Analysis of Intra- and Extracellular Levels of Purine Bases, Nucleosides, and Nucleotides in HepG2 Cells by High-performance Liquid Chromatography

Abstract

To evaluate cellular uptake and purine transport, we developed a high-performance liquid chromatography method for intra-and extracellular purine quantification. Our aim was to develop an effective method for simultaneously quantifying the substrate and metabolites with high sensitivity. C18 columns from different manufacturers were tested for simultaneous quantification of 22 different purine bases, nucleosides, and nucleotides. We used a YMC-Triart C18 column. The analysis conditions, including extraction solutions for the cells and cell culture medium, were optimized to achieve good quantification. Linearity, accuracy, determination limits, and recovery were assessed and showed good performance. The developed HPLC method was successfully applied to the qualitative analysis of 22 different intra-and extracellular purines, demonstrating that it is useful for studying the overall pattern of purine metabolism. This method could also be useful for evaluating metabolic dynamics of purines under a variety of stimulatory conditions of culture cells.

Improved Parkinsons disease motor score in a single-arm open-label trial of febuxostat and inosine

“…Additionally, measurements of Hyp and Xan in the peripheral blood were made using previously published methods. [ 39 , 43 ] Briefly, we collected peripheral blood into an EDTA-2A blood collection tube and then mixed 500 μl of blood with the same amount of ice cold 8% perchloric acid (PCA). The mixture was then immediately vortexed, after which, it was centrifuged at 12,000× g for 5 seconds at 4 C and the supernatant then stored at −80 C. Prior to analysis, we added 40 μl of 2 M K 2 CO 3 in 6 M KOH to 650 μl of the lysate.…”

<b>Clinical studies on changes in purine compounds in blood and urine by the simultaneous administration of febuxostat and inosine, or by single administration of each</b>

“…This was centrifuged at 12,000 x g for 10 minutes at 4 °C, and then 160 µL of mobile phase was added to 40 µL of the supernatant and subjected to HPLC. The conditions of HPLC are the same as described previously 14) . The amount of purine was expressed by the molar amount contained in the solution after removing PCA.…”

Xanthinuria Type 1 with a Novel Mutation in Xanthine Dehydrogenase and a Normal Endothelial Function

“…Concentrations of hypoxanthine and xanthine in plasma and urine were measured by high-pressure liquid chromatography ( 12 ). The plasma hypoxanthine level dropped to 0.8 mmol/L, which was below the normal range (1.1-3.0 mmol/L), whereas the plasma xanthine concentration increased to 2.8 mmol/L, which was above the normal range (0.7-1.2 mmol/L).…”