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• W2112248863 abstract "Reactive nitrogen oxide species (RN3333333333362) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that γ-tocopherol (γT), the major form of vitamin E in the North American diet, may play an important role in these diseases. γT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-γT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, αT, and γT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (∼10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing ∼4% of γT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to γT or corrected for total neutral lipids, while α- and γT levels remained unchanged. These results demonstrate that nitration of γT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase.The present HPLC method should be helpful in clarifying the precise physiological role of γT. Reactive nitrogen oxide species (RN3333333333362) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that γ-tocopherol (γT), the major form of vitamin E in the North American diet, may play an important role in these diseases. γT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-γT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, αT, and γT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (∼10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing ∼4% of γT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to γT or corrected for total neutral lipids, while α- and γT levels remained unchanged. These results demonstrate that nitration of γT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase. The present HPLC method should be helpful in clarifying the precise physiological role of γT. Reactive nitrogen oxide species (RNOS) have been implicated as important effector molecules in inflammatory diseases (1Christen S. Hagen T.M. Shigenaga M.K. Ames B.N. Chronic inflammation, mutation, and cancer.in: Parsonnet J. Microbes and Malignancy: Infection as a Cause of Human Cancers. Oxford University Press, New York, Oxford1999: 35-88Google Scholar, 2Laroux F.S. Pavlick K.P. Hines I.N. Kawachi S. Harada H. Bharwani S. Hoffman J.M. Grisham M.B. Role of nitric oxide in inflammation.Acta Physiol. Scand. 2001; 173: 113-118Google Scholar). On the other hand, antioxidants such as vitamin E have been proposed to modulate the effects mediated by RNOS and thus provide protection against inflammation-induced pathology.Vitamin E is a family of lipid-soluble chain-braking antioxidants that share a similar chromanol ring structure (Fig. 1). Members of this family differ only in the number and position of methyl substitutents on the phenolic ring, and the degree of saturation of the phytyl side chain. The two principal forms of vitamin E in the diet and tissues are α-tocopherol (αT, 2) and γ-tocopherol (γT, 1). αT is the most effective antioxidant in vitro (3Burton G.W. Ingold K.U. Autoxidation of biological molecules. 1. The antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in vitro.J. Am. Chem. Soc. 1981; 103: 6472-6477Google Scholar) and has the highest bioactivity in vivo among the different vitamers (4Brigelius-Flohe R. Traber M.G. Vitamin E: function and metabolism.FASEB J. 1999; 13: 1145-1155Google Scholar). Several human intervention studies have been conducted to demonstrate, despite generally inconclusive results, a protective effect of αT in inflammatory diseases, such as atherosclerosis (5Ricciarelli R. Zingg J.M. Azzi A. Vitamin E: protective role of a Janus molecule.FASEB J. 2001; 15: 2314-2325Google Scholar). However, a growing body of evidence suggests that γT may also play an important role in these diseases (6Jiang Q. Christen S. Shigenaga M.K. Ames B.N. gamma-tocopherol, the major form of vitamin E in the US diet, deserves more attention.Am. J. Clin. Nutr. 2001; 74: 714-722Google Scholar).In a seminal study, Cooney et al. found that γT is superior to αT in the detoxification of nitrogen dioxide (7Cooney R.V. Franke A.A. Harwood P.J. Hatch-Pigott V. Custer L.J. Mordan L.J. γ-Tocopherol detoxification of nitrogen dioxide: Superiority to α-tocopherol.Proc. Natl. Acad. Sci. USA. 1993; 90: 1771-1775Google Scholar). We later demonstrated that the reaction of γT with RNOS such as peroxynitrite is fundamentally different to that of αT (8Christen S. Woodall A.A. Shigenaga M.K. Southwell-Keely P.T. Duncan M.W. Ames B.N. gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications.Proc. Natl. Acad. Sci. USA. 1997; 94: 3217-3222Google Scholar). Thus, while RNOS oxidize αT near-quantitatively to the para-quinone, α-tocopheryl quinone, the major reaction product of γT with RNOS is the nitro-phenol, 5-nitro-γ-tocopherol (NGT, 3) (Fig. 1). Most importantly, nitration of γT is not affected by the simultaneous presence of αT, suggesting that the two forms of vitamin E may complement each other (8Christen S. Woodall A.A. Shigenaga M.K. Southwell-Keely P.T. Duncan M.W. Ames B.N. gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications.Proc. Natl. Acad. Sci. USA. 1997; 94: 3217-3222Google Scholar). Based on this observation, we speculated that γT is a target for nitration in vivo and may be involved in the detoxification of RNOS. To be able to test this hypothesis, we developed a highly specific and sensitive HPLC method for the detection of NGT in biological samples. The method is based on isocratic reversed-phase separation of extracted tocopherols and electrochemical detection using a dual-electrode coulometric cell. Using a zymosan-induced peritonitis model in rats, we found that NGT is present in normal blood plasma at a level of a few percent of its parent, γT, and is increased by 2-fold during acute inflammation. This relatively simple and highly specific HPLC method should be useful in clarifying the precise physiological role of γT.MATERIALS AND METHODSMaterialsαT and γT (purity >99.7%) were from Fluka. Their concentration was determined using ε294 nmEtOH = 3,265 M−1 cm−1 and ε298 nmEtOH = 3,810 M−1 cm−1 (9Bourgeois C. Determination of Vitamin E: Tocopherols and Tocotrienols. Elsevier Applied Science, London and New York1992Google Scholar), respectively. Butylated hydroxytoluene (BHT) and lithium acetate were from Aldrich, zymosan A from Sigma. Sodium dodecyl sulfate (SDS) was from US Biological and purified with hexane (10Lang J.K. Gohil K. Packer L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions.Anal. Biochem. 1986; 157: 106-116Google Scholar). All aqueous solutions were made with analytical-grade water prepared using a Milli-Q water purification system (Millipore). All solvents used were HPLC grade (Fisher Scientific), except for hexane (GC quality, from J. T. Baker) and ethanol [reagent grade, containing 5% (v/v) isopropanol, Fisher Scientific]. Mobile phases were filtered through 0.22 μm Durapore membranes (Millipore), degassed by sonication, and kept oxygen-free by continuous sparging with helium gas when in use.Zymosan peritonitis modelAnimal experiments were approved by the UC Berkeley Animal Care and Use Committee. In addition, experiments were carried out according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Male Fischer 344 rats obtained from Simonsen Laboratories (Gilroy, CA) were used at 15-weeks-of-age (∼320 g body weight) after acclimatization for 7 weeks on a standard Purina 5011 chow diet (Dyets, Bethlehem, PA). Peritonitis was induced as previously described (11Shigenaga M.K. Lee H.H. Blount B.C. Christen S. Shigeno E.T. Yip H. Ames B.N. Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection.Proc. Natl. Acad. Sci. USA. 1997; 94: 3211-3216Google Scholar). Briefly, zymosan A prepared in PBS and treated for 30 min at 95°C was injected intraperitoneally at 250 mg/kg body weight. Control animals received nonpyrogenic PBS. Since this treatment causes a marked decline in food consumption (12Demling R. LaLonde C. Ikegami K. Picard L. Nayak U. Alpha-tocopherol attenuates lung edema and lipid peroxidation caused by acute zymosan-induced peritonitis.Surgery. 1995; 117: 226-231Google Scholar), one control group consisted of animals fasted for the entire duration of the experiment. Twenty-four h after injection of zymosan, animals were anesthetized under ether and sacrificed by cervical dislocation after blood removal by subclavicular artery section. Heparinized plasma and excised tissues were stored immediately at −80°C until analysis.Synthesis of 5-nitro-γTNGT (2,7,8-trimethyl-2-[4′,8′,12′-trimethyltridecyl]-5-nitro-6-chromanol) was synthetized by the nitrous acid method as described previously (8Christen S. Woodall A.A. Shigenaga M.K. Southwell-Keely P.T. Duncan M.W. Ames B.N. gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications.Proc. Natl. Acad. Sci. USA. 1997; 94: 3217-3222Google Scholar). Briefly, an ethanolic solution of γT (1 mg/ml) was acidified with 0.04 vol glacial acetic acid and nitration induced by the addition of 0.6 vol of a 2% sodium nitrite solution. After 2 min, the reaction was stopped with 0.4 vol of 20% potassium hydroxide. Two volumes of water were added, the crude product extracted into hexane and further purified by semi-preparative reversed-phase HPLC using 100% methanol as the eluent, and peak-collected using diode-array detection (HP 1090). The concentration of the pooled fractions was determined using ε410 nmEtOH = 1,976 M−1 cm−1 (13Marcinkiewicz S. Complete analysis of tocopherol mixtures. III. New method of determination of tocopherols based on chromatography of their hydrogenation products and on spectrometry of their nitro derivatives.Chem. Anal. 1972; 17: 13-20Google Scholar). The purity of the product was >99%.Extraction procedureNGT and all other tocopherols were extracted from plasma into hexane based on previously published procedures (14Burton G.W. Webb A. Ingold K.U. A mild, rapid, and efficient method of lipid extraction for use in determining vitamin E/lipid ratios.Lipids. 1985; 20: 29-39Google Scholar, 15Sattler W. Mohr D. Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.Methods Enzymol. 1993; 233: 469-489Google Scholar). It is important that the extraction mixture has not been acidified, since even weak acidic conditions promote the nitration of γT when nitrite is present (cf. NGT synthesis). Generally, 0.2 ml of blood plasma was extracted with 7 ml of a 2:5 mixture (v/v) of methanol-hexane to which 20 μl of 0.1 M BHT was added. BHT is necessary to inhibit the formation of substances that interfere with NGT analysis. The hexane phase was evaporated under a stream of high-purity nitrogen gas. Extracts were redissolved in 160 μl reagent grade ethanol and immediately subjected to HPLC analysis. For low NGT levels, 0.4 ml of blood plasma can be extracted with double the volume of methanol-hexane, but redissolving in the same volume of ethanol. Although we recommend that extracts are analyzed immediately, they can be stored at −20°C for a few hours, without noticeable changes in analyte concentrations.HPLC analysisThe HPLC system consisted of a Hitachi (Tokyo, Japan) L-6200 gradient pump and a Hitachi L-7200 autosampler with built-in Peltier cooling unit set to 2°C. The electrochemical detection system consisted of a Model 5200A Coulochem II controller (ESA Inc., Chelmsford, MA) and a Model 5011 high sensitivity analytical cell. A membrane pulse damper (SSI) and Model 5020 guard cell were installed between the pump and the autosampler. The whole system was assembled with PEEK tubing and passivated with 20% nitric acid prior to use. UV detection was performed on a Spectroflow 757 detector (Applied Biosystems) connected in series after the electrochemical cell.NGT was separated from the other tocopherols on a 15 × 0.46 cm Supelcosil™ (Supelco, Bellefonte, PA) LC-18-DB column (deactivated octadecyl silane, 3 μm particle size) equipped with an LC-8 precolumn cartridge assembly and eluted isocratically with 95:5 (v/v) methanol-0.5 M lithium acetate (pH 4.75) at a flow rate of 1.3 ml/min. Generally, 50 μl of extract was injected. β-tocopherol is not separated from γT under reversed-phase conditions, but constitutes only a minor fraction (<10%) in blood plasma and other tissues of rats (16Schuep W. Rettenmaier R. Analysis of vitamin E homologs in plasma and tissue: high-performance liquid chromatography.Methods Enzymol. 1994; 234: 294-302Google Scholar) and humans (17Handelman G.J. Machlin L.J. Fitch K. Weiter J.J. Dratz E.A. Oral alpha-tocopherol supplements decrease plasma gamma-tocopherol levels in humans.J. Nutr. 1985; 115: 807-813Google Scholar). Also, 7-nitro-β-tocopherol elutes before NGT under reversed- phase conditions (18Morton L.W. Ward N.C. Croft K.D. Puddey I.B. Evidence for the nitration of gamma-tocopherol in vivo: 5-nitro-gamma-tocopherol is elevated in the plasma of subjects with coronary heart disease.Biochem. J. 2002; 364: 625-628Google Scholar). After the first 20 min, non-polar lipids and other interfering substances were washed off the column by running a linear gradient from the initial mobile phase to tert-butanol-methanol (50:50, v/v) within 20 min. After this step, initial conditions were restored within another 20 min. Coulometric detection was performed at +300 (E1; upstream) and +500 mV (E2; downstream electrode), unless indicated otherwise. Free cholesterol (FC) was monitored at 210 nm and used as an internal standard in the recovery studies.Lipid analysisThe quantitatively major cholesterol esters, cholesteryl oleate (Ch18:1), cholesteryl linoleate (Ch18:2), and cholesteryl arachidonate (Ch20:4) were determined together with FC in hexane extracts by HPLC with UV detection (15Sattler W. Mohr D. Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.Methods Enzymol. 1993; 233: 469-489Google Scholar). Total triglycerides (TG) were determined enzymatically (19Warnick G.R. Enzymatic methods for quantification of lipoprotein lipids.Methods Enzymol. 1986; 129: 101-123Google Scholar) using a commercial kit (Stanbio Laboratories, San Antonio, TX).Statistical analysisStatistical tests were performed using GraphPad Prism (San Diego, CA). Comparisons between the different groups were done by using the unpaired Student's t-test. A two-tailed P value <0.05 was considered statistically significant. All results are expressed as mean ± SD.RESULTSHPLC separation and electrochemical detectionSeparation of NGT was achieved by slight modification of methods described previously for analyzing unmodified tocopherols (10Lang J.K. Gohil K. Packer L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions.Anal. Biochem. 1986; 157: 106-116Google Scholar, 15Sattler W. Mohr D. Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.Methods Enzymol. 1993; 233: 469-489Google Scholar). NGT is conveniently separated on reversed-phase columns using short-chain alcohols as solvent, as are all other tocopherols (20Lang J.K. Schillaci M. Irvin B. Vitamin E.in: De Leenheer A.P. Lambert W.E. Nelis H.J. Modern Chromatographic Analysis of Vitamins. Marcel Dekker, Inc., New York1992: 153-195Google Scholar). Figure 2shows a typical chromatogram of authentic tocopherols resolved on a deactivated octadecylsilane column using a mobile phase containing methanol/water. NGT is retained much longer under reversed-phase conditions than its parent compound, similar to the situation with 3-nitrotyrosine. A deactivated column was required for adequate separation of biological samples. The cleaning cycle with tert-butanol/methanol after each injection was necessary for maintaining the high sensitivity of the assay.Fig. 2Chromatogram of a standard mixture of tocopherols using coulometric detection. A methanolic mixture of 16.6 pmol γ-tocopherol (γT) [1], 225 pmol α-tocopherol (αT) [2], and 3.4 pmol 5-nitro-γ-tocopherol (NGT) [3] was separated on a deactivated C18 column using 95:5 (v/v) methanol-0.5 M lithium acetate (pH 4.75) at a flow rate of 1.3 ml/min as described under Materials and Methods. Eluent was monitored by coulometric detection using a potential of +300 and +500 mV on the upstream (A) and downstream (B) electrode, respectively. After 20 min, the solvent was gradually changed to tert-butanol-methanol (50:50, v/v) to remove non-polar lipids and other interfering substances from the column.View Large Image Figure ViewerDownload (PPT)Electrochemical detection was chosen for analysis, because NGT is, in contrast to αT and γT, not fluorescent. Initially, we tried to chemically (using dithionite, borohydride, or Sn-borohydride under phase transfer conditions) or electrochemically reduce NGT to 5-amino-γT, a strategy previously employed for the detection of 3-nitrotyrosine (11Shigenaga M.K. Lee H.H. Blount B.C. Christen S. Shigeno E.T. Yip H. Ames B.N. Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection.Proc. Natl. Acad. Sci. USA. 1997; 94: 3211-3216Google Scholar, 21Liu H. Huang T. Kissinger C.B. Kissinger P.T. Comparison of detection methods for liquid chromatographic determination of 3-nitro-L-tyrosine.J. Chromatogr. B Biomed. Sci. Appl. 1998; 713: 289-295Google Scholar, 22Sodum R.S. Akerkar S.A. Fiala E.S. Determination of 3-nitrotyrosine by high-pressure liquid chromatography with a dual-mode electrochemical detector.Anal. Biochem. 2000; 280: 278-285Google Scholar), since nitration is known to increase the oxidation potential of phenolic compounds, and aminophenols are much more electrochemically active. However, none of these approaches nor UV photolysis (21Liu H. Huang T. Kissinger C.B. Kissinger P.T. Comparison of detection methods for liquid chromatographic determination of 3-nitro-L-tyrosine.J. Chromatogr. B Biomed. Sci. Appl. 1998; 713: 289-295Google Scholar) was successful. We therefore chose to analyze NGT directly in the oxidative mode. To achieve the highest possible sensitivity, we used coulometric detection using a high-sensitivity dual-electrode. One of the main advantages of using electrodes in series is that the near-quantitative conversion efficiency of coulometric detection can be used to selectively filter out interfering substances. Although amperometric detection can be readily used for measuring NGT in biochemical experiments (8Christen S. Woodall A.A. Shigenaga M.K. Southwell-Keely P.T. Duncan M.W. Ames B.N. gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications.Proc. Natl. Acad. Sci. USA. 1997; 94: 3217-3222Google Scholar), this type of detection was not sensitive enough to detect basal levels of NGT in biological samples.Sensitivity of coulometric detection (i.e., high signal to noise ratio) was greatly affected by the choice of electrolyte. Lithium acetate was found to be the best choice. Other electrolytes such as sodium perchlorate, lithium perchlorate, or ammonium acetate commonly used for electrochemical detection-based tocopherol HPLC assays (20Lang J.K. Schillaci M. Irvin B. Vitamin E.in: De Leenheer A.P. Lambert W.E. Nelis H.J. Modern Chromatographic Analysis of Vitamins. Marcel Dekker, Inc., New York1992: 153-195Google Scholar) gave inferior results. To determine the ideal working potentials, hydrodynamic voltammograms of NGT, αT, and γT were run. The voltammograms in Fig. 3clearly show that nitration increases the oxidation potential of γT. However, in contrast to 3-nitrotyrosine, this potential is still low enough to detect NGT directly in the oxidative mode without increasing background noise to unacceptably high levels. A potential of +300 mV was chosen on the first electrode to oxidize low-potential compounds such as αT, and to allow NGT to be selectively oxidized on the second electrode. A potential of +500 mV on the second electrode was chosen to give a good signal while maintaining high specificity for the analyte. Using these settings, detection of NGT was linear over more than four orders of magnitudes (Fig. 4, r 2 = 0.9999) and highly sensitive (∼10 fmol detection limit).Fig. 3Hydrodynamic voltammogram of tocopherols. A standard mixture containing 1 nmol each of αT (closed triangles), γT (open circles), and NGT (closed circles) was separated by HPLC as described in the legend to Fig. 2, and the eluant monitored with coulometric detection at increasing potentials up to +950 mV. Hydrodynamic voltammograms were obtained by calculating the percentage of the maximum peak area for each tocopherol and plotting these values versus the applied potential.View Large Image Figure ViewerDownload (PPT)Fig. 4Standard curve of NGT. NGT dissolved in methanol was analyzed under the same HPLC conditions as described in the legend to Fig. 2. Please note that detector response (peak area) was normalized to a full scale of 100 nA.View Large Image Figure ViewerDownload (PPT)Recovery, reproducibility, and stabilityUsing the extraction procedure described under Materials and Methods, NGT, γT, and αT were recovered from methanol with an efficiency of 94.6 ± 0.01, 95.3 ± 0.02, and 96.5 ± 0.01% (n = 4). NGT added to rat plasma and extracted several times on the same day was recovered with 84.7 ± 2.1% (n = 6) efficiency (2.5% CV). When the sample was extracted on different days, recovery was 84.6 ± 2.1% (n = 3) (2.5% CV). Repetitive analysis (n = 6) of the same plasma sample for endogenous NGT (∼0.01 μM) also had a low CV (5%). NGT added to rat plasma and incubated at 37°C was stable for at least 30 min (data not shown). Addition of physiological concentrations of nitrite (0.1 mM) to rat or human plasma supplemented with γT in vivo and incubation at 37°C did not result in the formation of artefactual NGT (data not shown). Evaporation of extracts by a stream of nitrogen did also not result in unwanted nitration of γT using the extraction procedure described under Materials and Methods. These results demonstrate that the extraction and analysis method described here are highly specific for NGT formed in vivo.Concentrations of tocopherols in rat blood plasmaWe next determined the levels of NGT, γT, and αT in rats injected intraperitoneally with zymosan. This treatment provokes a systemic inflammatory response characterized by bacterial translocation (23Deitch E.A. Specian R.D. Grisham M.B. Berg R.D. Zymosan-induced bacterial translocation: a study of mechanisms.Crit. Care Med. 1992; 20: 782-788Google Scholar) and marked increases in circulating cytokines (24Shayevitz J.R. Miller C. Johnson K.J. Rodriguez J.L. Multiple organ dysfunction syndrome: end organ and systemic inflammatory response in a mouse model of nonseptic origin.Shock. 1995; 4: 389-396Google Scholar), nitrite/nitrate (11Shigenaga M.K. Lee H.H. Blount B.C. Christen S. Shigeno E.T. Yip H. Ames B.N. Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection.Proc. Natl. Acad. Sci. USA. 1997; 94: 3211-3216Google Scholar, 25Cuzzocrea S. Filippelli A. Zingarelli B. Falciani M. Caputi A.P. Rossi F. Role of nitric oxide in a nonseptic shock model induced by zymosan in the rat.Shock. 1997; 7: 351-357Google Scholar), and 3-nitrotyrosine (11Shigenaga M.K. Lee H.H. Blount B.C. Christen S. Shigeno E.T. Yip H. Ames B.N. Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection.Proc. Natl. Acad. Sci. USA. 1997; 94: 3211-3216Google Scholar), and a profound decline in antioxidants such as ascorbate (26Demling R. Ikegami K. Picard L. Lalonde C. Administration of large doses of vitamin C does not decrease oxidant-induced lung lipid peroxidation caused by bacterial-independent acute peritonitis.Inflammation. 1994; 18: 499-510Google Scholar). Consistent with previous reports (12Demling R. LaLonde C. Ikegami K. Picard L. Nayak U. Alpha-tocopherol attenuates lung edema and lipid peroxidation caused by acute zymosan-induced peritonitis.Surgery. 1995; 117: 226-231Google Scholar), zymosan treatment led to a significant decrease in plasma αT (Table 1). The same was true for γT. However, this decrease could entirely be attributed to the complete inflammation-induced decline in food consumption, because plasma levels of αT and γT in zymosan-treated animals were identical to those in control animals fasted for 24 h. In contrast, plasma NGT levels were significantly higher in zymosan-treated animals compared to fasted controls. A representative HPLC chromatogram of plasma from a zymosan-treated animal is shown in Fig. 5. NGT (Fig. 5B, peak 3) was identified by coelution with an authentic standard and identical electrochemical behavior (i.e., voltammogram). The present method can also be used for the detection of NGT in tissue homogenates. The only difference is that SDS has to be added to the extraction mixture (14Burton G.W. Webb A. Ingold K.U. A mild, rapid, and efficient method of lipid extraction for use in determining vitamin E/lipid ratios.Lipids. 1985; 20: 29-39Google Scholar). Figure 6shows a typical chromatogram obtained from a liver homogenate.TABLE 1Plasma tocopherol and lipid concentrations 24 h after zymosan treatmentControl (5)Control, Fasted (4)Zymosan (4)μmol/lTocopherolsαT11.1 ± 2.26.9 ± 0.6aSignificantly different from control, P < 0.01. 6.9 ± 1.3γT0.42 ± 0.150.11 ± 0.02aSignificantly different from control, P < 0.01. 0.11 ± 0.03NGT0.012 ± 0.0040.005 ± 0.001aSignificantly different from control, P < 0.01.0.009 ± 0.002bSignificantly different from control, fasted, P < 0.01.mmol/lLipidsFC0.28 ± 0.040.20 ± 0.02aSignificantly different from control, P < 0.01. 0.36 ± 0.06bSignificantly different from control, fasted, P < 0.01.Ch18:10.05 ± 0.010.06 ± 0.01 0.05 ± 0.03Ch18:20.26 ± 0.040.19 ± 0.03cSignificantly different from control, P < 0.05. 0.09 ± 0.04bSignificantly different from control, fasted, P < 0.01.Ch20:40.43 ± 0.080.53 ± 0.04 0.23 ± 0.10bSignificantly different from control, fasted, P < 0.01.TC1.01 ± 0.140.98 ± 0.10 0.73 ± 0.19TG2.67 ± 1.601.61 ± 0.57 1.61 ± 0.18TNL3.62 ± 1.462.59 ± 0.65 2.34 ± 0.22Tocopherols, correctedαT (per 103 TNL)3.3 ± 0.72.8 ± 0.6 2.9 ± 0.3γT (per 103 TNL)0.12 ± 0.030.05 ± 0.00dSignificantly different from control, P < 0.001. 0.05 ± 0.01NGT (per 103 TNL)0.004 ± 0.0010.002 ± 0.000aSignificantly different from control, P < 0.01.0.004 ± 0.001bSignificantly different from control, fasted, P < 0.01.NGT (%γT)3.7 ± 0.44.0 ± 0.6 7.7 ± 0.5eSignificantly different from control, fasted, P < 0.001.αT, α-tocopherol; Ch18:1, cholesteryl oleate; Ch18:2, cholesteryl linoleate; Ch20:4, cholesteryl arachidonate; FC, free cholesterol; γT, γ-tocopherol; NGT, 5-nitro-γ-tocopherol; TC, total cholesterol; TG, triglycerides; TNL, total neutral lipids. Total cholesterol (TC) = [FC] + [Ch18:1] + [Ch18:2] + [Ch20:4]. Total neutral lipids (TNL) = [TC] + [TG]. Values represent mean ± SD. Number of animals used in each group are indicated in parentheses.a Significantly different from control, P < 0.01.b Significantly different from control, fasted, P < 0.01.c Significantly different from control, P < 0.05.d Significantly different from control, P < 0.001.e Significantly different from control, fasted, P < 0.001. Open table in a new tab Fig. 5Typical chromatogram of tocopherols in blood plasma of a zymosan-treated rat. Hexane phase of plasma extract was redissolved in ethanol and separated by HPLC as described in the legend to Fig. 2. Eluant was monitored simultaneously by coulometric (A and B) and UV detection at 210 nm (C). Peak identification: γT [1], αT [2], NGT [3], and free cholesterol (FC) [4]. The NGT peak in this chromatogram represents ∼0.5 pmol of analyte on column.View Large Image Figure ViewerDownload (PPT)Fig. 6Typical chromatogram of tocopherols in liver homogenate of a zymosan-treated rat. Tissue was homogenized in 10 vol (v/w) sodium acetate buffer (0.1 M, pH 7.2). One tenth of a milliliter SDS (1%, w/v) and 20 μl butylated hydroxytoluene (0.1 M) were added to 0.4 ml homogenate and mixed vigorously. Then, 2 ml of ice-cold methanol was added and mixed for 30 s. Finally, lipophilic antioxidants were extracted into 5 ml of ice-cold hexane b" @default.
• W2112248863 created "2016-06-24" @default.
• W2112248863 creator A5039848179 @default.
• W2112248863 creator A5047067531 @default.
• W2112248863 creator A5054567757 @default.
• W2112248863 creator A5060420474 @default.
• W2112248863 date "2002-11-01" @default.
• W2112248863 modified "2023-09-24" @default.
• W2112248863 title "Analysis of plasma tocopherols α, γ, and 5-nitro-γ in rats with inflammation by HPLC coulometric detection" @default.
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