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

Abstract

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 -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-[1-13 C]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-[1-13 C]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 [1-13 C]alanine in the kidney and in the blood can be distinguished, underscoring the potential of D-[1-13 C]alanine as a probe of D-amino acid metabolism. INTRODUCTIONWhile being less abundant than L-amino acids, D-amino acids are widespread in nature and take part in normal mammalian metabolism. Aside from D-serine and D-aspartate, which humans can synthesize endogenously, most D-amino acids are taken up through the diet, and their levels are regulated by D-amino acid oxidase (DAO) and D-aspartate oxidase. 1 DAO, a peroxisomal flavoenzyme, catalyzes the conversion of basic and neutral D-amino acids into the corresponding -keto acids in two steps. First, the D-amino acid is oxidized to an imino acid, and the flavin adenine dinucleotide coenzyme is concomitantly reduced. The imino acid then undergoes non-enzymatic hydrolysis to yield the -keto acid and ammonia. In mammals, DAO is abundantly present in the kidneys, where it is expressed in the epithelial cells of the S2 and S3 segments of the proximal tubules. Although it is also expressed in hepatocytes, in the central nervous system (CNS) and in polymorphonuclear (PMN) leukocytes, the physiological and pathological role of D-amino acids and DAO in these tissues is not completely understood. DAO has been shown to play a detoxifying role in the kidneys, as it helps maintain low D-amino acid levels. 2 Since it controls D-serine levels, DAO activity in the CNS has been associated with conditions affecting N-methyl-D-aspartate (NMDA) receptor signaling/dysfunction, including schizophrenia, 3 amyotrophic lateral sclerosis ...

Peptidoglycan-Targeted [18F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection

“…This process is remarkably efficient, as it converts hyperpolarized D-[1-13 C]-alanine to [1-13 C]-pyruvate in mice within seconds. 25 Following the conversion of the D-[3-11 C]-alanine PET tracer to [3-11 C]-pyruvate, numerous biomolecules in glycolysis, the TCA cycle, and fatty acid biosynthesis would be subsequently labeled (Figure 1C). These considerations motivated the design of a D-amino acid-derived PET tracer that (1) was fluorine-18 labeled, (2) had high structural homology to D-alanine, (3) was stable in vivo, and (4) was incorporated into bacterial peptidoglycan but was not a substrate for mammalian DAAO.…”

“…These background signals were likely due to d -amino acid oxidase (DAAO), an FAD-dependent flavoenzyme that catalyzes the oxidation of d -alanine to pyruvate. This process is remarkably efficient, as it converts hyperpolarized d -[1- 13 C]-alanine to [1- 13 C]-pyruvate in mice within seconds . Following the conversion of the d -[3- 11 C]-alanine PET tracer to [3- 11 C]-pyruvate, numerous biomolecules in glycolysis, the TCA cycle, and fatty acid biosynthesis would be subsequently labeled (Figure C).…”