Introduction

Aminopeptidase N (APN) is an enzyme that removes the N-terminal amino acid residue from peptides or proteins. It plays different physiological roles, and it has been recognized as a tumor biomarker¹ as it is involved in tumor angiogenesis. Because of the challenges presented by the detection of APN activity with fluorescent or luminescent probes in opaque biological samples, NMR probes are good candidates to monitor its enzymatic activity. Hata et al.², have recently demonstrated the specificity of alaninamide for APN activity in kidney homogenate. The present study aims to explore the possibility of using hyperpolarized L-[1-¹³C]alaninamide to detect APN activity in vivo in the rat kidney.

Methods

Samples of 4.4 M of L-[1-¹³C]alaninamide∙HCl doped with 25 mM of OX063 radical were polarized in a custom-built 7 T polarizer at 1 K by shining microwave radiation at 196.59 GHz, 50 mW in power. The polarization buildup was monitored for approximately 2 hours, after which the samples were rapidly dissolved in 5.5 mL of a hot buffer solution (pH = 7.4) and transferred (3 s) to a separator infusion pump located in a 9.4 T/ 31 cm animal scanner. n = 4 male Sprague Dawley rats were anaesthetized using 1-2% isoflurane. 1.2 mL of the hyperpolarized alaninamide solution was injected through the femoral vein over 9 s. The bolus concentration prior to infusion was ≈ 6.4 mM. The ¹³C signal acquisition was performed with respiration and cardiac gating. ¹H-decoupled ¹³C FIDs were acquired by applying 30° BIR4 pulses using a single loop ¹H / quadrature ¹³C surface coil placed over the left kidney of the animal. The repetition time between each scan was ≈ 3 s. Blood gases, pH and physiological parameters were monitored shortly after each infusion. For each infusion, the acquired FIDs were fitted with Bayes (Washington University, St. Louis) to determine the spectral peak amplitudes and the ratios between metabolites. The alaninamide titration curve was determined by performing ¹³C NMR measurements at 9.4 T, 37° on a set of 1 M Ala-NH₂∙HCl samples with pH ranging between 3.55 and 10.16, referenced to 15 mM ¹³C urea. Carbamate formation was monitored via ¹³C NMR (9.4 T, 37°) in a pH 8 solution of 10 mM L-[1-¹³C]alaninamide ∙HCl, 100 mM NaH¹³CO₃, and 5 mM ¹³C urea.

Results and discussion

The most striking feature of the ¹³C spectrum of alaninamide is the presence of a three-fold split alaninamide peak (Fig. 1), which arises from the 1-¹³C sensitivity to protonation and deprotonation of the amine. The alaninamide acid-base titration curve (Fig. 2) shows a ¹³C chemical shift change of ≈ 8.4 ppm and pK­_a of 7.9. The three peaks correspond to three different extracellular pH compartments within the kidney: pH = 7.46 ± 0.05, pH = 7.22 ± 0.09 and pH 6.58 ± 0.05. Based on literature^3,4, the three compartments can be tentatively assigned to the cortex/blood, medulla and calyx/ureter. Because of the overlap in chemical shift with the pH = 7.46 compartment peak, the resolution of alanine is limited and strongly depends on the pH value. A second metabolic product appears from the very first scan at 183.67 ppm: the metabolite was identified as carbamate forming from the reaction of alaninamide with CO₂ present in the blood (Fig. 3).

Conclusions

The in vivo renal metabolism of hyperpolarized L-[1-¹³C]alaninamide was studied. The probe was observed to be extremely sensitive to pH variations within the kidney. Alanine production was observed, as well as carbamate formation from the reaction of the parent compound with CO₂. Although the poor resolution of the alanine peak limits its use as APN probe in the kidney, lower-pH tissues may be better for this application.

Acknowledgements

This work was supported by the Swiss State Secretariat for Education, Research and Innovation (SERI) within the Marie Curie Initial Training Network EUROPOL project (n° SERI: 15.0164), and by the Centre d'Imagerie Biomédicale (CIBM) of the UNIL, UNIGE, HUG, CHUV, EPFL, and the Leenards and Jeantet Foundations.