Malondialdehyde In Biological Samples Research Articles

A combined surface-enhanced Raman spectroscopy (SERS)/colorimetric approach for the sensitive detection of malondialdehyde in biological samples

Variations of malondialdehyde (MDA) level in biological samples often induce pathological changes, which is associated with various diseases. Here, we developed a combined surface-enhanced Raman spectroscopy (SERS) and colorimetric strategy for MDA quantitation. The methodology is based on the condensation reaction between 4-aminothiophenol (4-ATP)-modified Au nanoflowers (Au NFs) with the aldehyde groups of MDA, which causes the aggregation of the Au NFs and a concomitant change in the solution color from purple to blue and shifts in the local surface plasmon resonance band to longer wavelengths compared with monodisperse NFs. Additionally, after the condensation reaction, a new Raman peak ascribable to the CN vibration appeared at 1630 cm−1. The intensity of this peak was directly related to the concentration of MDA in solution, which allowed establishing the quantitative measurement of MDA based on SERS. The developed SERS assay displayed satisfactory sensitivity and selectivity with a broad linear range from 1.0 × 10−12 to 1.0 × 10−7 M and a low detection limit (∼3.6 × 10−13 M), outperforming other reported optical and electrochemical methods. Furthermore, the use of 4-ATP-modified Au NF probes to monitor MDA in human serum demonstrates the applicability of this combined SERS/colorimetric approach in a real environment.

Robust and reliable detection of malondialdehyde in biological samples via microprobe-triggered surface-enhanced Raman spectroscopy

Malondialdehyde (MDA) is an endogenous genotoxic product of lipid peroxidation and its accumulation in the human body is associated with various diseases. To accurately understand the role of MDA in physiological and pathological processes, it is of great significance to develop a robust and reliable method for rapid detection of MDA. Hence, we proposed a surface-enhanced Raman spectroscopy (SERS) based method for MDA analysis, which employed 4-aminophenylthiophenol (PATP) as a SERS microprobe to recognize and quantify MDA. Qualitative analysis of MDA was performed using the fingerprint Raman spectrum of MDA adducts, and quantitative analysis of MDA was conducted using the characteristic peak of PATP at 1083 cm−1 as the internal standard. MDA detection can be performed in a mild reaction environment and the detection limit was below 0.50 μM. The proposed method successfully detected MDA in fresh blood samples and found satisfactory recovery rates in spiked samples. The proposed method allows reliable detection of endogenous toxic compounds in blood plasma, which is of great significance for monitoring oxidative stress indicators, such as lipid peroxidation rate and intensity, potential antioxidant capacity, and the degree of tissue peroxidation damage.

Effects of Analytical Procedures on the Repeatability of Malondialdehyde Determinations in Biological Samples

Background: Malondialdehyde (MDA) is a commonly used biomarker of oxidative stress in clinical studies and has been measured in many pathological conditions during last decades. Different analytical methods have been reported for determination of MDA in biological samples in which MDA was adducted with thiobarbituric acid (TBA) to produce more sensitive chromophore and also convert it to a fluorescent compound. In spite of the routine applications of this derivatization and subsequent analysis of MDA in biomedical studies, its reliability, repeatability and reproducibility is questionable. The aim of this work is to investigate the effects of some factors on the repeatability of MDA determinations in standard solutions and also in plasma samples using spectroscopic method. Methods: MDA-TBA adduct is prepared in standard solutions and the effects of pH, temperature, reaction time, open, closed and reflux systems and the ratio of MDA and TBA is investigated by measuring the absorbance of the solution at 532 nm. These effects are also investigated in human plasma samples. Results: The best results are obtained at pH 2.5, temperature of 70 °C, reaction time of 150 minutes, reflux system and ratio of 2. Conclusion: Using the optimized conditions are resulted in better repeatability.

Simple and sensitive determination of malondialdehyde in human urine and saliva using UHPLC-MS/MS after derivatization with 3,4-diaminobenzophenone.

Malondialdehyde has been used as a biomarker for lipid peroxidation in biological samples. An ultra-high performance liquid chromatography with tandem mass spectrometry method was developed to determine the levels of malondialdehyde in human urine and saliva samples. To select the optimum derivatization reagent from four diamino compounds, the reactivity and sensitivity of their derivatives were compared, and 3,4-diaminobenzophenone was selected. The optimum reaction conditions for malondialdehyde with 3,4-diaminobenzophenone were as follows: a reagent dosage of 50mg/L, pH of 4, and reaction for 30min at 50°C. The formed derivative product was analyzed using ultra-high performance liquid chromatography with tandem mass spectrometry without additional extraction or concentration steps. In the optimal conditions, the method was used to determine malondialdehyde concentration in human urine and saliva samples. The limits of quantification for malondialdehyde in biological samples were over a concentration range of 0.1-0.3μg/L. Additionally, the calibration curve showed a linearity greater than r2 =0.997. The method was used to analyze 14 human urine and saliva samples from healthy volunteers. Malondialdehyde was detected in the concentration range of 1.7-33.6μg/g creatinine in all human urine samples and 0.1-1.3μg/L in all human saliva samples.

Development of 4-hydrazinyl-7-nitrobenzofurazan as a fluorogenic probe for detecting malondialdehyde in biological samples

Malondialdehyde (MDA) is a critical product of polyunsaturated fatty acid peroxidation and represents a common biomarker of oxidative stress. In this study, we have developed a fluorogenic MDA probe, 4-hydrazinyl-7-nitrobenzofurazan (NBDH). The reaction between NBDH and MDA in acidic conditions yields a highly fluorescent product, 4,5-dihydro-1H-pyrazole derivative (NBD-P1). The MDA detection range of our probe NBDH falls between 0.1 and 20μM, and the corresponding detection limit amounts to 7.2nM (3δ). NBDH also demonstrates a high selectivity toward MDA over other aldehydes and biologically relevant molecules, and proves to be an excellent MDA assay in biological fluids.

Salivary malondialdehyde as an oxidative stress biomarker in oral and systemic diseases.

Salivary malondialdehyde as an oxidative stress biomarker in oral and systemic diseases.

Critical Review of Malondialdehyde Analysis in Biological Samples

Malondialdehyde (MDA) is one of the most common biomarkers of oxidative stress and is used for the diagnosis of many diseases. This work aims to review the reaction used for the derivatization of MDA, the parameters affecting adduct formation, and a number of recently published analytical methods for quantification of MDA in biological samples. A brief discussion of the reliability of MDA as an oxidative stress biomarker is also provided. Keywords: Oxidative stress, biomarker, malondialdehyde, serum analysis, bioanalysis, review.

Convergence of the 5-LOX and COX-2 pathways: heme-catalyzed cleavage of the 5S-HETE-derived di-endoperoxide into aldehyde fragments

Oxygenation of the 5-lipoxygenase product 5S-hydroxyeicosatetraenoic acid by cyclooxygenase-2 yields a bicyclic di-endoperoxide. The di-endoperoxide contains two peroxides spanning from carbons 9 to 11 and 8 to 12, and two hydroxyls at carbons 5 and 15 of arachidonic acid (Schneider C., et al. 2006. Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2. J. Am. Chem. Soc. 128: 720). Here, we report that treatment of the di-endoperoxide with hematin or ferrous chloride results in cleavage of both peroxide O-O bonds and of the bonds between the carbons that carry the peroxide groups, producing the aldehydes 4-hydroxy-2E-nonenal (4-HNE), 8-oxo-5S-hydroxy-6E-octenoic acid, and malondialdehyde (MDA). The hematin- and ferrous iron-catalyzed transformation of the di-endoperoxide proceeded with a similar yield of products as the cleavage of the prostaglandin endoperoxide PGH(2) to 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid and MDA. Chiral phase HPLC analysis of the 4-HNE cleavage product showed greater than 98% 4S and thus established the S configuration of the 15-carbon of the di-endoperoxide that had not previously been assigned. This transformation of the 5-lipoxygenase/cyclooxygenase-2 derived di-endoperoxide invokes the possibility of a novel pathway to formation of the classic lipid peroxidation products 4-HNE and MDA.

Age-related impairment of HDL-mediated cholesterol efflux

Our aim in this study was to investigate the effect of aging on the capacity of HDLs to promote reverse cholesterol transport. HDLs were isolated from plasma of young (Y-HDL) and elderly (E-HDL) subjects. HDL-mediated cholesterol efflux was studied using THP-1 and J774 macrophages. Our results show that E-HDLs present a lower capacity to promote cholesterol efflux than Y-HDLs (41.7 +/- 1.4% vs. 49.0 +/- 2.2%, respectively; P = 0.013). Reduction in the HDL-mediated cholesterol efflux capacity with aging was more significant with HDL(3) than HDL(2) (Y-HDL(3), 57.3 +/- 1% vs. E-HDL(3), 50.9 +/- 2%; P = 0.012). Moreover, our results show that ABCA1-mediated cholesterol efflux is the more affected pathway in terms of cholesterol-removing capacity. Interestingly, the composition and structure of HDL revealed a reduction in the phosphatidylcholine-sphingomyelin ratio (E-HDL, 32.7 +/- 2.7 vs. Y-HDL, 40.0 +/- 1.9; P = 0.029) and in the phospholipidic layer membrane fluidity in E-HDL compared with Y-HDL as well as an alteration in the apolipoprotein A-I structure and charge. In conclusion, our results shown that E-HDLs present a reduced capacity to promote cholesterol efflux, principally through the ABCA1 pathway, and this may explain the increase of the incidence of cardiovascular diseases observed during aging.

Use of Methyl Malondialdehyde as an Internal Standard for Malondialdehyde Detection: Validation by Isotope-Dilution Gas Chromatography–Mass Spectrometry

Malondialdehyde (MDA), a compound derived from lipid peroxidation and from eicosanoid biosynthesis, exists in biological matrices both in the free form and bound to SH and/or NH2 groups of various biomolecules (1). Although other compounds (isoprostanes) have been proposed as more reliable indicators of oxidative damage (2), MDA is still widely used in clinical chemistry laboratories to monitor oxidative stress (3). Several methods have been developed to evaluate MDA in biological samples (1)(4), but the different analytical conditions used and the lack of a suitable internal standard have led to large discrepancies in measurements even at physiologic MDA concentrations in human plasma (5). We (6) recently reported a “reference method” for free and total plasma MDA quantification, as the phenylpyrazole derivative, by isotope-dilution gas chromatography–mass spectrometry (ID-GC-MS) with dideuterated MDA (d2-MDA) as internal standard. This method, used for clinical MDA detection (7)(8)(9), offers the possibility of validating other proposed internal standards that differ from MDA in structure, stability, and reactivity. Unfortunately, the major limitations of d2-MDA include its difficult synthesis (10) and that it is detectable only by GC-MS, a method not always available in clinical laboratories. A compound that appears to be more suitable as an internal standard is methyl malondialdehyde (MMDA) because it is structurally close to MDA, is absent from biological matrices, is easily obtainable from a commercial compound, and is detectable by common methods such as HPLC, GC, and capillary electrophoresis. MMDA was first evaluated as an internal standard for MDA determinations by Bull and Marnett (11), who unfortunately experienced difficulties in resolving the underivatized MDA and MMDA by HPLC. Recently, Claeson et al. (12) reported the use of MMDA as an internal standard for measurement of MDA in rat brain by capillary electrophoresis, …

Validation of methyl malondialdehyde as internal standard for malondialdehyde detection by capillary electrophoresis

The aim of this study was to validate, by capillary electrophoresis, the use of synthesized methyl malondialdehyde as the internal standard for the direct quantification of free and total (free + bound) malondialdehyde in biological samples. All analyses were performed in 20 cm×50 μm uncoated capillaries at 20 °C, using 25 mmol/L borax (pH 9.3) and 5 mmol/L tetradecyltrimethylammonium bromide as running buffer. The applied voltage was −4 kV (about 8 μA ), the detector being set at 260 nm for a total run time of 8 min per sample. Free malondialdehyde was evaluated after acetonitrile extraction, while the samples evaluated for total malondialdehyde were, before extraction, hydrolyzed for 1 h at 60 °C in the presence of 1 mol/L NaOH. The detection threshold was 0.2 μmol/ L in microsomes and 0.4 μmol/ L in plasma. As an application of the method, three pools of rat liver microsomes were quantified before (0.35±0.1 and 1.1±0.5 nmol/mg protein, free and total malondialdehyde, respectively, mean±SD) and after lipoperoxidation induction using systems able to generate oxygen free radicals (18.4±3.2 and 19.7±2.0 nmol/mg protein). The results were confirmed by isotopic dilution gas chromatography-mass spectrometry, used as the reference method. The feasibility of capillary electrophoresis for malondialdehyde determination in normal and pathological human plasma was also investigated.

Determination of malondialdehyde in biological samples by solid-phase extraction and high-performance liquid chromatography.

An analytical procedure for determination of malondialdehyde in tissue homogenates and blood serum was developed. A reaction with 2,4-dinitrophenylhydrazine is used followed by cleaning up of the derivative by solid-phase extraction. The samples were analyzed by isocratic high-performance liquid chromatography (HPLC) using a narrow-bore HPLC-column. A good separation of the 1-pyrazole peak from that of 2,4-dinitrophenylhydrazine was observed. A high linear dependence was established by the concentration of 1-pyrazole in the range of 10-5000 ng/ml. The detection limit of the method applied for tissue homogenates and blood serum was approximately 10 ng/ml or lower, and RSD of the method was 9% (n = 8). The peak of 1-pyrazole for these samples was well separated from the other matrix peaks. Experiments carried out evaluated that the solid-phase extraction might be an effective step of the sample preparation, significantly increasing the selectivity of the analysis and the life-time of the column. The method seems to be applicable for determination of malondialdehyde in different biological samples.

Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation.

Under acidic and mild-temperature conditions, 1-methyl-2-phenylindole was found to react with malondialdehyde (MDA) and 4-hydroxyalkenals to yield a stable chromophore with intense maximal absorbance at 586 nm. The use of methanesulfonic acid results in optimal yields of chromophore produced from MDA as well as from 4-hydroxynonenal. By contrast, the use of hydrochloric acid results in an optimal yield of chromophore produced from MDA and a negligible reaction of 4-hydroxynonenal. Taking advantage of such chromogenic reactions, we developed a new colorimetric assay of lipid peroxidation. Using a methanesulfonic acid-based medium, MDA and 4-hydroxyalkenals can be measured at the 586 nm wavelength. However, the presence of endogenous inhibitors of the reaction with 4-hydroxyalkenals is common, and this means that the latter may be underestimated in some biological samples. The assay performed in a hydrochloric acid-based medium enables the specific measurement of MDA in the presence of 4-hydroxyalkenals. Upon hydrolysis of Schiff bases in hydrochloric acid (pH 1.5), either assay can be used to specifically measure the amount of total MDA in biological samples because 4-hydroxyalkenals undergo an irreversible cyclization reaction under the hydrochloric acid-based conditions of hydrolysis. The two assays were applied to the determination of the amount of MDA alone and of MDA and 4-hydroxyalkenals in an in vitro model of lipid peroxidation. This methodology was also used to clarify complex patterns of tissue-specific MDA production in vivo, following hydrolysis of Schiff bases, in rodents treated with doxorubicin.

Analysis of Malondialdehyde in Biological Samples by Capillary Gas Chromatography

An analytical method to measure malondialdehyde (MDA) was developed. MDA was derivatized with N-methylhydrazine (NMH) to 1-methylpyrazole (1-MP). 1-MP was extracted and analyzed by capillary gas chromatography with nitrogen-phosphorus detection. Analyte concentration, pH, and matrix effects of 1-MP-spiked samples were investigated to determine optimal recovery conditions. Efficiencies for solid-phase extraction ranged from 95.6 ± 0.9 to 81.6 ± 3.5% compared to 75.0 ± 6.4 to 67.5 ± 9.6% for liquid-liquid extraction for 20 to 1 nmol/ml 1-MP-spiked samples, respectively. Solid-phase extraction of 1-MP was more effective than liquid-liquid extraction over a range of pH 2-8.5 and in various aqueous matrices. Addition of methanol to the matrix decreased the solid-phase extraction efficiency. Reaction yield at pH 2-8.5 showed full conversion of MDA to 1-MP following reaction with NMH. Recovery of bound MDA was investigated by incubating bovine serum albumin (BSA) spiked with MDA at 37°C for 18 h and separating the free MDA and MDA-bound protein. The recovery of bound MDA from BSA increased by increasing the acidity and temperature. Specific applications of this method for biological samples are given for the analysis of endogenous MDA in the plasma and red blood cells of mice and the formation of MDA in ultraviolet-irradiated cells in culture.

An improved method for the measurement of malondialdehyde in biological samples.

An improved method was developed for measuring malondialdehyde (MDA) as its thiobarbituric acid (TBA) complex. Samples were initially incubated with 1% potassium iodide and 0.1% butylated hydroxytoluene at 50 degrees C for 20 min, and then with 0.4% TBA at 60 degrees C for 60 min. The MDA-TBA complex formed was extracted with isobutyl alcohol and measured by high-performance liquid chromatography with fluorescence detection. The improved method allows for a more specific determination of MDA present in biological samples.

Highly sensitive high-performance liquid chromatography for the measurement of malondialdehyde in biological samples

A highly sensitive method for the measurement of malondialdehyde as thiobarbituric acid—malondialdehyde complex by reversed-phase high-performance liquid chromatography with fluorescence detection in biological samples is described. As samples, 20 μl of rat plasma or 10% (w/v) liver homogenate mixed with 2.0% thiobarbituric acid in 2 M sodium acetate buffer containing 0.05% butyl hydroxytoluene (pH 3.5) were heated at 95°C for 45 min to give the complex. The complex, extracted with n-butanol, was chromatographed on a system equipped with a reversed-phase column, and the eluted peak was monitored with a fluorescence detector (excitation wavelength 515 nm, emission wavelength 553 nm). The mobile phase was a acetonitrile—water (2:8, v/v) under isocratic conditions at ambient temperature, and a single analysis was done in ca. 4 min. The minimum detection level for malondialdehyde was as low as 0.05 pmol. The n-butanol extract was stable at least for 3 days. The simple mobile phase, the extremely sensitive detection limit, and the stability of the complex make this system applicable to routine clinical analysis with a small amount of tissue or biopsy sample.