LC–MS assay for quantifying ornithine decarboxylase activity in the biological matrix and cultured cells using stable isotope‑labeled ornithine

Abstract

Polyamines (putrescine, spermidine, and spermine) are essential for animal health and development, and their intracellular levels must be tightly regulated to maintain normal cellular functions. Ornithine decarboxylase (ODC) catalyzes the rate-limiting decarboxylation step in polyamine biosynthesis, and thus accurate assessment of its activity is vital for studies of polyamine metabolism. However, conventional ODC assays rely on radiolabeled substrates and require specialized facilities for radioactive handling. To overcome these limitations, we developed a sensitive and non-radioactive ODC assay using stable isotope-labeled ornithine in combination with liquid chromatography–mass spectrometry (LC–MS). In this protocol, animal tissues (e.g., 0.5 g) or cultured cells (e.g., 2.0 × 10⁶ cells) are homogenized, and cytosolic fractions are prepared by centrifugation. These fractions are incubated with d₇-ornithine under 37 °C conditions, and the produced d₇-putrescine is derivatized with dansyl chloride and quantified by LC–MS analysis. Reaction samples showed a distinct peak corresponding to d₇-putrescine, whereas negative control displayed negligible signals. Moreover, d₇-spermidine and d₇-spermine were not detectable under these conditions, indicating d₇-putrescine production directly reflects ODC activity. We optimized reaction time and substrate concentrations to ensure linearity and precision, and confirmed that the assay responds appropriately to pharmacological inhibition of ODC. Collectively, this protocol provides a practical, sensitive, and non-radioactive method for quantifying ODC activity in both animal tissues and cultured cells, and it offers an accessible tool for polyamine metabolism research.