Reactions of lipid-derived malondialdehyde with collagen.

Malondialdehyde is a product of fatty acid oxidation (e.g. from low density lipoprotein) implicated in the damage of proteins such as collagen in the cardiovascular system (Chio, K. J., and Tappel, A. L. (1969) Biochemistry 8, 2821-2827). Its concentration is raised in diabetic subjects probably as a side effect of increased protein glycation. Collagen has enzyme-catalyzed cross-links formed between its individual molecules that are essential for maintaining the structure and flexibility of the collagen fiber. The cross-link dehydro-hydroxylysinonorleucine reacts irreversibly with 10 mM malondialdehyde at least 3 orders of magnitude faster than glucose reactions with lysine or arginine, such that there is little cross-link left after 1 h at 37 degrees C. Other cross-links and glycated elements of collagen are also vulnerable. Several possible products of malondialdehyde with collagen cross-links are proposed, and the potential involvement of collagenous histidine in these reactions is discussed. We have also isolated Ndelta-(2-pyrimidyl)-L-ornithine from collagenous arginine reacted with malondialdehyde. The yields of this product were considerably higher than those from model reactions, being approximately 2 molecules/collagen molecule after 1 day at 37 degrees C in 10 mM malondialdehyde. Collagenous lysine-derived malondialdehyde products may have been present but were not protected from protein acid hydrolysis by standard reduction techniques, thus resulting in a multitude of fragmented products.     

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.     

Hydroxyl-radical-induced iron-catalysed degradation of 2-deoxyribose. Quantitative determination of malondialdehyde.

The degradation of 2-deoxyribose to thiobarbituric acid-reactive material was investigated with two hydroxyl-radical-generating systems: (i) a defined gamma-radiolysis method and (ii) incubation with FeSO4 in phosphate buffer. In each case the thiobarbituric acid-reactive material can be accounted for by malondialdehyde, as measured by an h.p.l.c. method for free malondialdehyde. In the radiolysis system there is a large post-irradiation increase in free malondialdehyde if iron ions are added to the samples. It is proposed that this is due to iron ions catalysing the formation of hydroxyl radicals from radiolytically generated H2O2 as well as stimulating the breakdown of an intermediate deoxyribose degradation product. A mechanism for the formation of malondialdehyde during deoxyribose degradation is proposed.     

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.     

Lipofuscin-like fluorophores originated from malondialdehyde

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of post-mitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. In the present mini-review, I provide a comprehensive summary of fluorescent adducts originated from malondialdehyde (MDA).     

Lipid peroxidation-DNA damage by malondialdehyde

Malondialdehyde is a naturally occurring product of lipid peroxidation and prostaglandin biosynthesis that is mutagenic and carcinogenic. It reacts with DNA to form adducts to deoxyguanosine and deoxyadenosine. The major adduct to DNA is a pyrimidopurinone called M1G. Site-specific mutagenesis experiments indicate that M1G is mutagenic in bacteria and is repaired by the nucleotide excision repair pathway. M1G has been detected in liver, white blood cells, pancreas, and breast from healthy human beings at levels ranging from 1-120 per 108 nucleotides. Several different assays for M1G have been described that are based on mass spectrometry, 32P-postlabeling, or immunochemical techniques. Each technique offers advantages and disadvantages based on a combination of sensitivity and specificity. Application of each of these techniques to the analysis of M1G is reviewed and future needs for improvements are identified. M1G appears to be a major endogenous DNA adduct in human beings that may contribute significantly to cancer linked to lifestyle and dietary factors. High throughput methods for its detection and quantitation will be extremely useful for screening large populations.     

Improved method for plasma malondialdehyde measurement by high-performance liquid chromatography using methyl malondialdehyde as an internal standard

Measurement of malondialdehyde (MDA) is an important contribution to the assessment of oxidative stress. We report a sensitive and reproducible high-performance liquid chromatography (HPLC) method for measurement of plasma MDA in the assessment of lipid peroxidation. Using methyl malondialdehyde (Me-MDA) as an internal standard with reversed-phase HPLC and UV detection and derivatisation with 2,4 dinitrophenylhydrazine (DNPH), we obtained maximum MDA values with 60-min incubation of 10% plasma with 1 M NaOH at 60 degrees C. The dilution of the plasma and a longer incubation time in the alkaline hydrolysis step greatly improved recovery of MDA from its bound form. Ratios of peak height of MDA/Me-MDA were linear over a range of 0-100 microM with correlation coefficients >0.99. The recovery was 88.5%. Within and between run variations were <4 and <7%, respectively. The mean MDA value measured in 20 healthy volunteers was 13.8 microM (+/-1.32).     

Subpicogram determination of malondialdehyde by gas-liquid chromatography with nitrogen phosphorus detector. I. Standardization

A method for the measurement of malondialdehyde (MDA) using GLC with a nitrogen phosphorus detector (GLC/NPD) is described and evaluated. The method uses 2-hydrazinobenzothiazole (HBT) which forms condensation derivatives with MDA, acetoacetaldehyde, and acetylacetone (AA). GC/MS and 13C NMR studies of the three derivatives obtained showed that they are 2-(pyrazol-1'-yl)benzothiazoles and that they can be separated by GLC/NPD. Any one of these derivatives can be used as an internal standard for the measurement of the other two. The optimal conditions for the measurement of MDA were studied. At pH 2.5 and 70 degrees C, the condensation derivative is quantitatively formed in 30 min. Its extraction is obtained by a mixture of n-hexane/isoamyl alcohol 98/2 (v/v) containing HBT-AA as internal standard. The GLC detection limit is 0.04 pmol. Inter- and intrassay coefficients of variation were 2.9 and an average of 4.0%, respectively. The method is specific, and there was no interference from other carbonyl compounds. The artifactual formation of MDA from carbohydrates during the derivatization reaction is negligible. The method is proposed as a reference method for the standardization of working solutions of MDA or MDA-generating solutions.     

Distribution and oxidation of malondialdehyde in mice

The in vivo metabolism of malondialdehyde (MDA) by male and female Swiss mice was investigated. Distribution of an i.p. dose of MDA is rapid and uniform throughout the body. Conversion of 14C-labeled MDA to CO2 is complete 4 hours after an i.p. dose of 5 mumol to 200 mumol with no signs of short term toxicity. The yields of CO2 from [1-14C]-beta-alanine, [3-14C]-beta-alanine, [1-14C]-sodium acetate, and [2-14C]-sodium acetate were also determined. Comparison of the yields of CO2 from this series of compounds suggests the intermediacy of malonic semialdehyde in the metabolism of MDA. High doses (600 mumol) of beta-alanine or acetate given prior to 14C-MDA reduced the yield of 14CO2. Ethanol and disulfiram were both inhibitors of MDA metabolism, indicating the involvement of aldehyde dehydrogenase in the oxidation of MDA. These data demonstrate the ability of animal tissues to rapidly remove exogenously administered MDA. They also have implications with respect to the possible pathological consequences of in vivo MDA generation.     

High-throughput determination of malondialdehyde in plant tissues

Malondialdehyde (MDA) is a widely used marker of oxidative lipid injury whose concentration varies in response to biotic and abiotic stress. Commonly, MDA is quantified as a strong light-absorbing and fluorescing adduct following reaction with thiobarbituric acid (TBA). However, plant tissues in particular contain many compounds that potentially interfere with this reaction and whose concentrations also vary according to the tissue type and stress conditions. As part of our studies into the stress responses of plant tissues, we were interested in developing a rapid, accurate, and robust protocol for MDA analysis using reverse-phased HPLC to avoid these problems with reaction specificity. We demonstrate that a partitioning step into n-butanol during sample preparation is essential and that gradient HPLC analysis is necessary to prevent sample carryover between injections. Furthermore, the starting composition of the mobile phase must be sufficiently hydrophobic to allow direct injection of the n-butanol extracts without peak splitting, tailing, and other artifacts. To minimize analysis times, we used a short, so-called "Rocket" HPLC column and high flow rates. The optimized HPLC separation has a turnaround time of 2.5 min per sample. Butanolic extracts of MDA(TBA)(2) were stable for at least 48 h, and recoveries were linear between 0.38 and 7.5 pmol MDA added. Importantly, this procedure proved to be compatible with existing extraction procedures for l-ascorbate and glutathione analysis in different plant species, allowing multiple "stress metabolite" analyses to be carried out on a single tissue extract.     

Distribution and oxidation of malondialdehyde in mice

The metabolism of melondialdehyde (MDA) by male and female Swiss mice was investigated. Distribution of an i.p. dose of MDA is rapid and uniform throughout the body. Conversion of ¹⁴C-labeled MDA to CO₂ is complete 4 hours after an i.p. dose of 5 μmol to 200 μmol with no signs of short term toxicity. The yields of CO₂ from [1-¹⁴C]-β-alanine, [3-¹⁴C]-β-alanine, [1-¹⁴C]-sodium acetate, and [2-¹⁴C]-sodium acetate were also determined. Comparison of the yields of CO₂ from this series of compounds suggests the intermediacy of malonic semialdehyde in the metabolism of MDA. High doses (600 μmol) of β-alanine or acetate given prior to ¹⁴C-MDA reduced the yield of ¹⁴CO₂. Ethanol and disulfiram were both inhibitors of MDA metabolism, indicating the involvement of aldehyde dehydrogenase in the oxidation of MDA.These data demonstrate the ability of animal tissues to rapidly remove exogeneously administered MDA. They also have implications with respect to the possible pathological consequences of MDA generation.     

Free radical formation in the oxidation of malondialdehyde and acetylacetone by peroxidase enzymes.

Malondialdehyde, a product of lipid peroxidation, and acetylacetone undergo one-electron oxidation by peroxidase enzymes to form free radical metabolites, which were detected with ESR using the spin-trapping technique. The structures of the radical adducts were assigned using isotope substitution. With both malondialdehyde and acetylacetone and the enzymes myeloperoxidase and chloroperoxidase, carbon-centered radicals were detected. With horseradish peroxidase, a carbon-centered radical was initially trapped and then disappeared with the concomitant appearance of an iminoxyl radical.     

Methyl malondialdehyde is not suitable as an internal standard for malondialdehyde detection in urine after derivatisation with 2,4-dinitrophenylhydrazine

A previously described method of measurement of malondialdehyde (MDA) in human urine after derivatisation with 2,4-dinitrophenylhydrazine (DNPH) was tested for a possibility of using methyl malondialdehyde (MeMDA) as an internal standard. Despite structural similarity, those compounds were found to produce different yields of derivatisation under the same conditions depending on urine matrix. We conclude, that MeMDA is not suitable as an internal standard for the measurement of MDA in urine under previously reported conditions when DNPH is used as a deriviatising agent.     

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.     

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.     

Specific recognition of malondialdehyde and malondialdehyde acetaldehyde adducts on oxidized LDL and apoptotic cells by complement anaphylatoxin C3a

Oxidatively modified low-density lipoproteins (Ox-LDL) and complement anaphylatoxins C3a and C5a are colocalized in atherosclerotic lesions. Anaphylatoxin C3a also binds and breaks bacterial lipid membranes and phosphatidylcholine liposomes. The role of oxidized lipid adducts in C3a binding to Ox-LDL and apoptotic cells was investigated. Recombinant human C3a bound specifically to low-density lipoprotein and bovine serum albumin modified with malondialdehyde (MDA) and malondialdehyde acetaldehyde (MAA) in chemiluminescence immunoassays. No binding was observed to native proteins, LDL oxidized with copper ions (CuOx-LDL), or phosphocholine. C3a binding to MAA-LDL was inhibited by two monoclonal antibodies specific for MAA-LDL. On agarose gel electrophoresis, C3a comigrated with MDA-LDL and MAA-LDL, but not with native LDL or CuOx-LDL. C3a bound to apoptotic cells in flow cytometry. C3a opsonized MAA-LDL and was taken up by J774A.1 macrophages in immunofluorescence analysis. Complement-activated human serum samples (n=30) showed increased C3a binding to MAA-LDL (P<0.001) and MDA-LDL (P<0.001) compared to nonactivated samples. The amount of C3a bound to MAA-LDL was associated with total complement activity, C3a desArg concentration, and IgG antibody levels to MAA-LDL. Proteins containing MDA adducts or MAA adducts may bind C3a in vivo and contribute to inflammatory processes involving activation of the complement system in atherosclerosis.     

超氧化物歧化酶、丙二醛、脯氨酸作为裂殖壶菌菌种性能评价的新指标

[目的]对不同Schizochytrium sp.HX-308种子期超氧化物歧化酶(SOD)、丙二醛(MDA)和脯氨酸(Pro)进行测定,旨在寻找出一种评价菌种质量优劣的新方法.[方法]选用Schizochytrium sp.HX-308中的1株驯化高产菌株和1株原始菌株在正常条件下活化,以及同一原始菌株分别在正常条件和恶劣条件下活化的2组实验,分别通过WST-1法、巴比妥酸比色法和比色法测定SOD、MDA和Pro含量,探讨不同菌株中这3个指标与裂殖壶菌Schizochytrium sp.HX-308最终发酵性能之间的关系.[结果]发酵性能最佳的驯化菌株整个种子期的SOD、MDA和Pro含量都最低,平均仅为0.025 U/g、26.20 mmol/g· Fw和0.098 mg/g· Fw,而发酵性能最差的恶劣条件下菌株的SOD已达到了它的6倍以上,MDA和Pro也达到了2倍以上.[结论]本研究最终证实,这3个指标与菌株的发酵性能呈负相关关系,可以作为评价裂殖壶菌菌株发酵性能优劣的新指标,为菌株选育的优化提供了指导.