Studies of phospholipid oxidation by electrospray mass spectrometry: from analysis in cells to biological effects

The oxidation of lipids is important in many pathological conditions and lipid peroxidation products such as 4-hydroxynonenal (HNE) and other aldehydes are commonly measured as biomarkers of oxidative stress. However, it is often useful to complement this with analysis of the original oxidized phospholipid. Electrospray mass spectrometry (ESMS) provides an informative method for detecting oxidative alterations to phospholipids, and has been used to investigate oxidative damage to cells, and low-density lipoprotein, as well as for the analysis of oxidized phosphatidylcholines present in atherosclerotic plaque material. There is increasing evidence that intact oxidized phospholipids have biological effects; in particular, oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerophosphocholine (PAPC) have been found to cause inflammatory responses, which could be potentially important in the progression of atherosclerosis. The effects of chlorohydrin derivatives of lipids have been much less studied, but it is clear that free fatty acid chlorohydrins and phosphatidylcholine chlorohydrins are toxic to cells at concentrations above 10 micromolar, a range comparable to that of HNE and oxidized PAPC. There is some evidence that chlorohydrins have biological effects that may be relevant to atherosclerosis, but further work is needed to elucidate their pro-inflammatory properties, and to understand the mechanisms and balance of biological effects that could result from oxidation of complex mixtures of lipids in a pathophysiological situation.     

Separation and quantitation of molecular species from plant lipids by high-performance liquid chromatography

Monogalactosyl-, digalactosyl-, and sulfoquinovosyl diacylglycerol as well as phosphatidyl glycerol were isolated by conventional TLC and then separately subjected to HPLC for resolution of molecular species. Molecular species emerge in groups from reversed-phase columns during gradient elution. The groups are separated according to the sum of carbon and double bond numbers in fatty acyl pairs in linear relation to elution times. Therefore, it is possible to identify a species group with respect to carbon and double bond numbers by its retention time. The separation is monitored by recording the absorbance at 200 nm which depends on double bond combinations in acyl pairs. Diacylglycerols released from glyco- and phospholipids were separated as rho-anisoyl derivatives according to similar criteria. In this case separation was monitored at 250 nm, at which wavelength the absorbance is directly related to molar proportions. By calculating corrected 200-nm/250-nm absorbance ratios for different molecular species of rho-anisoyl diacylglycerols, relative response factors for different double bond combinations were obtained. The 200-nm absorbances of intact lipid species can be converted to molar proportions by division with these factors.     

Preparative scale separation of neutral lipids and phospholipids by centrifugally accelerated thin-layer chromatography

Mixtures of lipids and phospholipids were separated by centrifugally accelerated thin-layer chromatography on a preparative scale (300-500 mg lipid mixture per run). The isolated lipids and phospholipids were identified by 1H and 13C NMR spectroscopy and their fatty acid composition was determined by GLC and GLC-MS of their methyl esters.     

UV-visible spectral identification of the solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid

Solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid were investigated by spectroscopy. The spectral data showed the formation of a stable organomanganese intermediate, which was responsible for the rise in the absorbance at 420 nm. This result enables unambiguous interpretation of the absorbance change at 420 nm, as the intermediate permanganate ions could be isolated on the solid supports.     

Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

The oxidation of phenylhydrazine: superoxide and mechanism.

The oxidation of phenylhydrazine in buffered aqueous solutions is a complex process involving several intermediates. It can be initiated by metal cations, such as Cu2+; in which case EDTA acts as an inhibitor. It can also be intiated by oxyhemoglobin; in which case chelating agents do not interfere. Superoxide radical is both a product of this reaction and a chain propagator. The formation of O2- could be demonstrated in terms of a reduction of nitroblue tetrazolium, which was prevented by superoxide dismutase. The importance of O2- in carrying the reaction chains was shown by the inhibition of phenylhydrazine oxidation by superoxide dismutase. Hydrogen peroxide accumulated during the reaction and could be detected with catalase. The progress of this oxidation could be monitored in terms of oxygen consumption and by following increases in absorbance at 280 or 320 nm. The oxidation was markedly autocatalytic and superoxide dismutase had the effect of extending the lag period. The absorbance at 280 nm was due to an intermediate which first accumulated and was then consumed. This intermediate appears to be benzendiazonium ion. The absorbance at 320 nm was due to a stable product, which was not identified. The time course of oxygen consumption paralleled the increase in absorbance at 320 nm and lagged behind the changes at 280 nm. Exogenous benzenediazonium ion accelerated the oxidation of phenylhydrazine and eliminated the lag phase. Benzenediazonium ion must therefore react with phenylhydrazine to produce a very reactive intermediate, possibly phenyldiazene. A mechanism was proposed which is consistent with the data. The intermediates and products of the oxidation of phenylhydrazine include superoxide radical, hydrogen peroxide, phenylhydrazyl radical, phenyldiazene, and benzenediazonium ion. This is a minimal list: others remain to be detected and identified. It appears likely that the diverse biological effects of phenylhydrazine are largely due to the reactivities of these intermediates and products.     

Conjugated dienes detected in tissue lipid extracts by second derivative spectrophotometry

By studying lipid peroxidation induced by tetrachloromethane in rat liver microsomal PUFA, it has recently been shown that the primary products formed are conjugated diene hydroperoxides having either cis, trans (c,t) or trans,trans (t,t) stereochemistry. Both c,t and t,t hydroperoxidienes present distinct absorbances at 242 nm and 233 nm, respectively. The reaction is kinetically controlled in relation to the total H-atom donating ability of the cell environment. These results have been confirmed in vivo and in vitro experiments performed under different experimental conditions. The need for a precise and objective method to detect conjugated diene signals, the inherent difficulties with current techniques, and the availability of new spectrophotometric techniques have led us to devise a new method based on the second derivatization of the spectrum.     

Substrate specificity of acetyl coenzyme A synthetase

Acetyl coenzyme A synthetase (EC 6.2.1.1) has been examined for its ability to accept various carboxylic acids as substrates in place of acetic acid. The activity of the enzyme with these substrates was monitored using a coupled enzyme assay and high pressure liquid chromatography (HPLC) analysis. Short chain carboxylic acids were found to be active including: propionic, acrylic, fluoroacetic, methacrylic, 3-chloropropionic, 3-bromopropionic, and propiolic. The kinetic parameters, Km and % Vmax of the carboxylic acid substrates, are reported and show that these acids are poorer substrates than acetic acid. Several of the acyl CoAs were synthesized on a preparative scale using enzyme catalysis, purified using preparative HPLC, and characterized using proton NMR spectroscopy. In the course of the NMR identification, a complete and fully resolved spectral assignment for all the protons of coenzyme A was made and is reported. The acyl-CoA analogs should be useful as substrate analogs and as potential affinity labels for enzymes that bind acetyl-CoA.     

A methodology for determination of phospholipids

A simple method for the determination of phospholipids in an aqueous dispersion and in amniotic fluid was developed. The procedure is based on the observation that dispersed phospholipids promoted the solubilization of an insoluble dye--detergent complex. The solubilization of the complex between the negatively charged dye, Coomassie brilliant blue (CBB), and a positively charged detergent, cetyltrimethylammonium bromide (CTAB), produced a blue solution having a visible absorbance maximum above 600 nm. A linear increase in absorbance intensity occurs with an increase in phospholipid concentration. An assay using the CBB-CTAB reagent adsorbed on 3-mm glass beads is used to estimate total dispersed phospholipids between 2 and 25 micrograms/ml. Thereby, a two-phase water-methanol-chloroform system is formed. The products of zwitterionic phospholipids (such as phosphatidylcholine and phosphatidylethanolamine) partition to the organic phase while the dye complex solubilized in anionic phospholipids (such as phosphatidylglycerol and phosphatidylinositol) partitions to the aqueous phase. This procedure results in a convenient, sensitive, and rapid method for the simultaneous determination of the total phospholipid, zwitterionic phospholipid, and anionic phospholipid concentrations. Application of the new assay for determination of phospholipids in amniotic fluid is described.     

A convenient method for preparative peptide separation

A chromatographic system has been developed for the separation of peptides on a preparative scale. The principle of the method is to use the peptide bond absorbance in the 200–250 nm wavelength range as the basis for the peptide detection, and therefore to use only inorganic buffers that are transparent at these low wavelengths. Chromatography on phosphocellulose and on triethylaminoethyl-(TEAE-)cellulose leading to complete separation of the tryptic peptides of oxidized pancreatic ribonuclease is reported as an illustration of the method. The new system, which can be used both rapidly and conveniently and with simple equipment, appears to have broad applicability to peptide separation.     

Single plate separation of lung phospholipids including disaturated phosphatidylcholine

We have developed an improved thin-layer chromatographic method for separation of lung phospholipids. Individual phospholipids are completely separated in the first dimension. All phospholipids, except phosphatidylcholine, are then removed. The phosphatidylcholine-containing area is reacted with osmium tetroxide and saturated phosphatidylcholine species are separated from the unsaturated oxidation products by subsequent chromatography in the second dimension. This method should prove useful in studies on lung surfactant metabolism and secretion, in prediction of fetal lung maturity by analysis of amniotic fluid phospholipids, and in studies on surfactant obtained by bronchoscopy in adult lung diseases.     

High-performance liquid chromatography of phospholipids with UV detection: optimization of separations on silica

Chromatography of phospholipids was performed on silica columns with detection by absorbance at 205 nm using mixtures of hexane—isopropanol—water in which the role of water and isopropanol in elution was investigated. One system was developed which provided adequate separation of most major phospholipid species. However, lipids with several ionizable groups were not well separated and gave multiple broad peaks. A second system was developed utilizing sulfuric acid for ion suppression. The behavior of phospholipids in this system was found to be dependent on the presence of quaternary ammonium, amino, or hydroxyl groups. Except for plasmalogen, phospholipids were recovered intact. This system was optimized to provide baseline resolution of essentially all phospholipid species commonly found in mammalian tissues.     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

Chemistry of phospholipid oxidation

The oxidation of lipids has long been a topic of interest in biological and food sciences, and the fundamental principles of non-enzymatic free radical attack on phospholipids are well established, although questions about detail of the mechanisms remain. The number of end products that are formed following the initiation of phospholipid peroxidation is large, and is continually growing as new structures of oxidized phospholipids are elucidated. Common products are phospholipids with esterified isoprostane-like structures and chain-shortened products containing hydroxy, carbonyl or carboxylic acid groups; the carbonyl-containing compounds are reactive and readily form adducts with proteins and other biomolecules. Phospholipids can also be attacked by reactive nitrogen and chlorine species, further expanding the range of products to nitrated and chlorinated phospholipids. Key to understanding the mechanisms of oxidation is the development of advanced and sensitive technologies that enable structural elucidation. Tandem mass spectrometry has proved invaluable in this respect and is generally the method of choice for structural work. A number of studies have investigated whether individual oxidized phospholipid products occur in vivo, and mass spectrometry techniques have been instrumental in detecting a variety of oxidation products in biological samples such as atherosclerotic plaque material, brain tissue, intestinal tissue and plasma, although relatively few have achieved an absolute quantitative analysis. The levels of oxidized phospholipids in vivo is a critical question, as there is now substantial evidence that many of these compounds are bioactive and could contribute to pathology. The challenges for the future will be to adopt lipidomic approaches to map the profile of oxidized phospholipid formation in different biological conditions, and relate this to their effects in vivo. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.     

Lipid analysis of human HDL and LDL by MALDI-TOF mass spectrometry and (31)P-NMR

The analysis of HDL and LDL is important for the further understanding of atherosclerosis because changes of the protein and lipid moieties occur under pathological conditions. Because destruction of lipids leads to the formation of well-defined products such as lysophospholipids or chlorohydrins, methods that allow their fast and reliable determination would be useful. In this study, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for the analysis of the lipid composition of human lipoproteins. These data were compared with high resolution (31)P-NMR spectroscopy. Differences between LDL and HDL in sphingomyelin and phosphatidylcholine content could be monitored by NMR and mass spectrometry, and differences with respect to the extraction efficiency were found by MALDI-TOF MS. Additionally, treatment of LDL with hypochlorite and phospholipase A(2) resulted in marked changes (formation of chlorohydrines and lysolipids). Lysophosphatidylcholines were detectable by both methods, whereas MALDI-TOF MS failed to detect chlorohydrines of phospholipids.We conclude that MALDI-TOF MS provides rapidly a reliable lipid profile of lipoproteins. However, a previous lipid separation must be performed to detect lipid oxidation products. NMR can be directly applied, but suffers from lower sensitivity, and provides only limited information on fatty acid composition.     

The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL

For more than two decades, there has been continuing evidence of lipid oxidation playing a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways. The failure of antioxidant vitamins to influence clinical outcomes may have many explanations, including the inability of vitamin E to prevent the formation of these oxidized phospholipids and other lipid oxidation products of the myeloperoxidase pathway. Preliminary data suggest that the oxidation hypothesis of atherogenesis and the reverse cholesterol transport hypothesis of atherogenesis may have a common biological basis. The levels of specific oxidized lipids in plasma and lipoproteins, the levels of antibodies to these lipids, and the inflammatory/anti-inflammatory properties of HDL may be useful markers of susceptibility to atherogenesis. Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides may both promote a reduction in oxidized lipids and enhance reverse cholesterol transport and therefore may have therapeutic potential.     

Differential scanning calorimetry of dispersions of products of oxidation of 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine

Ultraviolet light was used to promote the autoxidation of 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC). The extent of oxidation was monitored by ultraviolet spectroscopy, reaction with thiobarbituric acid, fatty acid analysis, and thin-layer chromatography. Fatty acid analysis and thin-layer chromatography appeared to provide the most consistent estimates of oxidation, especially when extensive oxidation had taken place. The oxidized samples were separated by flash chromatography into fractions enriched in different oxidation products. Differential scanning calorimetry of aqueous dispersions of these fractions indicated that oxidation products had higher transition temperatures than the original SLPC.     

New colorimetric method for the quantitative estimation of phospholipids without acid digestion

A unique colorimetric method for the quantitative determination of phospholipids that does not involve the acid digestion of the lipid is described. The phospholipids, after separation by thin-layer chromatography and elution from the silica gel, are heated with a chromogenic solution that is a modification of a spray reagent formulated by Vaskovsky and Kostetsky (1968, J. Lipid Res., 9: 396). The absorbance of the colored complex was read at 710 nm, and it followed Beer's law in the range of 1-10 micro g of phospholipid phosphorus.