A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Baseline levels of adenine nucleotides (AMP, ADP, ATP) in primary cell cultures are typically in the low picomolar range.
• Conventional High-Performance Liquid Chromatography (HPLC) methods struggle to accurately quantify these low concentrations due to sensitivity limitations, often requiring sample pooling or indirect detection.

Purpose of the Study:

• To develop a highly sensitive and selective ion-pairing HPLC method with fluorescence detection for quantifying adenine nucleotides.
• To measure adenine nucleotides and adenylate energy charge in mono-layered primary astrocyte cell cultures.

Main Methods:

• Adenine nucleotides were derivatized to their 1, N(6)-etheno derivatives using chloroacetaldehyde.
• Optimized fluorescence derivatization conditions (pH 4.5, 60°C for 60 min) and HPLC parameters, including a 0.8mM tetrabutylammonium phosphate ion-pairing reagent concentration.
• Achieved baseline separation and quantification with limits of detection and quantification around 0.08 and 0.16 pmol, respectively.

Main Results:

• The method demonstrated high sensitivity, with linear calibration curves from 0.15-20.6 pmol for various adenine nucleotides.
• Achieved excellent derivatization yield (94.1% for 1, N(6)-ethenoadenosine) and minimized nucleotide hydrolysis.
• Exhibited low intra- and inter-day variability (<5.1% and <3.4%, respectively).

Conclusions:

• The developed ion-pairing HPLC method with fluorescence detection is highly sensitive and selective for quantifying adenine nucleotides in primary cell cultures.
• Successfully applied the method to measure adenine nucleotides and determine an adenylate energy charge of 0.92±0.02 in primary astrocyte cultures.
• This technique overcomes the limitations of conventional methods for analyzing low-abundance nucleotides in biological samples.