Measurement of the acute metabolic response to hypoxia in rat tumours in vivo using magnetic resonance spectroscopy and hyperpolarised pyruvate.

PURPOSE: To estimate the rate constant for pyruvate to lactate conversion in tumours in response to a hypoxic challenge, using hyperpolarised (13)C1-pyruvate and magnetic resonance spectroscopy. METHODS AND MATERIALS: Hypoxic inspired gas was used to manipulate rat P22 fibrosarcoma oxygen tension (pO2), confirmed by luminescence decay of oxygen-sensitive probes. Hyperpolarised (13)C1-pyruvate was injected into the femoral vein of anaesthetised rats and slice-localised (13)C magnetic resonance (MR) spectra acquired. Spectral integral versus time curves for pyruvate and lactate were fitted to a precursor-product model to estimate the rate constant for tumour conversion of pyruvate to lactate (kpl). Mean arterial blood pressure (MABP) and oxygen tension (ArtpO2) were monitored. Pyruvate and lactate concentrations were measured in freeze-clamped tumours. RESULTS: MABP, ArtpO2 and tumour pO2 decreased significantly during hypoxia. kpl increased significantly (p<0.01) from 0.029±0.002s(-1) to 0.049±0.006s(-1) (mean±SEM) when animals breathing air were switched to hypoxic conditions, whereas pyruvate and lactate concentrations were minimally affected by hypoxia. Both ArtpO2 and MABP influenced the estimate of kpl, with a strong negative correlation between kpl and the product of ArtpO2 and MABP under hypoxia. CONCLUSION: The rate constant for pyruvate to lactate conversion, kpl, responds significantly to a rapid reduction in tumour oxygenation.

NMR-based evaluation of the metabolic profile and response to dichloroacetate of human prostate cancer cells.

The aim of this study was to evaluate the metabolic profile of human prostate cancer cells that have different metastatic potential and to determine their response to dichloroacetate (DCA) using NMR technology. Two isogenic human prostate cancer cell lines, differing in their metastatic potential [LNCaP (poorly metastatic) and LNCaP-LN3 (highly metastatic)], were studied. Metabolite ratios from NMR spectral integrals acquired at a field strength of 9.4 T using a 5-mm broadband probe with an NMR-compatible bioreactor were compared in the presence and absence of the pyruvate dehydrogenase kinase inhibitor DCA. Lactate dehydrogenase (LDH) isoenzymes were assessed by zymography. Following the treatment of cells with 50 mm DCA, there was a significant reduction in the lactate/choline, lactate/creatine, lactate/alanine and the combined lactate/(choline + creatine + alanine) ratios in LNCaP-LN3 cells relative to LNCaP cells. No significant changes in metabolite ratios were found in LNCaP cells following DCA treatment. As expected, LDH zymography assays showed an absence of the LDH-B subunit in LNCaP-LN3 cells, whereas both LDH-A and LDH-B subunits were present in LNCaP cells. DCA was shown to significantly modify the metabolite ratios in highly metastatic LNCaP-LN3 cells, but not in poorly metastatic LNCaP cells. This effect was probably related to the absence of the LDH-B subunit in LNCaP-LN3 cells, and could have a bearing on cancer treatment with DCA and related compounds.

A system for accurate and automated injection of hyperpolarized substrate with minimal dead time and scalable volumes over a large range.

Over recent years hyperpolarization by dissolution dynamic nuclear polarization has become an established technique for studying metabolism in vivo in animal models. Temporal signal plots obtained from the injected metabolite and daughter products, e.g. pyruvate and lactate, can be fitted to compartmental models to estimate kinetic rate constants. Modeling and physiological parameter estimation can be made more robust by consistent and reproducible injections through automation. An injection system previously developed by us was limited in the injectable volume to between 0.6 and 2.4ml and injection was delayed due to a required syringe filling step. An improved MR-compatible injector system has been developed that measures the pH of injected substrate, uses flow control to reduce dead volume within the injection cannula and can be operated over a larger volume range. The delay time to injection has been minimized by removing the syringe filling step by use of a peristaltic pump. For 100µl to 10.000ml, the volume range typically used for mice to rabbits, the average delivered volume was 97.8% of the demand volume. The standard deviation of delivered volumes was 7µl for 100µl and 20µl for 10.000ml demand volumes (mean S.D. was 9 ul in this range). In three repeat injections through a fixed 0.96mm O.D. tube the coefficient of variation for the area under the curve was 2%. For in vivo injections of hyperpolarized pyruvate in tumor-bearing rats, signal was first detected in the input femoral vein cannula at 3-4s post-injection trigger signal and at 9-12s in tumor tissue. The pH of the injected pyruvate was 7.1±0.3 (mean±S.D., n=10). For small injection volumes, e.g. less than 100µl, the internal diameter of the tubing contained within the peristaltic pump could be reduced to improve accuracy. Larger injection volumes are limited only by the size of the receiving vessel connected to the pump.

Quantification method using the Morlet wavelet for magnetic resonance spectroscopic signals with macromolecular contamination.

We study the Morlet wavelet transform on characterizing Magnetic Resonance Spectroscopic (MRS) signals acquired at short echo-time. These signals contain contributions from metabolites, water and a baseline which mainly originates from large molecules, known as macromolecules, and lipids. The baseline signal decays faster than the metabolite ones. Therefore, by making use of the time-scale representation of the wavelet, the two signals can be distinguished without any additional pre-processing. This is confirmed by the experimental results which show that the Morlet wavelet can correctly quantify the metabolite contributions even when a baseline is embedded in the MRS signals.