Potential thiobarbituric acid-reactive substances in peroxidized lipids

This paper describes what components produce a red pigment in the thiobarbituric acid (TBA) test of peroxidized lipids. Alk-2-enals, alka-2,4-dienals and hydroperoxide functions are potential candidates for the red pigment in the TBA test. The TBA test can be regarded as an excellent method to reflect the combined effect of these components and thus the degree of lipid oxidation.     

Determination of thiopental in urine sample with high-performance liquid chromatography using iodine-azide reaction as a postcolumn detection system

The reaction between iodine and azide ions induced by thiopental was utilized as a postcolumn reaction for chromatographic determination of thiopental. The method is based on the separation of thiopental on an Nova-Pak CN HP column with an acetonitrile-aqueous solution of sodium azide as a mobile phase, followed by spectrophotometric measurement of the residual iodine (lambda=350 nm) from the postcolumn iodine-azide reaction induced by thiopental after mixing an iodine solution containing iodide ions with the column effluent containing azide ions and thiopental. Chromatograms obtained for thiopental showed negative peaks as a result of the decrease in background absorbance. The detection limit (defined as S/N=3) was 20 nM (0.4 pmol injected amount) for thiopental. Calibration graphs, plotted as peak area versus concentrations, were linear from 40 nM. The elaborated method was applied to determine thiopental in urine samples. The detection limit (defined as S/N=3) was 0.025 nmol/ml urine. Calibration graphs, plotted as peak area versus concentrations, were linear from 0.05 nmol/ml urine. Authentic urine samples were analyzed, thiopental was determined at nmol/ml urine level.     

Determination of a guanosine—malonaldehyde adduct in urine by high-performance liquid chromatography with a thiobarbituric acid reaction detector

A new method for the determination of a guanosine—malonaldehyde adduct, β--ribofuranosylpyrimido[1,2-a]purin-10(3H)-one (GMA), in rat and human urine is described. The method involves rapid pretreatment using, in sequence, polyamide, ion-exchange and reversed-phase cartridges; determination is by means of high-performance liquid chromatography with a thiobarbituric acid reactor in series with a fluorescence detector. This device can quantitatively determine the adduct at the sub-picomole level. This rapid, selective and sensitive method is suitable for the determination of guanine—malonaldehyde adducts in biological samples, such as human and rat urine. A semi-preparative method for the extraction and purification of these adducts from rat urine and for their identification by mass spectrometry and high-performance liquid chromatography with ultraviolet detection is also reported.     

Beta-carotene generates thiobarbituric acid-reactive substances by interaction with nitrogen dioxide in air

Generation of thiobarbituric acid-reactive substances (TBARS) from methyl linoleate in the exposure of nitrogen dioxide/air was inhibited by β-carotene in a dose-dependent manner. However, introduction of nitrogen dioxide/air or oxygen into a solution of β-carotene generated a significant amount of TBARS accompanying loss of its characteristic yellow color. Storing β-carotene in a solid state at ambient temperatures in air generated a large amount of TBARS accompanying loss of its yellow color. TBARS from β-carotene may interfere the measurement of TBARS from polyunsaturted fatty acids, and may give undesirable effects on biomaterials.     

Assay of biological thiols by a combination of high-performance liquid chromatography and postcolumn reaction with 6,6'-dithiodinicotinic acid

A simple and rapid method for the determination of nanomole levels of biological thiols is described. The analysis is based on the combination of reverse-phase high-performance liquid chromatography with a postcolumn reaction with 6,6'-dithiodinicotinic acid. Thiols, including cysteine, cysteamine, thiolhistidine, homocysteine, glutathione, penicillamine, ergothioneine, and thiouracil were separated by eluting with 33 mM KH2PO4 at pH 2.2. Glutathione, cysteine, cysteamine, homocysteine, and penicillamine were quantitatively determined with detection limits of 0.1 nmol, while the quantitative detection of thiolhistidine, ergothioneine, and thiouracil was not successful. The method was applied to the assay of glutathione in human erythrocytes and Escherichia coli.     

Determination of methylguanidine in plasma and urine by high-performance liquid chromatography with fluorescence detection following postcolumn derivatization

A high-performance liquid chromatographic method was developed for the determination of methylguanidine in biological fluids. Methylguanidine and the internal standard were isolated from plasma by cation-exchange solid-phase extraction prior to chromatographic analysis. Urine samples were diluted and injected directly onto the analytical column. Chromatographic separation was carried out on an Ultrasil cation-exchange column using a mixture of methanol and monochloroacetate (15/85, v/v) as the mobile phase. Postcolumn derivatization of methylguanidine was carried out using alkaline ninhydrin reagent and the resulting fluorescent product was detected on-line. The method was specific, sensitive, reproducible, and linear over a wide a range of concentrations. The lower limit of detection for methylguanidine in plasma and urine was 1 and 100 ng/ml, respectively. The method was successfully employed for quantification of the levels of methylguanidine in normal and uremic human subjects, normal dogs, and dogs with ischemic-induced acute or spontaneous chronic renal failure.     

Acetylcholine measurement by high-performance liquid chromatography using an enzyme-loaded postcolumn reactor

Choline oxidase and cholinesterase were found to retain their activity for 1-2 weeks at room temperature while adsorbed to a commercially available anion-exchange cartridge. These enzymes convert acetylcholine to H2O2. Acetylcholine can be measured in tissue extracts by separation at pH 7 on a polymeric reverse-phase high-performance liquid chromatography column, conversion of acetylcholine to H2O2 on a postcolumn enzyme-loaded anion-exchange cartridge, and electrochemical detection of the H2O2 formed.     

A selective postcolumn o-phthalaldehyde-derivatization system for the determination of histamine in biological material by high-performance liquid chromatography

Based on studies of the reaction between histamine and o-phthalaldehyde in alkaline solution, a method optimized for the determination of histamine in biological samples by means of HPLC and postcolumn o-phthalaldehyde derivatization has been developed. The method permits determination of histamine even at low-picomolar levels. By means of a valve, placed immediately after the column outlet, the eluent stream can be switched between the fluorimetric and an electrochemical detector system whereby electroactive biogenic amines may also be studied under the same chromatographic conditions.     

A method to reduce interference by sucrose in the detection of thiobarbituric acid-reactive substances

A thiobarbituric acid (TBA) reaction for measuring lipid peroxidation products was evaluated for interference by several ingredients commonly used in solutions to prepare or analyze tissue homogenates or subcellular organelles. These included sucrose (up to 100 mm final concentration in the assay medium), Tris-maleate (up to 40 mm), imidazole (up to 20 mm), inorganic phosphate (up to 10 mm), and 4-morpholinepropanesulfonic acid (up to 20 mm). When the samples were heated at 95°C as recommended in some procedures, only sucrose significantly affected color development. Sucrose concentrations as low as 10 mm significantly increased absorbance at 532 nm of aqueous tetramethoxypropane (TMP) standards, and so the assay could not be applied reliably to tissue samples prepared in sucrose. Sucrose interference was only partially reduced by subsequent organic extraction (n-butanol plus pyridine), with measured absorbances remaining significantly greater (50–100%) than sucrose-free controls at sucrose concentrations of 20 mm or more. Modifying the assay to include sucrose in blanks and TMP standards failed to adequately correct for interference when the absorbance of unextracted (aqueous) solutions was measured. Further modification by adding sucrose to blanks and TMP standards, followed by butanolpyridine extraction, gave standard curves that were linear, through the origin, and had slopes equivalent to those of sucrose-free standards. This modification enabled almost complete recovery (average 2% error) of known amounts of TMP added to aliquots of tissue homogenates containing amounts of sucrose that otherwise significantly interfered. Also, with the modified method the content of TBA-reactive substances in tissues homogenized in sucrose was found to be not significantly different from that measured in tissues homogenized in a noninterfering substance, KCl.     

Analysis of tamoxifen-DNA adducts by high-performance liquid chromatography using postcolumn online photochemical activation

Tamoxifen, a widely used nonsteroidal antiestrogen in the treatment of breast cancer, forms several metabolites. 4-Hydroxytamoxifen (4-OHTam), a metabolite found in the bloodstream, has much higher affinity for the estrogen receptor than tamoxifen itself. Oxidative activation of 4-OHTam induces DNA damage. DNA isolated from HL-60 cells exposed to 10 microM 4-OHTam in the presence of 1 microM hydrogen peroxide was digested enzymatically to release both normal and modified nucleosides. The modified nucleosides were enriched by butanol extraction. Using UV detection, HPLC analysis of the butanol extract from 200 microg DNA digest detected approximately 4 4-OHTam-dG adducts per 10(7) nucleotides (n = 3). Online postcolumn UV irradiation in HPLC and fluorescence detection improved the detection sensitivity by 3 x 10(2) times. Using 4-OHTam as an example, this report demonstrated for the first time the power of the technique to assay tamoxifen-DNA adducts directly in the DNA digest without relying on postlabeling.     

Determination of cystamine by high-performance liquid chromatography

A highly sensitive and specific assay method for cystamine using high-performance liquid chromatography has been developed. The method is based on postcolumn derivatization of cystamine with o-phthaladehyde in the presence of 2-mercaptoethanol and sodium hypochlorite. The separation of cystamine was achieved using a cation exchange column (ISC-05/S0504). The assay was linear over the concentration range of 2 to 200 pmol. For the application of this assay method to biological materials, the pretreatment with a cation exchange column (Dowex 50W X 8) was necessary to remove interfering o-phthaladehyde-reactive substances. Since cysteamine in biological materials was quantitatively converted to cystamine during these sampling procedures, this method was found to be suitable for assaying the cysteamine plus cystamine content in various organs and tissues. The cysteamine-cystamine content in various tissues of rat determined by the present assay method has been presented.     

Microdetermination of hyaluronan in human plasma by high-performance liquid chromatography with a graphitized carbon column and postcolumn fluorometric detection

A chemical method for the determination of hyaluronan (hyaluronic acid, HA) has been developed and applied to the human blood plasma. Human blood plasma HA was converted to the ΔDi-HA by digestion with hyaluronidase SD and determined by a sensitive and selective high-performance liquid chromatography (HPLC). The HPLC includes the separation and detection of ΔDi-HA using a graphitized carbon column and fluorometric reaction with 2-cyanoacetamide in an alkaline eluent. The calibration graph for ΔDi-HA was linear over the range 0.2 ng-1 μg. It was revealed that the concentration of HA in normal human blood plasma is very low levels (about 24 ng/ml) in comparison to low-sulfated chondroitin 4-sulfate (about 13 μg/ml).     

Detection of oxidized and reduced glutathione with a recycling postcolumn reaction

A rapid, sensitive, and selective method for the quantitation of both oxidized (GSSG) and reduced (GSH) glutathione in biological materials is described. Oxidized and reduced glutathione are resolved by anion-exchange high-performance liquid chromatography and detected with an in-line, recycling postcolumn reaction. The recycling reaction specifically amplifies the response to oxidized and reduced glutathione 20-100 times over that obtained with a stoichiometric reaction, permitting the detection of 2 pmol glutathione. Oxidized and reduced glutathione levels were measured in rat liver and in dog heart mitochondria. Special precautions are necessary to avoid artifacts which lead to either underestimation or overestimation of GSSG levels. GSH/GSSG ratios of approximately 100-300 were observed in samples prepared from rapidly frozen rat liver. Somewhat higher GSH/GSSG ratios were observed in isolated dog heart mitochondria.     

Polychlorinated biphenyls-induced lipid peroxidation as measured by thiobarbituric acid-reactive substances in liver subcellular fractions of rats

Rats were given a 0.05% polychlorinated biphenyls (PCB) diet supplemented with adequate nutrients for 10 days and not only PCB-induced lipid peroxidation as measured by thiobarbituric acid (TBA)-reactive substances but also variations of lipid peroxides scavengers in liver and its subcellular fractions (nuclei and cell debris, mitochondrial, microsomal and cytosolic fractions) were investigated. The lipid peroxidation in liver and subcellular fractions in the PCB-treated group increased significantly except in the nuclei and cell debris fraction. The increase in lipid peroxidation in the microsomal fraction appeared to be associated in part with the decrease in vitamin E (alpha-tocopherol) content and induction of drug-metabolizing enzymes. In the cytosolic fraction, the total lipid content increased, glutathione peroxidase (GSHPx) activity decreased and the quantity of free radical-reactive substances suppressing lipid peroxidation was low as measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) value. From these results, the increase in lipid peroxidation in the cytosolic fraction in the PCB-treated group was ascribed to the abundance and availability of oxidizable substrate attended with fatty liver, to the decline in GSHPx activity, and to the insufficiency in antioxygenic activity as observed by the decrease in the DPPH value.     

Determination of multiple redox-active compounds by high-performance liquid chromatography with coulometric multi-electrode array system

Studies of the antioxidant defense system and the related metabolic pathways are often complicated by cumbersome analytical methods, which require separate and multi-step extraction and chemical reaction procedures. Further, assaying multiple parameters is limited because of the usual small sample amounts. High-performance liquid chromatography coupled with a coulometric multi-electrode array system provides us high specificity and sensitivity to measure electrochemically oxidizable compounds in biological samples. In contrast to previously reported methods with two columns in series and a complex gradient elution profile, we have developed an automated procedure to simultaneously measure multiple redox-active low-molecular weight compounds that utilizes a single column with a simplified binary gradient profile. No other chemical reactions are necessary. In order to reduce the running time and yet achieve a reproducible retention time by the auto sampler injection, our gradient elution profile was modified to produce a shorter equilibration time, stable retention time, and a reduced cost per test.     

Determination of metformin in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of metformin, an oral antidiabetic agent, in plasma is described. Plasma samples containing the internal standard, phenformin, are eluted through Amprep extraction columns before injection into the chromatographic column, packed with μBondapak phenyl. The eluent is monitored at 236 nm. At a mobile phase flow-rate of 1.35 ml/min, the retention times of metformin and phenformin are 2.8 and 5.6 min, respectively. The intra-day coefficients of variation are 1.5 and 4.3% at metformin concentrations of 0.05 and 1 mg/l, respectively.