5-羟色胺和胃动素在狗小肠移行性复合运动调控中的作用

应用慢性植入小肠腔外应力传感器记录清醒狗小肠移行性复合运动（ＭＭＣ）。在颈外静脉和支配１０￣１５ｃｍ肠段的空肠动脉内分别放置－硅胶管以备灌流药物的取血样品用。本研究观察了静脉和动脉注射药物对小肠ＭＭＣ的影响及ＭＭＣ不同时相血浆５－羟色胺（５－ＨＴ）和胃动素的浓度变化。结果表明：（１）ＭＭＣ不同时相血浆５－ＨＴ和胃动素浓度呈周期性变化,５－ＨＴ峰值在胃动素峰值之前出现。血浆５－ＨＴ浓度在ＭＭＣⅡ相后     

High level production of a peptide hormone analogue [Leu13]motilin inE. coli

Summary A peptide hormone analogue [Leu¹³]motilin has been produced in high yield by recombinant DNA techniques. The peptide was expressed from a multicopied [Leu¹³]motilin gene fused to a salmon growth hormone gene fragment. The monomeric [Leu¹³]motilin was obtained by treatment of the fusion protein with cyanogen bromide, carboxypeptidase A and B. [Leu¹³]motilin showed the equivalent biological activity to that of the natural form.     

Angiotensin stimulates oxytocin release

A sensitive and specific radioimmunoassay for oxytocin (OT) was developed to study the effect of angiotensin II (ANG II) upon neurohypophyseal OT release in conscious rats. ANG II injected into the lateral cerebral ventricle (i.c.v.) produced within 60 seconds a steep increase in plasma OT concentration from a control value of 10.46 ± 1.35 fmol/ml to 88.95 ± 5.06 fmol/ml and 119.56 ± 11.46 fmol/ml following 10 and 100 ng i.c.v. ANG II, respectively. Inhibition of OT release by simultaneous application of the ANG II antagonist {Sar¹, Ile⁸}-ANG II suggests that ANG II acted via specific angiotensin receptors in the brain.     

Electron-capture gas chromatography of plasma sulphonylureas after extractive methylation

Conditions for the extractive alkylation of eight sulphonylurea hypoglycemic drugs have been evaluated. Extractive methylation of the compounds was achieved within 90 min using tetrabutylammonium as counter-ion (0.1 M at pH = 6.9) with 5% methyl iodide in dichloromethane as organic phase. Mass spectral analysis showed derivatives methylated at the sulphonamide nitrogen. A higher pH or use of tetrapentylammonium as counter-ion caused hydrolysis of the sulphonylureas.The derivatives showed a high electron-capture response with minimum concentrations detectable in the range 1–4 × 10^?16 moles sec^?1.Therapeutic plasma concentrations of glipizide and tolbutamide were determined by direct extractive methylation of the compounds from the plasma sample. The glipizide derivative was determined by electron-capture gas chromatography down to about 20 ng/ml in a 0.5-ml plasma sample. The relative standard deviation at the 0.2 μg/ml level of glipizide was 6% (n=6). The corresponding figure in the determination of tolbutamide at the 10 μg/ml level was 3% (n=10).     

Variations in plasma motilin, somatostatin, and pancreatic polypeptide concentrations and the interdigestive myoelectric complex in dog

We have looked at the plasma concentrations of motilin, pancreatic polypeptide (PP), and somatostatin (STS) during the various phases of the interdigestive motor complex (IDMC) in dogs. As expected, motilin cyclical increase was always associated with the phase III of the IDMC. Statistical analysis of PP variations revealed a significant rise 10 min before duodenal phase III; however, in individual animals, this relationship was inconsistent. Although a dose-related increase in PP blood levels was induced by administration of synthetic canine motilin (0-200 ng kg-1 iv), fasting plasma levels of PP were not correlated with the concentrations of circulating endogenous motilin. After truncal vagotomy, while motilin release and the intestinal motility pattern remained unaltered, the phase III associated cyclical increases of PP disappeared. Infusion of physiological amounts of PP (1 microgram kg-1 h-1 for 3 h) mimicking the postprandial release failed to reproduce a fed pattern type of intestinal motility and of motilin secretion. No statistical correlation could be established between STS plasma levels and the motor activity of the intestine. STS plasma levels were not correlated with circulating concentrations of motilin and the exogenous administration of physiological doses of synthetic canine motilin failed to modify STS plasma levels. Morphine (200 micrograms kg-1 iv) stimulated only the release of motilin. These data suggest that the role played by circulating concentrations of PP and STS in the control of the IDMC in dog is at most minimal.     

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg6, d-Trp7,9, mePhe8]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg⁶, d-Trp^7,9, mePhe⁸]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met¹¹ (M2) and G[Met¹¹(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C₁₈, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C₂-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.     

Interaction of circulating cell-derived and plasma growth factors in stimulating cultured smooth muscle cell replication

Multiple growth factors that circulate in plasma have been shown to stimulate cellular growth in vitro. The plasma growth factors appear to stimulate DNA synthesis in cultured fibroblasts only after prior exposure of cell growth factors derived from circulating cell types, such as platelets and macrophages. The purpose of these studies was to investigate the role of the plasma growth factors in stimulating smooth muscle cell replication following exposure to platelet-derived growth factor (PDGF). Following transient exposure to PDGF, insulin stimulated smooth muscle cell replication but only when supraphysiologic concentrations were used (i.e., greater than 1.0 μg/ml). Somatomedin-C (Sm-C), in contrast, was found to stimulate a 320% increase in [³H]thymidine incorporation when concentrations that are present in extracellular fluids were used (i.e., 0.5–10 ng/ml). Epidermal growth factor (EGF), an important mitogen for multiple cell types, caused a 70% increase in [³H]thymidine incorporation when added to quiescent cells following PDGF exposure, and EGF caused a substantial increase in the absolute level of [³H]thymidine incorporation when coincubated with Sm-C. When EGF (1 ng/ml) was incubated simultaneously with concentrations of Sm-C between 1 and 10 ng/ml plus Sm-C-deficient plasma, maximal [³H]thymidine incorporation was 2.1-fold greater in the presence of EGF. In contrast, insulin (20 ng/ml), when coincubated with Sm-C under similar conditions, had no enhancing effect on the cellular response to Sm-C. None of the plasma factors tested was an effective stimultant of replication when incubated either in serum-free medium or in the presence of Sm-C-deficient plasma without prior PDGF exposure. Hydrocortisone was shown to inhibit smooth muscle cell replication in concentrations between 10^?7 and 10^?5M. In summary, multiple plasma growth factors can stimulate the smooth muscle cell replication, and Sm-C appears to be most effective of those tested. Insulin and EGF appear to work by different mechanisms; that is, EGF can facilitate the cellular response to Sm-C, whereas insulin is effective only at supraphysiologic concentrations at which it will directly bind to Sm-C receptors.     

Critical considerations in teh development of an assay for sulfidopeptide leukotrienes in plasma

A sensitive and specific assay has been developed for measurement of total sulfidopeptide leukotriense (LT) in plasma. LTC₄ and LTD₄ in plasma are converted to LTE₄ which is then extracted by C₁₈ Sep-Pak binding and elution. Total LTE₄ in resolved by reverse phase high performance liquid chromatography (RP-HPLC) and quantitated by radioimmunoassay (RIA). A [³H]LTE₄ internal standard is added to the starting plasma sample to allow RP-HPLC to be assayed for LTE₄-like immunoreactivity. The correlation between the measured increase in LTE₄ concentration after addition of incremental amounts of LTC₄ and LTE₄ to plasma was 0.989 and 0.978, respectively, with slopes of 1.05 and 1.11. Addition of 51 pg/ml LTE₄ to 5 ml plasma was detectable; the measured increase was 48 ± 12 pg/ml (mean ± SE, n = 7). The intra-assay coefficient of variation for 341 pg/ml of added LTC₄ was 3.2% (n = 6). Sulfidopeptide leukotrienes could not be detected in blood samples taken from 12 normal volunteers in whom the theoretical detection limit, calculated from the sensitivity of the RIA, the overall recovery of LTE₄, and the volume of plasma extracted, was 83 ± 4 pg LTE₄/ml plasma (0.19 ± 0.01 pmol sulfidopeptide leukotriene/ml plasma; mean ± SE).     

Radioimmunoassay of secretin in plasma.

A sensitive, specific and reproducible radioimmunoassay for secretin is described. Antibodies were readily produced against low microgram quantities of synthetic secretin. The secretin antibodies did not cross-react with the structurally similar G.I.P., V.I.P., or glucagon. Synthetic secretin was iodinated using Chloramine "T" and purified by a two-state procedure incorporating gel filtration and radient elution from a cation exchange column. Plasma samples were found to produce variable interference in the assay necessitating the incorporation of secretin-free "blands" for each patient's plasma. Production of secretin-free plasma was by incubation of plasma samples at 37 degrees C for 96 hours. The sensitivity of the assay was 12.5-25 pg/ml. Normal fasting secretin levels were 21 +/- S.E. 7 pg/ml. A mean rise in plasma secretin to 220 pg/ml was observed after intraduodenal acidification.     

Motilin effects on the proximal stomach in patients with functional dyspepsia and healthy volunteers

This study investigates motilin effects on the proximal stomach in patients with functional dyspepsia (FD) and healthy volunteers. Eight healthy volunteers and 12 patients with FD were infused with synthetic motilin or placebo. Proximal gastric volume was measured with a barostat at constant pressure and during isobaric distensions. Abdominal symptoms were scored by visual analog scales. Plasma motilin concentrations were measured by radioimmunoassay. Motilin concentrations and baseline gastric volumes were similar for patients and healthy volunteers. Motilin, compared with placebo, reduced gastric volume by 112 ml [F(29,195); confidence interval (CI) 95%] in patients and by 96 ml [F(-7,200); CI 95%] in healthy volunteers. In patients, motilin decreased compliance by 76 ml/mmHg [F(9,143); CI 95%] compared with placebo, which was similar in volunteers [66 ml/mmHg; F(11,120); CI 95%]. Patients were more nauseous during motilin compared with placebo (P = 0.04), whereas healthy volunteers did not experience nausea. We conclude that in a fasted condition, FD patients have a similar proximal gastric motor response to motilin as healthy volunteers, but experience an exaggerated sensation of nausea.     

Determination of rifampin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifampin in human plasma. Rifampin and sulindac (internal standard) are extracted from human plasma using a C₂ Bond Elut extraction column. A 100-μl volume of 0.1 M HCl is added to the plasma before extraction to increase the retenction of the compounds on the extraction column. Methanol (1 ml) is used to elute the compounds and 0.5 ml of 3 mg/ml ascorbic acid in water is added to the final eluate to reduce the oxidation of rifampin. Separation is achieved by reversed-phase chromatography on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate-acetonitrile (55:45, v/v). Detection is by ultraviolet absorbance at 340 nm. The retention times of rifampin and internal standard are approximately 4.4 and 7.8 min, respectively. The assay is linear in concentration ranges of 50 to 35 000 ng/ml. The quantitation limit is 50 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Sensitive and specific method for the determination of josamycin in human plasma by liquid chromatography–mass spectrometry

A highly sensitive and specific method for the determination of josamycin in human plasma by LC–MS was developed and validated. Josamycin was extracted from human plasma by a single-step liquid–liquid extraction and analyzed by LC–MS via an electrospray ionization interface. Selected ion monitoring was used to detect josamycin and its internal standard. The intra-day precision and accuracy, expressed as C.V. and R.E., ranged from 2.8% to 13.5% and −10.3% to 7.6%, respectively. The lower limit of detection was 0.1 ng/ml and the lower limit of quantitation was set at 1 ng/ml when 0.5 ml of plasma was used. No endogenous interference was observed in human plasma obtained from drug-free volunteers.     

Plasma Testosterone in Bulls

Levels of peripheral plasma testosterone and LH were studied in 4 bulls hourly during a 12 hr. sampling period at 5 times of the year. The average plasma testosterone levels were significantly lower in October (1.8 ng/ml, Ρ < 0.001) and December (2.5 ng/ml, Ρ < 0.05) than in February, June and August (3.5, 3.7 and 3.7 ng/ml respectively). LH showed a slight fluctuation during the day, with values ranging between 0.8 and 3.8 ng/ml, but underwent no significant seasonal variation. A significant increase in average plasma testosterone was observed 1 hr. after the LH peaks (P < 0.001).     

TCDD causes stimulation of c-ras expression in the hepatic plasma membranes in vivo and in vitro

A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21^ras protein, or indirectly measured by the specific binding of ³H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P < 0.05, Student t test) levels of hepatic p21^ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the differencebecame statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21^ras protein which did not become statistically significant even at 24 hr post-dosing. TCDD evoked increases in hepatic p21^ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a ³H-GTP binding protein with MW of approximately 21 Kd. ³H-GTP binding in total hepatic membranes was also elevated (P<0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of ≈ 21 Kd.     

The Effects of Ziprasidone, Clozapine and Haloperidol on Lipid Peroxidation in Human Plasma (in vitro): Comparison

Oxidative injury in schizophrenia can be caused by the disease itself and probably by antipsychotics treatment. The aim of the study was to establish whether there is a difference between ziprasidone, clozapine and haloperidol effect on lipid peroxidation in human plasma, measured by the level of thiobarbituric acid reactive substances (TBARS). The samples of plasma from healthy subjects were incubated with the drugs (1 and 24 h) and compared with control samples. The levels of TBARS were measured spectrophotometrically, according to the Rice-Evans method. The multifactorial variance analysis ANOVA II test showed that the differences in TBARS levels significantly depended on the studied drugs (ziprasidone 40 ng/ml, haloperidol 4 ng/ml and clozapine 350 ng/ml) (F = 3.248 p = 0.047) and (ziprasidone 139 ng/ml, haloperidol 20 ng/ml and clozapine 420 ng/ml) (F = 2.248, p = 2.9 × 10^?5). Statistically increased levels of TBARS after 24 h incubation of plasma with ziprasidone 139 ng/ml and haloperidol 20 ng/ml (p < 0.001, p < 0.05 respectively) in comparison with control samples were observed. Clozapine did not significantly (p > 0.05) increase TBARS level in plasma in comparison with control samples. The results obtained in the study showed that ziprasidone and haloperidol contrary to clozapine induced a significant increase in plasma lipid peroxidation.     

Ontogeny of insulin binding during chick skeletal myogenesis in vitro.

Our studies show that insulin receptors exist on chicken skeletal muscle cells at all developmental stages in culture. ¹²⁵I-labeled insulin binding at physiological concentrations to mature myotubes demonstrated saturability, binding proportional to cell number, reversibility, and specificity by competition with native hormone which reduced specific binding by 40% with 1 ng/ml and was maximal with 10 μg/ml. Further evidence for specificity was shown by no competition of insulin specific binding with insulin A chain, insulin B chain, growth hormone, and thyrotropin. Two binding sites were detected, with affinity constants of 10¹⁰M^?1 and 2 × 10⁹M^?1. The hormone receptor complex showed rapid dissociation (70% in 30 min) after equilibrium binding. During myogenesis, an increase in insulin receptors occurs from 500 per proliferating myoblast to 3000 per cell equivalent in mature (6 day) myotubes. Since these studies demonstrate that insulin receptors are present and other studies have shown that insulin is present during most of chicken embryogenesis, insulin may regulate muscle development in vivo to a greater degree than previously suspected.     

NMR solution structure and dynamics of motilin in isotropic phospholipid bicellar solution

The structure and dynamics of the gastrointestinal peptide hormone motilin, consisting of 22 amino acid residues, have been studied in the presence of isotropic q=0.5 phospholipid bicelles. The NMR solution structure of the peptide in acidic bicelle solution was determined from 203 NOE-derived distance constraints and six backbone torsion angle constraints. Dynamic properties for the ¹³C-¹H vector in Leu10 were determined for motilin specifically labeled with ¹³C at this position by analysis of multiple-field relaxation data. The structure reveals an ordered -helical conformation between Glu9 and Lys20. The N-terminus is also well structured with a turn resembling that of a classical -turn. The ¹³C dynamics clearly show that motilin tumbles slowly in solution, with a correlation time characteristic of a large object. It was also found that motilin has a large degree of local flexibility as compared with what has previously been reported in SDS micelles. The results show that motilin interacts with the bicelle, displaying motional properties of a peptide bound to a membrane. In comparison, motilin in neutral bicelles seems less structured and more flexible. This study shows that the small isotropic bicelles are well suited for use as membrane-mimetic for structural as well as dynamical investigations of membrane-bound peptides by high-resolution NMR.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

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-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.     

Measurement of plasma uracil using gas chromatography–mass spectrometry in normal individuals and in patients receiving inhibitors of dihydropyrimidine dehydrogenase

A sensitive gas chromatographic–mass spectrometric method is described for reliably measuring endogenous uracil in 100 μl of human plasma. Validation of this assay over a wide concentration range, 0.025 μM to 250 μM (0.0028 μg/ml to 28 μg/ml), allowed for the determination of plasma uracil in patients treated with agents such as eniluracil, an inhibitor of the pyrimidine catabolic enzyme, dihydropyrimidine dehydrogenase. Calibration standards were prepared in human plasma using the stable isotope, [¹⁵N₂]uracil, to avoid interference from endogenous uracil and 10 μM 5-chlorouracil was added as the internal standard.     

Determination of acetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatography

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C₈ columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water–phosphate buffer (pH 2.5)–acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 μg/cm² and of SA from 0.1 to 5 μg/cm² in tape and to quantities of ASA 0.1 to 2 μg/ml and 1 to 50 μg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 μg/cm²) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 μg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100–200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.