Habitat monitoring and genotoxicity in Ucides cordatus (Crustacea: Ucididae), as tools to manage a mangrove reserve in southeastern Brazil.

In Brazil, the state of São Paulo contains both preserved areas (Juréia-Itatins Ecological Station) and extremely impacted ones (Cubatão Municipality). This study evaluated the concentrations of five metals (Cu, Cd, Cr, Pb, and Hg) in two mangroves with different levels of anthropogenic impact and the apparent genotoxicity to Ucides cordatus. Water and sediment samples were obtained, and metal concentrations were determined with an atomic absorption spectrophotometer. The genotoxic impact was quantified based on the number of micronucleated cells per 1,000 analyzed (MN‰), using hemolymph slides stained with Giemsa. Metal concentrations in water were below the detection limit, except for lead, although no significant difference was observed between the areas (P > 0.05). Sediment from Cubatão had higher concentrations of Cd, Pb, Cr, and Cu than sediment from Juréia-Itatins (P < 0.05), but no significant differences in metal concentrations were detected among depth strata of the sediment (P > 0.05). Crabs from Cubatão had a 2.6 times higher mean frequency of micronucleated cells (5.2 ± 1.8 MN‰) than those from Juréia-Itatins (2.0 ± 1.0 MN‰; P < 0.0001). The more-polluted conditions found in the mangrove sediments of Cubatão were reflected in the micronucleus assay, demonstrating their genotoxic effect; however, genetic damage should be attributed to a synergistic effect with other kinds of pollutants previously recorded in different environments of Cubatão. U. cordatus proved to be an excellent bioindicator of mangrove pollution. This study established, for the first time, the normal frequency of MN‰ in a population of this species within an ecological station.

Follicular-phase concentrations of progesterone, estradiol-17ß, LH, FSH, and a PGF2α metabolite and daily clustering of prolactin pulses, based on hourly blood sampling and hourly detection of ovulation in heifers.

Circulating concentrations of hormones were determined each hour in 13 heifers from the end of the luteolytic period to ovulation (follicular phase, 3.5 days). Diameter of the preovulatory follicle was determined every 8 hours, and the time of ovulation was determined hourly. The diameter of the preovulatory follicle decreased 0.8 ± 0.1 mm/h in heifers when there was 1 to 3 hours between the last two diameter measurements before ovulation. The concentration of progesterone (P4) after the end of the luteolytic period (P4 < 1 ng/mL) changed (P < 0.0001), as shown by a continued decrease until Hour -57 (Hour 0 = ovulation), then was maintained at approximately 0.2 ng/mL until 2 hours before the peak of the LH surge at Hour -26, and then a decrease to 0.1 ng/mL along with a decrease in estradiol-17ß. Concentrations of LH gradually increased (P < 0.007) and concentrations of FSH gradually decreased (P < 0.0001) after the end of luteolysis until the beginning nadirs of the respective preovulatory surges. A cluster of prolactin (PRL) pulses occurred (P < 0.0001) each day with approximately 24 hours between the maximum value of successive clusters. Hourly concentrations of a PGF2α metabolite decreased (P < 0.007) until Hour -40, but did not differ among hours thereafter. Novel observations included the gradual increase in LH and decrease in FSH until the beginning of the preovulatory surges and follicle diameter decrease a few hours before ovulation. Results supported the following hypotheses: (1) change in the low circulating P4 concentrations during the follicular phase are temporally associated with change in LH concentrations; and (2) PRL pulses occur in a cluster each day during the follicular phase of the estrous cycle.

Circadian influence on the preovulatory LH surge, ovulation, and prolactin concentrations in heifers.

A novel circadian study of the effect of clock hours on the preovulatory LH surge, ovulation, and maximal PRL concentration was done in 13 nontreated Holstein heifers. Hourly blood sampling and hourly ultrasound examinations to detect the hour of ovulation began at 8 and 48 hours, respectively, after CL area (cm(2)) had decreased 15% from the area at 15 days postovulation. The resulting experimental period began at the beginning of postluteolysis (progesterone, <1 ng/mL) and encompassed a mean of 3.5 days until ovulation. The frequency of the peak of the preovulatory LH surge for the three 8-hour periods of a 24-hour day was different (P < 0.02) between 2:00 AM to 9:00 AM (N = 9), 10:00 AM to 5:00 PM (N = 3), and 6:00 PM to 1:00 AM (N = 1). The median was 6:00 AM. The frequency of ovulations for 8-hour periods was different (P < 0.02) between 3:00 AM to 10:00 AM (N = 9), 11:00 AM to 6:00 PM (N = 3), and 7:00 PM to 2:00 AM (N = 1). The median was 7:30 AM. Two or three clusters of PRL pulses occurred during the 3.5 days. Based on all available PRL pulse clusters (N = 36), the clock hours of the maximal concentration/cluster was greater (P < 0.0001) for 9:00 AM to 2:00 PM (N = 33 clusters) than for each of the three other 6-hour periods (N = 0, 1, or 2 per period). The median was 11:30 AM. The hypothesis was supported that the peak of the preovulatory LH surge, ovulation, and maximal PRL concentration during pulse clusters occur with greater frequency during certain clock hours in heifers.

Stimulatory effect of PGF2α on PRL based on experimental inhibition of each hormone in mares.

During the luteolytic period in mares, the peak of 65% of pulses of a PGF2α metabolite (PGFM) and the peak of a pulse of PRL have been reported to occur at the same hour. It is unknown whether the synchrony reflects an effect of PGF2α on PRL or vice versa. Controls, a flunixin meglumine (FM)-treated group (to inhibit PGF2α), and a bromocriptine-treated group (to inhibit PRL), were used at 14 days postovulation in June and in September (n = 6 mares/group/mo). Blood samples were collected hourly from just before treatment (Hour 0) to Hour 10. Concentrations of PGFM in the FM group were lower (P < 0.05) at Hours 4 to 6 than in the controls in each month, but bromocriptine had no detected effects on PGFM. Concentrations of PGFM averaged over all groups and within each group did not differ between June and September. Compared to the controls, concentrations of PRL in June were lower (P < 0.05) in the FM group at Hours 4 to 8 and in the bromocriptine group at Hours 4 to 10. Concentration of PRL averaged over groups was lower (P < 0.0001) in September (0.9 ± 0.05 ng/mL, mean ± SEM) than in June (3.0 ± 0.3 ng/mL). Results supported the hypothesis that the positive association between PGFM and PRL concentrations in mares represents an effect of PGF2α on PRL rather than an effect of PRL on PGF2α.

Follicle suppression of circulating follicle-stimulating hormone and luteinizing hormone before versus after emergence of the ovulatory wave in mares.

The effect of the ovarian follicles on plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) before versus after the expected emergence of the ovulatory follicular wave was studied on Days 0 to 18 (Day 0=ovulation) in four groups of mares (n=6/group). In addition to a control group, all follicles >/=6mm in diameter were ablated on Days 0.5, 6.5, or 12.5 in a herd of mares with reported emergence at 6mm of the future ovulatory follicle on mean Day 10.5. Concentrations of FSH were not different between the Day-0.5 or Day-6.5 ablation groups and the corresponding controls. However, ablation on Day 12.5 resulted in an immediate FSH increase (group-by-day interaction, P<0.003). For LH, ablation on Day 0.5 resulted in an interaction (P<0.02), partially from lower (P<0.05) concentrations on each of Days 15.5 to 18.0 than that in the controls, whereas ablation on Days 6.5 or 12.5 did not result in a significant group effect or interaction. Testosterone concentration, but not progesterone or estradiol concentration, was lower (P<0.04) on Day 2 in the Day-0.5 ablation group than that in the controls. We inferred that follicles did not contain adequate FSH suppressors on Days 0.5 and 6.5 and that they were present only in the Day-12.5 ablation group or after the expected emergence of the ovulatory wave. The hypothesis of an association between low postovulatory concentrations of an ovarian steroid and low concentrations of LH after Day 15 was supported.

Validation of a selective method for determination of paroxetine in human plasma by LC-MS/MS.

PURPOSE: A sensitive, robust, and selective liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) was developed and validated for paroxetine quantification in human EDTA plasma. METHODS: Sample preparation was based on liquid-liquid extraction using a mixture of ethyl acetate/hexane (50/50; v/v) to extract the drug and internal standard from plasma. Chromatography was performed on a C-18 analytical column and the retention times were 1.6 and 1.7 for paroxetine and fluoxetine (IS), respectively. The ionization was optimized using ESI(+) and selectivity was achieved by tandem mass spectrometric analysis using MRM functions, 330.0 --> 70.0 and 310 --> 43.9 for paroxetine and fluoxetine. RESULTS: Analytical curve ranged from 0.2 to 20.0 ng/mL. Inter-day precision and accuracy of the quality control (QC) samples were < 15% relative standard deviation (RSD). Analyte stability during sampling processing and storage were established. CONCLUSION: Validation results on linearity, specificity, accuracy, precision as well as application to the analysis of samples taken up to 120 h after oral administration of 20 mg of paroxetine in 28 healthy volunteers were found to be of good performance in bioequivalence study.

Validated method for determination of bromopride in human plasma by liquid chromatography--electrospray tandem mass spectrometry: application to the bioequivalence study.

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method for the quantification of bromopride I in human plasma is presented. Sample preparation consisted of the addition of procainamide II as the internal standard, liquid-liquid extraction in alkaline conditions using hexane-ethyl acetate (1 : 1, v/v) as the extracting solvent, followed by centrifugation, evaporation of the solvent and sample reconstitution in acetonitrile. Both I and II (internal standard, IS) were analyzed using a C18 column and the mobile-phase acetonitrile-water (formic acid 0.1%). The eluted compounds were monitored using electrospray tandem mass spectrometry. The analyses were carried out by multiple reaction monitoring (MRM) using the parent-to-daughter combinations of m/z 344.20 > 271.00 and m/z 236.30 > 163.10. The areas of peaks from analyte and IS were used for quantification of I. The achieved limit of quantification was 1.0 ng/ml and the assay exhibited a linear dynamic range of 1-100.0 ng/ml and gave a correlation coefficient (r) of 0.995 or better. Validation results on linearity, specificity, accuracy, precision and stability, as well as application to the analysis of samples taken up to 24 h after oral administration of 10 mg of I in healthy volunteers demonstrated the applicability to bioequivalence studies.

Detection of viable myocardium by dobutamine and dipyridamole stress echocardiography.

The basic aspect of cell behaviour that is the clue for viability recognition by stress echo is regional function. By definition, the function is depressed both in viable and in necrotic segments. However, only viable segments retain a contractile reserve; which can be evoked by an inotropic challenge, either cathecolaminic or flow-mediated, as consistently shown by several experimental studies. This is the basis of viability recognition by pharmacological stress echocardiography. Both dobutamine and dipyridamole exploit the same pathophysiological principle: viable tissue has a residual contractile reserve, which can be elicited by an appropriate inotropic stimulus. With dobutamine, the stimulus is a beta-receptor mediated effect on myocardial cell, which is later matched by an increase in flow. With dipyridamole, the primary stimulus is an adenosine A2-receptor mediated effect on the coronary arteriole smooth muscle cell, leading to an increase in flow. At that point, the increase in function is to be expected on the basis of the known relationship between myocardial contractility and coronary perfusion. From the technical point of view, the "viability window" is dose-related with dobutamine, and time-related with dipyridamole--where the same dose is associated to an early inotropic phase followed by a later ischemic response. In spite of the different pathophysiological background and technical modalities, dobutamine and and dipyridamole have shown a similar sensitivity and specificity for prediction of viability, with an overall accuracy only marginally lower than resting thallium. The practical, clinical impact of these observations is still blunted by relatively small numbers of observations in highly selected populations including a limited number of patients and studies in very few centers.(ABSTRACT TRUNCATED AT 250 WORDS)