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

SIMULTANEOUS DETERMINATION OF ATP, ITS METABOLITES AND NAD+ INBLOOD BY HPLC WITH PHOTODIODE ARRAY DETECTORGueli Maria Concetta, Cusimano Vincenza, Lo Re Marianna, Giuseppe SalemiDipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (BioNEC), Universitàdegli Studi di Palermo.Nucleotides are major high-energy phosphate carriers, subunits of nucleic acids andprecursors for the synthesis of nucleotide cofactors such as NAD+ and SAM. The study ofpurine nucleotides metabolism is very important topic for a right understanding for thecellular life. Living cells rely on ATP for growth, differentiation, and response tophysiological stimuli and environmental stress.We propose a fast isocratic HPLC system with photodiode array detector (PDA) for thesimultaneous 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). Forstability 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 T3analitycal column (10μL loop). The m.f. was a 40 mmol/L potassium phosphate buffer, pH2.2 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, includingNAD+ 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 wereidentified 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 insmall amounts of plasma extracts. Therefore, this HPLC-PDA system is suitable to evaluatethe energetic state in a variety of cell types, both under normal and pathological events and isvery useful tool both in research and in clinical laboratories. 57° SIB FERRARA 18-19 Settembre 2013