A low-cost mass spectrometry-based approach for quantifying purines in placental explants

Abstract

Purine nucleotides serve as immediate building blocks for the synthesis of RNAs and DNA, energy carriers and co-substrates in the activation of many metabolites, regulators in cellular metabolism, and signaling molecules. Knowledge of the actual tissue-specific concentration of these metabolites and their synthesis rate is essential to studies focused on a tissue-specific metabolic rewiring in the pathogenesis of different diseases, including carcinogenesis. The existing data mainly characterize the purine concentration in body fluids and malignant cells. The protocols for estimating de novo purine biosynthesis usually required the usage of radioactive precursors and costly facilities like HPLC with tandem mass spectrometry or nuclear magnetic resonance. We developed a new simplified method to measure the level of purines by HPLC/ESI-MS, using a stable isotope glycine to label and measure newly synthesized purines. The developed method proved to be highly sensitive, interday repeatable, and intraday reproducible. We applied the method to measure purine concentration and the rate of purine synthesis in placental explants from the first and third trimesters of gestation and MCF7 cells cultivated with 20 μM and 40 μM homocysteine. Corresponding homocysteine concentration is typical for breast cancer and pregnancy-associated complications affecting the human placenta. The content of total purines in placental explants from the first trimester of gestation was around 9.0 μmol/g wet tissues at specified cultivation conditions. The newly formed purines comprised approximately 1% of total purines and decreased steadily in explants cultivated with 20 mM and 40 μM homocysteine. They reached the values characteristic for explants from the third trimester grown without homocysteine, 4.0 μmol/g wet tissues. In MCF7 cells, the newly formed purines comprised around 50% of total purines and decreased under homocysteine treatment. A low-cost mass spectrometry-based approach may be used to estimate the level and the rate of purine synthesis in eukaryotes’ tissues in a wide range of purines concentrations.