancement obtained by hyperpolarization requires improved ¹³C MR in vivo imaging techniques for their optimum utility. Materials and Methods: ¹³C imaging sequences, including ¹³C 3D FIESTA, were compiled for a GE LX 1.5 T clinical MR scanner. Two water soluble ¹³C imaging agents were hyperpolarized utilizing parahydrogen and an automated polarizer. ¹³C polarization was quantified in flow phantoms and in rats with jugular vein catheters. Results: Fast 3D FIESTA ¹³C MR imaging technique acquired sequential 3D images (3.66 s/acquisition) with superior SNR. Hyperpolarized ¹³C solutions and vascular phantoms achieved a maximum signal of 26,624±593. In vivo ¹³C MR images of the cardiopulmonary circulation showed maximum ¹³C signal of 2,402±158. ¹³C images acquired within 3.66 s showed signal enhancement over 10,000 compared to equilibrium polarization. Conclusion: 3D-FIESTA was effective for sub-second in vivo imaging of hyperpolarized ¹³C reagents produced in a custom-built parahydrogen polarizer. Application to ¹³C hyperpolarized by parahydrogen is demonstrated in vitro and in vivo     

Quantification of metabolites from single-voxelin vivo 1H NMR data of normal human brain by means of time-domain data analysis

We present here a combination of time-domain signal analysis procedures for quantification of human brainin vivo ¹H NMR spectroscopy (MRS) data. The method is based on a separate removal of a residual water resonance followed by a frequency-selective time-domain line-shape fitting analysis of metabolite signals. Calculation of absolute metabolite concentrations was based on the internal water concentration as a reference. The estimated average metabolite concentrations acquired from six regions of normal human brain with a single-voxel spin-echo technique for theN-acetylaspartate, creatine, and choline-containing compounds were 11.4±1.0,6.5±0.5, and 1.7±0.2 mmol kg^–1 wet weight, respectively. The time-domain analyses ofin vivo ¹H MRS data from different brain regions with their specific characteristics demonstrate a case in which the use of frequency-domain methods pose serious difficulties.     

31P MR spectroscopy and in vitro markers of oxidative capacity in type 2 diabetes patients

Background: Skeletal muscle mitochondrial function in type 2 diabetes (T2D) is currently being studied intensively. In vivo ³¹P magnetic resonance spectroscopy (³¹P MRS) is a noninvasive tool used to measure mitochondrial respiratory function (MIFU) in skeletal muscle tissue. However, microvascular co-morbidity in long-standing T2D can interfere with the ³¹P MRS methodology. Aim: To compare ³¹P MRS-derived parameters describing in vivo MIFU with an in vitro assessment of muscle respiratory capacity and muscle fiber-type composition in T2D patients. Methods: ³¹P MRS was applied in long-standing, insulin-treated T2D patients. ³¹P MRS markers of MIFU were measured in the M. vastus lateralis. Muscle biopsy samples were collected from the same muscle and analyzed for succinate dehydrogenase activity (SDH) and fiber-type distribution. Results: Several ³¹P MRS parameters of MIFU showed moderate to good correlations with the percentage of type I fibers and type I fiber-specific SDH activity (Pearson’s R between 0.70 and 0.75). In vivo and in vitro parameters of local mitochondrial respiration also correlated well with whole-body fitness levels (VO _2peak) in these patients (Pearson’s R between 0.62 and 0.90). Conclusion: Good correlations exist between in vivo and in vitro measurements of MIFU in long-standing insulin-treated T2D subjects, which are qualitatively and quantitatively consistent with previous results measured in healthy subjects. This justifies the use of ³¹P MRS to measure MIFU in relation to T2D.     

Assignment of the 2.03 ppm resonance in in vivo <Superscript>1</Superscript>H MRS of human brain tumour cystic fluid: contribution of macromolecules

MRI and MRS are established techniques for the evaluation of intracranial mass lesions and cysts. The 2.03 ppm signal recorded in their ¹H-MRS spectra is often assigned to NAA from outer volume contamination, although it has also been detected in non-infiltrating tumours and large cysts. We have investigated the molecular origin of this resonance in ten samples of cystic fluids from human brain tumours. The NMR detected content of the 2.03 ppm resonance in 136 ms echo time spectra, assuming an N- CH₃ origin, was 3.19 ± 1.01 mM. Only one third (34 ± 12%) of the N-acetyl containing compound (NAC) signal could be extracted by perchloric acid (PCA) indicating that most of it originated in a macromolecular PCA-insoluble component. Chemical analysis of the cyst fluids showed that sialic acid bound to macromolecules would account for 64.3% and hexuronic containing compounds for 29.2% of the NMR-detectable ex vivo signal, 93.4% of the signal at TE 136 ms. Lactate content measured by NMR (6.4 ± 4.4 mM) and the predominance of NAC originating in sialic acid point to a major origin from tumour rather than from plasma for this 2.03 ppm resonance.     

Comparison of in vivo1H MRS of human brain tumours with1H HR-MAS spectroscopy of intact biopsy samples in vitro

High resolution magic angle spinning (MAS)¹H nuclear magnetic resonance (NMR) spectroscopy has been employed to study intact human brain tumour tissue and comparison with the corresponding in vivo spectrum has been made. Two dimensional¹H MAS-NMR measurements, including J-resolved and homonuclear shift correlation spectra, were obtained to aid metabolite signal assignment. MAS gave greatly improved line-shape and reduced line-width in comparison to conventional high resolution in vivo¹H MRS of intact tissue, permitting the simultaneous detection of cellular lipids and metabolites. The technique provides the most direct method for comparison of in vivo spectra with high resolution spectra in vitro and hence allows more reliable peak assignment of in vivo¹H MRS spectra.     

Experimental31p nmr study of the influence of ionic strength on the apparent dissociation constant of mgatp

The classical method for³¹P NMR determination of intracellular free magnesium concentration ([Mg _free ²⁺ ]) requires an accurate knowledge of the apparent dissociation constant (K _D ) of MgATP. There is a large difference between the previously determined values ofK _D . Although the value of 50 µM, determined by a³¹P NMR method, is now largely accepted, a value of 86 µM has more recently been measured with a fitting method derived from the original one, and with a different ionic strength. The purpose of our study was to assess if the cause of the difference between these two previously reportedK _D values was due to the measuring method or to the ionic strength value used.Working at pH=7.2,T=37°C, and [KCl]=0.25 M, we performedK _D measurements with the original³¹P NMR method and with the fitting method. The results (67±13 µM and 61±20 µM, respectively) were not significantly different. Then, with the first method, we measured K_D at [KCl]=0.12 M and found a value of 19±5 µM. We conclude that the main cause of difference between theK _D values measured by³¹P NMR reside in the disparity of ionic strength values used for their measurement. OurK _D measurements at [KCl]=0.25 and 0.12 M demonstrate the importance of the ionic strength value used for imitating the intracellular medium on the absolute value of ([Mg _free ²⁺ ]) measured by³¹P NMR spectroscopy.Address for correspondence: Université Catholique de Louvain, Unité CPMC, Bâtiment Lavoisier, Place Louis Pasteur n°1, B-1348 Louvain-la-Neuve, Belgium. Additional reprints of this chapter may be obtained from the Reprints Department, Chapman & Hall, One Penn Plaza, New York, NY 10119.     

Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays

KTCTL-26 and KTCTL-2 are renal cell carcinoma (RCC) lines with high and lowexpression of P-170 glycoprotein, respectively. Inherent differences between the two cell lines in terms of phosphate metabolites and growth characteristics in culture were examined for possible association with multidrug resistance (MDR). Differences in response to drug treatment were investigated for 40 h incubations with various doses of vinblastine (VBL) alone or as cotreatments with various concentrations of the calcium antagonist diltiazem (DIL) and/or interferon–α (IFN-α). Treatment effects were quantitated using the MTT survival assay and ³¹P magnetic resonance spectroscopy (MRS) to determine phosphate metabolite profiles in intact cells. KTCTL-2 and KTCTL-26 cells exhibited significant inherent differences in phosphocholine, glycerophosphocholine, glycerophosphoethanolamine, and phosphocreatine levels. KTCTL-26 cells were more sensitive than KTCTL-2 to 0.011μM VBL alone (87% vs. 102% survival) or to 0.011μM BL + 10μM DIL (55% vs. 80% survival). The latter treatment resulted in a significant decrease in the ratio of phosphocholine to glycerophosphocholine in KTCTL-26 cells but no significant changes in phosphate metabolites in KTCTL-2 cells. Metabolomic ³¹P MRS detects different metabolite profiles for RCC cell lines with different MDR phenotypes and may be useful for noninvasive characterization of tumors in a clinical setting.This revised version was published online in August 2005 with a corrected sequence of authors.     

Large increases in1H metaboliteT 2's after cerebral hypoxia-ischemia correlate with ATP depletion

In vivo proton (¹H) magnetic resonance spectroscopy (MRS) can measure cerebral metabolite concentrations and nuclear relaxation times. Function of the sodium (Na⁺)/potassium (K⁺) pump in cell membranes depends on adequate adenosine triphosphate (ATP) levels: intracellular Na⁺ is normally extruded in exchange for extracellular K⁺. Low ATP will cause pump dysfunction and loss of K⁺ accompanied by influx of Na⁺and water. Raised intracellular water may increase molecular mobility and this might be detectable as increased apparent transverse relaxation times (T ₂'s).¹H-MRS of the brains of newborn piglets during acute hypoxia-ischemia revealed enigmatic increases in the peak area of creatine + phosphocreatine (Cr) relative to those of choline-containing compounds (Cho) andN-acetylaspartate (NAA). Interleaved¹H and phosphorus (³¹P) MRS showed that theT ₂'s of both Cr and lactate (Lac) increased during acute hypoxia-ischemia and these changes correlated with reductions in nucleotide triphosphate (NTP; largely ATP). Within 50 h of metabolic recovery from the primary insult, as delayed energy failure developed, theT ₂'s of Cho, Cr, NAA, and Lac increased greatly. TheseT ₂ changes also correlated with NTP depletion. These observations demonstrate important relationships betweenT ₂'s and function of the ATP-dependent Na⁺/K⁺ pump.     

Potential pitfall in the determination of free [Mg2+] by31P NMR when using the β/α-ATP peak height ratio method

Recently, Clarke et al., (Clarke K, Kashiwaya Y, King MT, Gates D, Keon CA, Cross HR, Radda GK, Veech RL. The β/α peak height ratio of ATP. A measure of free [Mg _free ²⁺ ] using³¹ P NMR, J. Biol. Chem. 1996;271:21142–21150.) reported a new method to noninvasively determine the concentration of intracellular free magnesium ([Mg _free ²⁺ ]) based on the measurement of the peak height ratioh _β/α of the β- and α-ATP signals in³¹P NMR spectra.h _β/α varies with Mg_free ²⁺], however, the study presented here shows thath _β/α also strongly depends on the homogeneity of the static magnetic field. For this reason, we performed at a magnetic field strength of 1.5 T³¹P NMR measurements of solutions that mimic intracellular medium. The magnetic field homogeneity was varied by changing the currents in the shim coils, and the effect onh _β/α is demonstrated with and without proton decoupling. In both cases,h _β/α strongly depends on the magnetic field homogeneity and can therefore lead to a pitfall in the determination of [Mg_free ²⁺].     

Severity of delayed (“secondary”) cerebral energy failure after acute hypoxia-ischemia is related to the time integral of acute ATP depletion

The aim of this study was to reproduce the delayed (secondary) cerebral energy failure previously described in birth-asphyxiated newborn infants and to investigate relationships between primary insult severity and the extent of the delayed energy failure. Phosphorus (³¹P) magnetic resonance spectroscopy (MRS) at 7 T was used to study the brains of 12 newborn piglets during an acute, reversible, cerebral hypoxic-ischemic episode which continued until nucleotide triphosphates (NTP) were depleted. After reperfusion and reoxygenation, spectroscopy was continued for 48 h. High-energy metabolite concentrations returned to near normal levels after the insult, but later they fell as delayed energy failure developed. The time integral of NTP depletion in the primary insult correlated strongly with the minimum [phosphocreatine (PCr)]/[inorganic orthophosphate (P_i)] observed 24–48 h after the insult. (Linear regression analysis gave slope –8.04 h^–1; ordinate intercept=1.23;r=0.92;P<0.0001.) This model is currently being used to investigate the therapeutic potential of various cerebroprotective strategies including hypothermia.     

ESMRMB 2009 Congress,Antalya, Turkey, 1–3 October: Abstracts,Thursday

Object  To determine whether glycine can be measured at 7 T in human brain with ¹H magnetic resonance spectroscopy (MRS). Materials and methods  The glycine singlet is overlapped by the larger signal of myo-inositol. Density matrix simulations were performed to determine the TE at which the myo-inositol signal was reduced the most, following a single spin-echo excitation. ¹H MRS was performed on an actively shielded 7 T scanner, in five healthy volunteers. Results  At the TE of 30 ms, the myo-inositol signal intensity was substantially reduced. Quantification using LCModel yielded a glycine-to-creatine ratio of 0.14 ± 0.01, with a Cramér–Rao lower bound (CRLB) of 7 ± 1%. Furthermore, quantification of metabolites other than glycine was possible as well, with a CRLB mostly below 10%. Conclusion  It is possible to detect glycine at 7 T in human brain, at the short TE of 30 ms with a single spin-echo excitation scheme.     

In vivo comparison of atherosclerotic plaque progression with vessel wall strain and blood flow velocity in apoE−/− mice with MR microscopy at 17.6 T

Object  At present, in vivo plaque characterization in mice by MRI is typically limited to the visualization of vascular lesions with no accompanying analysis of vessel wall function. The aim of this study was to analyze the influence of atherosclerotic plaque development on the morphological and mechanical characteristics of the aortic vessel wall in a pre-clinical murine model of atherosclerosis. Materials and methods  Groups of apolipoprotein E-deficient (apoE^−/−) and C57BL/6J control mice fed a high-fat diet were monitored over a 12-week time period by high-field MRI. Multi-Slice-Multi-Spin-Echo and Phase-Contrast MRI sequences were employed to track changes to aortic vessel wall area, blood flow velocity and distensibility. Results  After 6- and 12-weeks, significant changes in vessel wall area and circumferential strain were detected in the apoE^−/− mice relative to the control animals. Blood flow velocity and intravascular lumen remained unchanged in both groups, findings that are in agreement with the theory of positive remodeling of the ascending aorta during plaque progression. Conclusion  This study has demonstrated the application of high-field MRI for characterizing the temporal progression of morphological and mechanical changes to murine aortic vasculature associated with atherosclerotic lesion development.     

The C-neu mammary carcinoma in Oncomice; characterization and monitoring response to treatment with herceptin by magnetic resonance methods

To characterize spontaneously occurring c-neu/HER2 overexpressing tumours in oncomice and their response to herceptin by non-invasive magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). Oncomice were monitored by localized ³¹P MRS during unperturbed growth and before and after treatment with 10 mg/kg herceptin (Hoffman La Roche) intraperitoneally for up to 21 days post-treatment. Vascular morphology and function was assessed by quantitation of tumour magnetic resonance (MR) relaxation rates R₂* and R₂ prior to and either during carbogen (95% O₂/5% CO₂) breathing or following administration of the blood-pool contrast agent NC100150 (Clariscan, Amersham Health). Immunohistochemistry showed strong membrane staining for HER2 protein overexpression. The ³¹P MRS showed only a significant (p<0.01) increase of phosphomonoester / total phosphate ratio over 21 days of growth. Herceptin increased the tumour volume doubling time compared to untreated tumours and significantly increased the phosphomonoester / -nucleoside triphosphate ratio 2 days after treatment (p=0.01). Tumours showed a highly heterogeneous yet significant (p<0.01) decrease or increase in R₂* in response to carbogen or NC100150 respectively. The absence of a decline in tumour bioenergetics with growth, commonly seen in ³¹P MRS studies of transplanted rodent tumour models, coupled with the heterogeneous blood volume revealed by ¹H MRI, suggest a metabolic and vascular phenotype similar to that found in human tumours.     

Magnetic resonance spectroscopy data analysis: time or frequency domain?

Comparisons of time and frequency domain methods are presented forin vitro ¹H andin vivo ³¹P magnetic resonance spectroscopy (MRS) data. Many distortions in the MR spectrum, introduced by applying the Fourier transform to a nonideal free-induction decay (FID), can be handled more elegantly in the time domain, where operations are carried out directly on the measured signal. It was found that if the measured signal is well conditioned—high signal-to-noise ratio (S/N), no truncation, no baseline problems—then both time and frequency domain methods give the same results within the error limits. However, distortions in the measured signal make analysis by time domain methods preferable over frequency domain methods. In all applications, the use of prior knowledge appeared to play an important role.     

Signal enhancement through heteronuclear polarisation transfer in in-vivo <Superscript>31</Superscript>P MR spectroscopy of the human brain

Significant ³¹P NMR signal enhancement through heteronuclear polarisation transfer was obtained in model solutions and in vivo on a 1.5-T whole-body MR scanner equipped with two RF channels. The much higher population differences involved in proton Zeeman energy levels can be transferred to the ³¹P levels with the refocused INEPT (insensitive nucleus enhancement by polarisation transfer) double-resonance experiment by means of a series of simultaneously applied broadband RF pulses. INEPT achieves a polarisation transfer from ¹H to ³¹P spin states by directly reordering the populations in spin systems with heteronuclear scalar coupling. Thus, only the ³¹P NMR signal of metabolites with scalar ¹H–³¹P coupling is amplified, while the other metabolite signals in the spectra are suppressed. Compared to Ernst-angle excitation, a repetition-time-dependent signal enhancement of η=(29±3)% for methylene diphosphonic acid (MDPA) and η=(56±1)% for phosphorylethanolamine (PE) was obtained on model solutions through optimisation of the temporal parameters of the pulse experiment. The results are in good agreement with numerical calculations of the theoretical model for the studied spin systems. With optimised echo times, in-vivo ³¹P signal enhancement of the same order was obtained in studies of the human brain.     

In vivo quantification of response to treatment in patients with multiple myeloma by <Superscript>1</Superscript>H magnetic resonance spectroscopy of bone marrow

Object Magnetic resonance imaging (MRI) is the gold standard non-invasive technique to detect malignant disease in the bone marrow. Proton magnetic resonance spectroscopy (MRS) can be performed as a quick adjunct to routine spinal MRI. We performed proton MRS to patients with multiple myeloma (MM) at diagnosis and after treatment to investigate the possible correlation of MRS data with response to therapy. Patients and methods Twenty-one patients with newly diagnosed MM underwent combined MRI/MRS explorations of a transverse center section in the fifth lumbar vertebral body. MRS was acquired with STEAM and 40 ms TE. Areas of unsuppressed water and lipid resonances were used to calculate the lipid-to-water ratio (LWR). Results No association was detected between initial LWRs and the clinical characteristics of patients. Post treatment MRS was available in 16 patients of whom 11 (69%) presented an LWR increase, this included all complete responders (8/8, 100%, P = 0.012). A post-treatment LWR value equal to or larger than one is proposed as a non-invasive marker of complete response to treatment. Conclusion Only patients responding to treatment presented a significant increase in bone marrow LWR after therapy. MRS may provide an adequate quantification of response to chemotherapy in patients with MM.     

31P NMR studies of human soleus and gastrocnemius show differences in theJ γβ coupling constant of ATP and in intracellular free magnesium

Localized proton decoupled³¹Pin vivo NMR spectroscopy of the human calf muscle was performed using a 1.5-T whole-body imager and the slice selective two-dimensional chemical-shift-imaging (2D-CSI) technique. The³¹P-³¹P coupling constants and the chemical shifts of ATP were compared in gastrocnemius and soleus. Significant differences were found in the coupling constantJ : (18.1±0.7) Hz versus (17.1±0.6) Hz (means ± SD,P<10^–5). Differences were also observed in the chemical shift separation between the - and -ATP signal: (8.498±0.023) ppm versus (8.522 ± 0.022) ppm (p<0.001) in gastrocnemius and soleus, respectively. Ahigher [MgATP]/[ATP_free] ratio and a significantly higher level of intracellular free magnesium of (0.52±0.06) mM in gastrocnemius versus (0.46 ± 0.05) mM in soleus (p<0.001) can be derived based on and K _D ^MgATP . Heterogeneity needs to be taken into account in clinical studies on magnesium by NMR methods in calf muscle. The coupling constantJ provides additional information, possibly on enzymatic processes, and correlates with [Mg _free ^2– ]. The detailed analysis of muscles with different fiber type characteristics lends support to the significance of this parameter in evaluating metabolism. The data reported can be used as prior knowledge for fits in which the coupling constants are set to a fixed value.     

Tissue oxygen tension measurements in the Shionogi model of prostate cancer using 19 F MRS and MRI

Objectives: To investigate changes in tumour tissue oxygenation throughout the tumour growth–regression–relapse cycle in an androgen-dependent animal tumour model. Materials and methods: ¹⁹F T₁ relaxometry of Perfluoro-15-Crown-5-Ether was used to measure in vivo partial oxygen pressure (pO₂) of Shionogi tumours on a 2.35-T MR scanner. Perfluoro-15-Crown-5-Ether was administered as an emulsion injected intravenously or as a neat compound injected directly into the tumour. Non-localized, tumour ¹⁹F T₁ measurements, made at multiple time points throughout the tumour cycle, were translated into pO₂ levels. Results: No correlation between tumour size and pO₂ values was found. Values of pO₂ for growing tumours (50 ± 30 torr) were significantly lower than for regressing and relapsing tumours after 9 days post-castration (70 ± 10 torr, p<0.05). Maximum pO₂ values (90 ± 30 torr) were reached between fifth and eighth day post-castration, when tumour pO₂ was significantly higher than both pre-castration (p<0.001) and after 9 days post-castration (p<0.05). Conclusion: We demonstrate that longitudinal pO₂ measurements in vivo are feasible. Values of pO₂ for growing androgen-dependent tumours were significantly lower than for regressing and relapsing androgen-independent tumours. These results have potential clinical importance in optimizing the timing of chemotherapy and/or radiotherapy of hormone dependent tumours.