Facilitated nitration and oxidation of LDL in cigarette smokers.

BACKGROUND: Cigarette smoking increases the risk of developing atherosclerosis and ischaemic heart disease. Smoking-induced oxidative stress is considered to favour oxidation of low-density lipoprotein (LDL) and subsequently promotes the atherogenic process. We investigated whether peroxynitrite, a reaction product of cigarette smoke, is involved in facilitated oxidation of LDL in smokers. MATERIALS AND METHODS: Plasma LDL was obtained from 10 healthy asymptomatic cigarette smokers and 10 healthy nonsmokers. The state of enhanced oxidative stress in the plasma was assessed by LDL subfraction assay using anion-exchange high-performance liquid chromatography (AE-HPLC) and measurements of thiobarbituric acid-reactive substances (TBARS), 8-hydroxydeoxyguanosine (8-OHdG), vitamin E, 3-nitrotyrosine and 3-chlorotyrosine. RESULTS: Smokers showed a significantly higher level of TBARS and 8-OHdG as well as a significantly lower level of vitamin E than nonsmokers, even after stopping smoking for 10 h or more. The LDL subfraction assay demonstrated an increase in oxidatively modified LDL, as expressed by lower levels of LDL1 and higher levels of LDL2. The 3-nitrotyrosine levels in apolipoprotein B in LDL were significantly higher in smokers than nonsmokers, while the 3-chlorotyrosine levels remained unchanged. In addition, these changes observed in the smokers were further accelerated within 30 min after resumption of cigarette smoking when compared with the levels before smoking resumption. CONCLUSION: The present study suggests that peroxynitrite plays a significant role in oxidative modification of plasma LDL induced by cigarette smoking.

Separation of basic drug enantiomers by capillary zone electrophoresis using glucuronyl glucosyl beta-cyclodextrin as a chiral selector.

Separations of basic drug enantiomers have been investigated using glucuronyl glucosyl beta-cyclodextrin (GUG beta-CD) as a chiral selector in the background electrolyte by capillary zone electrophoresis. The effects of GUG beta-CD concentration and running buffer pH on the migration times and resolution of 16 basic drug enantiomers were precisely examined using a linear polyacrylamide-coated capillary. High resolution of 16 basic drug enantiomers was generally attained with a running buffer pH 2.5 or 3.5 containing 10 mM GUG beta-CD. Next, we compared the chiral resolution abilities of GUG beta-CD with those of beta-CD and maltosyl beta-CD (G2 beta-CD). GUG beta-CD showed higher resolution for basic drug enantiomers tested than beta-CD and G2 beta-CD. This could be due to that hydrogen bonding or ionic interactions of uncharged and charged glucuronyl glucosyl groups of GUG beta-CD with an analyte could stabilize the inclusion complex.

Separation of basic drug enantiomers by capillary electrophoresis using ovoglycoprotein as a chiral selector: comparison of chiral resolution ability of ovoglycoprotein and completely deglycosylated ovoglycoprotein.

Separations of basic drug enantiomers by capillary electrophoresis (CE) using ovoglycoprotein (OGCHI) as a chiral selector are described. The effects of running buffer pH and 2-propanol content on the migration times and resolution of basic drug enantiomers were examined using a linear polyacrylamide-coated capillary. High resolution of basic drug enantiomers was attained using a mixture of 50 mM sodium phosphate buffer (pH 4.5-6.0) and 2-propanol (5-30%) including 50 microM OGCHI. It was found that ionic and hydrophobic interactions could work for the recognition of basic drug enantiomers. Further, we compared the chiral resolution ability of OGCHI with that of completely deglycosylated OGCHI (cd-OGCHI) using them as chiral selectors in CE. OGCHI showed higher resolution for basic drug enantiomers tested than cd-OGCHI. The results suggest that the chiral recognition site(s) for OGCHI exists on the protein domain of OGCHI.

Uniformly sized molecularly imprinted polymer for (S)-naproxen retention and molecular recognition properties in aqueous mobile phase.

A uniformly sized molecularly imprinted polymer (MIP) for (S)-naproxen has been prepared by a multi-step swelling and polymerization method using 4-vinylpyridine (4-VPY) and ethylene glycol dimethacrylate (EDMA) as a functional monomer and cross-linker, respectively. We optimized the preparation method of the MIP by changing the molar amounts of the template molecule and functional monomer. Further, we examined the effects of organic modifier type, column temperature and flow-rate on the retentivity and enantioselectivity for naproxen using a mixture of phosphate buffer and organic modifier (acetonitrile, ethanol and 2-propanol) as an eluent. When the amounts of (S)-naproxen, 4-VPY and EDMA used were 4, 6 and 25 mmol, respectively, the enantioselectivity and resolution for naproxen were good despite the shorter retention. When acetonitrile was used as an organic modifier, the highest column efficiency was obtained for the separation of naproxen enantiomers. With regard to the effects of column temperature and flow-rate, the column performance was improved by elevating a column temperature and decreasing a flow-rate.

Oxidants in cigarette smoke extract modify low-density lipoprotein in the plasma and facilitate atherogenesis in the aorta of Watanabe heritable hyperlipidemic rabbits.

Epidemiological studies have shown that cigarette smoking is a major cause of atherosclerosis. Oxidants as well as nicotine in cigarette smoke have been implicated in atherogenesis. To clarify the mechanism involved, we examined the chronic effects of nicotine and nicotine-free cigarette smoke extracts (CSE) on oxidative modification of low-density lipoprotein (LDL) in the plasma of Watanabe heritable hyperlipidemic rabbits and atherogenesis in the aorta. CSE was prepared by bubbling the gas phase of smoke (1 ml/three cigarettes) into phosphate buffer saline, and 3 ml of this CSE was injected daily into the ear vein of the rabbit for five months. The rabbits treated with CSE showed an increase in lipid peroxide levels, estimated as thiobarbituric acid reactive substances (TBARS), with a corresponding decrease in vitamin E levels in the plasma. They also showed enhanced oxidative modification of LDL, assessed by anion-exchange HPLC, incorporation into macrophages and measurement of TBARS. These events could be efficiently prevented by administering vitamin E (150 mg/kg/day, p.o.). Nicotine alone (0.5 mg/kg/day, s.c.) led to a temporary increase in the plasma triglyceride level. At the end of the experiment, CSE but not nicotine had caused progression of atherosclerotic lesions together with accumulation of cholesteryl ester in the thoracic aorta, while vitamin E had significantly prevented such atheromatous formation. These results indicate that oxidants in CSE can promote the development of atherosclerosis through oxidative modification of plasma LDL, particularly in hypercholesterolemia, and offer evidence for increased vitamin E utilization in smokers.

Anion-exchange high-performance liquid chromatography assays of plasma lipoproteins and modified low-density lipoproteins using a ProtEx-DEAE column.

Previously [Anal. Biochem., 232 (1995) 163-171], we reported a high-performance liquid chromatography (HPLC) assay method for human plasma lipoproteins using a diethylaminoethyl (DEAE)-glucomannan column, which is not commercially available. In this study, HPLC assay methods for lipoproteins in plasma samples of human and experimental animals, and modified low-density lipoproteins (LDLs) of rabbits have been developed using a commercially available anion-exchange ProtEx-DEAE column. For the assays of plasma lipoproteins, the method includes complete separation of high-density lipoproteins, LDLs and very low-density lipoproteins within 20 min using stepwise elution, and determination by post-column reaction with an enzymatic cholesterol reagent as the total cholesterol (TC) level. Similarly, mild oxidative and artificially oxidised LDLs were separated into their subfractions using stepwise elution, and determined based on the TC level. The methods using the DEAE-glucomannan and ProtEx-DEAE columns were cross-validated. There was an excellent correlation between the two methods. The obtained results reveal that the anion-exchange HPLC method using the ProtEx-DEAE column could be useful for the assays of plasma lipoproteins and modified LDLs.

Protein-based chiral stationary phases for high-performance liquid chromatography enantioseparations.

The enantioseparations of various compounds using proteins as the chiral selectors in high-performance liquid chromatography (HPLC) are considered in this review. The proteins used include albumins such as bovine serum albumin and human serum albumin, glycoproteins such as alpha1-acid glycoprotein, ovomucoid, ovoglycoprotein, avidin and riboflavin binding protein, enzymes such as trypsin, alpha-chymotrypsin, cellobiohydrolase I, lysozyme, pepsin and amyloglucosidase, and other proteins such as ovotransferrin and beta-lactoglobulin. This review deals with the properties of HPLC chiral stationary phases based on proteins, and the enantioselective properties and chiral recognition mechanisms of these stationary phases.

HPLC-based bioseparations using molecularly imprinted polymers.

HPLC-based separations of amino acids and peptides, nucleotide bases, drugs, sugars and steroids using molecularly imprinted polymers (MIPs) have been reviewed in this article. The molecular recognition mechanisms of the template molecules on the MIPs in organic and aqueous eluents were discussed. Furthermore, new polymerization methods suitable for preparations of HPLC columns and packing materials using molecular imprinting techniques, and their applications to HPLC-based separations are also dealt with.

Effects of trimethylsilylation of copper(II)-phthalocyanine sulfonyl-aminopropyl silica gels on the separation of pi-electron-rich compounds by high-performance liquid chromatography.

As an attempt to elucidate the factor(s) responsible for the poor performance of a copper(II)-phthalocyanine aminopropylsilica gels (CU-PCSD) column for HPLC, the silanol and/or amino groups remaining on Cu-PCSD were endcapped with trimethylchlorosilane (TMCS) or N-trimethylsilylimidazole (TMSI). The trimethylsilylated Cu-PCS(D)S (Cu-PCSD-TMCS and -TMSI) were investigated concerning their performance as an HPLC-stationary phase in the separation of pi-electron-rich polyaromatic hydrocarbons (PAHs), such as mutagenic anthracene and pyrene. As a result, trimethylsilylation with TMSI, which reacts only with silanol-groups, was not effective to improve the column efficiency. In contrast, trimethylsilylation by TMCS, which reacts with both the silanol- and amino-groups, improved the theoretical plate numbers (N) for PAHs separation with the Cu-PCS(D) column, indicating that the low N values on the Cu-PCSD column were caused by electrostatic interactions between PAHs and the remaining amino-groups on Cu-PCS(D). Furthermore, the retention data of mutagenic heterocyclic amines (HCAs) indicated that the remaining amino groups interact with the polar groups of HCAs.

Uniform-sized molecularly imprinted polymers for 2-arylpropionic acid derivatives selectively modified with hydrophilic external layer and their applications to direct serum injection analysis.

Uniform-sized molecularly imprinted polymers (MIPs) for (S)-naproxen and -ibuprofen selectively modified with hydrophilic external layer, restricted access media (RAM)-MIPs, have been prepared. First, the MIP for (S)-naproxen or -ibuprofen was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multistep swelling and thermal polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and it was added directly to the MIP for (S)-naproxen or -ibuprofen 4 h after the start of molecular imprinting. The obtained RAM-MIP material for (S)-naproxen or -ibuprofen was applied for direct serum injection assays of the drug by a column-switching system, consisting of a RAM-MIP material and conventional C18-silica column. However, leakage of the imprint molecule prevented accurate and precise assays of the drug. This problem has been overcome by using the RAM-MIP for (S)-naproxen for the assays of ibuprofen in rat plasma. The optimized column-switching system was applied successfully to the assay of ibuprofen in rat plasma after oral administration.

Evidence of modified LDL in the plasma of hypercholesterolemic WHHL rabbits injected with aqueous extracts of cigarette smoke.

Cigarette smoking is a major risk factor for atherosclerosis, and oxidative modification of low density lipoprotein (LDL) is a key event in the development of atherosclerosis. The aim of the present study was to determine whether modified LDL would be formed in the plasma of Watanabe heritable hyperlipidemic (WHHL) rabbits injected with nicotine-free cigarette smoke extracts (CSE). In order to assess this, cigarette smoke-modified LDL (CS-LDL) was prepared by incubation of rabbit native LDL (N-LDL) with CSE for 24 h. The oxidative modification in CS-LDL was well established by the reduced ratio between two LDL subfractions (LDL2/LDL3) separated by anion-exchange HPLC, together with the fast migration in the anodic direction in agarose gel electrophoresis and the increased lipid peroxide levels. Very similar modification was noted with mildly oxidatively modified LDL prepared by incubation of N-LDL with 5 µM CuCl(2) for 1 h. When WHHL rabbits (n=4) intravenously received a single injection of CSE, the ratio of LDL2/LDL3 was markedly reduced compared with the control rabbits (n=4) while total cholesterol levels in the plasma gradually decreased until 24 h after the injection. These results suggest that oxidatively modified LDL, probably like CS-LDL, is produced in the plasma of WHHL rabbits injected with CSE.

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. VIII. Chiral recognition ability of partially and completely deglycosylated ovoglycoprotein.

The influence of sugar moieties of ovoglycoprotein from chicken egg white (OGCHI) on chiral discrimination of various solutes has been investigated. Partially deglycosylated OGCHI (pd-OGCHI) and completely deglycosylated OGCHI (cd-OGCHI) were obtained by treatments of OGCHI with N-glycosidase, and a mixture of endoglycosidase and N-glycosidase, respectively. The average molecular masses of OGCHI, pd-OGCHI and cd-OGCHI were estimated to be about 30 000, 28 400 and 21 400, respectively, by matrix-assisted laser desorption time-of-flight mass spectrometry. The isoelectric points of OGCHI, pd-OGCHI and cd-OGCHI were in the ranges 4.37-4.51, 4.34-4.44 and 4.17-4.43, respectively, by isoelectric focusing. The OGCHI, pd-OGCHI and cd-OGCHI were bound to aminopropyl-silica gels activated with N,N'-disuccinimidylcarbonate to compare retentive and enantioselective properties of the three columns. It was found that pd-OGCHI showed excellent chiral recognition abilities comparable to OGCHI, and that the retentivity and enantioselectivity of basic solutes tested on the pd-OGCHI column were higher than those on the OGCHI column, while those of acidic solutes tested on the pd-OGCHI column were lower. Further, cd-OGCHI still showed chiral recognition abilities for various solutes tested. These results reveal that the chiral recognition site(s) for OGCHI exists on the protein domain of OGCHI.

Uniform-sized molecularly imprinted polymer material for (S)-propranolol.

A uniform-sized molecularly imprinted polymer for (S)-propranolol has been prepared by a multi-step swelling and thermal polymerization method using methacrylic acid (MAA) and ethylene glycol dimethacrylate (EDMA) as a host functional monomer and cross-linker, respectively. The obtained (S)-propranolol imprinted MAA-EDMA materials were evaluated using aqueous-rich eluents by HPLC. The (S)-propranolol imprinted MAA-EDMA materials had specific recognition for (S)-propranolol and moderate recognition for some structurally related beta-adrenergic antagonists (especially, pindolol and alprenolol), but had no recognition for other basic compounds, and neutral and acidic compounds. Enantioseparations of propranolol and some structurally related beta-adrenergic antagonists were attained with the imprinted MAA-EDMA materials. Hydrophobic interaction of the naphthyloxy and isopropyl groups of propranolol, and ionic interaction of the isopropylamino group of propranolol with the (S)-propranolol imprinted MAA-EDMA materials could play an important role in enantioselective recognition of propranolol.

Enantiomer separation of drugs by capillary electrophoresis using proteins as chiral selectors.

The separation of drug enantiomers using proteins as the chiral selectors in capillary electrophoresis (CE) is considered in this review. The proteins used include albumins such as bovine serum albumin, human serum albumin and serum albumins from other species, glycoproteins such as alpha1-acid glycoprotein, crude ovomucoid, ovoglycoprotein, avidin and riboflavin binding protein, enzymes such as fungal cellulase, cellobiohydrolase I, pepsin and lysozyme and other proteins such as casein, human serum transferrin and ovotransferrin. Protein-based CE is carried out in two modes: in one proteins are immobilized or adsorbed within the capillary, or protein-immobilized silica gels are packed into the capillary (affinity capillary electrochromatography mode), and in the other proteins are dissolved in the running buffer (affinity CE mode). Furthermore, the advantages and limitations of the two modes and the factors affecting the chiral separations of various drugs by protein-based CE are discussed.

Peroxynitrite-generating species: good candidate oxidants in aqueous extracts of cigarette smoke.

Cigarette smoking is a well-known risk factor for atherosclerosis, but the mechanism of the adverse biological effect of smoking remains to be established. Cigarette smoke contains high concentrations of free radicals and oxidants. We show here that cigarette smoke extracts (CSE), prepared by bubbling the gas phase of smoke into phosphate-buffered saline, could convert tyrosine to 3-nitrotyrosine. The tyrosine nitration terminated 6 h after incubating tyrosine with CSE at 37 degrees C. These results indicate that the active oxidants in CSE are peroxynitrite-generating species like 3-morpholinosydnonimine (SIN-1), suggesting that they modify plasma lipoproteins and contribute to the pathogenesis of atherosclerosis.

Separation of enantiomers on HPLC chiral stationary phases based on human plasma alpha1-acid glycoprotein: effect of sugar moiety on chiral recognition ability.

HPLC chiral stationary phases based on human plasma alpha1-acid glycoprotein (AGP) and partially deglycosylated AGP (pd-AGP) were prepared to investigate the effects of sugar moiety of AGP on chiral discrimination of various solutes. Removal of a sugar moiety of AGP by treatment with N-glycosidase was confirmed by high-performance capillary electrophoresis, reversed-phase HPLC and matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrometry. The average molecular weights of AGP and pd-AGP were estimated to be ca. 33,000 and 30,600, respectively, by MALDI-TOF mass spectrometry. Next, AGP and pd-AGP were bound to aminopropyl-silica gels activated with N,N '-disuccinimidylcarbonate. The retentivity+ and enantioselectivity of the neutral, acidic and basic solutes tested on the pd-AGP column were significantly or not significantly larger in most solutes than those on the AGP column. This is ascribable to that by cleavage of a sugar chain(s) by N-glycosidase, pd-AGP could become more hydrophobic than AGP, and/ or that a solute could be easily accessible to the specific and/or non-specific binding sites of pd-AGP. It is interesting that warfarin enantiomers are not resolved on the pd-AGP column, but resolved on the AGP column. A sugar chain(s) of AGP cleaved by N-glycosidase might be involved in the enantioselective binding of warfarin enantiomers.

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. VII. Comparison of chiral recognition ability of ovoglycoprotein from chicken and Japanese quail egg whites.

Ovoglycoproteins from chicken and Japanese quail egg whites (OGCHI and OGJPQ, respectively) were isolated, and characterized by isoelectric focusing, high-performance capillary electrophoresis, reversed-phase HPLC and matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS. The isoelectric point (pI) of natural OGCHI was 4.37-4.51 by isoelectric focusing, while natural OGJPQ showed two discrete bands at pI 4.68 and 4.77. The average molecular masses of natural OGCHI and OGJPQ were estimated to be about 30000 and 27400 by MALDI-TOF-MS. Both natural OGCHI and OGJPQ were either fully or partially glycosylated with a ratio of ca. 3:1. Next, natural OGCHI and OGJPQ were bound to aminopropyl-silica gels activated with N,N'-disuccinimidylcarbonate to compare retentive and enantioselective properties of the two columns. The OGCHI column is suitable for chiral resolution of basic compounds, while the OGJPQ column is suitable for that of acidic compounds. With regard to chiral resolution of neutral compounds, it is dependent on a compound resolved which column could be suitable. Differences in the retentivity and enantioselectivity between OGCHI and OGJPQ columns are due to differences in the enantioselective binding properties. The results obtained reveal that chiral recognition of various solutes could be efficiently attained by using both columns complementarily.

High-performance liquid chromatographic assay of hydroperoxide levels in oxidatively modified lipoproteins.

An anion-exchange HPLC method has been developed for the chemiluminescence (CL) assay of hydroperoxide (HPO) levels in native and oxidized low density lipoproteins (N- and Ox-LDLs, respectively) of Watanabe heritable hyperlipidemic (WHHL) rabbits. The method involves anion-exchange HPLC separation in N- and Ox-LDLs using a DEAE-glucomannan gel, and direct CL detection of HPOs in them without extraction of the lipids following postcolumn reaction with isoluminol, microperoxidase and Triton X-100. Addition of Triton X-100, which could solubilize lipids, were essential for the detection of HPOs in N- and Ox-LDLs. With an increase in the degree of oxidation, Ox-LDL was more retained on the DEAE-glucomannan gel with a concomitant increase in the CL intensity. The proposed method could analyze the HPO levels in N- and Ox-LDLs of WHHL rabbits without extraction of the lipids.