Journal of Clinical Chemistry and Laboratory Medicine
Open Access

Abstract

Free and Total Malondialdehyde Measured as 2,4-dinitrophenylhydrazine Adduct by HPLC-UV in Hemodialysis Patient Serum

Roberto Biondi, Stefano Brancorsini, Maria Giulia Egidi, Giulia Poli, Enrico Capodicasa, Isabella Tritto, Gian Carlo Di Renzo, Florentina Duica, Dragos Cretoiu and Nicolae Suciu

Objective: Malondialdehyde (MDA) is an index of oxidative stress. In biological matrices MDA exists in both free (f-MDA) and bound (b-MDA) forms. In this report, a method to detect f-MDA and t-MDA was developed in human serum, based on MDA derivatization with 2,4-dinitrophenylhydrazine (DNPH) and HPLC separation. Methods: This method gave high sensitivity, with Limits of Detection (LOD) and Limits of Quantification (LOQ) of 3.5 pmol/ml and 10 pmol/ml, respectively; while recoveries of t-MDA from spiked matrices (R%) reached 98.1 ± 1.8 and 96.51 ± 1.8 for f-MDA. Considering the lower levels of f-MDA in biological fluids, this method improves derivatization conditions (DNPH medium solution, time and temperature) and yielded 47 ± 12 pmol/ml of f-MDA in serum of 17 controls. Our method succeeded to determine f-MDA and t-MDA in hemodialysed patients. Results: Results indicated that f-MDA levels increased, b-MDA even doubled respect to controls. After dialytic treatment, b-MDA did not change, while f-MDA decreased up to pre-dialytic values, before the following cycle. Data suggest that patients on chronic Hemodialysis (HD) presented a remarkable oxidative stress status highlighted by lipid peroxidation increase. Conclusion: In conclusion, this method proposes a simple and sensitive alternative to preexisting protocols that could be suitable for a non-invasive evaluation of oxidative stress in human diseases.