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At least 343 records · Page 19Linked to original sources
PubMed1993

Characterization of absorption and scattering properties of small-volume biological samples using time-resolved spectroscopy.

With time-resolved spectroscopy, we develop an experimental approach by sample substitution to measure the absorption (mu a) and reduced scattering (mu's) coefficients of small-volume biological samples. To investigate the method, small-volume control samples are substituted into a large-size host medium during increases in the absorber (or scatterer) concentration of the host. By characterizing the deviation of the spectra taken with and without the sample, we determine the matching points where the sample and surrounding medium are optically identical. We show that this method can result in correct values of the mu a and mu's for the sample within 6% error if the matching conditions for both the mu a and mu's are fully realized. The results also indicate that this method can give approximate values of the mu a and mu's in a reasonable range if either the mu a or the mu's matching between the two media is realized. This method has been applied to the studies of absorption properties of a human finger and of scattering properties of yeast.

Absorption↗
PubMedDate not supplied

Glutathione and cysteine measurement in biological samples by HPLC with a glassy carbon working detector.

An HPLC method using a glassy carbon working detector for measuring cysteine and reduced glutathione (GSH) in biological samples was developed. Oxidized glutathione (GSSG) was also measured after reduction with glutathione reductase. This study was conducted in human plasma. Cysteine and GSH standard curves were linear in the physiological range, presenting detection limits of 22.0 pmol and 6.0 pmol respectively. Plasmatic results found were 43.92 +/- 4.15, 4.50 +/- 0.65, and 0.19 +/- 0.12 mM (means +/- SE), for cysteine, GSH and GSSG, respectively. Peak specificity for cysteine and reduced glutathione was certified by their disappearance after treatment with N-ethylmaleimide.

Adult↗
PubMed1996

Enzymatic method to determine dehydroascorbic acid in biological samples and in bread dough at various stages of mixing.

An enzymatic method is described for measuring L-dehydroascorbic acid in perchloric acid extracts of biological samples. The enzyme used in the assay was glutathione dehydrogenase (glutathione:dehydroascorbate oxidoreductase), which was purified from wheat flour using three column chromatography steps. The enzyme catalyzes the reduction of dehydroascorbic acid by glutathione, and the ascorbic acid product is measured spectrophotometrically at 265 nm. The assay is a fast (about 2 min) and simple two-step procedure. First, a mixture of extract and buffer is set to zero absorbance against air in a spectrophotometer. Second, a solution of glutathione dehydrogenase and glutathione is added and the absorbance after 2 min is used in a formula to calculate nmol dehydroascorbic acid/g or ml sample [513.8(Abs-0.013) x factor for dilution of extract]. The formula was derived from a calibration graph using pure L-dehydroascorbic acid standards. Pure L-dehydroascorbic acid was prepared from pure L-ascorbic acid, and the ascorbate oxidase used to catalyze the reaction was removed by ultrafiltration. Commercial L-dehydroascorbic acid (Aldrich) was unsuitable for use as a standard because the purity was only 56-67% in comparison to laboratory-prepared L-dehydroascorbic acid. The sensitivity of the assay was such that when dehydroascorbic acid was added to healthy human blood plasma during extraction with perchloric acid at the level of 7.5 nmol/ml plasma the dehydroascorbic acid could be measured with complete recovery. Low levels of dehydroascorbic acid were detected in fresh fruits and vegetables. When samples were ground for several minutes before extraction with perchloric acid, the dehydroascorbic acid levels increased more than 10-fold. Dehydroascorbic acid increased rapidly during mixing of bread dough containing added L-ascorbic acid.

Ascorbic Acid↗
PubMed1991

Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography.

A method for a simultaneous separation of malondialdehyde (MDA), ascorbic acid and adenine nucleotide derivatives in biological samples by ion-pairing high-performance liquid chromatography is presented. The separation is obtained by an LC-18-T 15 cm x 4.6 mm 3 microns particle size column using tetrabutylammonium as the pairing ion. The starting buffer consists of 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 plus 1% methanol, pH 7.00. A step gradient is formed using a second buffer consisting of 2.8 mM tetrabutylammonium hydroxide, 100 mM KH2PO4 plus 30% methanol, pH 5.5. Under these chromatographic conditions a highly resolved separation of MDA, ATP, ADP, AMP, adenosine, ascorbic acid, GTP, GDP, IMP, inosine, Hypoxanthine, Xanthine, uric acid, NAD, and NADP can be performed in about 36 min. In addition, the separation of NADH and NADPH can also be obtained; this renders the present method suitable for the detection of these reduced coenzymes in alkaline extracts from tissue samples. Data referring to PCA extracts from ischemic and reperfused isolated rat hearts and from human erythrocytes peroxidized in vitro by a challenge with 1 mM NaN3 and various concentrations of H2O2 are reported. The relevance of this chromatographic method lies in the possibility to determine directly MDA concentrations avoiding the unspecific thiobarbituric acid colorimetric test, any other manipulation of the sample out of the PCA extraction, and any possible coelution of other acid soluble compounds. The simultaneous determination of MDA, ascorbic acid, and of ATP and its degradation products gives the opportunity to correlate, by a single chromatographic run, peroxidative damages with the energy state of the cell which is of great importance in studies of ischemic and reperfused tissues.

Adenine Nucleotides↗
PubMed2004

Spurious serotonin dimer formation using electrokinetic injection in capillary electrophoresis from small volume biological samples.

One normally assumes that the analytical measurement process does not introduce spurious compounds. Capillary electrophoresis is a separation method frequently used for small-volume biological measurements. We demonstrate the potential for creating new peaks in a capillary electropherogram when using electrokinetic injections and illustrate the potential deleterious effects with biological samples involving serotonin and nitric oxide measurements. Specifically, when measuring the serotonin content from individual neurons using electrokinetic injections from 360 nL stainless steel vials, we detect a new peak that we identify as a serotonin dimer. We do not observe this peak when using hydrodynamic injections.

Animals↗
PubMed1983

Polarographic assay of submicrogram quantities of cis-dichlorodiamineplatinum (II) in biological samples.

Differential pulse polarographic assay of the antineoplastic agent cis-dichlorodiamineplatinum II and its analogues was performed after acid oxidative hydrolysis (HClO4, HNO3, HCl) of biological samples (plasma, tissue homogenates, urine) and reaction with ethylenediamine. Platinum levels and kinetics were determined in blood and urine of patients with non-oat-cell lung carcinoma. Detection limit of the polarographic assay was 0.5 ng platinum; analytical error was +/- 3%. Levels of free cis-dichlorodiamineplatinum (II) in plasma fell in samples stored at -20 degrees C; the half-life of free drug was 38 h.

Carcinoma, Bronchogenic↗
PubMed1995

Borrelia burgdorferi DNA in biological samples from patients with sarcoidosis using the polymerase chain reaction technique.

Polymerase chain reaction (PCR) was used to detect the presence of Borrelia burgdoferi DNA in biological samples from patients with sarcoidosis. The target DNA sequence was of chromosomal origin. The amplified DNA sequence was analyzed by agarose gel electrophoresis, PAGE with silver staining, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and DNA-DNA hybridization with a 32P-labelled probe. The assay was sensitive to fewer than two copies of B. burgdorferi genome, even in the presence of a 10(4)-fold excess of human eukaryotic DNA, and was also specific to different B. burgdorferi strains tested. Sera serologically positive to B. burgdorferi (n = 26), bronchoalveolar lavage fluid and supernatant of BALF (n = 26) and peripheral blood (n = 9) from sarcoidosis patients were tested. The positive rate was low (4/26, 2/26, and 0/9, respectively). It was considered that DNA from B. burgdorferi may be identified in a minority of patients with sarcoidosis, and it may play a pathogenetic role in such cases. More studies need to be done before advancing the hypothesis of an etiologic role of B. burgdorferi in sarcoidosis.

Base Sequence↗
PubMedDate not supplied

Rapid analysis of halothane in biological samples using headspace solid-phase microextraction and gas chromatography-mass spectrometry--a case of a double homicide.

A simple, rapid, and sensitive method for the analysis of halothane in biological samples was developed. The procedure describes the extraction of halothane from blood, liver, kidney, brain, urine, bile, and stomach contents by headspace solid-phase microextraction (HS-SPME) followed by capillary gas chromatography coupled with mass spectrometry (GC-MS). The recovery in blood samples after addition of ammonium sulfate and sulfuric acid was 72% compared to a sample prepared in water (100%). Linearity was established over a concentration range of 0.1-100 mg/kg of spiked blood samples with an excellent coefficient of correlation (0.996) and a limit of detection of 0.004 mg/kg. The time for analysis was approximately 40 min per sample including the extraction step. The procedure was used for quantitation of halothane in various samples in a case of a double homicide. HS-SPME in combination with GC-MS was an effective method for the determination and quantitation of halothane in biological material.

Aged↗
PubMed1990

Determination of tin in biological samples using gaseous hydride generation-inductively coupled plasma-atomic emission spectrometry.

A highly sensitive method determining for sub-microgram/gram levels of tin in biological samples is described. Tin hydride reduced by sodium borohydride and trichloroacetic acid solution was introduced into inductively coupled plasma after separation of liquid and excess hydrogen by an improved gas/liquid separator, and emission intensity was measured at a wavelength of 189.989 nm. Samples were decomposed by a nitric acid-perchloric acid mixture and analyzed after dilution by a standard addition technique. The relative standard deviation was 1.2% for a 10 ng/ml tin standard solution with a detection limit of 30 pg/ml.

Animals↗
PubMed1979

Evaluation of a rapid, sensitive and specific assay for the determination of collagenolytic activity in biological samples.

Several methods for the determination of collagenolytic activity were compared from the point of view of sensitivity, selectivity, simplicity and practical value for large numbers of biological samples. A labelled collagen substrate was prepared using [3H]acetic anhydride. The specificity of the assay as well as conditions allowing an optimum detection limit were investigated. The influence of low temperatures, lyophilisation and salt concentration on Clostridium histolyticum collagenase have been investigated.

Clostridium↗
PubMed1995

Analysis of reduced and oxidized lipoic acid in biological samples by high-performance liquid chromatography.

Using HPLC with electrochemical detection at a dual Hg/Au electrode, both reduced and oxidized lipoic acid can be measured in biological samples after addition of lipoic acid. The method does not detect bound lipoic acid, which must be liberated by strong acid or base hydrolysis. The detection limit for this HPLC method is 0.01 nmol of dihydrolipoate and 0.05 nmol of lipoate. Baseline separation of lipoate and dihydrolipoate is achieved on a 10 cm octadecyl column. The analysis is rapid (8 min/sample) and uses a single HPLC pump with isocratic mobile phase. The method has been adapted to study cellular and whole animal reduction of lipoate, membrane transport of lipoate and dihydrolipoate, and subcellular enzymes that reduce lipoate.

Cell Line↗
PubMed1993

Rapid and highly sensitive high-performance liquid chromatographic method for the determination of histamine and 3-methylhistamine in biological samples using fluorescamine as the derivatizing agent.

A highly sensitive and rapid high-performance liquid chromatographic assay for the determination of histamine and 3-methylhistamine in biological samples using 1-methylhistamine as the internal standard is described. Samples were purified and concentrated on cation-exchange columns and derivatized with fluorescamine. The lower detection limit was 20 pg on-column. Linearity was demonstrated up to 20 ng on-column. The samples could be derivatized simultaneously before injection and were stable for 7 days. The method was used for the determination of histamine and related compounds in coronary perfusates, extracts of homogenized rat hearts, and supernatants of stimulated peritoneal mast cells.

Animals↗
PubMed1999

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. I. Determination of clenbuterol in urine.

The potential of the direct coupling of solid-phase extraction (SPE) with mass spectrometry (MS) for the analysis of biological samples is demonstrated. For SPE a cartridge exchanger is used and the eluate is directly introduced into the mass spectrometer. This system has been investigated for the determination of clenbuterol in urine. With mixed-mode cartridges, a considerable ion suppression has been obtained. The mass spectrum at the elution time of clenbuterol is dominated by that of creatinine and adduct formation of clenbuterol and creatinine has been observed. The whole procedure including injection of 1 ml urine, washing and desorption has been developed with cartridges containing 8-microm C18-bonded silica. If only a single MS step is used, the selectivity and, therefore, the sensitivity are insufficient. The detection limit is about 100 ng/ml. However, with atmospheric pressure chemical ionisation and the tandem MS mode the detection limit has been decreased to about 2 ng/ml and the ion suppression is only about 10%. For the electrospray ionisation the detection limit is about 10-times higher and the ion suppression is less favourable. The repeatability for the SPE-MS-MS procedure was 6.5% at 10 ng/ml (n=5) and the difference between the response factors at 10 ng/ml and 100 ng/ml was only 2.5%. The MS behaviour of clenbuterol and the matrix under the present conditions is discussed.

Adrenergic beta-Agonists↗
PubMed1997

Concurrent quantification of cellular cholesterol, cholesteryl esters and triglycerides in small biological samples. Reevaluation of thin layer chromatography using laser densitometry.

Absolute specificity and high accuracy is required for the quantitation of cholesterol, cholesteryl esters and triglycerides in small biological samples, particularly in a limited number of cells. Both can be achieved through thin-layer chromatography and molybdatophosphoric acid staining, while the shortcomings of traditional spot detection are overcome by laser densitometry. The major advantage of the proposed technique is the concurrent assay of nanogram quantities of cholesterol, cholesteryl esters and triglycerides. Our assay is at least ten-fold more sensitive than common thin-layer chromatography-based techniques and at least four-fold more sensitive than common enzymatic methods. The present low-cost assay is highly reproducible and may be particularly suitable for the routine lipid analysis of a 10% aliquot of relatively small tissue and cell samples, equivalent, for instance, to > or = 10(4) human monocytes.

Cholesterol↗
PubMed2003

A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples.

A liquid chromatography/mass spectrometric (LC/MS) method was developed for simultaneous detection and quantitation of glutathione (GSH), glutathione disulfide (GSSG), cysteine (CysSH), homocysteine (HCysSH) and homocystine in biological samples (rat brain, lung, liver, heart, kidneys, erythrocytes and plasma). Thiols were derivatized with a large excess of Ellman's reagent, a thiol-specific reagent, to ensure an instantaneous and complete derivatization. The derivatization blocked the oxidation of the thiols to disulfides, preventing errors caused by thiol oxidation. The samples were then analyzed by LC/MS. The method provides a highly selective and sensitive assay for these endogenous thiols and their corresponding disulfides. The detection limits for GSH, GSSG, CysSH, HCysSH and homocystine were 3.3, 3.3, 16.5, 29.6 and 14.9 pmol, respectively. An attempt for cystine analysis was unsuccessful due to earlier elution of the compound and strong interferences caused by other endogenous compounds. This method will be a useful tool in the investigation of the roles of these important thiol-containing compounds and their corresponding disulfides in physiological and pathological processes.

Animals↗
PubMedDate not supplied

Comprehensive label-free method for the relative quantification of proteins from biological samples.

Pharmaceutical companies and regulatory agencies are broadly pursuing biomarkers as a means to increase the productivity of drug development. Quantifying differential levels of proteins from complex biological samples such as plasma or cerebrospinal fluid is one specific approach being used to identify markers of drug action, efficacy, toxicity, etc. We have developed a comprehensive, fully automated, and label-free approach to relative protein quantification from LC-MS/MS experiments of proteolytic protein digests including: de-noising, mass and charge state estimation, chromatographic alignment, and peptide quantification via integration of extracted ion chromatograms. Results from a variance components study of the entire method indicate that most of the variability is attributable to the LC-MS injection, with a median peptide LC-MS injection coefficient of variation of 8% on a ThermoFinnigan LTQ mass spectrometer. Spiked recovery results suggest a quantifiable range of approximately 32-fold for a sample protein.

Amino Acid Sequence↗
PubMed1995

A simple method for overcoming some problems when observing thick reflective biological samples with a confocal scanning laser microscope.

A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips ('sandwich' style) and the optical tomography was performed from both sides by turning the 'sandwich' upside-down. The samples were impregnated using standard Golgi-Cox, 'rapid Golgi' or other silver methods. The ability to turn the 'sandwich' upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e., near the lower surface of the specimen, or when it is opaque to the laser beam or excessively reflective.

Animals↗