Hyperpolarized ¹³C Magnetic Resonance Spectroscopy Reveals the Rate-Limiting Role of the Blood-Brain Barrier in the Cerebral Uptake and Metabolism of l-Lactate in Vivo.

The dynamics of l-lactate transport across the blood-brain barrier (BBB) and its cerebral metabolism are still subject to debate. We studied lactate uptake and intracellular metabolism in the mouse brain using hyperpolarized <sup>13</sup> C magnetic resonance spectroscopy (MRS). Following the intravenous injection of hyperpolarized [1- <sup>13</sup> C]lactate, we observed that the distribution of the <sup>13</sup> C label between lactate and pyruvate, which has been shown to be representative of their pool size ratio, is different in NMRI and C57BL/6 mice, the latter exhibiting a higher level of cerebral lactate dehydrogenase A ( Ldha) expression. On the basis of this observation, and an additional set of experiments showing that the cerebral conversion of [1- <sup>13</sup> C]lactate to [1- <sup>13</sup> C]pyruvate increases after exposing the brain to ultrasound irradiation that reversibly opens the BBB, we concluded that lactate transport is rate-limited by the BBB, with a 30% increase in lactate uptake after its disruption. It was also deduced from these results that hyperpolarized <sup>13</sup> C MRS can be used to detect a variation in cerebral lactate uptake of <40 nmol in a healthy brain during an in vivo experiment lasting only 75 s, opening new opportunities to study the role of lactate in brain metabolism

Probing cardiac metabolism by hyperpolarized 13C MR using an exclusively endogenous substrate mixture and photo-induced nonpersistent radicals

Purpose To probe the cardiac metabolism of carbohydrates and short chain fatty acids simultaneously in vivo following the injection of a hyperpolarized 13C-labeled substrate mixture prepared using photo-induced nonpersistent radicals. Methods Droplets of mixed [1-13C]pyruvic and [1-13C]butyric acids were frozen into glassy beads in liquid nitrogen. Ethanol addition was investigated as a means to increase the polarization level. The beads were irradiated with ultraviolet light and the radical concentration was measured by ESR spectroscopy. Following dynamic nuclear polarization in a 7T polarizer, the beads were dissolved, and the radical-free hyperpolarized solution was rapidly transferred into an injection pump located inside a 9.4T scanner. The hyperpolarized solution was injected in healthy rats to measure cardiac metabolism in vivo. Results Ultraviolet irradiation created nonpersistent radicals in a mixture containing 13C-labeled pyruvic and butyric acids, and enabled the hyperpolarization of both substrates by dynamic nuclear polarization. Ethanol addition increased the radical concentration from 16 to 26 mM. Liquid-state 13C polarization was 3% inside the pump at the time of injection, and increased to 5% by addition of ethanol to the substrate mixture prior to ultraviolet irradiation. In the rat heart, the in vivo 13C signals from lactate, alanine, bicarbonate, and acetylcarnitine were detected following the metabolism of the injected substrate mixture. Conclusion Copolarization of two different 13C-labeled substrates and the detection of their myocardial metabolism in vivo was achieved without using persistent radicals. The absence of radicals in the solution containing the hyperpolarized 13C-substrates may simplify the translation to clinical use, as no radical filtration is required prior to injection

Noninvasive rapid detection of metabolic adaptation in activated human T lymphocytes by hyperpolarized 13 C magnetic resonance

Abstract: The metabolic shift induced in human CD4+ T lymphocytes by stimulation is characterized by an upregulation of glycolysis, leading to an augmentation in lactate production. This adaptation has already been highlighted with various techniques and reported in several previous studies. We herein propose a method to rapidly and noninvasively detect the associated increase in flux from pyruvate to lactate catalyzed by lactate dehydrogenase using hyperpolarized 13C magnetic resonance, a technique which can be used for in vivo imaging. It was shown that the conversion of hyperpolarized 13C-pyruvate to 13C-lactate during the one-minute measurement increased by a mean factor of 3.6 in T cells stimulated for 5 days as compared to resting T cells. This method can be extended to other metabolic substrates and is therefore a powerful tool to noninvasively analyze T cell metabolism, possibly in vivo

In vivo detection of d-amino acid oxidase with hyperpolarized d-[1-C-13]alanine

d-amino acid oxidase (DAO) is a peroxisomal enzyme that catalyzes the oxidative deamination of several neutral and basic d-amino acids to their corresponding alpha-keto acids. In most mammalian species studied, high DAO activity is found in the kidney, liver, brain and polymorphonuclear leukocytes, and its main function is to maintain low circulating d-amino acid levels. DAO expression and activity have been associated with acute and chronic kidney diseases and with several pathologies related to N-methyl-d-aspartate (NMDA) receptor hypo/hyper-function; however, its precise role is not completely understood. In the present study we show that DAO activity can be detected in vivo in the rat kidney using hyperpolarized d-[1-C-13]alanine. Following a bolus of hyperpolarized d-alanine, accumulation of pyruvate, lactate and bicarbonate was observed only when DAO activity was not inhibited. The measured lactate-to-d-alanine ratio was comparable to the values measured when the l-enantiomer was injected. Metabolites downstream of DAO were not observed when scanning the liver and brain. The conversion of hyperpolarized d-[1-C-13]alanine to lactate and pyruvate was detected in blood ex vivo, and lactate and bicarbonate were detected on scanning the blood pool in the heart in vivo; however, the bicarbonate-to-d-alanine ratio was significantly lower compared with the kidney. These results demonstrate that the specific metabolism of the two enantiomers of hyperpolarized [1-C-13]alanine in the kidney and in the blood can be distinguished, underscoring the potential of d-[1-C-13]alanine as a probe of d-amino acid metabolism

Detection of D-amino acid oxidase using hyperpolarized molecular probes

D-amino acid oxidase (DAO) is an enzyme that catalyzes the degradation of D-amino acids in the body. Here, we explored the possibility of detecting D-amino acid oxidase activity by monitoring its metabolism in the rat kidney after a bolus injection of hyperpolarized D-[1-13C]alanine. Our data show that D-alanine is readily converted to lactate only when the DAO enzyme is not inhibited, indicating that the observed metabolism is that of DAO

Probing perturbed hepatic metabolism in bile-duct-ligated rats with hyperpolarized 13C pyruvate and arginine

Detoxification of ammonia by the urea cycle and maintenance of glucose homeostasis by gluconeogenesis are two critical functions of the liver. The bile duct ligation (BDL) model of cirrhosis was used to test the ability of hyperpolarized [6-13C]arginine and [1-13C]pyruvate to detect changes in liver function. The conversion of hyperpolarized L-[6-13C]arginine to 13C-urea was observed in a sham-operated rat but not in BDL rats. Striking differences in pyruvate metabolism between the two groups were also noted, indicating that these probes can sense changes in hepatic mitochondrial and cytoplasmic metabolism associated with biliary cirrhosis

Hyperpolarization of 2-keto[1-13C]isocaproate for in vivo studies with photo-induced radicals

Hyperpolarized 2-keto[1-13C]isocaproate (KIC) provides a means to probe brain nitrogen homeostasis and to assess molecular signatures of tumors. The dynamic nuclear polarization process requires a free-radical polarizing agent, and samples are typically doped with persistent radicals. An alternative is to use photo-induced radicals of α-keto acids that recombine upon dissolution. [1-13C]KIC hyperpolarized with photo-induced radicals could be used to measure the alterations in amino acid metabolism that are linked to neurodegenerative diseases and cancer, and the aim of the present study is to identify the main features that influence the polarization dynami

Measurement of metabolic changes in acute doxorubicin-induced cardiotoxicity in mice using hyperpolarized [1-13C]pyruvate

Chemotherapy cocktails containing doxorubicin produce irreversible cardiotoxic side effects that may progress to heart failure, which can only be avoided through dose limitation of the chemotherapeutic agents. Increasing evidence suggest that cardiac dysfunction caused by doxorubicin is triggered by an energetic deficit and alterations in mitochondrial metabolism. We quantified metabolic changes in vivo in a mouse model of acute doxorubicin-induced cardiotoxicity using hyperpolarized 13C MRS

Effects of 3-MPA on in vivo hepatic metabolism of hyperpolarized [1-13C] pyruvate

Ex vivo and in vivo studies on liver metabolism using hyperpolarized [1-13C]pyruvate report do not agree on whether hyperpolarized bicarbonate metabolite production results from pyruvate oxidation or gluconeogenesis. This study tested the ability of hyperpolarized [1-13C]pyruvate to probe gluconeogenesis in the liver of intact rats. While conversion to hyperpolarized bicarbonate was detected in the liver of fasted rats, treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinc acid resulted in 7-fold lower levels. This result supports the notion that hepatic gluconeogenic metabolism can indeed be directly probed in vivo with hyperpolarized pyruvate