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PubMed2005

Major artifacts encountered in studying biological samples containing ferric protoporphyrin IX.

Heme (ferric protoporphyrin IX, FP) dissolves very rapidly into the lipid phase of membranes, and a large number of studies have focused attention on its possible toxic effect in whole cells or isolated membranes. However, because of its molecular structure and reactivity, different problems can be encountered during the course of studying biological samples containing FP. In this article, we discuss important interferences by FP and artifacts that can affect the experimental values. First, FP interferes with the Lowry's protein determination; therefore, membranes containing FP are overestimated in their protein content determined by this procedure. Second, freezing membranes at -20 degrees C artifactually increases the local concentration of FP, thereby enhancing FP-induced lipid peroxidation. Third, in the presence of thiol compounds such as N-acetyl cysteine, FP is degraded to products that interfere with the thiobarbituric acid assay, one of the most widely used methods to measure the extent of lipoperoxidation.

Acetylcysteine↗

PubMed2000

Improved method for simultaneous determination of L-arginine and its mono- and dimethylated metabolites in biological samples by high-performance liquid chromatography.

An improved method has been developed for the determination of L-arginine and its methylated metabolites, N(G)-monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (asymmetric DMA, ADMA) and N(G),N(G)'-dimethyl-L-arginine (symmetric DMA, SDMA) in biological samples. Extraction of these compounds with a strong cation-exchange resin AG50W-X8 with L-homoarginine (2-amino-6-guanidinohexanoic acid) as an internal standard gave a recovery of more than 70% except for SDMA from plasma samples. After extracted samples were converted to fluorescent derivatives with o-phthalaldehyde (OPA) in an alkaline medium, the following high-performance liquid chromatographic separation with a ODS column (wide-pore size, 300 A) was successfully performed with an isocratic mobile phase system. The method permits quantitative determination of L-arginine and its methylated metabolites at concentrations as low as 4 microM and 0.18 microM, respectively. Using this method, the levels of L-arginine, L-NMMA, ADMA and SDMA in human plasma, urine and rat tissue were determined.

Adult↗

PubMed1998

Highly sensitive measurement of lipid molecular species from biological samples by fluorimetric detection coupled to high-performance liquid chromatography.

As the molecular species composition of glycerophospholipids provides more valuable information than the corresponding fatty acid composition, we have applied a fluorimetric detection (360 and 460 nm for excitation and emission wavelengths, respectively) of anthroyl derivatives of diradylglycerol species to minor phospholipid classes and subclasses from biological samples. Diacylglycerol species were obtained by phospholipase C treatment of phosphatidylcholine subclasses and phosphatidic acid extracted from rat thymocytes. Subpicomole measurements of molecular species from the minor subclass alkenylacylglycerophosphocholine could be achieved (e.g. 0.4 pmol of the 18:1/20:5 species). Such a sensitivity allowed study of the molecular species composition of another minor phospholipid, phosphatidic acid, and to evaluation of its alteration in mitogen-stimulated thymocytes as compared to unstimulated ones. Finally, we report that such a measurement is also applicable to other minor bioactive lipids with a hydroxyl group available, namely hydroxyeicosatetraenoates (HETEs), with a similar gain of sensitivity over conventional UV detection. Overall, these measurements, especially those of phospholipid molecular species, are sensitive, reliable and meaningful for precursor-product relationship between phospholipids.

Animals↗

PubMed2004

High-resolution capillary electrophoresis of nitrite and nitrate in biological samples.

Nitrite and nitrate are widely used reporters of endogenous nitric oxide (NO) and nitric oxide synthase (NOS) activity, which are crucial for a broad spectrum of physiological and pathophysiological pathways. Because of the great variety in spatial expression and activity of NOS in animal tissues, a high-resolution analysis of nitrite/nitrate concentrations in very small biological samples, such as individual cells or homogeneous cell clusters, is required. A high-performance capillary zone electrophoresis (CZE) system, which includes a PrinCE-476 computerized capillary electrophoresis and Crystal-1000 conductivity detector, was optimized to analyze nitrite/nitrate concentrations in submicroliter samples of mammalian neuronal tissues and large individual cells of invertebrates. Solid-phase microextraction (SPME) and Isotachophoretic stacking (ITS) were used. The method is highly reproducible and yield excellent limits of detection (LODs): 8.9 nM (0.41 ppb) and 3.54 nM (0.22 ppb) for nitrite and nitrate, respectively, relative to undiluted samples.

Electrophoresis, Capillary↗

PubMed1985

A firefly luciferase assay for determination of cytidine 5'-triphosphate in biological samples.

A routine assay for CTP in cell and tissue extracts using crude firefly lantern preparations is described. ATP, GTP, and UTP are removed by incubation of the samples with a mixture of 3-phosphoglycerate kinase, hexokinase, glucose-6-phosphate dehydrogenase, and UDP-glucose pyrophosphorylase in the presence of ADP. The method is sensitive (greater than 30 nM CTP), inexpensive, and reproducible. No chromatographic purification of the biological samples or of the firefly extract is necessary. Corrections must be made for some loss of CTP during the enzymatic incubation and for the background luminescence of ADP. The applicability of the assay is tested with extracts from the cyanobacterium Anacystis nidulans.

Adenosine Diphosphate↗

PubMed1994

Utilization of high-performance liquid chromatography as an enrichment step for the determination of cyclic fatty acid monomers in heated fats and biological samples.

A method was developed to determine traces of cyclic fatty acid monomers (CFAM) in oils and animal tissues. This method is a combination of some techniques developed earlier but with the enrichment step being achieved by high-performance liquid chromatography (HPLC) instead of urea inclusion. After transformation of the lipids into methyl esters, the latter were hydrogenated after addition of an internal standard (methyl heptadecanoate or ethyl hexadecanoate). The mixture was enriched in CFAM by HPLC on a semi-preparative C18 reversed-phase column using acetonitrile-acetone (90:10, v/v) at 4 ml/min. The enriched fraction containing the CFAM and the internal standard was then analysed by gas chromatography on a polar column (cyanosilicone phase). This method was developed using known mixtures of CFAM isolated from both heated sunflower and linseed oils. Small amounts of CFAM (50 microg/g of sample) were determined with good reproducibility without any loss during the HPLC enrichment step and with no modification of the relative proportions of the CFAM in the mixture. This method can be applied to either heated fats and oils or biological samples (heart cell culture) that contain only traces of CFAM. Ethyl hexadecanoate (16:0 ethyl ester) can be used as an internal standard for samples containing small amounts of 17:0.

Animals↗

PubMed1982

Determination of ellipticine in biological samples by high-performance liquid chromatography.

Ellipticine, a plant alkaloid effective against murine leukemias and solid tumors, is presently undergoing toxicological assessment prior to clinical trial. A rapid, sensitive, reversed-phase high-performance liquid chromatographic method employing an internal standard was developed for the detection of ellipticine and its principal metabolite 9-hydroxyellipticine after extraction from biological samples. The method was successfully applied to the quantitation of ellipticine in mouse blood and tissues after intravenous administration of ellipticine and to mouse blood levels of drug after oral administration. Similar success was achieved in determinations of ellipticine and 9-hydroxyellipticine in samples of spiked human blood and plasma. Mouse blood ellipticine levels monitored over 3 h after the intravenous administration of drug demonstrated a biphasic decline with a terminal half-life of 52 min.

Alkaloids↗

PubMed2003

Chloride present in biological samples as a tool for enhancement of sensitivity in capillary zone electrophoretic analysis of anionic trace analytes.

Effects originating from the variability of the sample matrix can be efficiently eliminated when the separation conditions are selected so that compounds of like charge with high concentration referred to as macrocomponents are embodied into the system of transient isotachophoresis. For stacking and separation of anionic trace analytes in biological samples, the presence of chloride is shown to be important to balance out effects of other macrocomponents that act against isotachophoretic stacking. Having acetoacetate, malate, citrate, and some drug metabolites in untreated human serum samples, the stacking mechanism of these compounds in an electrolyte system comprising 5 mM mandelic acid and epsilon -aminocaproic acid, pH 3.8, is explained. Analytes are monitored by indirect UV-absorption detection. Attention is paid to the minimum chloride concentration required with respect to the concentration ratio of phosphate (stacker) and lactate (destacker) present in the sample so as to ensure both stacking and separation of trace analytes. Insight into the separation process is given both with computer simulations and experiments. For selected analytes, the effect of chloride concentration on quantitative evaluation, sensitivity and limit of detection is demonstrated as well. Moreover, the applicability of the mobility window between phosphate and lactate for an additional group of metabolites is sketched.

Anions↗

PubMed2003

Separation and quantitation of short-chain coenzyme A's in biological samples by capillary electrophoresis.

Because of the importance of coenzyme A's (CoA's or CoASH) in many metabolic processes and the biosynthesis of some carbohydrates and lipids, many methods have been developed to separate and determine their levels in various tissues for metabolism studies, including enzymatic assays, paper chromatography, and high-performance liquid chromatography (HPLC). However, inadequate separation of coexisting CoA's in biological samples was often encountered due to the similarity of their structures. In this paper, we demonstrated for the first time the separation and quantitation of 12 different CoA's by using capillary electrophoresis with UV detection at 254 nm. All 12 CoA's (CoASH, HMG CoA, methylmalonyl CoA, succinyl CoA, methylcrotonyl CoA, isobutyryl CoA, oxidized CoA, acetyl CoA, crotonoyl CoA, n-propzoyl CoA, acetoacetyl CoA, malonyl CoA) were completely separated at -30 kV in a 100 mM NaH2PO4 running buffer containing 0.1% beta-cyclodextrin at pH 6.0. The total separation time was less than 30 min. The signal response was linear over 2 orders of magnitudes (from 1 to 100 nmol), and the detection limits were in the picomole range. The effects of pH, buffer concentration, additives, and operation voltages on sensitivity and resolution were also discussed. This technique, described here, is much more sensitive, faster, and simpler than the published HPLC methods and can potentially be used for mechanistic study in biological systems involving CoA metabolism.

Animals↗

PubMed1978

Assay of retinoids in biological samples by reverse-phase high-pressure liquid chromatography.

A separation procedure for retinoids based on reversephase high-pressure liquid chromatography with solvent mixtures of acetonitrile and water is described. The method may be applied to the screening of synthetic retinoids, which have potential for use in the prevention of cancer. It is easily adapted to a variety of biological samples and can be applied to other conventional retinoid assays in liver and plasma, detecting as little as 1 nmol retinyl esters and less than 0.3 nmol retinol per g tissue. The one-step chromatography results in separation and simultaneous determination of many of the synthetic retinoids and all of the natural retinoids, including the retinyl esters that are separated into their major fatty acid components. The method has been applied to the analysis of retinoid levels in the liver and intestine of vitamin A-deficient hamsters following a p.o. dose (0.5 mg/day for 2 days) of retinyl acetate or of a synthetic vitamin A analog and is predictive of the degree to which various synthetic retinoids can be converted to retinol and stored in the liver as retinyl esters. Because of its speed, excellent recoveries, and high resolution, the method offers significant advantages over previous, more lengthy procedures.

Animals↗

PubMed2004

An improved method for analyzing coenzyme Q homologues and multiple detection of rare biological samples.

We have developed a simple method for the estimation of coenzyme Q homologues, neurotransmitters, metal ions, lipid peroxidation, gene expression, and DNA fragmentation simultaneously from genetically engineered mice brain regions and cultured neurons. The primary objective of this study was to improve conventional time-consuming, cumbersome, and less efficient procedures, and reduce the cost of conducting kinetic studies in rare biological samples. The improved method is novel, precise, efficient, accurate, sensitive, economical, versatile, and highly reproducible. The recovery and shelf life of coenzyme Q homologues was significantly increased and the chromatograms exhibited reduced background and retention times. It is envisaged that in addition to coenzyme Q homologues, the improved method could be utilized for the multiple analyses of DNA, RNA and proteins from clinically significant biopsy and autopsy samples.

Animals↗

PubMed1994

Development and validation of a sensitive and specific radioimmunoassay for the determination of cicaprost in biological samples.

Cicaprost is a potent, chemically and metabolically stable PGI2-mimetic. Pharmacodynamic effects were observed after oral administration of approximately 10 micrograms in man when plasma levels were in the low pg-range. The present report describes the development of a selective antiserum and a tracer with high specific activity and their use for the RIA determination of Cicaprost in biological samples. Cicaprost-[3H]-methylester with a specific activity of 819 GBq/mmol was used as a tracer. RIA-analyses were carried out with 0.05-0.5 ml plasma adjusted to pH 2 with 1 N HCl and extracted with 2.5 ml diethylether. Separation of antiserum bound and unbound Cicaprost was achieved by the charcoal method. Extraction recovery of Cicaprost was approximately 90% at pH approximately 2. The detection limit of the assay was 10-20 pg/ml plasma. Coefficients of variations were 6, 3 and 9% (within-day, n = 5) and 25, 12 and 10% (day-to-day, n = 11) at 50, 100 and 200 pg/ml. HPLC-chromatograms of plasma extracts did not reveal any peak apart from Cicaprost, demonstrating the specificity of the method. The present RIA for Cicaprost exhibits high specificity and sensitivity and will be used for further bioanalyses in pharmacokinetic study.

Animals↗

PubMed2006

Full-field quantitative phase imaging by white-light interferometry with active phase stabilization and its application to biological samples.

We report a Koehler-illumination-based full-field, actively stabilized, low-coherence phase-shifting interferometer, which is built on a white-light Michelson interferometer. By using a phase-stepping technique we can obtain full-field phase images of the sample. An actively stabilized phase-lock circuit is employed in the system to reduce phase noise. An application to human epithelial cells (HeLa cells) is achieved in our experiment. The advancement of this technique rests in its ability to take images of unstained biological samples quantitatively and on a nanometer scale.

Equipment Design↗

PubMed1984

Fluorometric measurement of tin-protoporphyrin in biological samples.

Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, can suppress neonatal and other forms of hyperbilirubinemia in laboratory animals, and represents a potential new approach to the treatment of neonatal jaundice in humans. In order to study the disposition of Sn-protoporphyrin in vivo we have developed a sensitive fluorometric method for the quantitation of this metalloporphyrin in biological samples. The method is sensitive to concentrations as low as 0.01 nmol/ml, and is specific for Sn-protoporphyrin even in the presence of other porphyrins such as protoporphyrin.

Animals↗

PubMed2000

Atomic absorption spectrometric determination of trace zinc in alloys and biological samples after preconcentration with [1-(2-pyridylazo)-2-naphthol] on microcrystalline naphthalene.

An atomic absorption spectrometric method for the determination of trace amounts of zinc after adsorption of its [1-(2-pyridylazo)-2-naphthol] complex on microcrystalline naphthalene has been developed. This complex is adsorbed on microcrystalline naphthalene in the pH range 3.5-7.5 from large volumes of aqueous solutions of various alloys and biological samples with a preconcentration factor of 40. After filtration, the solid mass consisting of the zinc complex and naphthalene was dissolved with 5 ml of dimethylformamide and the metal was determined by flame atomic absorption spectrometry. Zinc can alternatively be quantitatively adsorbed on [1-(2-pyridylazo)-2-naphthol]-naphthalene adsorbent packed in a column and determined similarly. About 0.5 ng of zinc can be concentrated in a column from 200 ml of aqueous sample, where its concentration is as low as 2.5 pg ml-1. The calibration curve is linear in the range 0.1-6.5 ng ml-1 in dimethylformamide solution. Eight replicate determinations of 2 ng ml-1 of zinc gave a mean absorbance of 0.145 with a relative standard deviation of 1.5%. The sensitivity for 1% absorption was 0.061 ng ml-1. Various parameters, such as the effect of pH and the interference of a number of metal ions on the determination of zinc, have been studied in detail to optimize the conditions for the determination of zinc in various standard complex materials.

Adsorption↗

PubMed1993

Micellar electrokinetic chromatography of hydroxyproline and other secondary amino acids in biological samples with laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) was used for the rapid and sensitive detection of hydroxyproline in serum and hydrolyzed urine that were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC). The application of the combined o-phthalaldehyde (OPA)/FMOC derivatization in MEKC for the selective detection of secondary amino acids in biological samples is investigated.

Amino Acids↗

PubMed2001

Determination of captopril in biological samples by high-performance liquid chromatography with ThioGlo 3 derivatization.

Captopril, a well-known angiotensin converting enzyme (ACE) inhibitor, is widely used for treatment of arterial hypertension. Recent studies suggest that it may also act as a scavenger of free radicals because of its thiol group. Therefore, the present study describes a rapid, sensitive and relatively simple method for the detection of captopril in biological tissues with reverse-phase HPLC. Captopril was first derivatized with ThioGlo 3 [3H-Naphto[2,1-b]pyran,9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl-3-oxo-)]. It was then detected by fluorescence-HPLC using an Astec C(18) column as the stationary phase and a water:acetonitrile:acetic acid:phosphoric acid mixture (50:50; 1 mL/L acids) as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). The calibration curve for captopril was linear over a range of 10-2500 nM and the coefficient of variation acquired for the within- and between-run precision for captopril was 0.5 and 3.8%, respectively. The detection limit of captopril with this method was found to be 200 fmol/20 microL injection volume. Its relative recovery from biological samples was determined to the range from 93.3 to 105.3%. Based on these results, we believe that our method is advantageous for captopril determination.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1984

Study of chlorinated diphenyl ethers and chlorinated 2-phenoxyphenols as interferences in the determination of chlorinated dibenzo-p-dioxins and chlorinated dibenzo furans in biological samples.

Two classes of environmentally occurring chlorinated aromatic hydrocarbons which have mass spectral properties similar to the chlorinated dibenzo-p-dioxins (dioxins) and chlorinated dibenzofurans (furans) were studied. Standards of chlorinated diphenyl ethers (CDEs), and chlorinated 2-phenoxyphenols (CPPs) and their methyl ethers, along with the dioxins and furans were passed in steps through a simple method for the analysis of the latter compounds in biological samples. The CDEs, which interfere with the determination of furans by mass spectrometry, had similar extraction, high-performance liquid chromatographic and gas chromatographic properties as the furans but, in all cases studied, were well separated from them on an activated Florisil column using combinations of hexane and dichloromethane as eluting solvents. The higher CPPs on the other hand tended to generate dioxin residues by ring closure when exposed to strong hydrochloric acid solution during sample preparation. In addition, their methyl ethers containing four to six chlorines tracked completely through all stages of the method with the dioxins. Thus, if the methyl ethers of CPPs were present in a sample extract, additional mass spectral information would be needed to further differentiate them from dioxins in environmental samples.

Benzofurans↗