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PubMed2006

Micellar electrokinetic capillary chromatography for fast separation and sensitive determination of melatonin and related indoleamines using end-column amperometric detection.

MEKC was used in conjunction with end-column amperometric detection (AD) at a carbon disc electrode (0.3 mm diameter) for the selective and sensitive determination of melatonin and its five related indoleamines including its precursors and metabolites in the pineal gland. The introduction of a sample stacking technique in injection and the buffer additive SDS in the buffer solution system provided the rapid and sensitive analysis. Optimal buffer conditions (10 mmol/L phosphate containing 20 mmol/L SDS, pH 7.2), detection potential (+1.0 V vs. Ag/AgCl), and electrokinetic injection 10 s with the separation voltage of 24 kV were employed to achieve the baseline separation of six pineal hormones within 15 min. The peak currents and the analyte concentrations have a good linear relationship over the range of 6.0 x 10(-8) 6.0 x 10(-5 )mol/L. The detection limits for six pineal hormones by AD are 9.7 to 41.8 nmol/L (equal to 2.0 to 9.7 ng/mL) (S/N = 3), respectively. It is proved to provide about 30- to 250-fold improvement over UV, and be comparable with the sensitive fluorescence detection, which needs pre-column derivatization. The proposed method has been applied for analysis of melatonin and related indoleamines in rat pineal glands. A very simple sample pretreatment procedure, merely involving the homogenization step in perchloric acid, was enough to achieve recoveries in the range of 71 to 127% for all the analytes in the pineal gland.

Animals↗
PubMed1979

[Determination of aluminum in foods].

In connection with studies on the changes of functional properties of food constituents (e. g. solubility of proteins) by means of aluminium compounds, the elaboration of an appropriate method for the quantitative determination of aluminium has been necessary. The organic samples are mineralized with 80% perchloric acid; in case of fat-containing foods, after fat removal. After reaction with aluminon (ammonium salt of aurin tricarboxylic acid), aluminium is determined photometrically at 530 nm. The limit of detection lies between 1 and 2 ppm; the recovery rate is 103%; the standard deviation is +/- 10%. The determination (without mineralization) requires about 1 hours. The authors analyzed, inter alia, milk (1.2--1.7 ppm A1), fat cheese (9 ppm), micora (4 ppm), aluminium-stabilized protein texturates (300--1500 ppm).

Aluminum↗
PubMed1994

Selenium content of chicken meat in Croatia.

The selenium content in dark muscle and liver was determined in chicken meat. All samples had been collected from three large meat producing companies in Croatia. The samples were wet digested with a mixture of nitric and perchloric acids. The selenium content was analyzed by hydride generation atomic absorption spectrometry with the recovery from 97.8 to 98.2%. The correlation coefficient of 0.976 was determined after comparison with results of the fluorimetric method. Mean +/- standard deviation of the selenium concentration (ng Se/g dry matter) was 1330 +/- 20, 2000 +/- 30 and 2090 +/- 30 in liver, and 560 +/- 50, 520 +/- 70 and 740 +/- 50 in dark muscle.

Animal Feed↗
PubMed1984

Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD.

During the selection of monoclonal antibodies (MAb) raised against purified carcinoembryonic antigen (CEA), two MAbs were identified which immunoprecipitated a glycoprotein of 95 kD present both in perchloric acid extracts of normal lung and on the surface of normal granulocytes. This antigen was distinct from the previously reported normal glycoprotein crossreacting with CEA (NCA) which had a molecular weight of 55 kD. The difference between the smaller and the larger crossreacting antigens termed NCA-55 and NCA-95, respectively, was demonstrated by SDS-polyacrylamide gel electrophoresis, by elution from Sephadex-G200 and by selective binding to a series of anti-CEA MAb. Out of six MAb which all bound CEA purified from colon carcinoma, three did not react with these two crossreacting antigens, one bound only NCA-95, one reacted only with NCA-55 and one reacted with both NCA-55 and NCA-95. Immunoadsorbent purified preparations of 125I labelled NCA-95 and NCA-55 were found useful for the screening of new anti-CEA MAb. In addition, when tested on frozen sections of colon carcinoma, normal spleen, normal lung and pancreas, each type of MAb gave a clearly different pattern of reactivity. The three anti-CEA MAb which did not bind any of the crossreacting antigens stained only the colon carcinoma cells; the MAb binding to either one of the two types of NCA gave a similar pattern of reactivity both on colon carcinoma cells and on granulocytes. However, on normal lung and pancreas, the MAb binding NCA-55 stained granulocytes as well as bronchiolar and alveolar epithelial cells in lung and inter- and intra-lobular duct epithelial cells in pancreas, whereas the MAb binding only NCA-95 stained only the granulocytes. Thus, the newly identified NCA-95 appears to differ from NCA-55 not only in terms of molecular size and antigenicity but also by the fact that in normal lung and pancreas it is found in granulocytes but not in epithelial cells.

Animals↗
PubMed1987

Growth of a human colonic adenocarcinoma cell line (HT 29) on microcarrier beads: metabolic studies by 31phosphorus nuclear magnetic resonance spectroscopy.

A method allowing the growth of a human colon adenocarcinoma cell line (HT 29) on beaded polystyrene microcarriers has been developed by modifying the culture conditions used in monolayer cultures. Under optimized conditions, the cells became confluent 7 days after seeding and reached a density of 2.8 X 10(5) cells/cm2 of microcarrier (65% of the available area occupied). 31P NMR spectra were typically recorded on 300 X 10(6) cells continuously perfused at a flow rate of 15 ml/min in a specially designed NMR chamber in which the microcarrier beads were sequestered within the receiver coil volume. The in vivo spectrum displays a series of resonances assigned to nucleoside triphosphates (ATP and GTP), inorganic phosphate and various phosphomonoesters (mainly glucose-6-P and phosphorylcholine). Diphosphodiester resonances (DPDE, mainly UDP-N-acetyl-glucosamine and UDP-N-acetylgalactosamine) were not detected in the in vivo spectrum and were only apparent in the spectrum of the perchloric acid extract of the cells, indicating that these compounds have a restricted mobility in the intracellular compartment. The intracellular pH of HT 29 cells was 7.2 during the perfusion with a medium buffered at pH 7.3. The internal pH decreased slowly (2 X 10(-3) pH unit/min) during anoxic perfusion, but severe intracellular acidosis occurred after 40 min of ischemia (2.7 X 10(-2) pH unit/min). Sequential recording of 31P NMR spectra has shown that HT 29 cells are able to maintain their high energy phosphorylated compound levels (ATP) when subjected to 100 min of anoxia and 40 min of total ischemia.

Adenocarcinoma↗
PubMed1989

Biochemical and immunological properties of the human carcinoma antigen CAR-5 defined by the monoclonal antibody BD-5.

The monoclonal antibody (MAb) BD-5 reacts with an epitope (CAR-5) expressed in 83% of the gastric carcinomas and in 51% of the ductal pancreatic carcinomas. This MAb reacts also with epithelial cells of colorectal mucosa, but does not react at all with normal adult gastric mucosa or normal adult pancreas. We report the biochemical and immunochemical characterization of CAR-5-bearing molecule. The epitope was found to be carried on a mucin of more than 400 kDa with a density of 1.45 g/ml, metabolically labelled with 35S-sulfate, 3H-glucosamine, 3H-mannose and 35S-methionine. Antigenicity survived metaperiodate oxidation and alkalinization, while it was fully destroyed by pronase or papain. Trypsin, although cleaving the molecule, did not affect its antigenic activity. CAR-5 epitope is thus carried on the protein moiety of a sulfo-mucin. On the basis of its biochemical properties, the antigen was purified by a 3-step procedure, consisting of perchloric acid extraction, molecular sieving on Sepharose CL-4B and affinity chromatography on wheat-germ agglutinin coupled to Sepharose 4B. Cross-competition experiments, together with the chemical properties displayed by the different epitopes, clearly indicate that CAR-5 is different from all previously characterized carcinoma-associated determinants. Cross-DDIRMA experiments performed with different "catcher" and "tracer" antibody combinations showed that CAR-5 epitope may be expressed on the same mucin bearing CA 19-9, MOv2, DU-PAN-2, Lewisa and Lewisb epitopes.

Animals↗
PubMed1991

Histological and urinary reactivity of monoclonal antibody 1BE12 in bladder carcinoma. Purification of the antigen from urine.

We have previously reported the production of monoclonal antibody (MAb) 1BE12, which recognizes a glycoprotein in breast-cancer cells. In the present work, 1BE12 reactivity was tested by immunohistochemistry in bladder carcinoma (92 cases) and in non-tumoral bladder samples (15 cases). In 71% of bladder tumors, more than 30% of cells were intensely stained by 1BE12. The percentage of reactive cells was higher in cancers invading the muscle than in more superficial tumors (p = 0.039). In non-tumoral bladder, immuno-staining, when present, was usually confined to the superficial layers with a low number of cells stained (less than 30%) in 13/15 cases. Slot blots, performed on urine samples from 43 bladder-cancer patients and 21 healthy controls, were quantified by densitometry scanning. We found higher optical density (OD) values in urine from muscle-invasive-cancer patients than in urine from more superficial tumors and healthy controls, with a significantly different distribution (p = 0.005). The urinary antigen was detected by immunoblotting with 1BE12 as high-molecular-weight species (greater than 150 kDa). The reactive glycoprotein could thus be purified by immunoaffinity and FPLC filtration from the perchloric-acid-soluble fraction of urine from patients with invasive bladder carcinoma. The availability of purified antigen will allow us to quantitate our assay, in order to evaluate its potential use as a prognostic indicator in bladder-cancer patients.

Antibodies, Monoclonal↗
PubMed1993

Monoclonal antibody 4D3 detects small intestinal mucin antigen (SIMA)--glycoprotein in the serum of patients with colorectal cancer.

We have developed a sensitive ELISA using MAb 4D3 for the detection of a novel epitope on Small Intestinal Mucin Antigen (SIMA) and report here that SIMA is present in the serum of patients with colorectal cancer. SIMA has been shown to occur in tissue from a high proportion of patients with colorectal cancer. SIMA derived from serum was similar to tissue-derived SIMA: both eluted in the void volume of a Superose 6 column indicating a molecular weight above 5,000 kDa and they exhibited similar buoyant densities on CsCl gradients. The ELISA was most reliable after pre-treatment of serum with 0.4 M perchloric acid to remove interfering substances. The upper limit for SIMA in normal serum was set as the mean plus 2 standard deviations determined from a group of 97 healthy control subjects. In a sample of 113 patients with colorectal cancer, SIMA serum levels were elevated in 15% of patients with Dukes' Stage A, 38% with Stage B, 32% with Stage C and 75% with Stage D colorectal cancer. SIMA serum levels were compared with those of the widely used tumor marker, carcinoembryonic antigen (CEA). The SIMA assay detected a significant number of sera that were not detected by the test for CEA. We propose that SIMA will prove to be a valuable serological tumor marker, in combination with CEA and other tumor markers, for the detection of colorectal cancer.

Adenocarcinoma↗
PubMed1984

Quantitation of specific myeloid cells in rat bone marrow measured by in vitro 35S-sulphate incorporation.

A biochemical measurement which can be used for quantitation of specific early myeloid cells in rat bone marrow has been developed. This measurement consists of a rapid, simple assay for the in vitro quantitation of 35S-sulphate incorporation into rat bone marrow cells. Incubation of bone marrow cells with 35S-sulphate led to a time-dependent increase in radioactivity obtained in perchloric acid insoluble fractions of bone marrow cell suspensions. This incorporation was inhibited by cyanide and puromycin. Autoradiography has demonstrated the radiolabel to be specifically associated with immature cells of the myeloid series. The cells most active in this respect were eosinophils. When rats were treated with endotoxin, the rate of 35S-sulphate incorporation was increased. Cell number measurements, using conventional histopathology and a Coulter Counter, demonstrated that endotoxin caused an initial release of mature granulocytes from the bone marrow. The regeneration of this mature population in the marrow was rapid, and was characterized by an increase in the number of immature cells and a concomitant increase in the rate of 35S-sulphate incorporation measured in preparations of bone marrow cells in vitro. Furthermore, this response to endotoxin has demonstrated that Coulter Counting techniques can be used to distinguish specific populations of cells (e.g. mature granulocytes) within the bone marrow.

Animals↗
PubMed1987

The determination of Al, Cr, Co, Fe, and Ni in whole blood by electrothermal atomic absorption spectrophotometry.

Procedures for the determination of aluminum, chromium, cobalt, nickel, and iron in whole blood are presented and discussed. Chromium, cobalt, nickel, and iron were determined from one sample after decomposition of the blood with a mixture of nitric and perchloric acids. A graphite furnace was used for the determination of chromium, cobalt and nickel, and flame AAS was used for iron. The determination of Al was done from separate samples by GFAAS after dilution with 1% Triton TX-100. The normal concentration of these elements was measured in rabbit and dog blood.

Aluminum↗
PubMed1988

Fluoride content in human iliac bone: results in controls, patients with fluorosis, and osteoporotic patients treated with fluoride.

The major part of fluoride ingested is fixed on calcified tissues, mainly in bone tissue, and then is progressively but slowly recycled during bone remodeling. Thus, the measurement of bone fluoride content allows the determination of the extent of bone fluoride retention, and this parameter constitutes a useful complement to bone histology for the diagnosis of skeletal fluorosis and could also be used for the management of fluoride treatment of osteoporosis. A simple method is described to measure the fluoride content in calcined human iliac bone samples. Bone ashes were diluted in perchloric acid, and the measurement of the bone fluoride content was performed using a specific ion electrode combined with a reference electrode. Reference values are given for bone tissue from 76 control subjects (0.08 +/- 0.05% of bone ash), from two groups of 117 and 102 untreated osteoporotic patients (0.05 +/- 0.03% and 0.08 +/- 0.05%, respectively), from 166 sodium fluoride-treated osteoporotic patients (mean bone fluoride content varying from 0.24 to 0.67%, depending on the duration of therapy), and from 96 patients showing typical skeletal fluorosis (mean bone fluoride content varying from 0.56 to 1.33%, depending on the etiology of fluorosis and the relationship with the amount of fluoride ingested as well as with the duration of fluoride exposure). During a prolonged exposure of adult bone tissue to fluoride, the early bone fluoride uptake is variable and depends on the remodeling activity; then it increases rapidly before becoming more or less stable at a maximum level.

Female↗
PubMed1994

Analysis of monoclonal antibodies reactive with meconium- and amniotic fluid-derived mucin.

The present study was undertaken to determine whether monoclonal antibodies (moABs TKH-2, MA54, MA61, B72.3, and CC49) directed toward O-linked mucin-type glycoprotein detect the NeuAc alpha 2-6GalNAc (sialyl Tn) epitope in meconium- and amniotic fluid-derived mucin. Fetal colonic mucosal cells express the sialyl Tn antigen, particularly in goblet cell mucin. The reactivities of these moABs with a perchloric acid extract of meconium (meconium extract) and different native and neuraminidase treated glycoproteins were examined by solid-phase enzyme-linked immunosorbent assay (ELISA). All the moABs react with the meconium supernatant and meconium extract. The reactivities of TKH-2, MA54, and MA61 are neuraminidase sensitive, and the reactivity of TKH-2 with meconium extract was specifically inhibited by ovine submaxillary mucin (OSM). A NeuAc alpha 2-6GalNAc epitope is the characteristic component in meconium. Mucin released from the fetal respiratory tract could, in part, provide an alternative source in the amniotic fluid. TKH-2 is the most sensitive antibody directed to sialyl Tn antigen in meconium supernatant. The likelihood of TKH-2 serving as the basis for a sensitive assay to detect sialyl Tn in meconium- and amniotic fluid-derived mucin is provided.

Amniotic Fluid↗
PubMed2001

Effect of exogenous lactate on rat glioma metabolism.

Glioma-bearing rats were infused intravenously with a solution containing either [3-(13)C]lactate or both glucose and [3-(13)C]lactate for 20 min or 1 hr. Perchloric acid extracts of healthy and tumoral brain tissues were prepared and analyzed by (13)C- and (1)H-observed (13)C-edited nuclear magnetic resonance (NMR) spectroscopy to determine (13)C-label incorporation into brain tissue and glioma metabolites. Moreover, (13)C enrichments in blood lactate and glucose were determined from (1)H-NMR spectra. In the nontumoral tissue, (13)C labeling of amino acids indicated that [3-(13)C]lactate entered the brain and was metabolized. There was no labeling difference between the contralateral and the ipsilateral hemispheres. Lactate metabolism appeared more specifically neuronal, in agreement with our previous results obtained with normal rat brain (Bouzier et al. [2000] J. Neurochem. 75:480-486). In the glioma tissue, comparison of Ala C3, Glu C4, and Gln C4 labeling indicated that the contributions of blood glutamine and tricarboxylic acid (TCA) cycle to glutamate labeling were about 80% and 20%, respectively, after 1 hr of [3-(13)C]lactate infusion. In contrast, these contributions were about 10% and 90%, respectively, when [1-(13)C]glucose was infused in the absence of lactate. This indicated a major effect of the exogenous lactate on glioma metabolism, which may be due to the following process: The high blood lactate level might hinder the drain of glycolytic lactate produced inside the glioma and thus generate a change in redox potential such that the tumor cells are unable to restore it with oxidative phosphorylation. Thereafter, the high NADH level might inhibit glycolysis and the TCA cycle, and glutamine could become the major carbon source for glutamate labeling.

Animals↗
PubMed1994

13C NMR spectroscopy study of cortical nerve cell cultures exposed to hypoxia.

Primary cultures of cerebral cortical GABA-ergic neurons growing on top of a preformed layer of astrocytes (co-cultures) were incubated with [1-13C]glucose and exposed to a low oxygen atmosphere (2% O2) for 17 hr. 13C, 1H, and 31P nuclear magnetic resonance (NMR) spectroscopy was performed on perchloric acid (PCA) extracts of cells and of media collected from these cultures. In the control groups incorporation of 13C label into glutamine, citrate, and lactate could be demonstrated in both cell extracts and culture media. Labeled GABA and glutamate were only observed in cell extracts. During hypoxia high energy phosphates decreased but lactate production and glucose consumption increased. There was a decreased amount of citrate and glutamine in cell extracts and media of the hypoxic co-cultures. There was a change in distribution of the 13C label within the GABA molecule, with an increase of labeling in the C-2 position. This change in 13C distribution was not found in glutamine present in the media where it is a precursor for GABA in neurons. Instead a decrease in the corresponding C-4 position was observed. These results suggest that energy depletion during hypoxia leads to reduced export from the astrocytic tricarboxylic acid (TCA) cycle as demonstrated by a decreased amount of citrate and changed distribution of 13C in glutamine. The change in the distribution of label in GABA from cell extracts as compared to glutamine in the medium may indicate that neurons are synthesizing GABA using precursors supplied from their own TCA cycle and not from precursors supplied by astrocytes.

Animals↗
PubMed1977

Rapid GLD determination of ibuprofen in serum.

A rapid procedure for the determination of ibuprofen in human serum was developed using a single extraction with carbon tetrachloride after deproteinization with perchloric acid. The internal standard, 3-methyl-3-phenylbutyric acid, was added directly to the serum. Gas chromatograms were free of interfering peaks. Calibration curves (0--40 micron g/ml) were linear with a sensitivity of 0.5 micron of ibuprofen/ml of serum. Relative standard deviations ranged from 1.1 to 25%.

Chromatography, Gas↗
PubMed1981

High-performance liquid chromatographic analysis of iodochlorhydroxyquin in plasma.

A simple isocratic high-performance liquid chromatographic procedure for the analysis of iodochlorhydroxyquin in human plasma is described. Protein was precipitated using perchloric acid, and the supernatant and precipitated protein fractions were extracted with ether. The ether phases were evaporated to dryness, reconstituted in mobile phase, and chromatographed. A reversed-phase microparticulate C18 column, a precolumn, and a UV detector at 256 nm were used. A mobile phase containing 80% methanol and 20% of 0.05 M phosphoric acid was employed at a flow rate of 1 ml/min. Quantitation of iodochlorhydroxyquin in the 1--15-microgram/ml range in human plasma was demonstrated with a coefficient of variance of 0.1--0.06. Hydrocortisone, which is used in combination with iodochlorhydroxyquin in ointments and creams, does not interfere in the assay.

Chromatography, High Pressure Liquid↗
PubMed1975

Structural changes in myosin during contraction and the state of ATP in the intact frog muscle.

The reactivity of myosin to [14C]-labeled N-ethylmaleimide ([14C]NEM) or to tritium was determined in functionally different frog muscles. The incorporation of [14C]NEM into myosin decreased during isotonic or isometric contractions, as compared to resting muscle. The cysteine residues which were protected during contraction were not involved in the ATPase activity or the actin-binding ability of myosin. Peptide mapping revealed that several residues were protected simultaneously. The incorporation of tritium into the peptide N-H groups of myosin was also decreased during muscle activity. These data support the idea that activation and subsequent contraction of muscle are correlated with structural changes in the myosin molecule. The reactivity of myosin to [14C]NEM was increased when the muscle was stretched to 140% rest length and treated with iodoacetate to deplete ATP. Based on in vitro experiments and on literature data, it is suggested that in the resting muscle myosin contains bound MgATP which decreases the rate of incorporation of [14C]NEM into myosin and that upon the irreversible loss of ATP the rate increases. 31P nuclear magnetic resonance signals from a number of phosphates were detected in the intact frog muscle. The data indicated that the minimum concentration of ATP in the muscle is 3 mM, a value which agrees with that of chemical determination. The characteristic chemical shifts, coupling constants, and line widths of ATP in the muscle were considerably altered from that of either free ATP in aqueous solutions or ATP in perchloric acid extracts of muscle.

Actins↗
PubMed2002

Histone H1-like protein and a testis-specific variant in the reproductive tracts of Octopus vulgaris.

In this study, we have identified a 28-kDa protein resembling the linker H1 in the testis and prostate of the reproductive system of Octopus vulgaris. This protein, OvH1, was partially purified by reverse phase high-pressure liquid chromatography (HPLC) of the perchloric acid extract from testis nuclei. It showed electrophoretic mobility, CD spectrum and amino acid composition highly comparable with those of the mammalian histone. Moreover, it was microheterogeneous, as resulted from prostate and testis HPLC and mass spectrometry analyses. Such analysis showed that in testis there are two H1 subfractions, which do not appear in the prostate. Amino acid composition of the major testis specific variant (OvH1t) showed high similarity with rat testis specific H1t. The histone-like nature of OvH1 was confirmed by its ability to bind DNA as tested both by circular dichroism and protection of the nucleic acid toward deoxyribonuclease I activity. The circular dichroism spectra of Octopus DNA in the absence and presence of increasing amounts of the protein showed a dose-dependent effect, leading to a progressive compactness of the polynucleotide. OvH1/DNA complexes were also resistant to nuclease digestion. The presence of H1 in the testis and prostate of the reproductive system of Octopus is discussed in light of the fact that there is a similarity between its behavior and that of vertebrates.

Amino Acids↗