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Objectives: Norepinephrine is usually used in emergency situations such as in intensive care units (ICUs) for the restoration of blood pressure. The objective was to study the stability of highly-concentrated solutions of norepinephrine at 0.50mg/mL and 1.16mg/mL, diluted in glucose 5% (G5%) in polypropylene syringes, protected or not from light, up to 48h., Materials and Methods: Chemical stability was analysed by high-performance liquid chromatography coupled to photodiode array detection at each time of the analysis. The method was validated according to the International Conference on Harmonisation Q2(R1). Physical stability was evaluated by visual and subvisual inspection. Three syringes for each condition were prepared. At each time of the analysis, three samples were analysed for each syringe. pH values were evaluated at each moment of the analysis., Results: Solutions of norepinephrine at 0.50 and 1.16mg/mL, diluted in G5%, with or without protection from light, retained more than 95.0% of the initial concentration after a 48-hour storage at 20-25°C. No visual and subvisual modification occured during the stability study. No degradation product appearing during the stressed degradation was observed during the study but an additional peak with a relative retention at 0.66 was observed and constant. This peak was identified as 5-hydroxymethylfurfural, a degradation product of glucose., Conclusion: Norepinephrine diluted in G5% at 0.50mg/mL and 1.16mg/mL was physically and chemically stable over a period of 48hours at room temperature. These stability data of highly concentrated solutions provide additional knowledge to assist intensive care services in daily practice., (Copyright © 2019 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.)

Two-dimensional correlation spectroscopy (2DCOS) was employed for the identification of ephedrine (Ep) and pseudoephedrine (Ps) present in illegally adulterated slimming herbal products (SHPs). Second derivative (SD) spectral pretreatment was used prior to 2DCOS analysis to highlight specific features not readily observable by Fourier transform infrared spectroscopy (FTIR), SD-FTIR, or original 2DCOS, leading to enhanced resolution and a reduced lower limit of detection (<1% in this study). After examining the power spectra of suspicious SHPs, bands containing characteristic peaks for Ep (701, 747, 1042, 1363, 1375, 1451, 1478 cm -1 etc) and/or Ps (703, 767, 1037, 1375, 1428, 1455, 1590 cm - 1, etc.) were selected to construct synchronous and asynchronous maps for further analysis, while the latter was applied to discriminate positive SHPs adulterated simultaneously with Ep and Ps. The proposed method is simple and economical and has the potential to identify other chemicals in illegally adulterated herbal products. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Marinobufagenin (MBG), a steroid compound belonging to the bufadienolide cardiac inotropes, is a molecule enjoying a growing interest in the early diagnostic of volume expansion-mediated hypertensive states. This endogenous mammalian cardiotonic and natriuretic bufadienolide (characterized by vasoconstrictive activities) inhibits the α1 isoform of Na(+), K(+)-ATPase, implicating it in series of pathophysiological circumstances such as volume-expansion, essential hypertension and preeclampsia. Indeed, the enhanced production of MBG in preeclamptic patients has been confirmed in several studies, leading us to consider MBG as a biomarker for preeclampsia. The main source for MBG is located in the parotid and skin gland secretions of the toad Bufo marinus in which MBG is the major steroid cardiotonic component. This review emphasizes the key role of analytical development for dosage methods of MBG in biofluids, in the emergence of future perspectives in the diagnostic and therapeutic fields of preeclampsia (e.g. to investigate the biosynthetic origin of MBG and to better understand its implications)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: The idea that reactive oxygen species (ROS) are associated with the pathogenesis of inflammatory periodontal diseases and have a role (direct or indirect) in tissue damage has become a major area of research over the last decade. The purpose of this study is to determine, presence of 8-isoprostane in gingival crevicular fluid (GCF) in healthy, gingivitis and chronic periodontitis (CP) subjects and to find an association, if any between GCF 8-isoprostane levels and clinical periodontal parameters., Materials and Methods: 78 subjects (40 males and 38 females) were selected based on their clinical parameters into three groups: Group 1 (26 healthy), Group 2 (26 gingivitis subjects) and Group 3 (26 CP subjects). GCF 8-isoprostane levels were estimated by ELISA., Results: The 8-isoprostane concentration in GCF was highest in subjects with chronic periodontitis as compared to gingivitis and healthy subjects and a significant association was observed between GCF 8-isoprostane levels and all periodontal parameters., Conclusions: There was increase in 8-isoprostane levels in GCF as the disease process progressed from health to gingivitis and chronic periodontitis, suggesting a role for increased oxidative stress in CP., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

F2-isoprostanes are a family of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid. Several F2-isoprostanes, but in particular 8-epi PGF(2alpha), are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8-epi PGF(2alpha) concentrations in human plasma, whole blood, erythrocytes and urine. 8-epi PGF(2alpha)-d4, a stable isotope derivative of 8-epi PGF(2alpha), was used as an internal standard (IS). A 50 microL sample was focused on-column and separated on two 3 microm particle size SUPELCOSIL ABZ+Plus HPLC columns (15 cm x 4.6 mm and 7.5 cm x 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 353.4 --> 193.1 (8-epi PGF(2alpha)), 357.4 --> 197.1 (8-epi PGF(2alpha)-d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Objectives: The aim of the present study is to determine anion and cation contents of the herbals used in Turkish folk remedy to explore the rationale of their use in hemorrhoid treatment in the context of the vasoactivity of these elements., Design: Herbs used in the treatment of hemorrhoid were determined by the way of literature search. These herbs were obtained from certified herb sellers. Ground herb samples were placed in individual tubes containing methanol and incubated for 48 hours at 30 degrees C. At the end of the incubation, supernatants were analyzed for their ion concentrations by using ion chromatography., Results: The difference between ion levels between systemic and locally used herbs, was not statistically significant (p>0.05). Anion concentrations (except nitrate) of locally used herbs were slightly higher than systemically used herbs (p>0.05). Cation levels (except magnesium) of systemically used herbs were slightly higher than locally used herbs (p>0.05). It was shown that the concentration of vasoconstrictor effective ions was higher than the concentration of vasodilator effective ions (p<0.001). While vasoconstrictor ion concentration of systemically used herbs was 88.06 +/- 147.42 mg, vasodilator ion concentration of locally used herbs was 90.15 +/- 136.94 mg. The difference between vasodilator concentrations of groups was more evident; 5.39 +/- 9.80 mg and 14.32 +/- 66.48 mg for locally and systemically used herbs respectively., Conclusions: This study showed that herbal remedies used for the treatment of hemorrhoid in Turkey contain vasoactive and especially vasoconstrictor ions. Vasoconstrictor agents could amplify each others' effects as it has been previously shown, therefore, it is probable that the vasoconstrictor ion contents could contribute to the curative effects of herbals in the treatment of hemorrhoids.

Millepora complanata is a normal resident of coral reefs in the Mexican Caribbean. In this study, we describe for the first time the vasoconstrictor, phospholipase A2 (PLA2), and hemolytic activities elicited by a crude extract obtained from M. complanata. This extract caused a concentration-dependent contraction of isolated rat aortic rings (EC50=22.4+/-1.1 microg protein/mL). This effect was endothelium independent and significantly reduced in the absence of extracellular Ca2+ and when the intracellular Ca2+ stores were depleted. In addition, the crude extract obtained from M. complanata showed PLA2 activity (7.231+/-0.092 mmol min(-1) mg(-1)) and hemolysis of rat erythrocytes (HU50=1.64+/-1.04 mug protein/mL). The hemolysis increased in the presence of Ca2+ and decreased in the presence of cholesterol. Furthermore, this hemolysis was significantly reduced after incubation with an inhibitor of PLA2 enzymes. The hemolytic and vasoconstrictor effects were abolished after incubating the extract under denaturing conditions. Reverse phase chromatography of the M. complanata extract afforded 19 fractions (F1 to F19). F4 induced hemolysis and contained mainly a protein of 30 kDa, probably a PLA2 enzyme, while F8 and F11, containing mainly proteins of 15 and 20 kDa respectively, produced vasoconstrictor effects mediated by different mechanisms of action.

Introduction: The lipid composition of the sperm membrane has a significant effect on the functional characteristics of spermatozoa., Patients and Methods: In the present study, fatty acid composition of spermatozoa and seminal plasma levels of free 15-F(2t)-Isoprostane and catalase were assayed in men with normozoospermia, asthenozoospermia, asthenoteratozoospermia, and oligoasthenoteratozoospermia., Results: In spermatozoa from asthenozoospermic men only oleic acid levels showed a significant difference from normozoospermic men. In spermatozoa from asthenoteratozoospermic and oligoasthenoteratozoospermic samples all of the tested fatty acids were significantly higher than those from normozoospermic samples. Seminal plasma levels of catalase were significantly lower in all patients while levels of free 15-F(2t)-Isoprostane were significantly higher in all patients compared with normozoospermic men., Discussion: Spermatozoa from pathological samples may have higher levels of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), than spermatozoa from normozoospermic men. Therefore, damage induced by lipid peroxidation would be higher in spermatozoa from pathological samples than those from normozoospermic men.

A simple and rapid procedure based on gas chromatography-mass spectrometry (GC-MS) is described for determination of synephrine, active principle of Citrus aurantium plant, in solid and liquid dietary supplements. After the addition of 3,4-methylenedioxypropylamphetamine as internal standard (I.S.), a liquid-liquid extraction procedure in alkaline conditions with chloroform/isopropanol (9:1, v/v) was applied to the samples prior to analysis. Chromatography was performed on a fused capillary column and synephrine and I.S., derivatized with pentafluoropropionic anhydride, were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 0.1-50 microg/mg or microg/mL synephrine. Mean recovery ranged between 89.3% and 90.5% in both solid and liquid dietary supplements. The quantification limit was 0.1 microg/mg or microg/ml. The method was applied to analysis of various dietary supplements promoted for aiding weight control containing, among other constituents such as ephedrine alkaloids and methylxanthines, Citrus aurantium. Amount of synephrine present in such products ranged from 3.1 microg/mg solid product to 480.2 microg/mL liquid product.

The aim of this study was to evaluate the chromatographic performance of a PEG stationary phase, in comparison with those of C18 columns, for the HPLC analysis of phenethylamine ((+/-)-synephrine) and indoloquinazoline (rutaecarpine and evodiamine) alkaloids in methanolic extracts of fruits of Evodia rutaecarpa (Juss.) Benth. and E. rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang (i.e., E. officinalis Dode) (Rutaceae family). The method was validated and showed good linearity, precision, accuracy, sensitivity, and specificity. The highest content of both phenethylamine and indoloquinazoline alkaloids was found in methanolic fruit extracts of E. rutaecarpa, and it was closely related to the degree of maturity. E. officinalis fruits displayed low amounts of both types of alkaloids. Furthermore, an enantioselective HPLC method for the enantioseparation of (+/-)-synephrine from Evodia fruits was applied, by using a protein-based chiral stationary phase with cellobiohydrolase (CBH) as the chiral selector (Chiral-CBH). Isolation of synephrine from Evodia aqueous fruit extracts was carried out by strong cation-exchange SPE. The results of the application of the method to the analysis of Evodia samples showed that (-)-synephrine was the main component while (+)-synephrine was present in low concentration.

Adrenergic reactions could mediate metabolic and circulatory changes in the synovial membrane following knee surgery. The interstitial fluid of the synovial membrane and subcutaneous adipose tissue (reference) was monitored in vivo with microdialysis following knee arthroscopy with adrenaline added to the dialysis solvent, adrenaline together with a local anestetic added intra-articularly and without. Local metabolism and blood flow were measured. There was a similar increase, about two fold, in dialysate lactate in all three experimental conditions in the synovial membrane but no change in adipose tissue. Glucose and blood flow decreased by approximately 50% and 10% in both tissues following addition of adrenaline to the dialysate but no changes in the glucose concentrations or blood flow were observed in the other two experimental situations. As regards glycerol the addition of adrenaline caused an approximate 20% increase of the concentration in adipose tissue but an approximate 20% decrease in the synovial membrane. The intra-articular injection caused an approximate 50% increase of the glycerol level in the synovial membrane but no change in adipose glycerol. Thus, the hypermetabolic state in the synovial membrane following standard arthroscopy and the tissue damage (increased glycerol level) in the synovial membrane following postoperative pain relief by intra-articularly injected local anesthetics together with adrenaline doesn't enhance the hypermetabolic state seen postoperatively without adrenaline. However, catecholamines have pronounced in vivo effects on metabolism and blood flow in the synovial membrane.

Familial risk and environmental stress promote the development of alcohol dependence. We investigated whether a positive family history of alcoholism affects the neuroendocrine response to a standardized laboratory stress test in healthy subjects without alcohol use disorders. Twenty-four high-risk subjects with a paternal history of alcoholism (PHA) and 16 family history negative (FHN) controls were evaluated. Psychosocial stress was induced by having subjects deliver a 5-min speech and mental arithmetics in front of an audience on separate days, after drinking either placebo or ethanol (0.6 g/kg) in a randomized sequence. Adrenocorticotropin (ACTH) was measured in 10 plasma samples covering up to 75 min after the stress test. Plasma arginine vasopressin (AVP) was determined before the stressor, at the time of maximum ACTH secretion, and at 75 min after stress onset. The stress test induced a phasic increase in ACTH secretion. At the time of maximum ACTH, AVP was significantly increased in relation to baseline. Compared to placebo, alcohol administration significantly attenuated maximum ACTH concentration in PHA but not FHN subjects, and decreased AVP measured in the same samples in PHA but not FHN subjects. We conclude that activation of the hypothalamic-pituitary-adrenal system by psychosocial stress is accompanied by an increase in peripheral plasma AVP levels. Secretion of both ACTH and AVP suggest that alcohol attenuates the stress response selectively in PHA but not FHN subjects. This might imply some short-term positive alcohol effect in sons of alcoholics, but also constitute a mechanism by which their risk to develop alcohol use disorders is increased.

New polar reversed-phase stationary phases in HPLC provide specific selectivities which can help to solve traditional chromatographic problems related to the development of chromatographic methods with widely different retention times for the sample components. One such case is the analysis of pharmaceutical formulations against the common cold. Acetaminophen, phenylephrine and chlorpheniramine, compounds with different polarities, are frequently associated in these drugs. An isocratic and rapid HPLC method for the simultaneous determination of the three compounds, acetaminophen, phenylephrine and chlorpheniramine, in capsules as pharmaceutical formulations, including the separation of impurities (4-aminophenol and 4-chloracetanilide) and excipients, has been developed and validated. The final chromatographic conditions employed a Supelco Discovery HS PEG column poly(ethyleneglycol) 15x0.46 cm, 5 microm. The mobile phase was 20 mM phosphate buffer, pH 7.0-acetonitrile (90:10, v/v) at a flow-rate of 1 ml/min. UV detection was performed at 215 nm for all the compounds except acetaminophen, which was measured at 310 nm. Validation parameters permit us to consider this method suitable., (Copyright 2002 Elsevier Science B.V.)

Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELISAs were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a pro prietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3 ,5,5, -tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 0.1 ng/ml for the HRP assay, and 0 5 ng/ml for the AP assay, with r2 = 0 99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (0.07%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (0.04%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.

Background and Objectives: No data are presently available concerning the frequency of illicit drug use in plasmapheresis donors. We therefore examined source plasma units produced in the United States (US) and in Germany for evidence of illicit drug use among donors., Materials and Methods: Seventy-five US plasma units from 10 different US states and 75 German plasma units that had been analysed principally for their protein composition were screened for drugs. Determinations were made, using automated immunoassays, of the presence of cannabis, cocaine, amphetamine, methamphetamine, methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDE) and opiates. Positive results were confirmed by gas chromatography-mass spectrometry., Results: Eleven US plasma units were found to be positive for cocaine (14.6%), whereas all German samples were cocaine negative (P = 0.0007). Fifteen US plasma units (20%) and one German unit (1.3%) were confirmed as positive for cannabis (P = 0.0003). Three out of 75 US plasma units were positive for both cannabis and cocaine. In none of the 150 samples were amphetamine, methamphetamine, MDMA, MDE or opiates detected., Conclusions: Our results strongly suggest differences in cocaine and cannabis consumption between US and German plasmapheresis donors. If these results are confirmed by larger-scale studies, random drug screening (including cocaine) of donors should be implemented in order to reduce the number of drug-containing plasma units, especially in the USA.

A modified bioassay system was designed to demonstrate the diffusible nature of endothelium-derived contracting factor(s) released by acetylcholine in the aorta of spontaneously hypertensive rat. In "sandwich"-like layered preparation, isometric tension was recorded from a bioassay strip (without endothelium) in the presence of N(G)-nitro-L-arginine and tetrahydrobiopterin to selectively potentiate endothelium-dependent contractions. A donor strip (with or without endothelium) was stitched on the bioassay tissue so that it did not directly contribute to the recorded contractions. Acetylcholine induced contractions that occurred only when the donor strip was with endothelium. Superoxide dismutase did not affect but catalase and the combination of superoxide dismutase plus catalase significantly decreased the endothelium-dependent contraction. The contractions in the layered preparations were abolished when the donor strip with endothelium was treated previously with valeryl salicylate, an irreversible cyclooxygenase-1 inhibitor, but remained unaffected when the bioassay strip was treated with the compound. Previous treatment of the bioassay strip alone with S 18886 abolished the contractile response, whereas treatment of the donor strip with endothelium by the selective TP receptor antagonist only produced a moderate inhibition. These results indicate that in the aorta of spontaneously hypertensive rats, endothelium-dependent contractions to acetylcholine involve a diffusible substance(s) released by the endothelium. The production of this contracting factor(s) requires the activation of endothelial cyclooxygenase-1, and its action the activation of TP receptors on the vascular smooth muscle cells.

The pathogenesis of aspirin-induced asthma (AIA) has not yet been clearly elucidated, although eicosanoid metabolites appear to play an important role. We hypothesized that levels of eicosanoids in exhaled air condensate are abnormal in patients with AIA and that they change in patients receiving steroid therapy. We measured cysteinyl-leukotrienes (cys-LTs), prostaglandin E(2) (PGE(2)), and leukotriene B(4) (LTB(4)), and also 8-isoprostane as a marker of oxidative stress, by enzyme immunoassay in exhaled breath condensate from patients with AIA (17 steroid naive; mean age, 41 +/- 23 years; FEV(1), 63%pred), 26 patients with aspirin-tolerant asthma (ATA) (11 steroid naive; mean age, 47 +/- 18 years; FEV(1), 69%pred), and 16 healthy subjects (mean age, 45 +/- 17 years; FEV(1), 93%pred). Cys-LTs were significantly higher in steroid-naive patients with AIA compared with steroid-naive patients with ATA and healthy subjects (152.3 +/- 30.4 and 36.6 +/- 7.1 versus 19.4 +/- 2.8 pg/ml; p < 0.05 and p < 0.05, respectively). Steroid-naive patients with AIA also had higher levels of 8-isoprostane than normal subjects (131.8 +/- 31.0 versus 21.9 +/- 4.5 pg/ml; p < 0.05). There were significantly lower levels of both cys-LTs and 8-isoprostanes in steroid-treated patients with AIA. There was no difference in either the PGE(2) or LTB(4) level between the patient groups. This is the first study to show that cys-LTs and 8-isoprostanes are elevated in expired breath condensate of steroid-naive patients with AIA, and that cys-LTs are decreased in steroid-treated patients. Exhaled PGE(2) levels are not reduced, so that it is unlikely that a deficiency of PGE(2) is an important mechanism, whereas exhaled LTB(4) levels are unchanged, indicating an abnormality beyond 5-lipoxygenase.

The present paper introduces a rapid HPLC method for the determination of dioxopromethazine and phenylephrine in eye drops. The method uses a modified C18 stationary phase optimized for the separation of basic compounds and a methanol/1.5 mM phosphoric acid (60/40 v/v, pH 3.02) mobile phase. The flow rate is set to 2 ml/min, sample volume 20 microliters, and compounds are detected at 275 nm. Prior to analysis, the eye drops are diluted with water in a ratio of 1:50. The elaborated HPLC method and the chromatographic system were validated according to the procedure for the validation of chromatographic systems and methods.

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11-26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11-26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11-26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.

The Seville orange extract Citrus aurantium contains m-synephrine (phenylephrine) and octopamine; it causes cardiac disturbances in animals and is used by humans for weight loss. Juice from the orange (Seville orange juice [SOJ]) is used to "knock out" intestinal cytochrome P450 (CYP) 3A4 in bioavailability studies. The purpose of this study was to determine synephrine and octopamine concentrations in SOJ and SOJ's cardiovascular effects in normotensive humans. Subjects consumed 8 ounces of SOJ and water in crossover fashion followed by a repeat ingestion 8 hours later. Hemodynamic (heart rate; systolic, diastolic, and mean arterial pressure) measurements followed. Synephrine and octopamine were determined by high-performance liquid chromatography. Hemodynamics did not differ significantly between water and SOJ groups. Mean synephrine concentration of SOJ samples was 56.9 +/- 0.52 microg/ml; octopamine was not detected. SOJ ingestion by normotensive subjects is expected to be safe. Individuals with severe hypertension, tachyarrhythmias, and narrow-angle glaucoma and monoamine oxidase inhibitor recipients should avoid SOJ consumption. Persons taking decongestant-containing cold preparations should also refrain from SOJ intake.

A relatively simple and inexpensive thin-layer chromatographic (TLC) method is described for the detection and semiquantitative measurement of ergovaline in leaf sheaths of tall fescue (Festuca arundinacea). Samples were finely ground and extracted with methanol. The extracts were filtered and the methanol was evaporated. The aqueous residue was extracted with hexane, followed by chloroform at pH 9. The chloroform extract was concentrated and further purified on a preparative silica gel TLC plate, developed with toluene/ethyl acetate/acetonitrile (50:10:40). The ergovaline band was scraped and eluted with methanol. The eluant was concentrated and an aliquot was applied to a silica gel TLC plate. The plate was developed successively with chloroform/acetone/acetic acid (90:10:5) and chloroform/ethanol (9:1). Ergovaline was visualized with p-dimethylaminobenzaldehyde and sulfuric acid. Semiquantitation of ergovaline was achieved by comparison with a known standard of ergotamine, which was shown to have the same Rf as ergovaline in this system. Spike recovery of ergotamine averaged 60%, with a limit of detection of 200 microg/kg of dry tall fescue leaf sheaths. The method was applied to 15 tall fescue samples with varying degrees of fungal infection, and ergovaline was identified in all contaminated samples with endophyte infection above 15%. Thin-layer chromatography may be also applicable for tall fescue seed, where the ergovaline content is usually higher and the amount of interfering pigments is much lower.

The varieties of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotiphodium lolii contain several classes of toxic alkaloids, including ergopeptide alkaloids and lolitrem alkaloids. Lolitrem B, a potent tremorgen, is generally considered to be the predominant alkaloid in endophyte-infected perennial ryegrass. Ergovaline, a vasoconstrictor normally associated with endophyte-infected tall fescue (Festuca arudinacea), is also present in endophyte infected perennial ryegrass. Clinical signs of animals ingesting endophyte-infected perennial ryegrass are consistent with the presence of lolitrem B. However, clinical signs normally associated with ergovaline poisoning are not usually observed in animals ingesting endophyte-infected perennial ryegrass. A survey was conducted to quantitate both lolitrem B and ergovaline in 459 perennial ryegrass straw samples received at the Oregon State University College of Veterinary Medicine. Samples were analyzed for each alkaloid using separate high-performance liquid chromatography analyses. A strong positive correlation between the 2 alkaloids (r2 = 0.7335) was observed, especially in the samples containing <3,000 ppb (ng/g) lolitrem B. The threshold levels above which clinical signs typically occur are 2,000 ppb lolitrem B and 300-400 ppb ergovaline. All of the samples analyzed contained <425 ppb ergovaline.

Background: Interstitial mononuclear cell infiltration is a feature of experimental models of salt-sensitive hypertension (SSHTN). Since several products of these cells are capable of modifying local vascular reactivity and sodium reabsorption, we investigated whether mycophenolate mofetil (MMF), a drug known to inhibit infiltration and proliferation of immune cells, would modify the SSHTN induced by angiotensin II (Ang II) infusion., Methods: Sprague-Dawley rats received Ang II for two weeks using subcutaneous minipumps. A high-sodium (4% NaCl) diet was started on the third week and was maintained until the eighth week. MMF (30 mg/kg, N = 15), an immunosuppressive drug, or vehicle (N = 15) was given daily by gastric gavage during the initial three weeks. Sham-operated rats (N = 9) were used as controls. Body weight, blood pressure (tail-cuff plethysmography), and serum creatinine were determined weekly. Urinary malondialdehyde (MDA) excretion, renal histology, and immunohistology, including the presence of Ang II and superoxide-producing cells, were analyzed at the end of Ang II infusion and at eight weeks., Results: MMF treatment did not modify hypertension induced during exogenous Ang II infusion, but prevented the subsequent SSHTN. Tubulointerstitial injury resulting from Ang II infusion was significantly reduced by MMF treatment, as were proliferative activity, T-cell infiltration and activation (interleukin-2 receptor expression), superoxide-producing cells, and urinary MDA excretion. Ang II-producing cells were present in the renal tubulointerstitium of rats with SSHTN (60 +/- 30 Ang II-positive cells/mm(2) at 8 weeks) and were reduced by two thirds in the MMF-treated group. Forty percent of lymphocytes infiltrating the tubulointerstitium stained positive for Ang II. The expression of Ang II receptors in the kidney was unmodified., Conclusions: SSHTN resulting from Ang II infusion is associated with infiltration and activation of immune cells that produce Ang II. MMF treatment reduces these features and prevents the development of SSHTN.

Catecholamines may play an important role in the control of intrapulpal pressure as mediators of vasoconstriction. A baseline level of catecholamines (dopamine, epinephrine, and norepinephrine) in the uninflamed human dental pulp was previously reported using high-performance liquid chromatography. The purpose of this study was to compare the level of catecholamines present in the inflamed human dental pulp with the baseline level established in virgin teeth. Twelve uninflamed pulps were analyzed as a control and to validate previous findings. Pulp tissue was obtained from 10 vital and inflamed teeth requiring endodontic treatment. Selective criteria for each patient included: absence of systemic disease, medications, and allergies; a vital response to ice, heat, and electric pulp tests; and periodontal probing < or = 3 mm. A prior history of pain associated with the tooth was an additional criterion for inflamed pulps. To avoid the presence of an exogenous catecholamine, local anesthesia without epinephrine was administered. Dopamine, epinephrine, and norepinephrine were chemically extracted and analyzed by high-performance liquid chromatography with ultraviolet detection. Catecholamine levels found to be present in the pulp during inflammation were greater than the baseline level established in uninflamed pulp tissue.

Background: A low dose solution of adrenaline 2 microg x ml(-1), fentanyl 2 microg x ml(-1) and bupivacaine 1 mg x ml(-1) has been reported to give superior pain control when used for epidural analgesia after major surgery. The present paper describes the compounding and chemical stability of this triple-component solution during storage and use., Methods: Sterile triple-component concentrates (11X) were diluted by the use of gas isolator technology to give ready-to-use infusion solutions. Eight solutions were analysed by reverse phase high-performance liquid chromatography (HPLC) methods, and assays were performed on 1, 45, 90 and 180 days after storage at 2-8 degrees C. After 180 days the solutions were subsequently stored at 22 degrees C for four days before they were reanalysed. HPLC quantification of adrenaline was also performed on samples from solutions given to 28 different patients., Results: The concentration of adrenaline and fentanyl decreased approximately 3.5% from 1 to 180 days at 4 degrees C and four days at 22 degrees C. The corresponding figure for bupivacaine was an apparent increase by 2.4% in concentration. No absorption to the polypropylene plastic bags of fentanyl and bupivacaine was detected. None of the 28 samples derived from used infusion bags contained less than 95% of the declared content of adrenaline., Conclusions: The triple-component epidural analgesic solution remained stable during six months of cold storage, followed by four days of storage at room temperature. No significant degradation of adrenaline was observed in infusion solutions returned from the wards.

Post hoc analysis of data obtained from a study designed to modulate oxidative damage by dietary intervention revealed consistently strong inverse correlations between plasma xanthophyll carotenoids and oxidative damage indices. Thirty-seven women participated in a 14-day dietary intervention that increased mean vegetable and fruit (VF) consumption to approximately 12 servings/day. An additional 10 subjects participated in an intervention that limited VF consumption to less than four servings per day. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) in DNA isolated from peripheral lymphocytes and 8-OHdG excreted in urine were measured as indices of oxidative DNA damage. Lipid peroxidation was assessed by measuring 8-epiprostaglandin F2alpha (8-EPG) in urine. Plasma levels of selected carotenoids were also determined, with the intention of using a-carotene as a biochemical index of VF consumption. Urinary 8-OHdG and 8-EPG were measured by ELISA, and plasma carotenoids were measured by high performance liquid chromatography. Lymphocyte 8-OHdG was measured by reverse phase high performance liquid chromatography with electrochemical detection. We observed that the structurally related xanthophyll carotenoids, lutein and beta-cryptoxanthin, which occur in dissimilar botanical families, were consistently inversely associated with these oxidative indices. Statistically significant inverse correlations were observed between plasma lutein and/or beta-cryptoxanthin levels and lymphocyte 8-OHdG and urinary 8-EPG. Moreover, an inverse correlation was observed between change in plasma xanthophylls and change in lymphocyte 8-OHdG concentration that occurred during the course of the study. These data lead us to hypothesize that lutein and beta-cryptoxanthin serve as markers for the antioxidant milieu provided by plants from which they are derived. Whether these carotenoids are directly responsible for the observed antioxidant phenomena merits further investigation.

In the study of the regulation of skeletal muscle blood flow during exercise it is useful to obtain information regarding the concentrations of vasoactive substances in the muscle interstitium, a site where the compounds act on the vascular and skeletal muscle cells. The microdialysis technique is a useful tool for measuring interstitial substances in the muscle at rest and during exercise in human subjects, and the technique can also be used to study the effect of both systemic and local interventions in a specific area of an exercising muscle. Probe recovery, which represents the relative amount of a substance that is diffusing to the dialysis membrane, changes from rest to exercise and can be determined by the internal-standard technique which allows for a relatively high time resolution (min). Furthermore, the use of electrodes at the microdialysis outlet makes it possible to perform continuous measurements of interstitial substances. The present review gives examples of how the microdialysis technique has been applied to study potentially important vasodilators such as adenosine, NO and K+ in human skeletal muscles and highlights areas for future research to establish the functional importance of these compounds.

Diadenosine polyphosphates have been identified as important regulators of vascular tone and blood pressure. In reference to the background of the well-known vasoconstriction induced by hemolysate, we questioned whether this action may be due in part to the presence of diadenosine polyphosphates in human erythrocytes. Therefore, erythrocytes were separated from other blood cells and deproteinated. To concentrate and purify nucleotides, the extract was chromatographed by anion exchange, affinity, and reversed-phase columns. In one of the purified fractions, diadenosine hexaphosphate (diadenosine 5', 5'-P(1), P(6) hexaphosphate [AP(6)A]) was identified by matrix-assisted laser desorption/ionization mass spectrometry, ultraviolet spectroscopy, and enzymatic analysis. Hemolysate of erythrocytes injected into the isolated perfused rat kidney induced a dose-dependent vasoconstriction, which was partially inhibited by P(2)-purinoceptor antagonist. The data document the existence of AP(6)A in erythrocytes. AP(6)A may be involved in the pathogenesis of vasospasm induced by free hemoglobin.

This review illustrates the evolution in the clinical use of prostate specific antigen as a prostate cancer tumour marker. Because PSA lacks specificity, several new PSA parameters have been investigated such as the age-referenced PSA and the PSA density of the transition zone. We discuss also the molecular forms of PSA and the complexed PSA and new tumour markers which could be of interest in the future such as the HK2.

Objective: To evaluate the occurrence and clinical significance of lipid peroxidation (oxidative stress) in rheumatic diseases characterized by vascular involvement., Patients and Methods: Plasma 8-epi-PGF2alpha (oxidative stress marker) was measured by gas chromatography-mass spectrometry in 36 patients with systemic lupus erythematosus (SLE), 13 with systemic sclerosis (SSc), 13 with systemic vasculitis [Wegener's granulomatosis (WG), n = 4; Churg Strauss syndrome (CSS), n = 3; Behcet syndrome, n = 6], 12 with rheumatoid arthritis (RA) and in 23 healthy controls (n = 23)., Results: 8-epi-PGF2alpha levels were higher in patients with SLE (P = 0.007), SSc (P < 0.001) and vasculitis (P = 0.001) than in controls. In SLE, a positive Coombs' test and arterial hypertension independently predicted 8-epi-PGF2alpha concentrations (P = 0.004 and P = 0.001, respectively). SLE patients not taking prednisolone showed higher 8-epi-PGF2alpha concentrations than SLE patients on prednisolone (P = 0.02). In the latter group, a dose response relationship was noted between 8-epi-PGF2alpha and steroid dosage (r = 0.6, P = 0.0003). In WG and CSS, 8-epi-PGF2alpha concentrations correlated with disease activity (r = 0.8, P = 0.01) and were higher than in patients with Behcet disease (P = 0.003)., Conclusions: Oxidative stress may be pathogenetically relevant in some autoimmune rheumatic diseases with vascular involvement. Amelioration of some clinical manifestations of these diseases may be envisaged by targeting lipid peroxidation with dietary or pharmacological antioxidants.

It has been demonstrated that prostate-specific antigen (PSA), in spite of its name, can be detected in body fluids and tumors from a variety of organs. Investigations have shown that human glandular kallikrein 2 (hK2), a related prostate-secreted protease, can activate the zymogen form of PSA, suggesting that the two enzymes might work as a functional unit, with hK2 as the activator molecule and PSA as the effector molecule. If this is true, then hK2 should be found together with PSA in body fluids other than seminal plasma, as well. Recently, a sensitive and specific assay was devised for hK2, enabling its measurement in picogram quantities. With this assay, the concentration of hK2 was determined in samples of seminal plasma, amniotic fluid, breast milk, and saliva. Simultaneously, the samples were assayed for molecular forms of PSA. In seminal plasma, the mean PSA concentration was 0.82 mg/ml, while the hK2 level was around two orders of magnitude lower: mean value, 6.4 microg/ml. Approximately the same ratio of PSA to hK2 as in seminal plasma was found in amniotic fluid and breast milk, but in most samples, the hK2 values were too low for direct measurements and had to be concentrated prior to analysis. Measurable levels of PSA, all in the free form, were detected in amniotic fluid at the thirteenth week of gestation and then gradually increased to levels around and over 1 microg/L from the twentieth week. Significant levels of PSA were detected in amniotic fluid collected at delivery, also. Measurable levels of mammary PSA were primarily detected in colostrum, with a range from less than 0.03 microg/L to 2.1 mg/L. Around half of the molecules were in complex with protease inhibitor. Most surprisingly, determinations on saliva samples showed that none of them had detectable PSA levels but had measurable concentrations of hK2 with a mean value, 0.09 microg/L. The presence in saliva suggests that hK2 can be the human equivalent to one of the mouse salivary kallikreins with important biological function, like the epidermal growth factor-binding protein or the gamma subunit of nerve growth factor. However, this was ruled out, as a phylogenetic analysis showed that the human and mouse glandular kallikreins evolved independently from a common precursor after the separation of the primate and rodent lineages. In conclusion, the measurements show that in addition to the previously known secretion in seminal plasma, hK2 is secreted in amniotic fluid, breast milk, and saliva. Furthermore, the concerted expression of PSA and hK2 in seminal plasma, amniotic fluid, and breast milk suggests that the two proteases might form a functional unit but not always as demonstrated by the sole presence of hK2 in saliva.

Estrogen stimulates the renin-angiotensin system by augmenting both tissue and circulating levels of angiotensinogen and renin. We show, however, that angiotensin converting enzyme (ACE) activity in the circulation and in tissues is reduced in two animal models of postmenopausal chronic hormone replacement. We observed a reduction of ACE activity in association with a significant increase in plasma angiotensin I (Ang I) and hyperreninemia in ovariectomized monkeys treated with Premarin (conjugated equine estrogen) replacement for 30 months. Plasma angiotensin II (Ang II) levels were not increased in monkeys treated with estrogen, suggesting that the decrease in ACE curtailed the formation of the peptide. The Ang II/Ang I ratio, an in vivo index of ACE activity, was significantly reduced by estrogen treatment, further supporting the biochemical significance of estrogen's inhibition of ACE. In ovariectomized transgenic hypertensive (mRen2)27 rats submitted to estrogen replacement treatment for 3 weeks, ACE activity in plasma and tissue (aorta and kidney) and circulating Ang II levels were reduced, whereas circulating levels of angiotensin-(1-7) (Ang-(1-7)) were increased. Ang-(1-7), the N-terminal fragment of Ang II, is a novel vasodilator and antihypertensive peptide. Thus, the net balance of these effects of estrogen on the reninangiotensin vasoconstrictor/vasodilator system is to promote the anti-hypertensive effect.

Background: Oxidant stress is a purported mechanism of tissue damage in patients with ARDS and acute lung injury (ALI). Isoprostanes, prostanoid compounds primarily formed nonenzymatically via lipid peroxidation, are precise markers of in vivo oxidant stress. Plasma levels of metabolites of 8-iso-prostaglandin-F2alpha (8-iso-PGF2alpha) correlate with outcome in patients with ARDS., Objective: To examine exhaled breath condensate levels of 8-iso-PGF2alpha as a noninvasive quantification of pulmonary oxidant stress in patients with, or at risk for, ARDS/ALI., Methods: Breath condensate was collected from 22 patients with, or at risk for, ARDS/ALI by placing Tygon tubing submerged in an ice bath in line with the expiratory limb of the ventilator circuit. Ten patients without lung disease, who were intubated while undergoing minor surgical procedures, served as control subjects. Between 1 and 3 mL of condensate was collected over a 30- to 60-min period, then immediately frozen and stored at -70 degrees C until analysis. The 8-iso-PGF2alpha was purified and derivatized, then quantified by stable isotope dilution in conjunction with gas chromatography/mass spectrometry., Results: The mean level of exhaled 8-iso-PGF2alpha in the patients with ALI/ARDS, 87+/-28 pg/mL, was significantly higher than the mean in the normal group, 7+/-4 pg/mL (p = 0.007). The 8-iso-PGF2alpha levels were greater than two standard deviations above the mean of the normal group in 12 of 22 patients with or at risk for ARDS/ALI., Conclusions: These results provide further evidence that lipid peroxidation does occur in patients with ARDS/ALI. The measurement of exhaled isoprostanes provides a novel, noninvasive method to quantify oxidant stress in such patients.

We provide herein mass spectrometric evidence for the existence of F2 isoprostanes in an epidermal secretion of the catfish from the Arabian Gulf. The compounds were extracted, purified by thin layer chromatography (TLC) and derivatized into the pentafluorobenzyl trimethylsilyl (PFB-TMSi) derivatives and quantified using SIM-negative ion gas chromatography-mass spectrometry based on the addition of tetradeuterated 8-epi PGF2alpha as internal standard prior to sample workup. Freshly collected secretions from the male catfish contained 5.63 and 4.17 ng/ml (two determinations) and from the female catfish 3.60 and 3.81 ng/ml (two determinations) of free 8-epi PGF2alpha. The level of these compounds increases approximately 8-fold upon storage of the extract at -20 degrees. In view of the potent reported actions of F2 isoprostanes on vasoconstriction and as thromboxane receptor antagonists, the presence of isoprostanes in the catfish secretions may contribute to the reported ulcer healing properties of these secretions.

Objective: Peri-operative ischemic episodes following coronary artery bypass grafting with the internal mammary artery (IMA) are thought to be due to a vasospasm of this conduit. Endothelin-1 (ET-1) is a potent vasoconstrictor by itself and increases the response to other vasoconstrictor stimuli. This study focused on the possible role of an enhanced tissue ET-1-like immunoreactivity in the perioperative reaction of the IMA in patients with diabetes or hypercholesterolemia., Methods: Specimens of the distal part of the IMA from 46 patients (mean age 58.5 years, four women, 42 men) were studied prospectively. Nine of those patients were diabetic and 26 had evidence of hypercholesterolemia. Another cohort of 20 IMA specimens was stained retrospectively; 10 of those biopsies were from patients that had experienced transient ischemic events peri-operatively in the myocardial area supplied by the IMA. The biopsies were examined histologically and immunohistochemically (rabbit polyclonal ET-1 antiserum, three-step avidin-biotin complex) with regard to their immunoreactivity to tissue ET-1., Results: An immunoreactivity to ET-1 (graded 0-3) was present in 89% of the biopsies. The reactivity was significantly higher in patients with hypercholesterolemia ( 1.92+/-0.74) when compared to controls (1.0+/-0.63) (P = 0.04). The reactivity was also increased in patients with non-insulin-dependent diabetes mellitus (2.1+/-0.79), when compared to controls (P = 0.02). Mostly transient ischemic events in the area supplied by the IMA seemed to occur more frequently when the biopsies revealed a higher immunoreactivity to ET-1. They showed an increased reactivity to ET-1 (2.27+/-0.76) compared to 10 patients with an uneventful peri-operative course (1.66+/-0.71 ) (P = 0.04)., Conclusions: This study provides evidence that the internal mammary artery is not a passive conduit. Vasospasm or vasoconstriction, in particular at its distal end, may occur more frequently in patients with hypercholesterolemia or diabetes, and may lead to post-operative ischemic events.

Recent studies on human kallikrein 2 (hK2) have revealed striking similarities and significant differences with the closely related kallikrein PSA. Both PSA and hK2 are primarily localized to the prostate and share close structural similarities. Although both kallikreins are produced by the same secretory epithelial cells in the prostate, hK2 is associated more with prostate tumors than PSA and is highly expressed in poorly differentiated cancer cells. The potent trypsin-like activity of hK2 contrasts with the weak chymotrypsin-like activity of PSA. The inactive precursor form of PSA, proPSA, is converted rapidly to active PSA by hK2, suggesting an important in vivo regulatory function by hK2 on PSA activity. The high homology between hK2 and PSA results in significant cross-reactivity to hK2 by polyclonal and some monoclonal antibodies to PSA. Future studies on both PSA and hK2 need to take into account this potential for cross-reactivity. Specific monoclonal antibodies to hK2 have now demonstrated that serum levels of hK2, like PSA, are correlated with prostate cancer. The production of hK2 protein in active protease form and specific monoclonal antibodies to the hK2 antigen will allow extensive future studies delineating the physiological and clinical utility of this new prostate antigen.

A simple and robust method for the separation and quantification of epinephrine in dental anesthetic solutions was developed. The method allows the direct injection of high salt solutions without sample pre-treatment. Large sample plugs (5.7% of the total capillary length) are used for epinephrine determination by selective analyte focusing in capillary electrophoresis. The concentration detection limit for epinephrine is about 5.0 x 10(-7) M (90 ng ml-1) with a commercial UV detector. The separation protocol was validated in terms of its precision, linearity, accuracy and specificity.

In addition to the circulating renin-angiotensin system (RAS), a local system has been postulated in the anterior pituitary because immunodetection of its components in various mammalian species. However, different cell types appear to be involved in different species, and there is no general consensus on the subcellular localization of prorenin, renin and angiotensinogen. In this ultrastructural study, we investigated and quantified the presence of these components using double or triple immunogold labeling methods, in all the immunologically identified glandular cell types of the rat anterior pituitary. In contrast to previous reports, all these components were identified not only in lactotropes and gonadotropes but also in somatotropes, corticotropes, and thyrotropes. The highest levels were detected in lactotropes and gonadotropes, and renin gave the greatest signal. Angiotensinogen, prorenin, and renin were co-localized in the secretory granules of all rat pituitary glandular cell types. The simultaneous detection of the substrate (angiotensinogen) and both its specific cleavage enzyme and its proenzyme within the same granule suggests intragranular processing of this component. Moreover, the localization of these three constituents in the secretory granules also suggests that, in the rat anterior pituitary, they follow the regulated secretory pathway.

Oxytocin (OT) and vasopressin (VP) release from the neurohypophysis are correlated with the electrical activity of magnocellular cells (MNCs) in the supraoptic (SON) and paraventricular nuclei. Synaptic inputs to MNCs influence their electrical activity and, hence, hormone release. During lactation OT neurons display a synchronized high-frequency bursting activity preceding each milk ejection. In parallel to the adoption of this pattern of electrical activity, an ultrastructural reorganization of the SON has been observed during lactation. In the present study we performed a light microscopic, morphometric analysis of identified OT and VP neurons in the SON to determine whether the dendrites of these neurons participate in the plasticity observed during lactation. The dendritic trees of OT neurons shrunk during lactation ( approximately 41% decrease in the total dendritic length) because of a decreased dendritic branching concentrated at a distance of 100-200 microm from the soma. No changes in the maximal distal extension were observed. The distribution pattern of dendritic length into branch orders also was affected. Strikingly, opposite effects were observed in VP neurons. The dendritic trees during lactation elongated ( approximately 48% increase in the total dendritic length) because of an increased branching close to the soma. No changes in the maximal distal extension were observed. These results indicate that the length and geometry of the dendritic trees of OT and VP neurons are altered in opposite ways during lactation. These changes would influence the availability of postsynaptic space and alter the electrotonic properties of the neurons, affecting the efficacy of synaptic inputs.

Our recent findings that IGF-I inhibits placental thromboxane (TxB2) release (1, 2) and that prostanoid release from placentas or certain pregnancies complicated by intrauterine growth retardation (IUGR) is decreased [Sorem KA, Siler-Khodr TM, Placenta, 16:503-515, 1995] led us to investigate the effect of IGF-I on prostanoid release from placentas of IUGR pregnancies. The placental response of 6-keto-prostaglandin F1a (6-keto-PGF1a) and thromboxane (TxB2) to IGF-I in severe IUGR (n = 5) was compared with the response in normal pregnancies (n = 6). Placentas were perifused with medium containing IGF-I at doses of 0, 5.2, 10.4, 20.8, and 83.3 ng/ml. In three of the five IUGR placentas (responsive group), incubation with IGF-I resulted in an inhibition of TxB2, attaining significantly greater inhibition at a lower dose of IGF-I than the normal placental response. However, in two of the five IUGR placentas (non-responsive group), the TxB2 was insensitive to the normal inhibitory action of IGF-I. The baseline production of prostanoids from the IUGR placentas was not predictive of their response to IGF-I. Moreover, 6-keto-PGF1a was not inhibited in any of the placentas, IUGRs, or normals. However, the ratio for TxB2 over 6-keto-PGF1a basal production rate from the zero treatment time to the fifth hour was significantly less in the nonresponsive IUGR placentas than for the responsive IUGR placentas or for the normal placentas. Certain IUGR placentas demonstrated a significant suppression of TxB2 with IGF-I, whereas other IUGR placentas were insensitive to exogenous IGF-I. A decreased and unchanging ratio of TxB2/6-keto-PGF1a production rate was characteristic of the nonresponders.

Polymorphonuclear leucocytes (PMNs) from the synovial fluid of patients with rheumatoid arthritis (RA) showed reduced tissue kallikrein and kinin immunoreactivity in comparison with blood PMNs from healthy individuals as judged visually using confocal microscopy. Similarly, synovial fluid PMNs exhibited reduced tissue kallikrein immunoreactivity as compared with blood PMNs from the same RA patients. Blood PMNs stimulated to degranulate in vitro also displayed less immunostaining for tissue kallikrein and kinin than non-stimulated PMNs. By contrast, no difference in kininogen immunostaining was detected between RA synovial fluid PMNs and blood PMNs from healthy people. It is considered that the results support the hypothesis that tissue kallikrein, released from the granules of RA synovial fluid PMNs, cleaves the kinin moiety from multifunctional kininogen protein on the surface of the PMNs.

A 28-year-old white female with a history of two prior suicide attempts was found dead in her home by her common law husband. Autopsy findings were unremarkable except for partially dissolved ephedrine tablets in the stomach contents. Quantitation of ephedrine was by gas chromatography/mass spectrometry (GC/MS) following liquid/liquid extraction from alkaline samples and pentafluoropropionic acid derivatization. Significant toxicological finding included ephedrine; blood, 11 mg/L; liver, 24 mg/kg; kidney, 14 mg/kg; brain, 8.9 mg/kg; and amitriptyline; blood, 0.33 mg/kg; liver 7.8 mg/kg. The ephedrine values found far exceed those associated with therapeutic administration and are consistent with the few reported cases of severe ephedrine intoxication. The cause of death was determined to be fatal ephedrine intoxication and manner of death suicide.

We investigated a putative local angiotensin II (AngII) system in the rabbit retina by examining AngII contents in the retina, vitreous humor, and choroid by radioimmunoassays and AngII synthesis in the retina and choroid by detection of angiotensin converting enzyme (ACE) mRNA. An antibody directed against AngII was used to localize possible cellular sources of AngII in the retina. To enhance immunoreactivity and to further examine AngII metabolism, tissues were preincubated in medium containing either protease inhibitors (PI), PI together with the AngII-precursor AngI, or PI and AngII. In some experiments the conversion of AngI to AngII was blocked by an ACE inhibitor. AngII concentration in the vitreous humor was only about 10% of the plasma concentration; in the retina and the choroid, however, AngII concentrations were 10 and 86 times higher, respectively, than in the plasma. ACE mRNA was present in both retina and choroid. Immunohistochemistry for AngII revealed faintly labeled amacrine cells at the inner border of the inner nuclear layer of the retina. Preincubation with PI resulted in an enhanced immunoreaction and in the labeling of fibers in the inner and outer plexiform layer; Müller cells and their processes as well as ganglion cells were now stained as well but the specificity of ganglion cell staining remains questionable. The immunoreaction was further enhanced when AngI or AngII was added to the incubation medium, whereas labeling totally disappeared when the conversion of AngI to AngII was blocked. No immunoreactive cells were detected in the choroid. In conclusion, the synthesizing enzyme for AngII is expressed in the retina and a specific AngII concentration is maintained there; AngII is localized in distinct cell types and can be metabolized within these cells. These data point to a local retinal AngII system that is protected and independent of blood-borne AngII.

In previous studies high luminal concentrations of angiotensin II (Ang II) were found in rat proximal tubules. The physiological role of intraluminal Ang II remains to be established. In the present study, we investigated whether the luminal angiotensin II concentration in the proximal tubules ([Ang II]prox) can be modulated. Micropuncture studies were performed in control rats (C, N = 8) and rats subjected to acute volume expansion (VE, N = 8) or reduced renal perfusion pressure (RRP, N = 7). Changes in [Ang II]prox were compared to changes in whole kidney Ang II content ([ANG II]kidney) and the plasma concentration ([Ang II] plasma). In C rats, [Ang II]prox was 460 +/- 48 pmol/liter (10 to 20 times lower than hitherto reported), while [Ang II]kidney and [Ang II]plasma were 369 +/- 81 pmol/kg and 90 +/- 29 pmol/liter, respectively. In agreement with previous data, VE failed to suppress [Ang II]prox (674 +/- 132 pmol/liter), while at the same time [Ang II]kidney (42 +/- 10 pmol/kg) and [Ang II]plasma (12 +/- 3 pmol/liter) were markedly suppressed. This points to dissociated regulation of [Ang II] in the renal luminal compartment on the one hand and the extraluminal renal and systemic plasma compartments on the other hand. During RRP, [Ang II]prox increased significantly to 1675 +/- 465 pmol/liter. No dissociation between the three compartments was observed in this situation, as [Ang II]kidney (969 +/- 85 pmol/kg) and [Ang II]plasma (245 +/- 72 pmol/liter) increased in parallel. In summary, we confirm that Ang II is present in proximal tubules of rat kidneys at concentrations which exceed those in plasma. Its concentration could be modulated (approximately 3.5 increase) by reduction of renal perfusion pressure but not by acute volume expansion. In the latter condition, we observed a clear dissociation from Ang II generation in the extraluminal renal compartment, as whole kidney Ang II content was markedly suppressed.

Vasoconstrictor and Na/K pump inhibitory properties of a bufodienolide Na/K-ATPase inhibitor, marinobufagenin, were studied in isolated rings of 2 to 3 order branches of human pulmonary arteries respectively. Marinobufagenin displayed concentration-dependent vasoconstrictor activity (0.01 to 10 mmol/L). In sarcolemma membranes prepared from pulmonary artery marinobufagenin inhibited Na/K-ATPase (IC50 = 50 nmol/L). In eight healthy male Caucasians, concentrations of marinobufagenin-like immunoreactive material in C-18 extracted plasma were 1.38 +/- 0.60 nmol/L. Twenty-four-hour urinary release of marinobufagenin-like immunoreactive material in eight healthy males was 1.20 +/- 0.95 nmol/day. Chloroform extract of human urine was fractionated using reverse-phase high-performance liquid chromatography (32% acetonitrile, Deltapak). The HPLC fraction coeluting with marinobufagenin in 7 min, cross reacted with antimarinobufagenin and antidigoxin, but not antiouabain antibody. These results demonstrate that human plasma and urine contains a bufodienolide vasoconstrictor EDLF, marinobufagenin-like immunoreactive Na,K pump inhibitor.

pH affects the efficacy of local anesthetics by determining the percentage of the lipid-soluble base form of the anesthetic available for diffusion and penetration of the nerve sheath. The purpose of this study was to determine the relationship between pH and the concentrations of antioxidant and vasoconstrictor in dental local anesthetic solutions over real-time and after accelerated aging. Several batches of lidocaine and mepivacaine with vasoconstrictors were tested. Results showed that, immediately upon receipt from the manufacturers, three batches were below the USP pH limit (pH 3.3), and two batches contained less than the minimum limit of vasoconstrictors (90%). Real-time tests on batches that were within normal limits revealed that solutions were stable past 4 yr. Accelerated aging tests revealed a strong correlation between a decrease in pH and loss of antioxidants and vasoconstrictors. In conclusion, a quality batch of local anesthetic should remain efficacious long past the manufacturer's stated shelf life; a batch that is less than optimal, or one that is exposed to environmental stresses, will degrade rapidly, and efficacy may be affected by decreases in pH and loss of vasoconstrictor. pH may be an inexpensive, readily available screening test for efficacy of local anesthetics.

Naloxone, the analyte and the internal standard, sumatriptan, are extracted from plasma using solid-phase extraction columns. Chromatography and detection are performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with coulometric end-point detection. The standard curve was linear over the range 0-50 ng/ml of naloxone in plasma. The reproducibility, the coefficient of variation (C.V.) of the method over the range of the standard curve was 6.2-11.2%. The recovery averaged 90.4 +/- 8.9%. A plasma profile following i.v. administration of naloxone in one normal healthy volunteer is presented.

The vein wall is nourished by diffusion of blood nutrients from the lumen and from the vasa vasorum. It is likely that drugs take the same ways to reach and diffuse through the vessel wall. Thus the uptake of a drug with affinity for the vein wall should give information on its transport to the tissue. This study aimed to explore troxerutin uptake by the long saphenous vein. Troxerutin is a naturally fluorescent flavonoid which has been known to improve subjective signs of patients with chronic venous insufficiency. Nine patients undergoing surgical treatment of varicosis were enrolled in the study. They received for the last 4 days before surgery either 3,500 mg of troxerutin (n = 4) or 1,000 mg twice daily (n = 2). Three patients as controls did not receive any drug. Two samples from thigh and calf long saphenous vein were harvested in each patients and investigated with a confocal laser scanning microscope developed by our institute measuring the fluorescence emitted by troxerutin after excitation by a 458 nm wavelength laser-beam. The intensity of the overall fluorescence was significantly higher in the treated groups (p < 0,001) and slightly higher in the patient who received 3,500 mg of troxerutin than with the lower dosage. The outer wall region provided the highest fluorescence in the treated group while a significant difference was observed in the fluorescence of the medial region between treated and control group. These results showed a marked affinity of troxerutin for the venous wall. The highest uptake in the outer wall region is likely to result from transport through vasa vasorum, owing to the rheologic properties of the drug. The significant medial fluorescence may account for the venous tone improvement with the drug.

Galanin is found in perivascular sympathetic neurons in a wide range of vertebrate species. In placental mammals, galanin has either no effect on blood pressure, or weak depressor effects, but in other vertebrates it has been shown to be a potent pressor agent. To investigate how extensive the vasoconstrictor effects of galanin may be in the vertebrates, the vascular effects of galanin were tested in two species of shark, Heterodontus portusjacksoni, and Hemiscyllium ocellatum, and a ray, Rhinobatos typus. Nerve fibres showing immunoreactivity to galanin were located surrounding gut blood vessels, but were absent from branchial efferent arteries in all three species. Intravenous injection of galanin caused a significant rise in caudal arterial blood pressure in H. portusjacksoni and H. ocellatum, but no change in R. typus. Contraction of segments of pancreatico-mesenteric artery were measured in an organ bath also. Galanin (10(-6) M) caused 21-38% of the maximum K+ induced contraction in all species, but no response in efferent branchial arteries from R. typus. In conclusion, in three elasmobranchs, a galanin-like peptide is present in perivascular nerve fibres, and galanin causes differential vasoconstriction in vascular beds. These data extend the number of vertebrate groups in which galanin has been shown to be a vasoconstrictor peptide.