High-performance Liquid Chromatography Eluent Research Articles

Amphibian toxins from the skin of <i>Bufo regularis</i> have inhibitory effect on electrically invoked contractile response and bimodal effects on tone of longitudinal muscle of guinea pig ileum

The skins of some amphibians contain potentially bioactive principles that may have pharmaceutical, medicinal, toxicological or chemical importance. In addition, such active principles can be used as tools in biomedical research. The present study aims at isolating and purifying bioactive principles from the skin of Bufo regularis and studying their effect on isolated longitudinal smooth muscle strip of guinea pig ileum. High performance liquid chromatography (HPLC) was used to isolate toad toxins. The effects of crude, semi-purified and purified extracts were tested on longitudinal smooth muscle of guinea pig ileum using organ bath method. Effect of the toxins was studied on electrically-induced contractile response and the basal tone of the longitudinal muscle strip. HPLC purification resulted in four different bioactive components with a λmax UV absorbance pattern of around 295 nm. When tested on guinea pig ileum they had persistent inhibitory effect on the electrically-induced contractile responses. The pattern of effect was initial excitatory followed by long lasting inhibitory effect on tone of longitudinal muscle. The HPLC eluate at 79th min in methanol preparative run corresponding to the eluate at 40th min in the acetonitrile run had the maximum bioactivity. Hence, it was concluded that the skin of B. regularis contains four different components which vary in their potency on isolated smooth muscle strip of guinea pig ileum.Keywords: Bufo regularis, organ bath, longitudinal muscle of ileum, toad toxin, electrical field stimulation

Detection of multiclass explosives and related compounds in soil and water by liquid chromatography-dielectric barrier discharge ionization-mass spectrometry.

In this work, the multiclass detection of explosives and related compounds has been studied by liquid chromatography-high-resolution mass spectrometry by means of a time-of-flight mass spectrometer (TOFMS) operated in the negative ion mode, using dielectric barrier discharge ionization (DBDI). Reversed-phase high-performance liquid chromatography (HPLC) separation was performed using water-methanol mobile phase without any modifier, although the effect of ammonium acetate was studied. DBD plasma was generated by applying a square wave voltage of 2.5kV to a 100-mLmin-1 He flow. The DBDI probe was adjusted to fit the commercial API source housing so that the HPLC eluent was nebulized and vaporized in the same manner as for atmospheric-pressure chemical ionization (APCI). The ionization process was affected by the temperature of the two nitrogen streams used to vaporize the solvent and the analytes, particularly for RDX and HMX, which are thermolabile compounds. The best approach in terms of sensitivity for all the studied compounds was the use of a gradient of temperatures in the ionization source, starting at 225/200°C (vaporizer/drying gas temp) for nitramines and ending at 350/325°C for nitroaromatic compounds. High-resolution full-scan spectra of individual selected compounds were recorded by LC-DBDI-TOFMS, and the results were compared to LC-APCI-TOFMS. A better sensitivity (slope of calibration curve) was obtained by DBDI for more than 70% of the studied compounds in both wastewater and soil extracts. Graphical abstract ᅟ.

Automated radiochemical synthesis of [18F]FBEM: A thiol reactive synthon for radiofluorination of peptides and proteins

The automated radiochemical synthesis of N-[2-(4-[(18)F]fluorobenzamido)ethyl]maleimide ([(18)F]FBEM, IUPAC name: N-maleoylethyl-4-[(18)F]fluorobenzamide), a prosthetic group for radiolabeling the free sulfhydryl groups of peptides and proteins, is herein described. 4-[(18)F]fluorobenzoic acid was first prepared by nucleophilic displacement of a trimethylammonium moiety on a pentamethylbenzyl benzoate ester with [(18)F]fluoride. In the second step the ester was cleaved under acidic conditions. Finally, 4-[(18)F]fluorobenzoic acid was coupled to N-(2-aminoethyl)maleimide using diethylcyanophosphate and diisopropylethyl amine. Following high-performance liquid chromatography (HPLC) purification, [(18)F]FBEM was obtained in 17.3±7.1% yield (not decay corrected) in approximately 95 min. Isolation from the HPLC eluate and preparation for subsequent use, which was conducted manually, required an additional 10-15 min. The measured specific activity for three batches was 181.3, 251.6, and 351.5 GBq/μmol at the end of bombardment (EOB).

Memory effects with the on-column interface for on-line coupled high performance liquid chromatography-gas chromatography: The Y-interface

When using the on-column interface for on-line high performance liquid chromatography (HPLC)-gas chromatography (GC), there is a memory effect typically equivalent to 0.5–3% of the previous transfer. The shape of peaks distorted as a result of incomplete reconcentration of the initial bands enabled mapping of the distribution of the solute material in the uncoated precolumn and deriving the mechanism which causes the memory effect. The relatively slow transfer of HPLC eluent causes liquid being sucked backwards into the narrow interspace between the transfer line and the precolumn wall. Solvent is evaporated into the passing carrier gas and is replaced by more eluent pulled into this zone, resulting in enrichment of solute material. At the end of the transfer, some of this solute material enters the transfer line and remains there up to the subsequent transfer of an HPLC fraction. This problem is avoided by replacing the on-column injector used as interface by a Y-piece in which the eluent flow from HPLC and the carrier gas are joined. The memory effect was reduced to below 0.02%.

The effect of re-dissolution solvents and HPLC columns on the analysis of mycosporine-like amino acids in the eulittoral macroalgae Prasiola crispa and Porphyra umbilicalis

Many macroalgal species that are regularly exposed to high solar radiation such as the eulittoral green alga Prasiola crispa and the red alga Porphyra umbilicalis synthesize and accumulate high concentrations of mycosporine-like amino acids (MAAs) as UV-sunscreen compounds. These substances are typically extracted with a widely used standard protocol following quantification by various high performance liquid chromatography (HPLC) techniques. However, further preparation steps prior to HPLC analysis as well as different HPLC column types have not been systematically checked regarding separation quality and reproducibility. Therefore pure methanol, distilled water and HPLC eluent were evaluated as re-dissolution solvent for dried Prasiola and Porphyra extracts, which were subsequently analyzed on three reversed-phase C8 and C18 HPLC columns. The data indicate that distilled water and the HPLC eluent gave almost identical peak patterns and MAA contents on the C8 and C18 columns. In contrast, the application of the widely used methanol led to double peaks or even the loss of specific peaks as well as to a strong decline in total MAA amounts ranging from about 35% of the maximum in P. crispa to 80% of the maximum in P. umbilicalis. Consequently, methanol should be avoided as re-dissolution solvent for the HPLC sample preparation. An improved protocol for the MAA analysis in macroalgae in combination with a reliable C18 column is suggested.

Cyclic AMP in Rat Ileum: Evidence for the Presence of an Extracellular Cyclic AMP-Adenosine Pathway

Extracellular adenosine plays a relevant role in regulating intestinal motility and preventing inflammatory processes. Adenosine 3',5'-cyclic monophosphate (cAMP) extruded from cells may be converted to adenosine monophosphate and then to adenosine by ecto-phosphodiesterase and CD73/ecto-5'nucleotidase, respectively, thus representing a source of adenosine. Our purpose was to assess the existence of a functional extracellular cAMP-adenosine pathway in intestinal tissue, obtaining evidence for CD73 expression and evaluating the effect of cAMP on ileum motility. The formation of cAMP metabolites in rat ileum strips incubated with exogenous cAMP or [(3)H]cAMP was monitored by high-performance liquid chromatography. CD73 was detected by immunoprecipitation and immunofluorescence. The functional activity of exogenous cAMP on ileum strips was recorded by measuring tension changes. In ileum strips, the generation of cAMP-derived adenosine monophosphate, adenosine, and inosine was time and concentration dependent and was blocked by phosphodiesterase or CD73 inhibitors in a manner consistent with exogenous cAMP being processed through the extracellular cAMP-adenosine pathway. Accordingly, [(3)H]cAMP uptake in ileum strips was negligible. Immunofluorescence revealed CD73 surface expression on intestinal smooth muscle cells and intact smooth muscle. Exogenous cAMP concentration-dependently increased ileum muscle tension partially inhibited by adenosine inactivation or receptor blockade. Forskolin-stimulated endogenous cAMP induced concentration-dependent ileum relaxations. A functioning extracellular cAMP-adenosine pathway featuring CD73 expression is present in rat ileum and affects intestinal motility. Extracellular cAMP may therefore act on intestinal muscle both directly by binding to specific smooth muscle cell membrane sites and indirectly through its degradation products.

Partial Purification from Hot Dogs of N-Nitroso Compound Precursors and Their Mutagenicity after Nitrosation

Hot dogs contain apparent N-nitroso compounds (ANC) and ANC precursors (ANCP). ANCP purification was followed by nitrosation, sulfamic acid treatment, and analysis for ANC. Aqueous hot dog extracts were adsorbed on silica gel, which was eluted with MeCN and MeOH. The MeOH eluate was adsorbed on cation exchange resin (H+ form) and eluted with NH4OH. Eluted ANCP traveled at moderate speeds in high-performance liquid chromatography (HPLC) on amino and Pb2+ columns. Gas chromatography-mass spectrometry (GC-MS) of trimethylsilyl (TMS) derivatives of crude water extract indicated the presence of glycerol, phosphate, lactic acid, and two monosaccharides. GC-MS of TMS derivatives of Pb2+ column HPLC eluates indicated that ANCP included 1-deoxy-N-1-glucosyl glycine. The nitrosated NH4OH eluate showed 4x background mutagenic activity for Salmonella typhimurium TA-100. Un-nitrosated fractions showed 2x background activity. Although tryptophan nitrosation gave 88% ANC yield, tryptophan is probably not a major ANCP in hot dogs. Hot dog patties prepared with or without sucrose or glucose showed similar ANC and ANCP levels. We discuss possible implications of these findings for the etiology of colon cancer.

Identification of kinetin and kinetin riboside in coconut (Cocos nucifera L.) water using a combined approach of liquid chromatography–tandem mass spectrometry, high performance liquid chromatography and capillary electrophoresis

Kinetin (free base and riboside), which was assumed by many scientists to be a synthetic cytokinin plant growth hormone, has been detected for the first time in the endosperm liquid of fresh young coconut fruits ("coconut water"). To facilitate the study, we developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of kinetin and kinetin riboside in purified coconut water extract sample. Following a solid-phase extraction of cytokinins in coconut water using C18 columns, the samples were further purified by Oasis MCX columns and analyzed by LC-MS/MS for kinetin and kinetin riboside. Detection by mass spectrometry was carried out using selected reaction monitoring (SRM) mode, by identifying the putative kinetin and kinetin riboside based on their characteristic fragments. Based on a signal-to-noise ratio of 3, the limits of detection in SRM mode were 0.02 microM and 0.005 microM for kinetin and kinetin riboside, respectively. Furthermore, optimal conditions for a baseline chromatographic separation of 18 cytokinin standards by high performance liquid chromatography (HPLC) were developed. The HPLC method had been employed for the confirmation and further fractionation of kinetin in coconut water extracts. The confirmation and fractionation of kinetin riboside was carried out using a further modified HPLC program due to the presence of other interfering material(s) in the sample matrix. Finally, fractions of putative kinetin and kinetin riboside collected from HPLC eluate of coconut water sample were further authenticated by independent capillary zone electrophoresis (CZE) experiment.

Metabolism of docetaxel in mice

Previous studies have shown by quantification of the parent drug and the known metabolites M-1, M-2, M-3 and M-4 that the mass balance of docetaxel in mice and humans is not complete. We therefore used reversed-phase high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection and tandem mass spectrometry to trace and identify putative metabolites in the feces and bile of mice injected intravenously with docetaxel. HPLC-PDA revealed two metabolic products in the feces and more than ten potential new metabolites in the bile. Mass spectrometry was performed on docetaxel reference compound, on the known metabolites M-1, M-2, M-3 and M-4, and on HPLC eluate fractions containing metabolic products, six fractions originating from the bile and two from the feces. The mass spectra of the most abundant unknown metabolite in the bile and the feces were identical, and indicated that this structure contained a carboxyl moiety at the tert-butyl group. Under the conditions of storage this product degraded to metabolite M-4. All other unknown metabolites found in the bile samples were oxidized products, with the oxidations in both the C-13 side chain and the baccatin structure, the latter being a new finding.

Comprehensive proteome analysis of ovarian cancers using liquid phase separation, mass mapping and tandem mass spectrometry: a strategy for identification of candidate cancer biomarkers.

A two-dimensional (2-D) liquid phase separation method, liquid isoelectric focusing followed by nonporous reversed-phase high performance liquid chromatography (HPLC), was used to separate proteins from human ovarian epithelial whole cell lysates. HPLC eluent was interfaced on-line to an electrospray ionization (ESI) time of flight (TOF) mass spectrometer to obtain accurate intact protein molecular weights (Mr). 2-D protein expression maps were generated displaying protein isoelectric point (pI) versus intact protein Mr. Resulting 2-D images effectively displayed quantitative differential protein expression in ovarian cancer cells versus non-neoplastic ovarian epithelial cells. Protein peak fractions were collected from the HPLC eluent, enzymatically digested, and analyzed by matrix-assisted laser desorption/ionization (MALDI) TOF-mass spectrometry (MS) peptide mass fingerprinting and by MALDI-quadrupole TOF tandem mass spectrometry peptide sequencing. Interlysate comparisons of differential protein expression between two ovarian adenocarcinoma cell lines, ES2 and MDAH-2774, and ovarian surface epithelial cells was performed. Five pI fractions from each sample were selected for comparative study and over 300 unique proteins were positively identified from the 2-D liquid expression maps using MS, which covered around 60% of proteins detected by on-line ESI-TOF-MS. This represents one of the most comprehensive proteomic analyses of ovarian cancer samples to date. Protein bands with significant up- or down-regulation in one cell line versus another as viewed in the 2-D expression maps were identified. This strategy may prove useful in identifying novel ovarian cancer marker proteins.

Kinetic study on the reactions of platinum drugs with glutathione.

The binding of platinum (Pt) drugs (oxaliplatin, carboplatin, and cisplatin) to glutathione (GSH, 6.75 mM) was investigated at 37 degrees C in Hepes (100 mM, pH approximately 7.4) or Tris-NO(3) (60 mM, pH 7.4) buffer and NaCl (4.62, 6.63, or 7.82 mM). The conditions were chosen to mimic passage of clinical concentrations of the drugs (135 microM) through the cytosol. The reactions were monitored by UV-absorption spectroscopy, high-performance liquid chromatography (HPLC), and atomic absorption spectroscopy. The initial rates, detected by UV absorbance, were similar for oxaliplatin and cisplatin reacting with GSH and were more than 5-fold faster than for carboplatin reacting with GSH. The Pt contents in HPLC eluates corresponding to unbound drug decreased exponentially with time, confirming that the reactions were first order in [Pt drug] and allowing determination of the pseudo first-order rate constants (k(1)). The second-order rate constants (k(2)) were calculated as k(1) divided by [GSH]. The k(2) value for oxaliplatin reacting with GSH was approximately 3.8 x 10(-2) M(-1) s(-1), for cisplatin reacting with GSH approximately 2.7 x 10(-2) M(-1) s(-1), and for carboplatin reacting with GSH approximately 1.2 x 10(-3) M(-1) s(-1) (approximately 32-fold slower than that of oxaliplatin and approximately 23-fold slower than that of cisplatin). These results demonstrate an influence of ligands surrounding the Pt coordination sphere on the reactivity of Pt(2+) with GSH.

High-performance liquid chromatography-electrospray ionization-mass spectrometry study of ginkgolic acid in the leaves and fruits of the ginkgo tree (Ginkgo biloba).

A method is developed for qualitative analysis of ginkgolic acids in the leaves and fruits of Ginkgo biloba by high-performance liquid chromatography (HPLC)-electrospray ionization-mass spectrometry technique. Negative ionization mode is successful in obtaining a very abundant deprotonated molecule [M - H]-. The mass detection sensitivity is higher than ultraviolet detection but relies heavily on the concentration of acetic acid in the HPLC eluent, which consists of acetonitrile-water-acetic acid. The method is also very specific for the analysis of ginkgolic acid with no interferences from the sample matrix.

Analysis of Tetracycline, Oxytetracycline and Chlortetracycline in Plasma Extracts by Electrospray Tandem Mass-Spectrometry and by Liquid Chromatography

Pure standards of tetracyclines (tetracycline, oxytetracycline and chlortetracycline) have been analyzed on a triple quadrupole mass spectrometer with Atmospheric Pressure Chemical Ionization (APCI) and Electrospray Ionization (ESI). ESI appeared to be considerably more sensitive than APCI. Collisional activation of the MH+ ions gave fragment ions at m/z values equal to MH+ - 35 which corresponds to loss of both H2O and NH3. The collisionally induced loss of 35 from MH+ was used in developing a mass spectrometric method based on loop injection and selected reaction monitoring (SRM) as the final analytical step. The method was tested on extracts from fortified plasma and the measurements from the MS-MS analysis were compared with results from High Performance Liquid Chromatography (HPLC) analysis of the same samples. The fortified plasma (from pig) samples were purified by chelate affinity chromatography (amberlite XAD columns). After filtration and evaporation of the solvent the redissolved residues were analyzed by HPLC and by MS-MS with ESI. The HPLC eluates (gradient of 0.01 M aqueous oxalic acid and CH3CN) were monitored at 356 and 369 nm. The signal to noise ratio in the analyses of extracts from plasma fortified to 20 ppb suggests a detection limit for the MS-MS method below 10 ppb of tetracylines in plasma.

Analysis of bacteriohopanepolyols in sediment and bacterial extracts by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

We report the new application of negative ion atmospheric pressure chemical ionization (APCI) to the detection of composite bacteriohopanepolyols in bacterial and sedimentary (or other natural environmental) samples. The method employs the addition of dichloromethane to the high performance liquid chromatography eluent prior to APCI to stimulate formation of chlorine addition/association ions from the hopanoid alcohols. This approach provided the first direct analysis of composite bacterial hopanoids in a sediment, identifying bacteriohopanetrol and bacteriohopanetetrol cyclitol ether. © 1998 John Wiley & Sons, Ltd.

A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGFs inhibits the mitogenic effects of IGF-I and insulin.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is increasingly becoming recognized as an essential mechanism in the regulation of insulin-like growth factor (IGF) bioavailability, both in the bloodstream and at cellular level. Plasmin generated on contact with various cell types provokes proteolytic cleavages that are similar to those induced in vivo by (as yet unidentified) IGFBP-3 proteases. Experimental conditions were determined to achieve plasmin-induced limited proteolysis of recombinant human nonglycosylated IGFBP-3. Two major fragments of 22/25 kilodaltons (kDa) and one of 16 kDa were identified by Western immunoblotting and isolated by reverse-phase chromatography. The 22/25-kDa fragments correspond to the major approximately 30-kDa glycosylated fragment of IGFBP-3 in serum and the 16-kDa fragment, to one of the same size, that is nonglycosylated. Western ligand blot analysis, affinity cross-linking, and competitive binding experiments using radiolabeled IGF and unlabeled IGF-I or -II showed that in the high performance liquid chromatography eluate containing the 16-kDa fragment, all affinity for IGFs had been lost, whereas the affinity of the 22/25-kDa fragments was considerably reduced. Scatchard analysis of the data indicated a 20-fold loss of affinity for IGF-II and an 50-fold loss for IGF-I compared with that of recombinant human IGFBP-3. In a chick embryo fibroblast assay in which DNA synthesis was stimulated both by IGF-I and by insulin (at 100-fold concentrations, so that interaction with the Type 1 IGF receptor would occur), IGFBP-3 was found to inhibit IGF-I-induced stimulation almost totally. It had no effect on stimulation by insulin, which has no affinity for the IGFBPs. With the 22/25-kDa fragments, barely 50% inhibition of IGF-I stimulation was achieved and no inhibition of insulin stimulation. Unexpectedly, with the fraction containing the 16-kDa fragment (despite the total lack of affinity for IGF-I), IGF-I-induced stimulation was inhibited to nearly the same extent as with intact IGFBP-3. In addition, insulin-induced stimulation was inhibited with similar potency. IGFBP-3 proteolysis therefore generates two types of fragment with different activities. One has weak affinity for IGF-I and is only a weak antagonist of IGF action. The other lacks affinity for the IGFs, but nevertheless inhibits IGF-stimulated mitogenesis, thus acting by a mechanism that is independent of the IGFs.

A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGFs inhibits the mitogenic effects of IGF- I and insulin

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is increasingly becoming recognized as an essential mechanism in the regulation of insulin-like growth factor (IGF) bioavailability, both in the bloodstream and at cellular level. Plasmin generated on contact with various cell types provokes proteolytic cleavages that are similar to those induced in vivo by (as yet unidentified) IGFBP-3 proteases. Experimental conditions were determined to achieve plasmin-induced limited proteolysis of recombinant human nonglycosylated IGFBP-3. Two major fragments of 22/25 kilodaltons (kDa) and one of 16 kDa were identified by Western immunoblotting and isolated by reverse-phase chromatography. The 22/25-kDa fragments correspond to the major approximately 30-kDa glycosylated fragment of IGFBP-3 in serum and the 16-kDa fragment, to one of the same size, that is nonglycosylated. Western ligand blot analysis, affinity cross-linking, and competitive binding experiments using radiolabeled IGF and unlabeled IGF-I or -II showed that in the high performance liquid chromatography eluate containing the 16-kDa fragment, all affinity for IGFs had been lost, whereas the affinity of the 22/25-kDa fragments was considerably reduced. Scatchard analysis of the data indicated a 20-fold loss of affinity for IGF-II and an 50-fold loss for IGF-I compared with that of recombinant human IGFBP-3. In a chick embryo fibroblast assay in which DNA synthesis was stimulated both by IGF-I and by insulin (at 100-fold concentrations, so that interaction with the Type 1 IGF receptor would occur), IGFBP-3 was found to inhibit IGF-I-induced stimulation almost totally. It had no effect on stimulation by insulin, which has no affinity for the IGFBPs. With the 22/25-kDa fragments, barely 50% inhibition of IGF-I stimulation was achieved and no inhibition of insulin stimulation. Unexpectedly, with the fraction containing the 16-kDa fragment (despite the total lack of affinity for IGF-I), IGF-I-induced stimulation was inhibited to nearly the same extent as with intact IGFBP-3. In addition, insulin-induced stimulation was inhibited with similar potency. IGFBP-3 proteolysis therefore generates two types of fragment with different activities. One has weak affinity for IGF-I and is only a weak antagonist of IGF action. The other lacks affinity for the IGFs, but nevertheless inhibits IGF-stimulated mitogenesis, thus acting by a mechanism that is independent of the IGFs.

The development and application of coupled HPLC-TLC for pharmaceutical analysis

Successful coupling of high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) facilitates correlation of the retention factor (Rf) of a component in a TLC separation with a peak observed in HPLC, thus eliminating the need for isolation of the component. Experiments were designed to optimize flow and spotting rates for typical HPLC mobile phases, evaluating such parameters as band broadening and on-plate loading. Also investigated were the application of flow splitting and semi-microbore HPLC columns. The problems encountered with the development of TLC plates upon which the HPLC eluent has been applied as a linear streak were studied, as were detection methods for the resulting spots. This paper describes the successful development of the technique, using model compounds of pharmaceutical interest.

Identification of endothelin-1, endothelin-2 and endothelin-3 in human endometrium

This study determined the presence of specific endothelin isoforms in human endometrium using high performance liquid chromatography (HPLC) combined with radioimmunoassay, and immunocytochemistry to detect the endothelin precursors (proendothelin-1, proendothelin-2 and proendothelin-3). Endothelin-like immunoreactivity was measured in HPLC eluates using antisera raised in rabbits against the carboxy-terminal heptapeptide of endothelin-1, which is common to the three endothelin isoforms. Of eight endometrial samples analysed by HPLC, three were in the proliferative phase of the cycle, and five in the secretory phase. Endothelin-1 was detected in seven samples, whereas endothelin-2 and endothelin-3 were seen in four and five specimens, respectively. No relationship was seen between endothelin isoforms and the stage of the cycle. Immunocytochemistry was performed on five proliferative and three secretory phase tissues. When present, staining for the precursor proendothelins was localized to endometrial glandular and luminal epithelium (proendothelin-1, 5 of 8; proendothelin-2, 5 of 8; proendothelin-3, 6 of 8). In two sections, staining was also seen in vascular endothelium using antibody raised against proendothelin-1 (n = 1) and proendothelin-3 (n = 1). These data provide evidence that the three endothelin isoforms are present in human endometrium, and suggest that these potent vasoactive agents may play a role in the paracrine control of the uterine vascular bed.

Simultaneous Automated Determination of Catecholamines, Serotonin, and Their Metabolites in Brain Tissue by HPLC and Electrochemical Detection

Abstract A robust procedure with minimal tissue sample pretreatment, based on reversed-phase, ion-pair high performance liquid chromatography (HPLC) combined with electrochemical detection has been developed for the automated and simultaneous determination of the biogenic amines norepinephrine (NE), epinephrine (E), dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), 3,4-dihydroxyacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (H I A A) in as little as 0.5 mg brain tissue. The chromatographic conditions remain stable for at least 8 weeks. In particular small amounts of samples which do not allow reexamination are routinely automatically analysed without close supervision. The sample pretreatment comprises only homogenization of the tissue samples in the HPLC eluent and centrifugation. The supernatant can be directly injected into the HPLC system without further purification. Moreover, optimal concentrations of octane sulfonic acid (230 μM) and methanol (6%; v/v) in the recirculating eluent, a reversed-phase analytical column filled with 3 μm Nucleosil 100 C18 material, constant column temperature (22 ± 1 °C) and an electrochemical detector with a dual electrode permitted baseline resolution without interference with the pharmacological dose of alpha-methyl-p-tyrosine frequently involved in catecholamine turnover studies as well as the determination of catecholamines, serotonin and their metabolites in femtomolar quantities.

Turnover and oxidation rates of plasma-free fatty acids in obese patients treated with dexfenfluramine.

In order to assess a possible direct metabolic effect of dexfenfluramine (dF) apart from its action on food intake reduction, 10 obese postmenopausal women and 10 obese men (BMI = 32.19 +/- 1.99 kg/m2) were studied in a single-blind fashion: 4 weeks on placebo (D-28 to D0) and 4 weeks on dF (D0 to D28). A balanced diet, computed on the basis of basal metabolic rate x 1.4, was followed throughout the study. The patients' alimentary diary was checked for compliance. FFA turnover and oxidation rate, using 1-14C palmitate intravenous infusion, was determined basally (D0), after single high-dose (30 mg, D1) and after long-term (15 mg b.i.d., D28) administration. Indirect calorimetric measurements were performed using a mass spectrometer, and the usual metabolic parameters were computed. The isotope method was used in order to assess plasma FFA oxidation rate. Plasma FFA were analyzed by high-performance liquid chromatography (HPLC) and specific activity was determined at 55, 60, 65 and 70 min by counting dpm in HPLC eluates corresponding to the retention time of palmitate. The turnover rate was not significantly modified after single high-dose dF administration when compared to basal values (6.24 +/- 1.62 at D1 vs. 5.63 +/- 2.07 mmol/kg/min at D0), but was significantly increased at D28 compared with D0 (6.35 +/- 1.96 mmol/kg/min; p < 0.05). A significant increase in FFA oxidation rate was observed with respect to basal values after dF administration both at D1 (0.81 +/- 0.33 at D1 vs. 0.61 +/- 0.21 mmol/kg/min at D0; p < 0.01) and at D28 (0.77 +/- 0.34 mmol/kg/min; p < 0.015).(ABSTRACT TRUNCATED AT 250 WORDS)