Methyl malondialdehyde as an internal standard for the determination of malondialdehyde

Methyl malondialdehyde (Me-MDA) is suggested as an internal standard for the determination of the lipid peroxidation product, malondialdehyde (MDA). A procedure for synthesising the Me-MDA sodium salt is described in detail. The purity and identity of the synthesised Me-MDA have been confirmed using nuclear magnetic resonance and UV spectroscopy, and by micellar electrokinetic chromatography. The applicability of Me-MDA as an internal standard has been demonstrated for rat brain homogenate samples. These samples were purified solely through ultrafiltration. The preferred analytical technique was capillary zone electrophoresis (CZE) with UV detection at 267 nm. The limits of detection (3 S/N) for the CZE separations of Me-MDA and MDA were 0.5 and 0.2 μM, respectively, and the total analysis time was approximately 10 min. Details of separations are also presented using high-performance liquid chromatography (HPLC) with UV detection at 245 nm, and gas chromatography, together with either electron capture or mass spectrometric detection. The GC separations require derivatisation of MDA and Me-MDA with pentafluorophenylhydrazine while the CZE and HPLC separations can be performed on the native molecules.     

Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative

We established a method for the detection of free and total (free and bound) malondialdehyde (MDA) in human plasma samples after derivatisation with 2,4-dinitrophenylhydrazine (DNPH). Free MDA was prepared by perchloric acid deproteinisation whereas an alkaline hydrolysation step for 30 min at 60°C was introduced prior to protein precipitation for the determination of total MDA. Derivatisation was accomplished in 10 min at room temperature subsequently chromatographed by HPLC on a reversed-phase 3 μm C₁₈ column with UV detection (310 nm). The detection limit was 25 pmol/ml for free and 0.3 nmol/ml for total MDA. The recovery of MDA added to different human plasma samples was 93.6% (n=11; RSD 7.1%) for the hydrolysation procedure. In samples from 12 healthy volunteers who underwent a hypoxic treatment (13% O₂ for 6 h) we estimated a baseline value of total MDA of 2.16 nmol/ml (SD 0.29) (ambient air) with a significant increase to 2.92 (nmol/ml, SD 0.57; P=0.01) after the end of this physiological oxidative stress challenge. Plasma values of free MDA in these samples were close to our detection limit. The presented technique can easily performed with an isocratic HPLC apparatus and provides highly specific results for MDA as do sophisticated GC–MS methods.     

Diaminonaphtalene, a new highly specific reagent for HPLC-UV measurement of total and free malondialdehyde in human plasma or serum.

We describe a new method to measure free and total malondialdehyde (MDA) in human plasma or serum, which is based on the derivatization of MDA with diaminonaphtalene (DAN) in an acidic medium at 37 degrees C. Derivatization is complete after 180 min at room temperature. By HPLC separation on a C18 column and diode array detection, the diazepinium thus formed exhibits a highly specific UV spectrum with a sharp maximum at 311 nm, which clearly distinguishes MDA from other short-chain aldehydes. Direct injection of deproteinized plasma avoids the use of an internal standard. The between-run imprecision is 9.1% (141 +/- 13 nM) for plasma and 6.6% (658 +/- 44 nM) for a commercial control. Typical within-day imprecision is 8% (93 +/- 7.5 nM) for total MDA, 3.2% (16 +/- 0.5 nM) for free MDA in plasma, and 1.6% (630 +/- 10 nM) for a commercial control. The recovery of MDA added to 10 different plasmas is 93-108% (mean = 100%). Plasma levels in healthy women (n = 79, 45-51 years) are 162 +/- 51 and 24 +/- 15 nM for total and free MDA, respectively. In younger men (n = 19, 21-37 years) total and free MDA are, respectively, 138 +/- 28 and 19 +/- 9 nM.     

Exposure to malondialdehyde induces an early redox unbalance preceding membrane toxicity in human erythrocytes

This work investigated the oxidative injury to human red blood cells (RBCs) by the exposure to exogenous malondialdehyde (MDA), in a physiological environment. When a 10% RBC suspension was incubated in autologous plasma, in the presence of 50 λμM MDA, 30% of MDA entered into the cells. A time-course study showed that MDA caused early (30-120 λmin) and delayed (3-18 λh) effects. MDA caused a fast depletion of reduced glutathione, and loss of the glucose-6-phosphate dehydrogenase activity, followed by a decrease of HbO 2 . Accumulation of methemoglobin, and formation of small amounts of hemichrome were later evident. Also, an HbO 2 -derived fluorescent product was measured in the membrane. The redox unbalance was followed by structural and functional damage to the membrane, evident as the formation of conjugated diene lipid hydroperoxides, concurrent with a sharp accumulation of MDA, consumption of membrane vitamin E, and egress of K + ions. SDS--PAGE of membrane proteins showed formation of high molecular weight aggregates. In spite of the marked oxidative alterations, the incubation plasma prevented a substantial hemolysis, even after a 18 λh incubation. On the contrary, the exposure of RBCs to 50 λμM MDA in glucose-containing phosphate saline buffer, resulted in a 16% hemolysis within 6 λh. These results indicate that the exposure to MDA causes a rapid intracellular oxidative stress and potentiates oxidative cascades on RBCs, resulting in their dysfunction.     

Determination of malondialdehyde by ion-pairing high-performance liquid chromatography

A method for the analysis of malondialdehyde (MDA) by ion-pairing HPLC is described. The method is direct, no derivitization is required, and sample preparation is minimal. After removal of particulates, the samples are injected directly onto an octadecylsilane column which is eluted with 14% (v/v) acetonitrile in 50 mM myristyltrimethylammonium bromide. 1 mM phosphate, pH 6.8. Detection is accomplished by monitoring absorbance at 254 nm or for greater sensitivity at 267 nm. The lower limit for reliable quantitation is 5 pmol MDA and the dynamic range extends to at least 4 nmol MDA. The method has been applied to the quantitation of MDA production during microsomal lipid peroxidation and to an assessment of the stability of MDA in microsomal and urine samples.     

Recovery of malondialdehyde in urine as a 2,4-dinitrophenylhydrazine derivative analyzed with high-performance liquid chromatography

Malondialdehyde (MDA) in urine was measured as a 2,4-dinitrophenylhydrazine (DNPH) derivative using high-performance liquid chromatography (HPLC) for the analysis. MDA standard coeluted with a peak obtained from rat urine after i.p. administration of MDA standard. This peak was also the only peak containing 14C after injection of a [14C]MDA standard, and was shown by mass spectrometry to contain 1-(2,4-dinitrophenyl)pyrazole, the derivative formed when MDA is treated with DNPH. Depending on the amount given (0.3-5.5 mumol), the recovery (after 24 h sampling period) in urine was 0.7-2.6%. This apparent non-linear kinetics may relate to several factors, such as dose-dependent metabolism. However, the peak urinary concentration approached the expected plasma concentration and reproducible recovery data were obtained, suggesting that MDA was passively excreted in a reasonably stable form. These data indicate that monitoring MDA excretion in urine can give useful information about lipid peroxidation in vivo.     

Quantitation of malondialdehyde (MDA) in plasma, by ion-pairing reverse phase high performance liquid chromatography

An ion-pairing high performance liquid chromatography method is described for the separation and quantitation of malondialdehyde in plasma. The MDA is determined as the thiobarbiturate chromogen formed by reaction of the plasma with 2-thiobarbituric acid under acid and heating conditions. However, under these conditions other interfering chromogens can also be formed. Using DEAE-cellulose chromatography followed by ion-pairing HPLC, we have been able to separate and quantitate the levels of MDA-TBA chromogen formed in plasma from other interfering chromogens. Measurements of MDA levels in the plasma of six normal individuals by HPLC gives a mean value of 4.57 +/- 0.33 nmole/ml, whereas the spectrophotometric determined value is 8.83 +/- 1.15 nmole/ml. These data suggest that some reevaluation of the numerous papers published on MDA levels in plasma using spectrophotometric methods may be necessary.     

Preparative steps necessary for the accurate measurement of malondialdehyde by high-performance liquid chromatography.

The need for a more specific, reliable, and reproducible technique for the measurement of malondialdehyde (MDA) has prompted modifications of currently available methods based on the formation and recovery of the complex between MDA and thiobarbituric acid (TBA). To 500 microliters of plasma or to 300 mg of liver homogenate, 2 ml of H2O and 500 microliters of 0.5% butylated hydroxytoluene in methanol were added to prevent further formation of MDA. Precipitation of proteins carried out with 200 microliters of 0.66 N H2SO4 and 150 microliters of 10% Na2WO4 (w/v) led to complete recovery of the MDA standard. Maximum formation of the MDA-TBA complex was obtained by adjusting the pH between 2.5 and 4.5 and heating the MDA-TBA mixture at 100 degrees C for 60 min. Extraction of the MDA-TBA complex was a critical step and proved complete with n-butanol at pH less than 0.75. It was then evaporated at 37 degrees C under nitrogen. The MDA-TBA complex solubilized in H2O was shown to be stable for at least 7 days. These preparative steps led to the detection of a single peak that on spectral analysis was identified as pure MDA-TBA. This procedure offers several advantages in terms of specificity, recovery, and reproducibility.     

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA–2-thiobarbituric acid (TBA) complex. The separation of MDA–TBA complex was performed using a 250×4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 μl (composed of 100 μl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 μl of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95°C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA–TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.     

Effects of carbon tetrachloride,menadione, and paraquat on the urinary excretion of malondialdehyde,formaldehyde, acetaldehyde,and acetone in rats

Excretions of the lipid peroxidation products, formaldehyde (FA), acetaldehyde (ACT), malondialdehyde (MDA), and acetone (ACON), were simultaneously identified and quantitated in the urine of female Sprague-Dawley rats by gas chromatography-mass spectroscopy (GC-MS) and high pressure liquid chromatography (HPLC) following the acute administration of carbon tetra-chloride, a model alkylating agent that does not induce glutathione depletion, and the redox cycling compounds paraquat and menadione. All three xenobiotics are well-known inducers of oxidative stress. Oxidative stress was induced by oral administration of single doses of 2.5 mL of carbon tetrachloride/kg, 60 mg menadione/kg, and 75 mg paraquat/kg. These doses are approximately 50% of the LD₅₀'s for the three xenobiotics. Urinary excretion of FA, ACT, MDA, and ACON increased relative to control animals following treatment with all xenobiotics. Over the 48 hours of the study, the greatest increases in the excretion of MDA, FA, ACT, and ACON occurred after paraquat administration, with increases of approximately 2.7-, 2.6-, 4.3-, and 11.0-fold, respectively. This technique may have wide-spread applicability as an effective biomarker for investigating altered lipid metabolism in disease states and exposure to environmental pollutants/xenobiotics.     

Estimation of lipoperoxidative damage and antioxidant status in diabetic children: relationship with individual antioxidants

Increased oxidative stress has emerged as a potential mechanism implicated in the pathogenesis, progression and cell dysfunction associated with many diseases including diabetes. In routine clinical practice, the estimation of the degree of oxidative damage and antioxidant status, even in paediatric patients, by appropriate techniques appears to be of interest. The aim of this study was to reliably identify patients with increased oxidant stress and/or reduced antioxidant defence mechanisms with a small blood sample and verify the applicability to the study of diabetic children (DC) at clinical onset of the disease. In 1-ml blood samples from 30 DC and 34 controls, techniques for accurately measuring malondialdehyde (MDA) concentrations in plasma and erythrocytes (using HPLC analysis with fluorometric detection), total radical antioxidant potential (TRAP) and blood plasma oxidizability were adapted and validated. Plasma alpha-tocopherol (HPLC), uric acid and sulfhydryl (SH) groups were also determined. At clinical onset of diabetes a significant reduction in plasma TRAP values (P<0.01) was observed in DC compared with controls. Similarly, a significant fall in individual antioxidant levels (alpha-tocopherol/total lipids, uric acid and protein SH) was noted in plasma of DC. Highly significant increases were found in both plasma and erythrocyte MDA levels in DC (p-MDA:1.7+/-0.2 microM; er-MDA: 7.2+/-0.7 nmol/g Hb) compared with controls (p-MDA:0.86+/-0.09 microM; P<0.0003; er-MDA:3.8+/-0.2 nmol/g Hb, P<0.0001). Plasma MDA and triglyceride levels correlated directly only in DC (P<0.001). Whole plasma oxidizability was significantly higher in DC than in controls (P<0.0001) and this parameter correlated significantly with plasma cholesterol and triglyceride concentrations (P<0.0001). The micromethods adapted and applied to the simultaneous detection of lipid peroxidation products and antioxidant status permit accurate and reliable assessment of the oxidative stress process in small plasma samples. Our results clearly show systemic peroxidative damage associated with insufficient defence mechanisms against ROS to be already present at clinical onset of type 1 diabetes mellitus in children and adolescents.     

Free malondialdehyde determination in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for quantification of malondialdehyde (MDA) in human plasma. Deproteinized samples were injected onto a Waters carbohydrate analysis column which was eluted with 20% (v/v) 0.03 M Tris buffer, pH 7.4, in acetonitrile. Peak absorbancy was measured at 267 nm. In contrast to data already published, we did not detect any free MDA in normal human plasma. This suggests that the classical thiobarbituric acid test is not suitable for the determination of MDA in human plasma.     

Mechanisms of action of malondialdehyde and 4-hydroxynonenal on xanthine oxidoreductase

Studies have been made on the possible involvement of malondialdehyde (MDA) and (E)-4-hydroxynon-2-enal (HNE), two terminal compounds of lipid peroxidation, in modifying xanthine oxidoreductase activity through interaction with the oxidase (XO) and/or dehydrogenase (XDH) forms. The effect of the two aldehydes on XO (reversible, XO(rev), and irreversible, XO(irr)) and XDH was studied using xanthine oxidase from milk and xanthine oxidoreductase partially purified from rat liver. The incubation of milk xanthine oxidase with these aldehydes resulted in the inactivation of the enzyme following pseudo-first-order kinetics: enzyme activity was completely abolished by MDA (0.5-4 mM), while residual activity (5% of the starting value) associated with an XO(irr) form was always observed when the enzyme was incubated in the presence of HNE (0.5-4 mM). The addition of glutathione to the incubation mixtures prevented enzyme inactivation by HNE. The study on the xanthine oxidoreductase partially purified from rat liver showed that MDA decreases the total enzyme activity, acting only with the XO forms. On the contrary HNE leaves the same level of total activity but causes the conversion of XDH into an XO(irr) form.     

Plasma carotenoid and malondialdehyde levels in ischemic stroke patients: relationship to early outcome

An association between ischemic stroke and increased oxidative stress has been suggested from animal studies. However, there is a lack of evidence with respect to this association in humans. Here, the time course of plasma levels of six carotenoids, which are lipophilic micronutrients with antioxidant properties, as well as of malondialdehyde (MDA), a marker of lipid peroxidation, was followed in ischemic stroke patients. Plasma levels of lutein, zeaxanthin, beta-cryptoxanthin, lycopene, alpha- and beta-carotene, as well as MDA were measured by high-performance liquid chromatography in 28 subjects (19 men and nine women aged 76.9+/-8.7 years) with an acute ischemic stroke of recent onset (<24h) on admission, after 6 and 24 h, and on days 3, 5, and 7. Carotenoid and MDA levels in patients on admission were compared with those of age- and sex-matched controls. Plasma levels of lutein, lycopene, alpha- and beta-carotene were significantly lower and levels of MDA were significantly higher in patients in comparison with controls. Significantly higher levels of MDA and lower levels of lutein were found in patients with a poor early-outcome (functional decline) after ischemic stroke as compared to patients who remained functionally stable. These findings suggest that the majority of plasma carotenoids are lowered immediately after an ischemic stroke, perhaps as a result of increased oxidative stress, as indicated by a concomitant rise in MDA concentrations. Among the carotenoids, only lutein plasma changes are associated with a poor early-outcome.     

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 x 50 microm uncoated capillaries at 20 degrees C, using 25 mmol/L borax (pH 9.3) and 5 mmol/L tetradecyltrimethylammonium bromide as running buffer. The applied voltage was -4kV (about 8 microA), 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 1h at 60 degrees C in the presence of 1 mol/L NaOH. The detection threshold was 0.2 micromol/L in microsomes and 0.4 micromol/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.     

Xenobiotic-metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 > 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.     

A chemiluminescence method to detect malondialdehyde in plasma and urine

A highly sensitive and convenient method to detect malondialdehyde (MDA) in specimens of plasma and urine was developed using high-performance liquid chromatography combining chemiluminescence detection. MDA was separated through a reverse-phase C18 column at a flow rate of 0.8 ml min⁻¹. It was then detected by a chemiluminescence detector. Variables that affected chemiluminescence reaction, including mobile phase, flow rate, chemiluminescence reagent, parameters of the photomultiplier tube, and temperature, were studied. This assay was linear from 0.50 to 50 μmol L⁻¹, with limits of detection and quantification of 0.08 and 0.30 μmol L⁻¹, respectively. The recoveries were in the range of 92.2 to 98.5%, and the intra- and interday reproducibilities were obtained with relative standard deviations of less than 4% and less than 6%, respectively. MDA in plasma and urine was stable for 12 h at 4 °C. MDA levels were 2.74, 3.19, and 3.20 μmol L⁻¹ for plasma and 1.25, 2.04, and 1.58 μmol L⁻¹ for urine, respectively. The proposed method is simple, rapid, and sensitive with low cost. Taken together, this method can be suitable for detecting MDA in tissue and feed specimens.     

Maternal and fetal plasma adenosine deaminase, xanthine oxidase and malondialdehyde levels in pre-eclampsia

The aim of this study was to evaluate maternal-fetal plasma adenosine deaminase, xanthine oxidase (ADA, XO) activity and malondialdehyde (MDA) levels and the relationship between them in pre-eclampsia. Maternal and umbilical cord whole blood samples were taken from 29 pre-eclamptic and 33 normal pregnants. The plasma ADA, XO activities as well as MDA levels were assayed by spectrophotometric methods. MDA levels and ADA, XO activities were found to be higher in maternal and fetal plasma in pre-eclamptics than in normal pregnancy. The differences were statistically significant between groups (p < 0.05). Increased maternal-fetal plasma XO and ADA activities, as a marker of immunological disorder, may be related to the pathogenesis of pre-eclampsia. In addition, increased MDA levels may be a reflection of increased oxidative stress in pre-eclamptics and their fetuses.     

Reaction of pyridoxamine with malondialdehyde: Mechanism of inhibition of formation of advanced lipoxidation end-products

Summary. Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) are implicated in many age-related chronic diseases and in protein aging. Recent studies suggest that pyridoxamine (PM) is an efficient AGEs/ALEs inhibitor in various biological systems. Because malondialdehyde (MDA) is an important intermediate in the formation of ALEs during lipid peroxidation, the purpose of this study is to determine whether PM can trap MDA directly and thereby prevent ALEs formation. PM reacted readily with MDA under physiological conditions. Within 6 h, a 1-pyridoxamino-propenal adduct derived from reaction of equimolar PM + MDA was detected. A 1-amino-3-iminopropene complex and a dihydropyridine-pyridinium complex were also identified after 7 d incubation. PM also greatly inhibited the lipofuscin-like fluorescence formation induced by MDA reaction with bovine serum albumin (BSA). Our results showed clearly that PM inhibited the formation of ALEs by trapping MDA directly under physiological condition, and provide insight into the mechanism of action of PM in protecting proteins against carbonyl stress.     

Comparison of low- and high-intensity resistance exercise on lipid peroxidation: role of muscle oxygenation

The purpose of this study was to examine the effect of low- vs. high-intensity resistance exercise on lipid peroxidation. In addition, the role of muscle oxygenation on plasma malondialdehyde (MDA) concentrations was explored. Eleven experienced resistance trained male athletes (age: 20.8 +/- 1.3 years; weight: 96.2 +/- 14.4 kg; height: 182.4 +/- 7.3 cm) performed 4 sets of the squat exercise using either a low-intensity, high-volume (LI; 15 repetitions at 60% 1 repetition maximum [1RM]) or high-intensity, low-volume (HI; 4 repetitions at 90% 1RM load). Venous blood samples were obtained before the exercise (PRE), immediately following the exercise (IP), and 20 (20P) and 40 minutes (40P) postexercise. Continuous wave near-infrared spectroscopy was used to measure muscle deoxygenation in the vastus lateralis during exercise. Deoxygenated Hb/Mb change was used to determine reoxygenation rate during recovery. No difference in MDA concentrations was seen between LI and HI at any time. Significant correlations were observed between plasma MDA concentrations at IP and the half-time recovery (T1/2 recovery) of muscle reoxygenation (r = 0.45) and between T1/2 recovery and the area under the curve for MDA concentrations (r = 0.44). Results suggest that increases in MDA occur independently of exercise intensity, but tissue acidosis may have a larger influence on MDA formation.