A specific, accurate, and sensitive measure of total plasma malondialdehyde by HPLC

Abstract

The process of lipid peroxidation results in a range of intermediates and end products including lipid hydroperoxides, aldyehydes, and malondialdehyde (MDA). These aldehydes and lipid hydroperoxides form DNA adducts and may result in extensive single-strand and doublestrand breaks ( 4 ). Various intermediates and end products generated during the lipid peroxidation cascade have been assayed, but the most-commonly employed approach continues to be the thiobarbituric acid (TBA) test ( 5 ).Although it is widely acknowledged that TBA reacts with a range of oxidized lipids, both saturated and unsaturated aldehydes, ( 6, 7 ) sucrose, and urea ( 8 ), to form various chromogens, referred to as TBA-reactive substances (TBARS), it is the reaction of TBA with MDA to produce a pink pigment ( 9 ) with an absorption maximum at 532 nm ( 10 ) and mass ion at 323 amu ( 11 ) that is a true indicator of lipid peroxidation. Historically, the TBARS test has been assayed by ultraviolet (UV) spectrophotometry or fl uorescence assays, but the specifi city of the TBA test, enabling the selective determination of MDA, may be achieved via chromatographic separation of the pink TBA 2 -MDA adduct ( 12 ). The adoption of HPLC techniques improves assay specifi city and the sensitivity of MDA determination, and we have previously confi rmed the validity of quantifying the TBA 2 -MDA adduct as a specifi c measure of lipid peroxidation in human biological fl uids ( 13 ).Despite the widely acknowledged limitations of the TBARS test ( 14 ), and in particular its lack of specifi city, it continues to be reported as a true measure of MDA in clinical disease ( 1,15,16 ). It is proposed that the poor assay specifi city associated with TBARS assays may lead to an overestimation of the levels of MDA in human plasma and other biological tissues and fl uids, and this, in turn, may Abstract Malondialdehyde (MDA) is one of the most commonly reported biomarkers of lipid peroxidation in clinical studies. The reaction of thiobarbituric acid (TBA) with MDA to yield a pink chromogen attributable to an MDA-TBA 2 adduct is a common assay approach with products being quantified by ultraviolet-Vis assay as nonspecific TBAreactive substances (TBARS) or chromatographically as MDA. The specifi city of the TBARS assay was compared with both chromatographic assays for total plasma MDA. The levels of total plasma MDA were signifi cantly lower than the plasma TBARS in each of the samples examined, and interestingly, the interindividual variation apparent in the level of plasma MDA was not evident in the plasma TBARS assay. Each of the four online chromatographic detectors yielded a precise, sensitive, and accurate determination of total plasma MDA, and selected-ion monitoring was the most-accurate assay (101.3%, n = 4). The online diode array detectors provided good assay specifi city (peak purity index of 999), sensitivity, precision, and accuracy. This research demonstrates the inaccuracy that is inherent in plasma TBARS assays, which claim to quantify MDA, and it is pro...

Annihilation of Leishmania by daylight responsive ZnO nanoparticles: a temporal relationship of reactive oxygen species-induced lipid and protein oxidation

“…28 Thiobarbituric acid reactive substances assay was used to detect and quantify any MDAs formed. 33,34 It is a common method to measure oxidative change in lipids due to free radicals. 35 It was found that there was a continuous increase in the MDA formation in each hour (Figure 7).…”

Oxidative stress affects sperm performance and ejaculate redox status in subordinate House Sparrows

“…60 µl), from which plasma was obtained by centrifuging the sample at 7000 rpm for 5 min. We determined the levels of malondialdehyde (MDA) from the plasma and erythrocyte samples following standard procedures using derivatization with thiobarbituric acid followed by ultra-highperformance liquid chromatography (UHPLC) coupled to fluorescence detection (adapted from Agarwal and Chase, 2002;Moselhy et al, 2013). Over the course of the diquat treatment, we found that MDA levels in plasma gradually increased ( Fig.…”

Liquid chromatography in clinical laboratory diagnostics

“…Малоновый диальдегид образуется при перекисном окислении ненасыщенных жир-ных кислот, таким образом являясь маркером ок-сидативного разрушения клеточных мембран [11]. Существует методика по хроматографическому определению малонового альдегида в плазме крови или сыворотке с помощью простой изократической жидкостной системы с флюоресцентной детекцией [12]. Флуорофор, получаемый в результате пробо-подготовки (осаждение белков и дериватизация), специфичен и детектируется в очень малых количе-ствах, при длине волны возбуждения 515 нм и длине волны эмиссии 553 нм.…”