Dietary antioxidants interfere with Amplex Red-coupled-fluorescence assays

Serrano, José; Jové, Mariona; Boada, Jordi; Bellmunt, Marı́a Josep; Pamplona, Reinald; Portero‐Otín, Manuel

doi:10.1016/j.bbrc.2009.08.041

Cited by 32 publications

(23 citation statements)

References 16 publications

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“…The rate of hydrogen peroxide production was calculated as a first derivation of the resorufin levels over time. The calibrations were performed by H 2 O 2 titrations in steps of 0.1 μ M at the beginning and throughout the whole measurement after each EGCG addition to eliminate possible interference of EGCG with Amplex Red assay, which was described before [43]. Due to the antioxidant properties of EGCG, we were not able to calibrate the signal at concentrations of EGCG higher than 20 μ M; therefore, the sensitivity of our H 2 O 2 measurements was limited by this EGCG concentration.…”

Section: Methodsmentioning

confidence: 99%

Effects of Epigallocatechin Gallate on Tert‐Butyl Hydroperoxide‐Induced Mitochondrial Dysfunction in Rat Liver Mitochondria and Hepatocytes

Mezera

Endlicher

Kučera

et al. 2016

Oxidative Medicine and Cellular Longevity

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Epigallocatechin gallate (EGCG) is a green tea antioxidant with adverse effects on rat liver mitochondria and hepatocytes at high doses. Here, we assessed whether low doses of EGCG would protect these systems from damage induced by tert-butyl hydroperoxide (tBHP). Rat liver mitochondria or permeabilized rat hepatocytes were pretreated with EGCG and then exposed to tBHP. Oxygen consumption, mitochondrial membrane potential (MMP), and mitochondrial retention capacity for calcium were measured. First, 50 μM EGCG or 0.25 mM tBHP alone increased State 4 Complex I-driven respiration, thus demonstrating uncoupling effects; tBHP also inhibited State 3 ADP-stimulated respiration. Then, the coexposure to 0.25 mM tBHP and 50 μM EGCG induced a trend of further decline in the respiratory control ratio beyond that observed upon tBHP exposure alone. EGCG had no effect on MMP and no effect, in concentrations up to 50 μM, on mitochondrial calcium retention capacity. tBHP led to a decline in both MMP and mitochondrial retention capacity for calcium; these effects were not changed by pretreatment with EGCG. In addition, EGCG dose-dependently enhanced hydrogen peroxide formation in a cell- and mitochondria-free medium. Conclusion. Moderate nontoxic doses of EGCG were not able to protect rat liver mitochondria and hepatocytes from tBHP-induced mitochondrial dysfunction.

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Section: Methodsmentioning

confidence: 99%

Effects of Epigallocatechin Gallate on Tert‐Butyl Hydroperoxide‐Induced Mitochondrial Dysfunction in Rat Liver Mitochondria and Hepatocytes

Mezera

Endlicher

Kučera

et al. 2016

Oxidative Medicine and Cellular Longevity

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“…21 Also, the presence of antioxidants such as anthracyclines, 22 gallic or ascorbic acids, is known to interfere as well. 23 At last but not least, various oxide nanoparticles such as TiO 2 or Fe 2 O 3 were found to markedly skew the accuracy of H 2 O 2 detection using the AR-based assay. 24 Very recently, the generation of RS from AR was also used for an indirect mapping of the photocurrent generated on single-crystalline rutile titanium oxide nanorods.…”

mentioning

confidence: 99%

Direct oxidative pathway from amplex red to resorufin revealed by in situ confocal imaging

Lefrançois

Vajrala

Arredondo

et al. 2016

Phys. Chem. Chem. Phys.

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Amplex Red (AR) is a very useful chemical probe that is employed in biochemical assays. In these assays, the non-fluorescent AR is converted to resorufin (RS), which strongly absorbs in the visible region (l abs = 572 nm) and yields strong fluorescence (l fluo = 583 nm). Even if AR is commonly used to report on enzymatic oxidase activities, an increasing number of possible interferences have been reported, thus lowering the accuracy of the so-called AR assay. As a redox-based reaction, we propose here to directly promote the conversion of AR to RS by means of electrochemistry. The process was first assessed by classic electrochemical and spectroelectrochemical investigations. In addition, we imaged the electrochemical conversion of AR to RS at the electrode surface by in situ confocal microscopy. The coupling of methodologies allowed to demonstrate that RS is directly formed from AR by an oxidation step, unlike what was previously reported. This gives a new insight in the deciphering of AR assays' mechanism and about their observed discrepancy.

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“…In phosphate buffered saline, we evidenced a highly intense cholesterol independent fluorescence that we could attribute partially (50%) to an exogenous H 2 O 2 production, as in those conditions it was diminished by catalase preincubation. The rest of the interference could be explained by a different mechanism recently described in our group [9], showing an interaction of vegetable antioxidants with peroxidases. Horseradish peroxidase is often used as final step for enzymatic-coupled reactions.…”

Section: Discussionmentioning

confidence: 92%

“…The potential contribution of vegetable derived peroxidases was ruled out by heat denaturation of vegetable extracts, even resulting in the increase of fluorescence (figure 2C). Finally, the inhibitory activity of catalase [1.11.1.6] (leading to fluorescence decreases to 50%) suggests that there is a vegetable extract dependent H 2 O 2 production which may be derived by the previously described interaction of sample antioxidants with horseradish peroxidase [9]. Moreover, in the presence of vegetable extracts, the fluorescence in both cholesterol-dependent and independent reactions offered similar values suggesting the possibility of using the cholesterol independent fluorescence as a blank to account the vegetable extract derived interferences (figures 2A and 2B).…”

Section: Resultsmentioning

confidence: 99%

When cholesterol is not cholesterol: a note on the enzymatic determination of its concentration in model systems containing vegetable extracts

et al. 2010

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BackgroundExperimental evidences demonstrate that vegetable derived extracts inhibit cholesterol absorption in the gastrointestinal tract. To further explore the mechanisms behind, we modeled duodenal contents with several vegetable extracts.ResultsBy employing a widely used cholesterol quantification method based on a cholesterol oxidase-peroxidase coupled reaction we analyzed the effects on cholesterol partition. Evidenced interferences were analyzed by studying specific and unspecific inhibitors of cholesterol oxidase-peroxidase coupled reaction. Cholesterol was also quantified by LC/MS. We found a significant interference of diverse (cocoa and tea-derived) extracts over this method. The interference was strongly dependent on model matrix: while as in phosphate buffered saline, the development of unspecific fluorescence was inhibitable by catalase (but not by heat denaturation), suggesting vegetable extract derived H2O2 production, in bile-containing model systems, this interference also comprised cholesterol-oxidase inhibition. Several strategies, such as cholesterol standard addition and use of suitable blanks containing vegetable extracts were tested. When those failed, the use of a mass-spectrometry based chromatographic assay allowed quantification of cholesterol in models of duodenal contents in the presence of vegetable extracts.ConclusionsWe propose that the use of cholesterol-oxidase and/or peroxidase based systems for cholesterol analyses in foodstuffs should be accurately monitored, as important interferences in all the components of the enzymatic chain were evident. The use of adequate controls, standard addition and finally, chromatographic analyses solve these issues.

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Dietary antioxidants interfere with Amplex Red-coupled-fluorescence assays

Cited by 32 publications

References 16 publications

Effects of Epigallocatechin Gallate on Tert‐Butyl Hydroperoxide‐Induced Mitochondrial Dysfunction in Rat Liver Mitochondria and Hepatocytes

Effects of Epigallocatechin Gallate on Tert‐Butyl Hydroperoxide‐Induced Mitochondrial Dysfunction in Rat Liver Mitochondria and Hepatocytes

Direct oxidative pathway from amplex red to resorufin revealed by in situ confocal imaging

When cholesterol is not cholesterol: a note on the enzymatic determination of its concentration in model systems containing vegetable extracts

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