Changes in plasma orexin A and orexin B concentrations during the estrous cycle of the pig

Orexin A (OXA) and orexin B (OXB) are neuropeptides synthesized mainly in the lateral hypothalamus, which are involved in the control of various physiological functions such as energy homeostasis, sleep, wakefulness and feeding behavior. The present study analyzes orexins A and B levels in the porcine plasma during the estrous cycle. The highest plasma concentrations of orexin A were observed on days 2–3 of the estrous cycle (p < 0.05 relative to days 10–12 and 14–16) and the lowest (p < 0.05) on days 14–16. The highest orexin B levels in the blood plasma were noted on days 17–19 (p < 0.05 vs. days 14–16). We demonstrated the presence of OXA and OXB in porcine blood plasma and the impact of the phase of the estrous cycle on the observed changes in plasma orexin levels.     

Immunolocalization of aquaporins 1, 3, and 5 in the nasal respiratory mucosa of a panting species,the sheep (Ovis aries)

The nasal respiratory mucosa is the primary site for evaporative water loss in panting species, necessitating the movement of water across the nasal epithelium. Aquaporins (AQP) are protein channels that facilitate water movement in various fluid transporting tissues of non-panting species. Whether the requirement for enhanced capacity for transepithelial water movement in the nasal respiratory mucosa of panting species has led to differences in AQP localization is unknown. Using immunohistochemistry, we report the localization of AQP1, 3, and 5 in the nasal respiratory mucosa of sheep being exposed to ambient temperatures of ~21 °C or ~38 °C for 4.5 h before death (n=3/treatment). Exposure to either treatment resulted in panting. While exposure to ~38 °C resulted in a higher respiratory frequency (mean difference: 82 breaths min⁻¹; P<0.001) than exposure to ~21 °C, there was no difference in the localization of AQPs. Connective tissue and vascular endothelial cells expressed AQP1. Glandular acini expressed AQP1 and apically localized AQP5, which was also present in glandular duct cells. Ciliated columnar epithelial cells expressed AQP5 apically and AQP3 basolaterally. Basal cells expressed AQP3. The distribution and co-localization of AQPs in the ovine nasal respiratory mucosa is different to that reported in non-panting species and may reflect the physiological demands associated with enhanced respiratory evaporation. We propose that AQP1, 3, and 5 may constitute a transepithelial water pathway via glandular secretions and across the surface epithelium, which provides a possible means for rapid and controllable water movement in the nasal respiratory mucosa of a panting species.     

Mitochondrial distribution patterns in canine oocytes as related to the reproductive cycle stage

This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n = 2), follicular phase (F, n = 4), ovulation (O, n = 2), early luteal (EL, n = 7) and mid/late luteal phase (MLL, n = 2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110 h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (I) small aggregates diffused throughout the cytoplasm; (II) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II + III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P < 0.05; O vs. A: P < 0.001), in EL (61%; O vs. EL: P < 0.01), or in MLL (0%; F vs. MLL: P < 0.05; O vs. MLL: P < 0.001). Fluorescence intensity did not vary according to mt distribution pattern except that it was lower in oocytes recovered in EL phase and showing small mt aggregations (P < 0.001). The majority of ovulated MII stage oocytes (79%) showed diffused tubular mt network. We conclude that mt distribution pattern of canine ovarian immature oocytes changes in relation to reproductive cycle stage and that patterns observed in oocytes recovered from bitches in periovulatory phases are heterogeneous and similar to those of in vivo matured oocytes.     

Coupled electron and proton transfer reactions during the O → E transition in bovine cytochrome c oxidase

A combined DFT/electrostatic approach is employed to study the coupling of proton and electron transfer reactions in cytochrome c oxidase (CcO) and its proton pumping mechanism. The coupling of the chemical proton to the internal electron transfer within the binuclear center is examined for the O → E transition. The novel features of the His291 pumping model are proposed, which involve timely well-synchronized sequence of the proton-coupled electron transfer reactions. The obtained pK_as and E_ms of the key ionizable and redox-active groups at the different stages of the O → E transition are consistent with available experimental data. The PT step from E242 to H291 is examined in detail for various redox states of the hemes and various conformations of E242 side-chain. Redox potential calculations of the successive steps in the reaction cycle during the O → E transition are able to explain a cascade of equilibria between the different intermediate states and electron redistribution between the metal centers during the course of the catalytic activity. All four electrometric phases are discussed in the light of the obtained results, providing a robust support for the His291 model of proton pumping in CcO. This article is part of a Special Issue entitled: Respiratory oxidases.     

Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 °C while the other was transported at 37 °C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 °C in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto–orcein staining.Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI + MII) than the oocytes from anestrual ovaries in the 37 °C group (p < 0.05). However, oocytes harvested from anestrual ovaries transported at 4 °C had the highest maturation (MI + MII) rate, and the difference between anestrual and luteal ovary groups was significant (p < 0.05). The oocytes from anestrual ovaries transported at 4 °C have significantly higher maturation rates than those transported at 37 °C (p < 0.0001). However, the transport temperature (37 or 4 °C) did not significantly affect the maturation (MI + MII) rates of oocytes harvested from the luteal (p = 0.61) and follicular (p = 0.48) stage ovaries.It can be concluded from this study that (1) both transport temperature and transport temperature × estrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 °C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries.     

Oestrus cycle characteristics and prediction of ovulation in Catalonian jennies

The Catalonian donkey breed is in danger of extinction, and much needs to be learned about the reproductive features of its females if breeding and conservation programmes are to be successful. This study reports the oestrous behaviour, oestrus cycle characteristics and dynamic ovarian events witnessed during 50 oestrous cycles (involving 106 ovulations) in 10 Catalonian jennies between March 2002 and January 2005. These jennies were teased, palpated transrectally and examined by ultrasound using a 5 MHz linear transducer—daily during oestrus and every other day during dioestrus. Predictors of ovulation were sought among the variables recorded.The most evident signs of oestrus were mouth clapping (the frequent vertical opening and closing of the mouth with ears depressed against the extended neck) and occasional urinating and winking of the vulval lips (homotypical behaviour). Interactions between jennies in oestrus were also recorded, including mounting, herding/chasing, the Flehmen response, and vocalization (heterotypical behaviour).Nine jennies ovulated regularly throughout the year; one had two anovulatory periods (54 and 35 days). The length of the oestrus cycle was 24.90 ± 0.26 days, with oestrus itself lasting 5.64 ± 0.20 days (mean ± S.E.M.) and dioestrus 19.83 ± 0.36 days. The incidence of single, double and triple ovulations was 55.66% (n = 59), 42.45% (n = 45) and 1.89% (n = 2), respectively. No significant difference was seen in the number of ovulations involving the left and right ovaries (52.63% [n = 70] compared to 47.37% [n = 63] respectively; P > 0.05). The mean interval between double ovulation was 1.44 ± 3.98 days. The mean diameter of the preovulatory follicle at day −1 was 44.9 ± 0.5 mm; the mean growth rate over the 5 days before ovulation was 3.7 mm/day.Data on preovulatory changes in oestrous behaviour, follicle size, follicle texture, the echographic appearance of the follicle and uterus, and uterine tone were subjected to stepwise logistic regression analysis to detect predictors of ovulation. The logit function showed the best predictors to be follicle size, follicular texture and oestrous behaviour. Certain combinations of these three variables allow the prediction of ovulation within 24 h with a probability of >75%.     

Segmentation d’images de lésions cérébrales primitives en TEP FET : influence du volume de travail

Purpose(1) Evaluate the reproducibility of segmentation methods depending on the preselection region for tumour volume determination on ¹⁸F-fluoro-ethyl-tyrosine (FET) PET. (2) Evaluate the intra and inter-operator reproducibility of the manual delineation. (3) Compare this delineation with the segmentation methods.Materials and methodsEighteen FET PET of patients with glioblastoma were analysed. Preselection regions were determined prior to any segmentation. Two physicians delineated the tumour volume manually. The tumour volume was also delineated with a threshold method (40 and 70% of SUV_max), and a random walk based method. Pearson coefficient (r) (P < 0.05 for r > 0.468) and Jaccard indices (JI) were used to compare the volumes.ResultsManual delineation was reproducible with r = 0.97 and IJ = 0.65 for intra-operator, and r = 0.76 and IJ = 0.45 for inter-operator reproducibility. The preselection regions for a given lesion were different and the segmentation varied with the preselection region: r = 0.55 JI = 0.58; r = 0.85 JI = 0.83; r = 0.70 JI = 0.39 respectively for the threshold of 40%, 70% and the random walk. The segmentation differed form de manual delineation with r = 0.37 and JI = 0.16; r = 0.54 and JI = 0.42; r = 0.43 and JI = 0.37 respectively for the threshold of 40%, 70% and the random walk.ConclusionThe reproducibility of the segmentation methods depends extensively on the preselection region. The intra-operator reproducibility of cerebral lesion delineation on FET PET is satisfactory. The inter-operator reproducibility could be improved.     

The effects of accelerometer placement on mechanomyographic amplitude and mean power frequency during cycle ergometry

The purposes of this study were threefold: (1) to compare the power output related patterns of absolute and normalized MMG amplitude and MPF responses for proximal and distal accelerometer placements on the vastus lateralis (VL) muscle during incremental cycle ergometry; (2) to examine the influence of accelerometer placements on mean absolute MMG amplitude and MPF values; and (3) to determine the effects of normalization on mean MMG amplitude and MPF values from proximal and distal accelerometer placements. Fifteen adults (10 men and 5 women; mean ± SD age = 23.9 ± 3.1 years) performed incremental cycle ergometry tests to exhaustion. Two accelerometers were placed proximal and distal on the VL muscle. Paired t-tests indicated that absolute MMG amplitude values for the proximal accelerometer were greater (p < 0.05) than the distal accelerometer at all power outputs. The normalized MMG amplitude also had greater values for the proximal accelerometer at all power outputs, except 50 W. There were no differences, however, between proximal and distal accelerometers for absolute MMG MPF, except at 75 W, and normalization eliminated this difference. Twenty-seven percent of the subjects exhibited different power output related patterns of responses between accelerometer placements for MMG amplitude and 47% exhibited different patterns for MPF. These findings indicated that normalization did not eliminate the influence of accelerometer placement on MMG amplitude and highlighted the importance of standardizing accelerometer placements to compare MMG values during cycle ergometry.     

Life cycle of the aquatic firefly Luciola ficta (Coleoptera: Lampyridae)

This research is the first to record the complete life history of the aquatic firefly Luciola ficta (Olivier) using a unique individual rearing method. Transparent containers (250 ml; height: 6 cm; bottom diameter: 8 cm; mouth diameter: 9.5 cm) were used to rear individuals from egg to adult in the laboratory, so that they could be observed throughout the whole life cycle. Larvae were fed on the meat of the water snail Cipangopaludina chinensis (Gray). Temperature ranged from 18 °C to 30 °C, relative humidity (RH) was 80 ± 5%, and the light:dark (L:D) ratio was 10:14. Of 80 eggs, 35 individuals completed their life cycle under these laboratory conditions in Jiji, Nantou County, Taiwan. The external morphological characteristics of each growing stage were described. Egg hatching rate was 95%. On average, one generation spanned 388.2 ± 25.7 days. The durations of egg, larva, climbing larva, cocoon, and adult stages were 19.1 ± 1.5 days, 328.9 ± 33.2 days, 10.9 ± 7.8 days, 14.7 ± 5.3 days, and 15.7 ± 5.2 days, respectively. The number of larval instars ranged from five to seven, with a modal value of six instars for males and seven instars for females. Female larval duration averaged 337.1 ± 31.2 days, which was higher than the 307.6 ± 34.1 days of the males. From January to December 2002, adult emergence peaked twice, with the main high peak appearing in April and the second peak occurring in August. The results of indoor rearing and of field investigations in Jiji, Nantou County, suggested that L. ficta is univoltine. Adult body length is negatively correlated with larval duration (P < 0.01). The life history traits of L. ficta show plasticity in adult occurrence, egg size, egg duration, larval duration, larval instars, and adult body length. Some variations were discussed in the context of survivorship in field habitats.     

Effect of 5-fluoro-2′-deoxyuridine on toxin production and cell cycle regulation in marine dinoflagellate,Alexandrium tamarense

The effect of metabolic inhibitor, 5-fluoro-2′-deoxyuridine (FUdR) on toxin production and the cell cycle of marine dinoflagellate, Alexandrium tamarense, was investigated. Compared to untreated cells, FUdR at 3 μM (p < 0.05) to 300 μM (p < 0.01) inhibited the cell proliferation and toxin production in a dose-dependent manner for A. tamarense cultured in modified T₁ medium. FUdR at 203 μM resulted in cell cycle arrest at the S phase at day 4 and toxigenesis was inhibited after day 2. The toxin profiles of the FUdR-treated cultures were similar to those of the control culture. These results suggest that FUdR inhibits saxitoxin (STX) biosynthesis in the early stage of the pathway. This report is the first to demonstrate the inhibition of toxin production in A. tamarense by a nucleoside analog.     

A quantitative HPLC–MS method for the simultaneous determination of testosterone, 11-ketotestosterone and 11-β hydroxyandrostenedione in fish serum

A simple and novel HPLC–MS method for the simultaneous quantification of testosterone, 11-ketotestosterone, and 11β-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water–acetonitrile mobile-phase with a cycle time of 12 min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2–50 ng/ml), limits of detection (0.1–0.2 ng/ml) and quantification (0.2–0.5 ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA).     

Expression of progesterone receptor membrane component (PGRMC) 1 and 2, serpine mRNA binding protein 1 (SERBP1) and nuclear progesterone receptor (PGR) in the bovine endometrium during the estrous cycle and the first trimester of pregnancy

Progesterone (P4) is involved in the regulation of essential reproductive functions affecting the target cells through both nuclear progesterone receptors (PGRs) and membrane progesterone receptors. The aim of this study was to determine the mRNA and protein expression for PGRMC1, PGRMC2, SERBP1 and PGR within the bovine endometrium during the estrous cycle and the first trimester of pregnancy. There were no changes in PGRMC1 and PGRMC2 mRNA and protein expression during the estrous cycle, however, mRNA levels of PGRMC1 and PGRMC2 were increased (P < 0.001) in pregnant animals. SERBP1 mRNA expression was increased (P < 0.05), while the level of this protein was decreased (P < 0.05) on days 11–16 of the estrous cycle. The expression of PGR mRNA was higher (P < 0.01) on days 17–20 compared to days 6–10 and 11–16 of the estrous cycle and pregnancy. PGR-A and PGR-B protein levels were elevated on days 1–5 and 17–20 of the estrous cycle as compared to other stages of the cycle and during pregnancy. In conclusion, our results indicate that P4 may influence endometrial cells through both genomic and nongenomic way. This mechanism may contribute to the regulation of the estrous cycle and provide protection during pregnancy.     

The effect of the absence or presence of a corpus luteum on the ovarian follicular population and serum oestradiol concentrations during the estrous cycle in Sanjabi ewes

The aim of this study was to determine if the presence or absence of a corpus luteum (CL) during estrous synchronization in ewes can affect the ovarian follicular population and the serum oestradiol concentrations. The estrous cycles of 197 Sanjabi ewes were synchronized using a 12-day treatment with intravaginal progestagen sponges (Chronogest^®). Estrus was detected in 144 ewes, 27–39 h after sponge removal. Blood samples were taken daily from day 2 and continued for 19 days and analyzed for serum oestradiol concentration. Nine ewes were slaughtered on each experimental day (days 1–16 after estrus) for ovary collection. The ovaries per ewe were classified as those without, or with one or two CL's, for each slaughter day. Visible follicles on the surface of the ovaries were classified, based on their diameter, into (i) very small (<2 mm), (ii) small (2–3.4 mm), (iii) medium (3.5–5 mm) and (iv) large (>5 mm) categories, and the respective numbers recorded. Results indicated, the number of ovarian follicles to decrease (P < 0.01) from days 1 to 5 of the cycle and showed a significant increase on day 7. Numbers were high again on day 11 and decreased (P < 0.01) on day 16 of the estrous cycle. The serum oestradiol concentrations were significantly higher (P < 0.001) in the double than in the single ovulating animals (one or two CL's, respectively) on days 2–0. However serum levels were also significantly higher (P < 0.001) in single, than twin ovulating animals on days 4–5 and 12–16 of the estrous cycle. There were no significant differences in the total number of very small follicles between animals without and those with two CL's. The number of small, medium and large follicles in ewes, with or without a CL on the ovary was significantly higher (P < 0.01) than ewes with two ovulations at certain stages of the estrous cycle. The present study provides evidence of differences in the follicular ovarian population in ovaries without CL's and double ovulations. The existence of an intraovarian effect of the CL numbers on follicular population is demonstrated.     

Discovery of a potent,selective, and orally bioavailable histamine H3 receptor antagonist SAR110068 for the treatment of sleep–wake disorders

Previous studies have shown that compound 1 displayed high affinity towards histamine H₃ receptor (H₃R), (human (h-H₃R), K_i = 8.6 nM, rhesus monkey (rh-H₃R), K_i = 1.2 nM, and rat (r-H₃R), K_i = 16.5 nM), but exhibited high affinity for hERG channel. Herein, we report the discovery of a novel, potent, and highly selective H₃R antagonist/inverse agonist 5a(SS) (SAR110068) with acceptable hERG channel selectivity and desirable pharmacological and pharmacokinetic properties through lead optimization sequence. The significant awakening effects of 5a(SS) on sleep–wake cycles studied by using EEG recording in rats during their light phase support its potential therapeutic utility in human sleep–wake disorders.     

Microinjection of melanin concentrating hormone into the lateral preoptic area promotes non-REM sleep in the rat

The ventrolateral preoptic area (VLPO) has been recognized as one of the key structures responsible for the generation of non-REM (NREM) sleep. The melanin-concentrating hormone (MCH)-containing neurons, which are located in the lateral hypothalamus and incerto-hypothalamic area, project widely throughout the central nervous system and include projections to the VLPO. The MCH has been associated with the central regulation of feeding and energy homeostasis. In addition, recent findings strongly suggest that the MCHergic system promotes sleep. The aim of the present study was to determine if MCH generates sleep by regulating VLPO neuronal activity. To this purpose, we characterized the effect of unilateral and bilateral microinjections of MCH into the VLPO on sleep and wakefulness in the rat. Unilateral administration of MCH into the VLPO and adjacent dorsal preoptic area did not modify sleep. On the contrary, bilateral microinjections of MCH (100 ng) into these areas significantly increased light sleep (LS, 39.2 ± 4.8 vs. 21.6 ± 2.5 min, P < 0.05) and total NREM sleep (142.4 ± 23.2 vs. 86.5 ± 10.5 min, P < 0.05) compared to control (saline) microinjections. No effect was observed on REM sleep. We conclude that MCH administration into the VLPO and adjacent dorsal lateral preoptic area promotes the generation of NREM sleep.     

Using short columns to speed up LC–MS quantification in MS binding assays

The present study describes the use of short columns to speed up LC–MS quantification in MS binding assays. The concept of MS binding assays follows closely the principle of traditional radioligand binding but uses MS for the quantification of bound marker thus eliminating the need for a radiolabelled ligand. The general strategy of increasing the throughput of this type of binding assay by the use of short columns is exemplified for NO 711 binding addressing GAT1, the most prevalent GABA transporter in the CNS. Employing short RP-18 columns with the dimension of 20 mm × 2 mm and 10 mm × 2 mm at flow rates up to 1000 μL/min in an isocratic mode retention times of 8–9 s and chromatographic cycle times of 18 s could be achieved. Based on the internal standard [²H₁₀]NO 711 fast chromatography methods were developed for four different columns that enabled quantification of NO 711 in a range from 50 pM up to 5 nM directly out of reconstituted matrix samples without further sample preparation. A validation of the established methods with respect to linearity, intra- and inter-batch accuracy and precision showed that the requirements according to the FDA guideline for bioanalytical methods are met. Furthermore the established short column methods were applied to the quantification of NO 711 in saturation experiments. The results obtained (i.e., K_d- and B_max-values) were almost identical as compared to those determined employing standard column dimension (55 mm × 2 mm).     

Reference database of the gait cycle for young healthy Tunisian adults

BackgroundQuantified gait analysis is a rising technology used increasingly to assess motor disorders. Normal reference data are required in order to evaluate patients, but there are no reference data available for the Tunisian healthy population.AimTo assess the features of normal Tunisian gait pattern, and examine the intrinsic reliability of spatio-temporal, kinematic and kinetic parameters within a new specific reference database.MethodsEighteen healthy active-young adults (age: 23.30 ± 2.54 years, height: 1.78 ± 0.04 m and, weight: 70.00 ± 4.80 kg) have participated to five trials of step gait where the dominant lower limb were recorded. Two over the five trials were randomly selected to be further analyzed. Twenty-three spatio-temporal, kinematic and kinetic parameters determined from 3-dimensional gait analysis. The intrinsic reliability was examined for each variable and our results were compared with those available in the literature.ResultsTwelve over 23 parameters have an excellent intrinsic reliability (P > 0.05, ICC > 0.9 and SEM < 5% of the grand mean). There are similarities with other studies (P < 0.05) but we noticed the existence of some specificity (the height of hip extension peak and the low cadence of gait) that could characterize the Tunisian population.ConclusionA specific reference database of the gait cycle has been established for healthy Tunisian active-young adults and excellent inter-trial reliability may be observed for different variables.     

The incidence of Streptococcus pneumoniae otitis media is affected by the polymicrobial environment particularly Moraxella catarrhalis in a mouse nasal colonisation model

Otitis media (OM) is a highly prevalent paediatric disease with both bacterial and viral triggers of infection. This study has investigated how combinations of bacteria associated with nasal colonisation and the occurrence and absence of viral infection (Sendai virus) induce OM in a mouse nasal colonisation model. The respiratory virus significantly contributed to bacterial OM for all bacterial combinations (p < 0.001). Streptococcus pneumoniae consistently dominated as the causative bacterium of OM and when co-infected with S. pneumoniae, Moraxella catarrhalis more significantly affected pneumococcal OM than did non-typeable Haemophilus influenzae (p < 0.001) by increasing the incidence rate, infection bacterial load and duration of infection. Nitric oxide levels in the middle ear, an indicator of inflammation, peaked at day 3 in single bacterium groups, but at day 1 in mixed bacterial groups and was produced in all bacteria inoculated groups even in the absence of viable bacterial recovery. Phagocytic cells were recruited rapidly to the ear following nasal inoculation but over time their numbers did not correlate with persistence of bacterial infection. The study has shown that the composition of bacteria in the nasal cavity and respiratory viral infection significantly affected the OM incidence rate, duration of infection and bacterial load (severity).     

Validation of an electrospray ionization LC/MS/MS method for quantitative analysis of vincristine in human plasma samples

Vincristine is a natural vinca alkaloid widely used in paediatric cancer treatment. Vincristine pharmacokinetics has been already studied, but few data are available in paediatric populations. A sensitive and specific liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of vincristine in plasma in order to investigate pharmacokinetics in a paediatric population. Two hundred microliters of plasma was added to vinblastine, used as internal standard. Chromatographic separation was achieved on a C8 HPLC column (Phenomenex Luna 50 mm × 2.0 mm, 3.0 μm) with a mobile phase gradient at a flow rate of 0.2 ml/min. Quantification was performed using the transition of 825.4 → 765.4 (m/z) for vincristine and 811.4 → 751.4 (m/z) for vinblastine. Chromatographic separation was achieved in 8 min. The limit of quantification was 0.25 ng/ml with a precision of 10.2% and an accuracy of 99.6%. The calibration curve was linear up to 50.0 ng/ml. Intra-day precision and accuracy ranged from 6.3% to 10% and from 91.9% to 100.8%, respectively. Inter-assay precision and accuracy ranged from 3.8% to 9.7% and from 93.5% to 100.5%, respectively. No significant matrix effect was observed for vincristine. A rapid, specific and sensitive LC/MS/MS method for quantification of vincristine in human plasma was developed and is now successfully applied for pharmacokinetic studies in paediatric patients.     

Association of urinary 15-F2t-isoprostane level with oxygen desaturation and carotid intima–media thickness in nonobese sleep apnea patients

Obstructive sleep apnea (OSA) is characterized by recurrent apnea during sleep that may unbalance oxidative stress, increasing atherosclerosis. Among oxidative stress markers, 15-F_2t-isoprostane is considered one of the most sensitive and specific metabolites of lipid peroxidation. To explore the relationship between urinary 15-F_2t-isoprostane with sleep apnea severity and carotid modifications in nonobese OSA patients, 31 nonobese sleep apnea patients were studied, along with 10 lean subjects without OSA. Patients were assessed by polysomnography, blood pressure measurement, and ultrasonography to determine the carotid intima–media thickness (IMT). Urinary 15-F_2t-isoprostanes were measured by liquid chromatography–tandem mass spectrometry. Urinary 15-F_2t-isoprostane concentrations were increased in severe OSA patients compared to control subjects (20.2 ± 7.3 vs 12.3 ± 2.8 ng/mmol creatinine; P = 0.020). Mean carotid IMT was correlated with 15-F_2t-isoprostane (r = 0.532; P < 0.001) and with the apnea–hypopnea index (r = 0.345; P = 0.029). 15-F_2t-Isoprostane level was related to the night time spent at SaO₂ < 90% (r = 0.478; P = 0.002), the apnea–hypopnea index (r = 0.465; P = 0.003), and the mean nocturnal SaO₂ (r = ? 0.424; P = 0.007). These results showed a relationship between lipid peroxidation, carotid intima–media thickness, and intermittent hypoxia in nonobese OSA patients, thus reinforcing the hypothesis that oxidative stress could be involved in the early atherosclerotic process.