Elsevier

Nuclear Medicine and Biology

Volumes 64–65, September–October 2018, Pages 28-33
Nuclear Medicine and Biology

One-pot enzymatic synthesis of l-[3-11C]lactate for pharmacokinetic analysis of lactate metabolism in rat brain

https://doi.org/10.1016/j.nucmedbio.2018.07.001Get rights and content

Abstract

Introduction

Lactate could serve as an energy source and signaling molecule in the brain, although there is insufficient in vivo evidence to support this possibility. Here we aimed to use a one-pot enzymatic synthetic procedure to synthesize l-[3-11C]lactate that can be used to evaluate chemical forms in the blood after intravenous administration, and as a probe for pharmacokinetic analysis of lactate metabolism in in vivo positron emission tomography (PET) scans with normal and fasted rats.

Methods

Racemic [3-11C]alanine obtained from 11C-methylation of a precursor and deprotection was reacted with an enzyme mixture consisting of alanine racemase, d-amino acid oxidase, catalase, and lactate dehydrogenase to yield l-[3-11C]lactate via [3-11C]pyruvate. The optical purity was measured by HPLC. Radioactive chemical forms in the arterial blood of Sprague Dawley rats with or without insulin pretreatment were evaluated by HPLC 10 min after bolus intravenous injection of l-[3-11C]lactate. PET scans were performed on normal and fasted rats administered with l-[3-11C]lactate.

Results

l-[3-11C]Lactate was synthesized within 50 min and had decay corrected radiochemical yield, radiochemical purity, and optical purity of 13.4%, >95%, and >99%, respectively. The blood radioactivity peaked immediately after l-[3-11C]lactate injection, rapidly decreased to the minimum value within 90 s, and slowly cleared thereafter. HPLC analysis of blood samples revealed the presence of [11C]glucose (78.9%) and l-[3-11C]lactate (12.1%) 10 min after administration of l-[3-11C]lactate. Insulin pretreatment partly inhibited glyconeogenesis conversion leading to 55.4% as [11C]glucose and 38.9% as l-[3-11C]lactate simultaneously. PET analysis showed a higher SUV in the brain tissue of fasted rats relative to non-fasted rats.

Conclusions

We successfully synthesized l-[3-11C]lactate in a one-pot enzymatic synthetic procedure and showed rapid metabolic conversion of l-[3-11C]lactate to [11C]glucose in the blood. PET analysis of l-[3-11C]lactate indicated the possible presence of active lactate usage in rat brains in vivo.

Introduction

Living brain tissues carry out needed functions using ATP molecules produced via oxidative phosphorylation of d-glucose and oxygen under normal physiological conditions. In the astrocyte-neuron lactate shuttle hypothesis, glycolysis mainly occurs in astrocytes located between neurons and capillary vessels. Monocarboxylic acid transporters move lactate produced through glycolysis to neighboring neurons that use lactate as an energy source in mitochondrial oxidative phosphorylation [1,2]. However, this hypothesis remains controversial [3,4]. Recent studies also reported that lactate could be used as an energy source under certain conditions [5,6] and as a signaling molecule [7] in living brain tissue.

Nuclear medical techniques such as Positron Emission Tomography (PET) are useful for noninvasive measurement of living functions and rely on the detection of radioactivity emitted from radiolabeled tracers administered in advance of the test. For instance, PET scans with a 18F-labeled glucose derivative, 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG), are useful not only for in vivo quantitative analysis of glucose metabolism in the brain and the heart [8,9], but also for noninvasive detection of disperse tumor sites (distant metastasis), atherosclerosis, and inflammation [[10], [11], [12]]. To clarify the role of lactate in living systems by PET, a previous study reported a synthetic procedure for l-[3-11C]lactate [13]. In that report, racemic [3-11C]alanine was first synthesized using a standard 11C-methylation method with [11C]CH3I and the corresponding precursor, which was then reacted with an enzyme mixture consisting of alanine transaminase (ALT), d-amino acid oxidase (DAAO), and catalase. ALT and DAAO converted l-[3-11C]alanine and d-[3-11C]alanine, respectively, to [3-11C]pyruvate, whereas catalase eliminated hydrogen peroxide generated as a byproduct. [3-11C]Pyruvate was next reacted with lactate dehydrogenase (LDH) to selectively produce l-[3-11C]lactate.

The usefulness of l-[3-11C]lactate has only been evaluated by PET in animal models of heart disease [14]. In this study, we aimed to use a novel enzyme reaction to synthesize l-[3-11C]lactate in a one-pot synthetic procedure. We used the resulting l-[3-11C]lactate to evaluate radioactive chemical forms in the blood after tracer administration, and performed in vivo brain PET scans of normal and fasted rats.

Section snippets

Radiochemical synthesis (Scheme 1)

All reagents were obtained commercially and used without further purification. The tracer l-[3-11C]lactate was synthesized following a 2-step enzymatic reaction as previously described [13,14] except that alanine racemase was used instead of ALT [15]. An enzyme mixture was prepared by mixing 1.0 ml 0.1 M phosphate buffer (pH 8) containing 500 U rabbit muscle LDH (Oriental Yeast Co., Ltd., Tokyo, Japan), 0.017 mM flavin adenine dinucleotide (FAD, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan),

Radiochemical synthesis

Prior to the animal experiments, the synthetic procedure for l-[3-11C]lactate was tested six times and each trial provided a constant and successful synthesis result. The non-decay corrected yield of l-[3-11C]lactate was 1.4 ± 0.2 GBq at the end of the synthesis time (EOS). The radiochemical yield was 13.4 ± 2.3% with decay-correcting to the end time of cyclotron bombardment (EOB). The radiochemical yield without decay correction was 2.5 ± 0.3%. The radiochemical purity at the EOS time was

Conclusions

We successfully synthesized l-[3-11C]lactate in a one-pot enzymatic reaction with high radiochemical and optical purities. In vivo rapid conversion of l-[3-11C]lactate to [11C]glucose was clearly demonstrated by HPLC analysis, and simple PET analysis suggested uptake of l-[3-11C]lactate in brains of fasted rats.

Acknowledgments

The authors have no competing interest to declare. This work was supported in part by JSPS KAKENHI Grant Number 15K09912 and Intramural Research Fund (27-6-18) for Cardiovascular Diseases of National Cerebral and Cardiovascular Center. The funding bodies had no role in study design, data collection and analysis, decision to publish, or manuscript preparation. The authors gratefully acknowledge Mr. Takahiro Shimada, Ms. Masako Kunimi, and Mr. Masashi Kaji for their contributions to probe

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