Due to reported instability of the basic glutamine prep, fresh preparations were used for each experiment made from 20 mg of [5-13C,4,4-2H2,5-15N]-L-glutamine in 14 μL NaOH (10 N), then mixed with 13.0 mg of a mixture of 8.2% OX063 (Oxford Chemicals) in glycerol. The final concentration of glutamine was 4 M and OX063 was 15 mM. The sample was sonicated in ice-chilled water for 10 min and subsequently polarized with dynamic nuclear polarization in a SPINlab Polarizer (General Electric) for over 2 h (5.0 T, 0.80 K, 139.995 GHz). Following polarization, the HP substrate was removed from the polarizer via rapid dissolution, during which the hyperpolarized sample was dissolved in a superheated aqueous solution of 200 mM phosphate buffer in H2O or D2O, 0.1 mM EDTA, pH 2.5, and collected into a vial at room temperature and directly used for further experiments.
Spinlab polarizer
Manufactured by GE Healthcare
Sourced in United States
The SPINlab polarizer is a specialized piece of lab equipment used to polarize samples for magnetic resonance (MR) imaging and spectroscopy applications. It uses a strong magnetic field to align the nuclear spin of samples, which is a critical step in the MR process. The SPINlab polarizer provides a consistent and reliable way to prepare samples for MR analysis, but its specific intended use and performance characteristics are not included in this factual description.
Automatically generated - may contain errors
Lab products found in correlation
Research fluid path, by GE Healthcare (2 mentions)
Ox063, by GE Healthcare (1 mentions)
Trityl radical, by GE Healthcare (1 mentions)
Gmp grade 14 m 1 13c pyruvic acid, by Merck Group (1 mentions)
Ah111501, by GE Healthcare (1 mentions)
1 13c pyruvic acid, by Merck Group (1 mentions)
Trizma hydrochloride solution, by Merck Group (1 mentions)
Ox063 radical, by GE Healthcare (1 mentions)
10 protocols using spinlab polarizer
1
Hyperpolarized Glutamine Preparation
2
Hyperpolarized [1-13C]-L-Lactate Production
3
Hyperpolarized [2-13C]-Pyruvate Imaging
4
Hyperpolarized Pyruvate MRS in Pigs
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The pigs were fed and received an oral glucose bolus (2 g/kg) 3.5 hours and 45 minutes before MRS, respectively. MRI and hyperpolarized [1-13C]pyruvate MRS were carried out at rest and under dobutamine-stress (10 µg/kg/min IV), which is used for mimicking exercise in the clinic36 (link). All examinations were carried out on a commercial 3 T MRI system (GE Healthcare). The [1-13C]pyruvate was hyperpolarized in a SPINLab polarizer (GE Healthcare)37 (link). At the start of pyruvate injection, 128 spectra were acquired. After post-processing38 (link), pyruvate to metabolite conversion was quantified using the area under the curve approach39 (link). Myocardial pH was estimated from the bicarbonate and lactate signals40 (link). Conventional MRI was analysed with segmentation and volume measures were normalised to body weight41 (link). The MRS stress data of one RVO animal and the stress data of one control animal were excluded due to equipment failure.
5
Hyperpolarized Pyruvate Production
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A SPINlab™ polarizer (GE Healthcare) that operates at ~0.8 K in a 5-T magnet was used for hyperpolarization. Two samples of pyruvic acid were assembled into clinical fluid paths (GE Healthcare) under a sterile environment and simultaneously polarized for each participant. Each pyruvate sample was prepared by mixing 1.47 g of GMP-grade 14-M [1-13C]pyruvic acid (Sigma Aldrich) with 27.7 mg of AH111501 radical (Syncom). The pyruvate sample was polarized for 3–4 hours before dissolving with 38 mL of sterile water at 130°C. The hyperpolarized pyruvate solution was immediately mixed with 36.5 mL of buffer media that contains 333-mM TRIS and 600-mM NaOH, resulting in 250-mM of pyruvate concentration, then examined by a dedicated quality control device (GE Healthcare) prior to the injection. In addition, terminal filtering, bubble point test, pH strip confirmation, and volume check were performed after the automated quality control. Hyperpolarized pyruvate (0.1 mmol/kg body weight) was intravenously administered with an injection rate of 5 mL/s, followed by a 25-mL saline flush.
6
Hyperpolarized [1-13C]KIC Preparation
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[1-13C] α-ketoisocaproic acid (KIC) was prepared from [1-13C] α-ketoisocaproic acid sodium salt (Cambridge Isotope Laboratories, Andover, MA, USA) by dissolving in 1-M HCl (pH < 1) and extracting the aqueous layer with diethyl ether. The combined organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to afford [1-13C]KIC (7 M, colorless oil, purity >95%, 94% yield)25 (link). For hyperpolarization, a 70-μL sample of the [1-13C]KIC mixed with 11-mM trityl radical (OX063, Oxford Instruments Molecular Biotools Ltd, Oxfordshire, UK) was placed in a sample vial, which was then assembled with a research fluid path (GE Healthcare, Waukesha, WI, USA) containing 16 mL of dissolution media (0.1 g/L Na2EDTA) in the dissolution syringe. After placed in a SPINlab® polarizer (GE Healthcare), the sample was polarized at ~0.8 K of temperature in a 5 T magnet by irradiating microwaves at a frequency of 139.93 GHz. The polarized sample (3 - 4 hrs) was dissolved and mixed with neutralization media (0.72 M NaOH, 0.4 M Trizma and 0.1 g/L Na2EDTA), resulting in 6.5–7.0 mL of 80-mM [1-13C]KIC solution (pH 7.4–8.2).
7
Polarized [1-13C]Pyruvate Preparation
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We used a SPINlab polarizer (GE) to polarize [1-13C]pyruvate. The preparation steps for polarization are as follows: (i) The 13C-enriched pyruvate sample was prepared with a stable organic radical: 15 mM AH-111501 (GE) was mixed in [1-13C]pyruvic acid (Sigma-Aldrich, 677175) thoroughly. (ii) Buffer solution for dissolution was prepared: 0.4 mM EDTA was added in 40 mM Trizma hydrochloride solution (Sigma-Aldrich, T2663). (iii) The pyruvate sample (100 μl) from step (i) and 20 ml of the buffer solution from step (ii) were loaded into the SPINlab polarizer (3.35 T, 0.98 K). After 90 min of polarization, the pyruvate sample was quickly dissolved into an ice-cold flask with 120 μl of 10 N sodium hydroxide solution (Fisher Scientific, SS255) to make the dissolved sample neutral (pH ~ 7.4) and reach 37°C faster. The dissolved pyruvate sample was added into the cell suspension with a ratio of 1:10, which made the concentration of pyruvate 14 mM.
8
Polarization Efficiency Optimization
9
Hyperpolarized 13C Urea Solution Preparation
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A 6.4 M 13C urea glycerol solution was prepared and doped with 18.5 mM AH111501 trityl radical. 1 mL samples were polarized for 4 h in a commercial 5 T SpinLab Polarizer (General Electric Healthcare, Waukesha, Wisconsin, USA) before dissolution using a buffer consisting of 30 mL 0.1% EDTA dissolved in water. Upon sample collection, the prepared solution was further diluted and cooled down with additional 4.5 mL of buffer at 0 °C to achieve body temperature for injection. 20 mL of the final 200 mM 13C urea solution were transferred to the scanner using an electromagnetic carrier device [34 (link)] built in-house, ensuring a magnetic field >7 mT over the whole syringe volume and transport period (15–20 s).
Polarization levels were established in separate experiments and amounted to 48 ± 5% at time of dissolution and 29 ± 3% at time of injection. T1 relaxation times inside the 3 T magnet were measured for the neat solutions (58 ± 1 s, n = 5) and after dilution in porcine heparinized venous blood samples within 15 min after sampling. Two urea concentrations (n = 5, each) were measured: 10% and 33%, with an observed T1 of 25 ± 2 s and 34 ± 0.5 s, respectively.
Polarization levels were established in separate experiments and amounted to 48 ± 5% at time of dissolution and 29 ± 3% at time of injection. T1 relaxation times inside the 3 T magnet were measured for the neat solutions (58 ± 1 s, n = 5) and after dilution in porcine heparinized venous blood samples within 15 min after sampling. Two urea concentrations (n = 5, each) were measured: 10% and 33%, with an observed T1 of 25 ± 2 s and 34 ± 0.5 s, respectively.
10
Optimizing Hyperpolarized Sample Preparation
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The co-polarization was made in a SPINlab polarizer (GE Healthcare, Denmark). Figure 1-a shows the fluid path assembly designed for use with the SPINlab. The fluid path consists of a vial (1) containing the sample and connected to the dissolution syringe (2) via two concentric tubes (3). The tubes can be pushed into the polarizer through the dynamic seal (4) without compromising the vacuum. The syringe has an exit port (5) that connects to a tube for transfer of the hyperpolarized solution.
The regular sample preparation procedure for the SPINlab fluid path requires that the vial, after adding the 13 C substrate, is frozen in liquid nitrogen (LN2). This freezing is necessary to perform pressure check and helium purging. Helium purging is needed to have a helium exchange gas inside the fluid path when the vial is cooled to <1 K.
The regular procedure to load the sample into the SPINlab involves lowering the sample vial into the sample pot (see Figure 1-c), were the hyperpolarization take place, through multiple steps (gradual insertion over 15 min) to avoid excessive increase in the helium temperature. This multi-steps lowering process was found to result in FA crystallization.
Therefore, to avoid the FA crystallization, the sample preparation and loading was modified as detailed in the next section.
The regular sample preparation procedure for the SPINlab fluid path requires that the vial, after adding the 13 C substrate, is frozen in liquid nitrogen (LN2). This freezing is necessary to perform pressure check and helium purging. Helium purging is needed to have a helium exchange gas inside the fluid path when the vial is cooled to <1 K.
The regular procedure to load the sample into the SPINlab involves lowering the sample vial into the sample pot (see Figure 1-c), were the hyperpolarization take place, through multiple steps (gradual insertion over 15 min) to avoid excessive increase in the helium temperature. This multi-steps lowering process was found to result in FA crystallization.
Therefore, to avoid the FA crystallization, the sample preparation and loading was modified as detailed in the next section.
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