SIMULTANEOUS DETERMINATION OF ATP, ITS METABOLITES AND NAD+ IN BLOOD BY HPLC WITH PHOTODIODE ARRAY DETECTOR

Abstract

SIMULTANEOUS DETERMINATION OF ATP, ITS METABOLITES AND NAD+ IN BLOOD BY HPLC WITH PHOTODIODE ARRAY DETECTOR Gueli Maria Concetta, Cusimano Vincenza, Lo Re Marianna, Giuseppe Salemi Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (BioNEC), Università degli Studi di Palermo. Nucleotides are major high-energy phosphate carriers, subunits of nucleic acids and precursors for the synthesis of nucleotide cofactors such as NAD+ and SAM. The study of purine nucleotides metabolism is very important topic for a right understanding for the cellular life. Living cells rely on ATP for growth, differentiation, and response to physiological stimuli and environmental stress. We propose a fast isocratic HPLC system with photodiode array detector (PDA) for the simultaneous separation and quantification of major components of high-energy metabolism: purine nucleotides (ATP, ADP, AMP), degradation products (nucleosides, bases) simultaneously with NAD+ in human plasma in a single run. Blood samples were obtained from the healthy adult volunteers (University workers/30). For stability study, 200 mL of plasma was deproteinized through a Millipore-Amicon Ultracel. Waters-HPLC system consisted of a 600E Pump; 2998 PDA; Empower TM2 DS; Atlantis T3 analitycal column (10μL loop). The m.f. was a 40 mmol/L potassium phosphate buffer, pH 2.2 with methanol 20% at a flow rate of 1.0 mL/min. The spectral range of the PDA was 200- 400 nm and the optimal wavelength was 254 nm. We have obtained an execellent base-line separation of high-energy phospates, including NAD+ as well as of their catabolic products. All the peaks were identified in order of elution: ADP, ATP, adenine, AMP, hypoxantine, uric acid, xanthine, NAD+ , adenosine and inosine (RT = 3.5; 3.6; 4.3; 5.4; 5.6; 6.3; 8.2; 10.2; 12.1; 19.1 min), respectively. Peaks were identified by spiking the samples with authentic standards. This method makes use of a fastsingle- step sample pre-treatment procedure and provides the assay of the key metabolites in small amounts of plasma extracts. Therefore, this HPLC-PDA system is suitable to evaluate the energetic state in a variety of cell types, both under normal and pathological events and is very useful tool both in research and in clinical laboratories. 57° SIB FERRARA 18-19 Settembre 2013