Kayvan R. Keshari

Quantitative analysis of prostate metabolites using 1H HR-MAS spectroscopy

A method was developed to quantify prostate metabolite concentrations using 1H high‐resolution magic angle spinning (HR‐MAS) spectroscopy. T1 and T2 relaxation times (in milliseconds) were determined for the major prostate metabolites and an internal TSP standard, and used to optimize the acquisition and repetition times (TRs) at 11.7 T. At 1°C, polyamines (PAs; T1mean = 100 ± 13, T2mean = 30.8 ± 7.4) and citrate (Cit; T1mean = 237 ± 39, T2mean = 68.1 ± 8.2) demonstrated the shortest relaxation times, while taurine (Tau; T1mean = 636 ± 78, T2mean = 331 ± 71) and choline (Cho; T1mean = 608 ± 60, T2mean = 393 ± 81) demonstrated the longest relaxation times. Millimolal metabolite concentrations were calculated for 60 postsurgical tissues using metabolite and TSP peak areas, and the mass of tissue and TSP. Phosphocholine plus glycerophosphocholine (PC+GPC), total choline (tCho), lactate (Lac), and alanine (Ala) concentrations were higher in prostate cancer ([PC+GPC]mean = 9.34 ± 6.43, [tCho]mean = 13.8 ± 7.4, [Lac]mean = 69.8 ± 27.1, [Ala]mean = 12.6 ± 6.8) than in healthy glandular ([PC+GPC]mean = 3.55 ± 1.53, P < 0.01; [tCho]mean = 7.06 ± 2.36, P < 0.01; [Lac]mean = 46.5 ± 17.4, P < 0.01; [Ala]mean = 8.63 ± 4.91, P = 0.051) and healthy stromal tissues ([PC+GPC]mean = 4.34 ± 2.46, P < 0.01; [tCho]mean = 7.04 ± 3.10, P < 0.01; [Lac]mean = 45.1 ± 18.6, P < 0.01; [Ala]mean = 6.80 ± 2.95, P < 0.01), while Cit and PA concentrations were significantly higher in healthy glandular tissues ([Cit]mean = 43.1 ± 21.2, [PAs]mean = 18.5 ± 15.6) than in healthy stromal ([Cit]mean = 16.1 ± 5.6, P < 0.01; [PAs]mean = 3.15 ± 1.81, P < 0.01) and prostate cancer tissues ([Cit]mean = 19.6 ± 12.7, P < 0.01; [PAs]mean = 5.28 ± 5.44, P < 0.01). Serial spectra acquired over 12 hr indicated that the degradation of Cho‐containing metabolites was minimized by acquiring HR‐MAS data at 1°C compared to 20°C. Magn Reson Med, 2006.

Evaluation of lactate and alanine as metabolic biomarkers of prostate cancer using 1H HR-MAS spectroscopy of biopsy tissues

The goal of this study was to investigate the use of lactate and alanine as metabolic biomarkers of prostate cancer using 1H high‐resolution magic angle spinning (HR‐MAS) spectroscopy of snap‐frozen transrectal ultrasound (TRUS)‐guided prostate biopsy tissues. A long‐echo‐time rotor‐synchronized Carr‐Purcell‐Meiboom‐Gill (CPMG) sequence including an electronic reference to access in vivo concentrations (ERETIC) standard was used to determine the concentrations of lactate and alanine in 82 benign and 16 malignant biopsies (mean 26.5% ± 17.2% of core). Low concentrations of lactate (0.61 ± 0.28 mmol/kg) and alanine (0.14 ± 0.06 mmol/kg) were observed in benign prostate biopsies, and there was no significant difference between benign predominantly glandular (N = 54) and stromal (N = 28) biopsies between patients with (N = 38) and without (N = 44) a positive clinical biopsy. In biopsies containing prostate cancer there was a highly significant (P < 0.0001) increase in lactate (1.59 ± 0.61 mmol/kg) and alanine (0.26 ± 0.07 mmol/kg), and minimal overlap with lactate concentrations in benign biopsies. This study demonstrates for the first time very low concentrations of lactate and alanine in benign prostate biopsy tissues. The significant increase in the concentration of both lactate and alanine in biopsy tissue containing as little as 5% cancer could be exploited in hyperpolarized 13C spectroscopic imaging (SI) studies of prostate cancer patients. Magn Reson Med 60:510–516, 2008.

Multi-compound Polarization by DNP Allows Simultaneous Assessment of Multiple Enzymatic Activities In Vivo

Methods for the simultaneous polarization of multiple 13C-enriched metabolites were developed to probe several enzymatic pathways and other physiologic properties in vivo, using a single intravenous bolus. A new method for polarization of 13C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13C sodium bicarbonate and [1-(13)C] pyruvate in the same sample was achieved, resulting in high solution-state polarizations (15.7% and 17.6%, respectively) and long spin-lattice relaxation times (T1) (46.7 s and 47.7 s respectively at 3 T). Consistent with chemical shift anisotropy dominating the T1 relaxation of carbonyls, T1 values for 13C bicarbonate and [1-(13)C] pyruvate were even longer at 3 T (49.7s and 67.3s, respectively). Co-polarized 13C bicarbonate and [1-(13)C] pyruvate were injected into normal mice and a murine prostate tumor model at 3T. Rapid equilibration of injected hyperpolarized 13C sodium bicarbonate with 13C CO2 allowed calculation of pH on a voxel by voxel basis, and simultaneous assessment of pyruvate metabolism with cellular uptake and conversion of [1-(13)C] pyruvate to its metabolic products. Initial studies in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model demonstrated higher levels of hyperpolarized lactate and lower pH within tumor, relative to surrounding benign tissues and to the abdominal viscera of normal controls. There was no significant difference observed in the tumor lactate/pyruvate ratio obtained after the injection of co-polarized 13C bicarbonate and [1-(13)C] pyruvate or polarized [1-(13)C] pyruvate alone. The technique was extended to polarize four 13C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1-(13)C]pyruvic acid, 13C sodium bicarbonate, [1,4-(13)C]fumaric acid, and 13C urea with high levels of solution polarization (17.5%, 10.3%, 15.6% and 11.6%, respectively) and spin-lattice relaxation values similar to those recorded for the individual metabolites. These studies demonstrated the feasibility of simultaneously measuring in vivo pH and tumor metabolism using nontoxic, endogenous species, and the potential to extend the multi-polarization approach to include up to four hyperpolarized probes providing multiple metabolic and physiologic measures in a single MR acquisition.

Hyperpolarized 13C dehydroascorbate as an endogenous redox sensor for in vivo metabolic imaging

Reduction and oxidation (redox) chemistry is involved in both normal and abnormal cellular function, in processes as diverse as circadian rhythms and neurotransmission. Intracellular redox is maintained by coupled reactions involving NADPH, glutathione (GSH), and vitamin C, as well as their corresponding oxidized counterparts. In addition to functioning as enzyme cofactors, these reducing agents have a critical role in dealing with reactive oxygen species (ROS), the toxic products of oxidative metabolism seen as culprits in aging, neurodegenerative disease, and ischemia/ reperfusion injury. Despite this strong relationship between redox and human disease, methods to interrogate a redox pair in vivo are limited. Here we report the development of [1-13C] dehydroascorbate [DHA], the oxidized form of Vitamin C, as an endogenous redox sensor for in vivo imaging using hyperpolarized 13C spectroscopy. In murine models, hyperpolarized [1-13C] DHA was rapidly converted to [1-13C] vitamin C within the liver, kidneys, and brain, as well as within tumor in a transgenic prostate cancer mouse. This result is consistent with what has been previously described for the DHA/Vitamin C redox pair, and points to a role for hyperpolarized [1-13C] DHA in characterizing the concentrations of key intracellular reducing agents, including GSH. More broadly, these findings suggest a prognostic role for this new redox sensor in determining vulnerability of both normal and abnormal tissues to ROS.

OCT1 is a high-capacity thiamine transporter that regulates hepatic steatosis and is a target of metformin

Significance This manuscript describes a previously unidentified mechanism for organic cation transporter 1 (OCT1), the major hepatic metformin transporter, in hepatic steatosis. Here we show that OCT1, long thought to function primarily as a transporter for drugs, functions as a major thiamine transporter in the liver, which has profound implications in cellular metabolism. Collectively, our results point to an important role of thiamine (through OCT1) in hepatic steatosis and suggest that the modulation of thiamine disposition by metformin may contribute to its pharmacologic effects. Organic cation transporter 1, OCT1 (SLC22A1), is the major hepatic uptake transporter for metformin, the most prescribed antidiabetic drug. However, its endogenous role is poorly understood. Here we show that similar to metformin treatment, loss of Oct1 caused an increase in the ratio of AMP to ATP, activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride (TG) levels in livers from healthy and leptin-deficient mice. Conversely, livers of human OCT1 transgenic mice fed high-fat diets were enlarged with high TG levels. Metabolomic and isotopic uptake methods identified thiamine as a principal endogenous substrate of OCT1. Thiamine deficiency enhanced the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase. Metformin and the biguanide analog, phenformin, competitively inhibited OCT1-mediated thiamine uptake. Acute administration of metformin to wild-type mice reduced intestinal accumulation of thiamine. These findings suggest that OCT1 plays a role in hepatic steatosis through modulation of energy status. The studies implicate OCT1 as well as metformin in thiamine disposition, suggesting an intriguing and parallel mechanism for metformin and its major hepatic transporter in metabolic function.

Quantification of choline- and ethanolamine-containing metabolites in human prostate tissues using 1H HR-MAS total correlation spectroscopy.

A fast and quantitative 2D high‐resolution magic angle spinning (HR‐MAS) total correlation spectroscopy (TOCSY) experiment was developed to resolve and quantify the choline‐ and ethanolamine‐containing metabolites in human prostate tissues in ≈1 hr prior to pathologic analysis. At a 40‐ms mixing time, magnetization transfer efficiency constants were empirically determined in solution and used to calculate metabolite concentrations in tissue. Phosphocholine (PC) was observed in 11/15 (73%) cancer tissues but only 6/32 (19%) benign tissues. PC was significantly higher (0.39 ± 0.40 mmol/kg vs. 0.02 ± 0.07 mmol/kg, z = 3.5), while ethanolamine (Eth) was significantly lower in cancer versus benign prostate tissues (1.0 ± 0.8 mmol/kg vs. 2.3 ± 1.9 mmol/kg, z = 3.3). Glycerophosphocholine (GPC) (0.57 ± 0.87 mmol/kg vs. 0.29 ± 0.26 mmol/kg, z = 1.2), phosphoethanolamine (PE) (4.4 ± 2.2 mmol/kg vs. 3.4 ± 2.6 mmol/kg, z = 1.4), and glycerophosphoethanolamine (GPE) (0.54 ± 0.82 mmol/kg vs. 0.15 ± 0.15 mmol/kg, z = 1.8) were higher in cancer versus benign prostate tissues. The ratios of PC/GPC (3.5 ± 4.5 vs. 0.32 ± 1.4, z = 2.6), PC/PE (0.08 ± 0.08 vs. 0.01 ± 0.03, z = 3.5), PE/Eth (16 ± 22 vs. 2.2 ± 2.0, z = 2.4), and GPE/Eth (0.41 ± 0.51 vs. 0.06 ± 0.06, z = 2.6) were also significantly higher in cancer versus benign tissues. All samples were pathologically interpretable following HR‐MAS analysis; however, degradation experiments showed that PC, GPC, PE, and GPE decreased 7.7 ± 2.2%, while Cho+mI and Eth increased 18% in 1 hr at 1°C and a 2250 Hz spin rate. Magn Reson Med 60:33–40, 2008.

Hyperpolarized [2-13C]-fructose: a hemiketal DNP substrate for in vivo metabolic imaging.

Hyperpolarized (13)C labeled molecular probes have been used to investigate metabolic pathways of interest as well as facilitate in vivo spectroscopic imaging by taking advantage of the dramatic signal enhancement provided by DNP. Due to the limited lifetime of the hyperpolarized nucleus, with signal decay dependent on T(1) relaxation, carboxylate carbons have been the primary targets for development of hyperpolarized metabolic probes. The use of these carbon nuclei makes it difficult to investigate upstream glycolytic processes, which have been related to both cancer metabolism as well as other metabolic abnormalities, such as fatty liver disease and diabetes. Glucose carbons have very short T(1)s (<1 s) and therefore cannot be used as an in vivo hyperpolarized metabolic probe of glycolysis. However, the pentose analogue fructose can also enter glycolysis through its phosphorylation by hexokinase and yield complementary information. The C(2) of fructose is a hemiketal that has a relatively longer relaxation time (approximately 16 s at 37 degrees C) and high solution state polarization (approximately 12%). Hyperpolarized [2-(13)C]-fructose was also injected into a transgenic model of prostate cancer (TRAMP) and demonstrated difference in uptake and metabolism in regions of tumor relative to surrounding tissue. Thus, this study demonstrates the first hyperpolarization of a carbohydrate carbon with a sufficient T(1) and solution state polarization for ex vivo spectroscopy and in vivo spectroscopic imaging studies.

A hydrogen peroxide-responsive hyperpolarized 13C MRI contrast agent.

We report a new reaction-based approach for the detection of hydrogen peroxide (H(2)O(2)) using hyperpolarized (13)C magnetic resonance imaging ((13)C MRI) and the H(2)O(2)-mediated oxidation of α-ketoacids to carboxylic acids. (13)C-Benzoylformic acid reacts selectively with H(2)O(2) over other reactive oxygen species to generate (13)C-benzoic acid and can be hyperpolarized using dynamic nuclear polarization, providing a method for dual-frequency detection of H(2)O(2). Phantom images collected using frequency-specific imaging sequences demonstrate the efficacy of this responsive contrast agent to monitor H(2)O(2) at pre-clinical field strengths. The combination of reaction-based detection chemistry and hyperpolarized (13)C MRI provides a potentially powerful new methodology for non-invasive multi-analyte imaging in living systems.

Recent Advances in the Molecular Imaging of Programmed Cell Death: Part II—Non–Probe-Based MRI, Ultrasound, and Optical Clinical Imaging Techniques

There is much that can be done to detect apoptosis and other forms of cell death with existing clinical modalities including ultrasound, MRI, and optical imaging without the need for current or new intravenous contrast agents. We will discuss how these widely available imaging technologies can readily be applied to the imaging of apoptosis in patients undergoing chemotherapy or radiation treatment. The limiting factor of course is the lack of knowledge of the optimal times after the start of treatment for the most accurate assessment of apoptosis and necrosis with each modality and specific technique. It is hoped that imaging studies that systematically look at treatment response can soon be performed to address these issues.

Imaging of blood flow using hyperpolarized [13C]Urea in preclinical cancer models

To demonstrate dynamic imaging of a diffusible perfusion tracer, hyperpolarized [13C]urea, for regional measurement of blood flow in preclinical cancer models.