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Query: UNIPROT:P01178 (oxytocin)
 15,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Experimental conditions and parameters involved in high performance liquid chromatography (HPLC) separations of the peptide hormone oxytocin and seven of its diastereoisomers, namely [1-hemi-D-cystine]-, [2-D-tyrosine]-, [4-D-glutamine]-, [5-D-asparagine]-, [6-hemi-D-cystine-], [7-D-proline]-, and [8-D-leucine]-oxytocin, on reverse phase columns were investigated. The effects of solvent, pH, and salt concentration were studied. Using the solvent systems 10% tetrahydrofuran-ammonium acetate buffer or 18% acetonitrile-ammonium acetate buffer and the muBondapak C18 support, oxytocin was separated from each of its diastereoisomers under all conditions studied, but the order of elution of diastereoisomers was highly dependent on solvent and to a lesser extent on pH. Separations of the hormone and its diastereoisomers on reverse phase HPLC and on classical partition chromatography on Sephadex G-25 were compared. The results are discussed in terms of the interactions of the solute with the reverse phase column and the solvent system. Implications of these findings in terms of the different solution conformations of the peptides are discussed.
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PMID:The separation of peptide hormone diastereoisomers by reverse phase high pressure liquid chromatography. Factors affecting separation of oxytocin and its diastereoisomers--structural implications. 3 28

A simple, isocratic, sensitive (1 ng), and specific high-performance liquid chromatographic (HPLC) method based on photodiode-array detection (PAD) is described for simultaneous quantitation of the bioactive peptides, lysine vasopressin (LVP), arginine vasopressin (AVP) and oxytocin (OXY). Acidified pig plasma and left ventricular (LV) tissue samples were first extracted with Sep-Pak C18 columns, and the bioactive peptides were eluted with methanol, then dried at 37 degrees C and reconstituted with HPLC mobile phase. The bioactive peptides were separated by HPLC on a Dynamax 3009-A C8 column with a mobile phase of 0.1% trichloroacetic acid-50 mM heptanesulfonic acid-30mM triethylamine-20% acetonitrile in water, pH 2.5 and identified with a Waters 990-PAD system (spectrum index plots in the range 200-400 nm). Standards of LVP, AVP and OXY and their mixtures showed a linear increase in the range 5 to 100 ng and were eluted at 6.1, 6.9 and 4.6 min, respectively. Spectrum analysis showed a distinct absorption peak at 280 nm, corresponding to peptide bonds. The reproducibility of the method coefficient of variation for standards is 6.9, 5.8 and 4.7% for LVP, AVP and OXY, respectively. In plasma and tissue it is much higher: 12.9% (LV tissue) and 18.6% (plasma) for LVP. Pig plasma contains negligible amounts of AVP and OXY; LVP is much higher (0.28 +/- 0.19 ng/ml). In pig tissue, LVP predominates (6.95 ng/g wet weight) compared to AVP (1.45) and OXY (1.50). Spectral analysis is necessary to identify the bioactive peptide peaks among interfering substances and to increase the sensitivity four-fold. The method described here is useful for the simultaneous determination of LVP, AVP and OXY in the nanogram range and can be extended to picogram levels by employing PAD spectral analysis techniques.
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PMID:Isocratic high-performance liquid chromatography-photodiode-array detection method for determination of lysine- and arginine-vasopressins and oxytocin in biological samples. 205 Jul 61

Although the posterior pituitary is known to contain the PRL releasing activity or factor (PRF), its chemical identification has been a matter of dispute. In the present study, we purified PRF in porcine posterior pituitary extracts to chemically determine the primary structure. PRF activity was assessed during purification by the release of immunoreactive PRL from superfused rat pituitary cells. Two hundred seventy porcine posterior pituitaries were boiled, homogenized, and extracted with 2 M acetic acid. The acid extract was precipitated with 67% acetone, and the supernatant was absorbed onto a C18 column. The column was eluted step-wise with 10, 20, 30, 40, 50, and 60% acetonitrile (CH3CN) in 0.1% trifluoroacetic acid (TFA). The greatest PRF activity was recovered in the 30% CH3CN/0.1% TFA fraction and was further purified by ion-exchange chromatography on SP-Sephadex, followed by gel-filtration on Sephadex G-50. The Sephadex G-50 fractions with major PRF activity were finally purified by two cycles of reverse phase HPLC, yielding a single peak of PRF. Amino acid, as well as sequence analyses, indicated that the highly purified PRF was oxytocin. Authentic oxytocin showed the same chromatographic behavior and biological activity as those of the isolated peptide. In another experiment, desalted crude extracts of rat and porcine posterior pituitary tissues were directly chromatographed by reverse phase HPLC, and each fraction was assayed for PRF activity. Only two areas showed PRF activity; the largest activity coeluted with oxytocin and the smaller one co-eluted with vasopressin. The fractions which coeluted with oxytocin also showed oxytocin immunoreactivity, as examined by RIA. The results clearly indicated that the major PRF in these posterior pituitary extracts was oxytocin.
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PMID:Oxytocin is the major prolactin releasing factor in the posterior pituitary. 229 52

Optimized C18 reversed-phase systems for oxytocin, desamino-oxytocin, lysine-vasopressin, ornithine-vasopressin and felypressin with gradient elution are discussed, focussing on precision, selectivity and ruggedness of the methods. Data from collaborative studies are presented, demonstrating the equivalence of high-performance liquid chromatography (HPLC) assays to bioassays. The findings suggest that HPLC is an excellent alternative to the time-consuming and less reliable animal testing.
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PMID:The use of high-performance liquid chromatography in the quality control of oxytocin, vasopressin and synthetic analogues. 248 22

A competitive, double antibody enzyme immunoassay for oxytocin in a heterologous system was developed. Horseradish peroxidase was conjugated with oxytocin using N-succinimidyl 3-(2-pyridyldithio) propionate, and rabbit anti-oxytocin serum was produced by immunization of oxytocin-bovine serum albumin complex which was prepared by the carbodiimide method. The sensitivity of the assay was 4 microIU/tube, which corresponded to 10 microIU per ml using 400 microliters of the sample which was extracted from the same volume of plasma by means of SEP-PAK C18 cartridges. The coefficients of variation for different levels of oxytocin ranged from 6.8-15.9% and 8.5-16.7%, for intra- and inter-assay. Recovery of oxytocin added to plasma after extraction was 99-117%. No or little cross-reaction with arginine- and lysine-vasopressin was found. Plasma oxytocin concentrations determined by the proposed enzyme immunoassay were well correlated with those determined by radioimmunoassay (r = 0.90).
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PMID:Enzyme immunoassay for oxytocin. 269 18

We describe a specific double-antibody radioimmunoassay for measuring arginine vasopressin (AVP) in human plasma. Antisera of high avidity were obtained from rabbits that had been injected with AVP coupled to bovine thyroglobulin. The antibody reacts with both the tripeptide tail and the pentapeptide ring of the molecule, thereby eliminating cross reaction with oxytocin. Synthetic AVP was labeled with 125I by a modification of the Chloramine-T technique. The specific activity of the labeled hormone was 29 MBq/micrograms of AVP, as estimated by self-displacement analysis. The assay involves Sep-Pak C18 extraction of acidified (pH 4) plasma. Recovery of [3H]AVP added to plasma averaged 86.6 (SD 6.1)% (n = 14). Dilution curves for plasma showed linearity of response with concentration. The overall sensitivity was 0.3 ng/L when 2-mL plasma samples were extracted. The intra-assay CV was 7.8% at 4.8 ng/L (n = 12) and the interassay CV was 12.3% (n = 16) and 6.3% (n = 14) at 2.7 and 4.1 ng/L concentrations, respectively.
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PMID:Development and evaluation of a radioimmunoassay for Arg8-vasopressin, after extraction with Sep-Pak C18. 298 81

Oxytocin (OT) and arginine vasopressin (AVP) are often secreted in response to the same stimuli. The hormones are seldom assayed together, however, because of labor intensive sample preparation and the duplicate volumes required. A method has been developed for the simultaneous extraction and separation of OT and AVP from a single serum sample. The method is suited for sample preparation prior to radioimmunoassay (RIA) and reduces sample volume and processing time by 50%. Serum, supplemented with labeled and unlabeled OT and AVP, was adsorbed onto C18 (octadecasilyl-silica, ODS) Sep-Pak cartridges. After washing with phosphosaline and 3% aqueous acetone, OT was eluted with 98% aqueous acetone followed by AVP with 80% acidified (0.02 mol/L HCl) acetone. The recoveries, determined by radioactivity and RIA measurements, were 86 +/- 3% (OT) and 71 +/- 7% (AVP). Cross contamination was less than 10%. Sep-Paks extracted up to 100 pg/mL of the hormones from 10 mL of serum. The method was employed to measure OT and AVP in the pregnant ewe. Both hormones were elevated during salt-loading and dehydration and were decreased by carotid infusions of ethanol.
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PMID:Preparation of serum oxytocin and arginine vasopressin prior to radioimmunoassay: simultaneous extraction and separation on C18 Sep-Pak cartridges. 323 35

The development and evaluation of a radioimmunoassay for N alpha-tri-glycyl-lysine8-vasopressin is described. The site of hapten conjugation of the immunogen has been controlled and the use of various radiolabelled tracers has been evaluated with special reference to the site of iodination. The most extensively studied antiserum showed specificity for the N-terminal triglycyl-extension as well as for several amino acid residues of the vasopressin ring. It crossreacted 27%, 28%, and 0.3% with Lys8-vasopressin, arg8-vasopressin and oxytocin respectively, and it was used to quantify triglycyl-lysine8-vasopressin in human plasma after SepPak C18 extraction. The sensitivity of the assay was 5 pg/tube with an intra-assay CV of 5-6% at 17 and 70 pg/tube. The identity of the immunoreactivity was studied by reversed phase chromatography.
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PMID:Development of an immunoassay for glypressin, an N-terminal extended vasopressin analogue. 336 Sep 20

A high-performance liquid chromatographic assay has been developed for quantitating oxytocin in common large volume parenteral intravenous solution matrices. Separation is accomplished by a reversed-phase mechanism using a C18 column. The analyte is detected fluorimetrically after post-column derivatization with fluorescamine. Reaction efficiency is controlled by the use of a reaction buffer which is added separately from the fluorescamine via a dual pump reactor system. Given the low analyte concentration [40 parts per billion (10(9)) or less], the samples are concentrated on-line through the use of a trapping column and switching valve. To improve productivity, pre-concentration and analysis of adjacent samples is timed to occur concurrently. Performance of the assay is characterized by a high degree of accuracy, precision and ruggedness; the system is capable of distinguishing between the analyte, matrix components, impurities and common degradates.
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PMID:Determination of trace levels of oxytocin in pharmaceutical solutions by high-performance liquid chromatography. 342 46

A chemical method has been established for the detection of carboxyl-terminally amidated peptides in tissue extracts. Tissue was homogenized in an acidic medium designed to solubilize peptides while precipitating high-molecular-weight protein. The homogenate supernatant was in turn subjected to reversed-phase extraction with C18 Sep-Pak cartridges. The eluates were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Individual fractions were exhaustively digested with thermolysin, derivatized with phenylisothiocyanate (PITC), and then subjected to ethyl acetate extraction under basic conditions. The phenylthiocarbamyl (PTC)-amino acid amide derivatives were selectively taken up into the organic phase, while the other digestion products remained in the aqueous phase. The organic phase was analyzed by RP-HPLC on a Pico-Tag amino acid analysis column, monitoring eluates at 254 nm. PTC-amino acid amides were identified and quantitated by comparing their elution positions and peak areas, respectively, with those of standards. Their identities were confirmed by amino acid analysis, following hydrolysis with hydriodic acid. The technique was applied to extracts of bovine posterior pituitaries and a human medullary thyroid carcinoma. Vasopressin (-Leu-Gly-amide), oxytocin (-Gly-amide), Lys1 gamma 1-melanotropin (-Phe-amide), and various acetylated and non-acetylated forms of alpha-melanotropin (-Val-amide) were identified in the posterior pituitary extract. Various forms of calcitonin (-Val-Gly-Ala-Pro-amide) were detected in the tumour extract. For vasopressin and calcitonin the thermolytic digest resulted in di- and tetra-peptides, respectively, reflecting thermolytic cleavage at more favoured sites.
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PMID:Use of Pico-Tag methodology in the chemical analysis of peptides with carboxyl-terminal amides. 373 29


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