Plasma Levels In Rats Research Articles

Effect of urinary excretion on the bladder tissue distribution of fluoroquinolones in rats.

The purpose of this study was to evaluate which of blood or urine has the greater effect on bladder tissue concentrations of fluoroquinolones important for the treatment of urinary tract infections by measuring concentrations of fluoroquinolones in the vesical tissue (chemically and immunohistochemically) and intravesical space (chemically). Thirty-minute incubation of isolated rat bladders with fluoroquinolones showed only a 1.9-fold difference in transferability among norfloxacin, levofloxacin, ciprofloxacin and sparfloxacin. Intravesical instillation of norfloxacin and sparfloxacin in rats yielded similar vesical tissue distributions. Thus, there were no large differences in vesical tissue transfer among the four fluoroquinolones. The bladder tissue/plasma concentration ratios of norfloxacin (high urinary excretion-type) and sparfloxacin (low urinary excretion-type) at 1 h after a single oral dose (10 mg/kg) to rats were 15.4 and 1.3, respectively. The bladder tissue/plasma concentration ratios of norfloxacin after an intravenous injection (10 mg/kg) to ureter-catheterized and sham-operated rats were 1.36 and 57.8. Thus the bladder tissue distribution was significantly higher in the urine-exposed bladder. Immunohistochemical examination of the vesical tissue localization of norfloxacin in rats given a single intravenous dose revealed the presence of the drug-positive image in the cytoplasm of surface layer cells (both in umbrella and cover cells) of the bladder transitional epithelium. In conclusion, the results suggest that norfloxacin and other fluoroquinolones are excreted into urine and then transferred to the surface layer of the bladder transitional epithelium. Therefore, the urine levels have a greater effect on the vesicle tissue distribution of fluoroquinolones than the plasma levels in rats.

Development and Validation of LC-MS/MS Assay for the Quantification of Progesterone in Rat Plasma and its Application to Pharmacokinetic Studies.

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of progesterone levels in rat plasma. Progesterone-d9 was used as an internal standard (IS). Samples were prepared using salting-out assisted liquid/liquid extraction (SALLE), and the extracts were injected directly onto the LC-MS/MS system. The chromatographic separation was achieved on a CAPCELL PAK C18 MGIII column (100 mm × 2.0 mm, i.d. 5 µm) using methanol and aqueous 0.1% formic acid solution gradient as the mobile phase with a constant flow rate of 0.45 mL/min. Electrospray ionization in the positive-ion mode was employed. Multiple reaction monitoring of the precursor to product ion pairs, from m/z 315.20 to m/z 109.10 for progesterone and from m/z 324.26 to m/z 113.07 for the IS, was used for quantification. Good linearity was observed over the concentration range of 0.05-20.00 ng/mL with a weighted (1/x(2)) linear regression. The intra- and inter-day precision (% relative standard deviation [RSD]) across 3 validation days over the entire concentration range was lower than 6.7%. Accuracy (% nominal) determined at 5 quality control concentrations was between 94.0 and 103.7%. The validation method was applied in a pharmacokinetic study evaluating progesterone levels after intramuscular or vaginal administration to ovariectomized (OVX) rats. The area under the plasma concentration-time curve (AUC) calculated after intramuscular administration was more than 4 times higher than the AUC measured following vaginal administration of a comparable dose.

Effect of Piperine, Quercetin, Polysorbate 80 on the Oral Bioavailability of Losartan in Male Wistar Rats

Scope: P-glycoprotein (P-gp) is found to play a very significant role in intestinal transport of losartan, an antihypertensive drug with variable and low oral bioavailability (33%), is a substrate for both CYP 3A4 and P-gp. This makes P-gp inhibition a logical strategy for improving losartan’s oral efficacy and reducing its toxicity. Thus, we investigated whether the co-administration of piperine, quercetin, and polysorbate 80 could increase losartan’s pharmacokinetic activity in vivo. Methods and results: We determined the effect of piperine, quercetin, and polysorbate 80 coadministered with losartan on plasma levels in rats by HPLC assay. Rats were administered losartan (10mg/kg; oral gavage) or losartan (10mg/kg; oral gavage) +piperine (10mg/kg; oral gavage) or losartan (10mg/kg; oral gavage) +quercetin (7.5mg/kg; oral gavage) or losartan (10mg/kg; oral gavage) +polysorbate 80 (1%w/v of drug solution; oral gavage), and the plasma levels of losartan were analyzed at different time intervals. Inhibition of p-gp activity resulted in an increased area under the curve (AUC), maximum plasma concentration (Cmax), and mean residence time (MRT) of losartan when compared to losartan alone. Conclusions: Losartan is one of the most widely used antihypertensive agents. Dietary constituents and pharmaceutical excipients are important agents modifying drug transport. In our studies, consumption of piperine, quercetin, and polysorbate 80 increases the therapeutic efficacy of losartan without inducing adverse effects on the treated mice.

The selective estrogen receptor modulator, bazedoxifene, reduces ischemic brain damage in male rat

While the estrogen treatment of stroke is under debate, selective estrogen receptor modulators (SERMs) arise as a promising alternative. We hypothesize that bazedoxifene (acetate, BZA), a third generation SERM approved for the treatment of postmenopausal osteoporosis, reduces ischemic brain damage in a rat model of transient focal cerebral ischemia. For comparative purposes, the neuroprotective effect of 17β-estradiol (E2) has also been assessed. Male Wistar rats underwent 60min middle cerebral artery occlusion (intraluminal thread technique), and grouped according to treatment: vehicle-, E2- and BZA-treated rats. Optimal plasma concentrations of E2 (45.6±7.8pg/ml) and BZA (20.7±2.1ng/ml) were achieved 4h after onset of ischemia, and maintained until the end of the procedure (24h). Neurofunctional score and volume of the damaged brain regions were the main end points. At 24h after ischemia–reperfusion, neurofunctional examination of the animals did not show significant differences among the three experimental groups. By contrast, both E2- and BZA-treated groups showed significantly lower total infarct volumes, BZA acting mainly in the cortical region and E2 acting mainly at the subcortical level. Our results demonstrate that: (1) E2 at physiological plasma levels in female rats is neuroprotective in male rats when given at the acute stage of the ischemic challenge and (2) BZA at clinically relevant plasma levels mimics the neuroprotective action of E2 and could be, therefore, a candidate in stroke treatment.

MP43-20 NANOTECHNOLOGY-BASED IMPLANT FOR LONG TERM TESTOSTERONE REPLACEMENT

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research I1 Apr 2014MP43-20 NANOTECHNOLOGY-BASED IMPLANT FOR LONG TERM TESTOSTERONE REPLACEMENT Eugenia Nicolov, Silvia Ferrati, Randy Goodall, Lee Hudson, Sharath Hosali, Michael Crowley, Ganesh Palapattu, Mohit Khera, and Alessandro Grattoni Eugenia NicolovEugenia Nicolov More articles by this author , Silvia FerratiSilvia Ferrati More articles by this author , Randy GoodallRandy Goodall More articles by this author , Lee HudsonLee Hudson More articles by this author , Sharath HosaliSharath Hosali More articles by this author , Michael CrowleyMichael Crowley More articles by this author , Ganesh PalapattuGanesh Palapattu More articles by this author , Mohit KheraMohit Khera More articles by this author , and Alessandro GrattoniAlessandro Grattoni More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1177AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Current delivery approaches in testosterone replacement therapy (TRT) generate fluctuations in testosterone plasma concentration. The objective of this study was to develop a constant, long-term TRT method. We have developed the implantable Personalized Molecular Drug-delivery System (PMDS) for the constant delivery of therapeutic agents using a silicon-based nanofluidics chip with nanochannels (density over 10,000,000/cm2) to control the rate of drug release via spatial and electrostatic nano-confinement with fabricated channel cross-section heights ranging from 2.5 to 250 nm. The sustained release is realized by constrained diffusion where the channel dimensions are precisely determined to create a steady diffusion of molecules. Here we demonstrate the use of the PMDS device to successfully delivery testosterone with long term constant plasma levels in rats. Methods In this study, the subcutaneous PMDS implant is made of machined titanium (see Figure) and contains the silicon-based nanofluidic chip, which is fabricated in a leading semiconductor foundry and acts as the rate controlling window. In vivo study: Three different testosterone formulations and a placebo formulation were aseptically loaded into the sterilized capsules and implanted into castrated male Sprague-Dawley rats (7-8 month old). A group of castrated rats received approximately 1/3 of a conventional testosterone pellet. A total of 54 rats were used including an intact control group. Blood samples were collected periodically for 126 days from the saphenous vein. Serum testosterone levels were measured by mass spectrometry. In vitro release of the formulations from identical capsules was measured in parallel by HPLC. Results The Figure shows the constant release of one testosterone formulation for 126 days. Intact and placebo serum testosterone levels are shown. Conclusions The PMDS implant delivers a relatively constant dose and mitigates the serum testosterone variability associated with implantable TRT modalities and will become an attractive alternative strategy for the long-term treatment of male hypogonadism. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e485-e486 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Eugenia Nicolov More articles by this author Silvia Ferrati More articles by this author Randy Goodall More articles by this author Lee Hudson More articles by this author Sharath Hosali More articles by this author Michael Crowley More articles by this author Ganesh Palapattu More articles by this author Mohit Khera More articles by this author Alessandro Grattoni More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Himbacine-derived thrombin receptor antagonists: c7-spirocyclic analogues of vorapaxar.

We have synthesized several C7-spirocyclic analogues of vorapaxar and evaluated their in vitro activities against PAR-1 receptor. Some of these analogues showed activities and rat plasma levels comparable to vorapaxar. Compound 5c from this series showed excellent PAR-1 activity (K i = 5.1 nM). We also present a model of these spirocyclic compounds docked to the PAR-1 receptor based on the X-ray crystal structure of vorapaxar bound to PAR-1 receptor. This model explains some of the structure-activity relationships in this series.

Vitamin A status regulates glucocorticoid availability in Wistar rats: consequences on cognitive functions and hippocampal neurogenesis?

A disruption of the vitamin A signaling pathway has been involved in age-related memory decline and hippocampal plasticity alterations. Using vitamin A deficiency (VAD), a nutritional model leading to a hyposignaling of the retinoid pathway, we have recently demonstrated that retinoic acid (RA), the active metabolite of vitamin A, is efficient to reverse VAD-induced spatial memory deficits and adult hippocampal neurogenesis alterations. Besides, excess of glucocorticoids (GCs) occurring with aging is known to strongly inhibit hippocampal plasticity and functions and few studies report on the counteracting effects of RA signaling pathway on GCs action. Here, we have addressed whether the modulation of brain GCs availability could be one of the biological mechanisms involved in the effects of vitamin A status on hippocampal plasticity and functions. Thus, we have studied the effects of a vitamin A-free diet for 14 weeks and a 4-week vitamin A supplementation on plasma and hippocampal corticosterone (CORT) levels in Wistar rats. We have also investigated corticosteroid binding globulin (CBG) binding capacity and 11beta-Hydrosteroid Dehydrogenase type 1 (11β-HSD1) activity, both important modulators of CORT availability at the peripheral and hippocampal levels respectively. Interestingly, we show that the vitamin A status regulates levels of free plasma CORT and hippocampal CORT levels, by acting through a regulation of CBG binding capacity and 11β-HSD1 activity. Moreover, our results suggest that increased CORT levels in VAD rats could have some deleterious consequences on spatial memory, anxiety-like behavior and adult hippocampal neurogenesis whereas these effects could be corrected by a vitamin A supplementation. Thus, the modulation of GCs availability by vitamin A status is an important biological mechanism that should be taken into account in order to prevent age-related cognitive decline and hippocampal plasticity alterations.

Development of a Supercritical Fluid Chromatography-Tandem Mass Spectrometry Method for the Determination of Azacitidine in Rat Plasma and Its Application to a Bioavailability Study

Azacitidine is widely used for the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukaemia (AML). The analysis of azacitidine in biological samples is subject to interference by endogenous compounds. Previously reported high-performance liquid chromatography/tandem mass spectrometric (HPLC-MS/MS) bioanalytical assays for azacitidine suffer from expensive sample preparation procedures or from long separation times to achieve the required selectivity. Herein, supercritical fluid chromatography with tandem mass spectrometry (SFC-MS/MS) was explored as a more promising technique for the selective analysis of structure-like or chiral drugs in biological matrices. In this study, a simple, rapid and specific SFC/MS/MS analytical method was developed for the determination of azacitidine levels in rat plasma. Azacitidine was completely separated from the endogenous compounds on an ACQUITY UPLC™ BEH C18 column (100 mm × 3.0 mm, 1.7 μm; Waters Corp., Milford, MA, USA) using isocratic elution with CO2/methanol as the mobile phase. The single-run analysis time was as short as 3.5 min. The sample preparation for protein removal was accomplished using a simple methanol precipitation method. The lower limit of quantification (LLOQ) of azacitidine was 20 ng/mL. The intra-day and inter-day precisions were less than 15%, and the relative error (RE) was within ±15% for the medium- and high-concentration quality control (QC) samples and within ±20% for the low-concentration QC samples. Finally, the developed method was successfully applied to a pharmacokinetic study in rats following the intravenous administration of azacitidine.

Development and Validation of Liquid Chromato­graphy-Tandem Mass Spectrometric Method for Quantification of Meropenem in Rat Plasma and its Application in a Preclinical Dose Proportionality Study

A simple, rapid, sensitive and selective assay based liquid chromatography-tandem mass spectrometric method was developed and validated for quantitative analysis of meropenem in rat plasma using rolipram as internal standard. Efficient chromatographic separation of analyte from matrix components was achieved by using Kromasil 100 C18 (150×4.6 mm, 5 µm) reversed phase column with mobile phase consisting of acetonitrile and 2 mM ammonium acetate buffer (80:20, v/v) delivered in isocratic mode with constant flow rate of 0.7 mL/min. Detection of meropenem and rolipram was performed in positive mode using multiple reaction monitoring (MRM). The mass transitions 384.1→141.0 and 276.2→191.1 were used to quantify meropenem and rolipram, respectively. A fast and simple solid phase extraction method was optimized for extraction of meropenem from rat plasma. The developed method was validated for selectivity, accuracy, precision, linearity, recovery, stability, matrix effect, dilution integrity as per regulatory guidelines. The developed method was selective with no interfering components at the retention time of meropenem and rolipram. The assay demonstrated acceptable linearity (R(2)>0.99) over a dynamic range of 0.19-201.40 µg/mL. The method exhibited excellent and acceptable precision and accuracy, and produced consistent recoveries. The method demonstrated excellent stability of meropenem in rat plasma under studied conditions investigated. Finally, the validated method was successfully applied to quantify meropenem levels in rat plasma of a dose escalation study.

Anti-obesity property of the brown seaweed, Sargassum polycystum using an in vivo animal model

Obesity has reached epidemic proportions in many countries around the world. Preventing and treating obesity is becoming an increasing priority due to dissatisfaction with high costs and hazardous side effects of anti-obesity drugs. This study was designed to investigate the anti-obesity properties of the Sabah brown seaweed, Sargassum polycystum, on body weight and blood plasma levels of rats fed a high-fat diet supplemented with different doses of the seaweed powder. Male Sprague Dawley rats were divided into five groups, representing control negative (CN) group, control positive (CP) group, low dosage group (LDG), medium dosage group (MDG) and high dosage group (HDG). The study duration was 8 weeks. All groups were fed high-fat diet throughout the study except for CN group, which was fed normal rat chow. LDG, MDG and HDG were supplemented high-fat diet with 2.5, 5.0 and 10.0 % seaweed powder, respectively. By comparing with the CP group, it was found that the HDG (10.0 % seaweed treatment diet) showed the greatest effect in suppressing weight gain, followed by the MDG (5.0 % seaweed treatment diet) and LDG (2.5 % seaweed treatment diet). The HDG decreased the levels of plasma total cholesterol and triglycerides. This finding shows that S. polycystum powder treatment had a positive effect on the inhibition of weight gain and has a promising value in preventing obesity.

Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation

Nitrite and nitrate are main stable products of nitric oxide, a pivotal cellular signaling molecule, in biological fluids. Therefore, accurate measurement of the two ions is profoundly important. Nitrite is difficult to be determined for a larger number of interferences and unstable in the presence of oxygen. In this paper, a simple, cost-effective and accurate HPLC method for the determination of nitrite and nitrate was developed. On the basis of the reaction that nitrite is oxidized rapidly to nitrate with the addition of acidic potassium permanganate, the determination of nitrite and nitrate was achieved by the following strategy: each sample was injected twice for HPLC analysis, i.e. the first injection was to measure nitrate, and the second injection was to measure total nitrate including initial nitrate and the nitrate from the conversion of nitrite with the addition of acid potassium permanganate in the sample. The amount of nitrite can be calculated as difference between injections 2 and 1. The HPLC separation was performed on a reversed phase C18 column for 15 min. The mobile phase consisted of methanol–water (2:98 by volume); the water in the mobile phase contained 0.60 mM phosphate salt (potassium dihydrogen and disodium hydrogen phosphate) and 2.5 mM tetrabutylammonium perchlorate (TBAP). The UV wavelength was set at 210 nm. Additionally, we systemically investigated the effects of the concentration of phosphate salt and TBAP in the mobile phase, the pH of the mobile phase, and the amount of acidic potassium permanganate added to the sample on the separation efficacy. The results showed that the limits of detection (LOD) and the limit of quantitation (LOQ) were 0.075 and 0.25 μM for nitrate (containing the oxidized nitrite), respectively. The linear range was 1–800 μM. This developed approach was successfully applied to assay nitrite/nitrate levels in cell culture medium, cell lysate, rat plasma and urine.

Interaction study of acetylcholinestrase inhibitors on pharmacokinetics of memantine in rat plasma by HPLC‐fluorescence method

The present study aims to investigate the possibility of interaction of donepezil (DP) and galantamine (GAL) as acetylcholinestrase inhibitors, on memantine (MT) hydrochloride in rat plasma by HPLC-fluorescence detection. The separation of MT was achieved within 12 min without interference of DP and GAL on the chromatogram. MT levels in rat plasma with a single administration of MT (2.5 mg/kg, i.p.) and those with a co-administration of DP (5.0 mg/kg, i.p.) and GAL (3 mg/kg, i.p.) were monitored. MT concentrations determined in rat plasma ranged from 10.0 to 245.6 ng/mL. Significant difference was observed in the behavior of MT with a co-administration of DP, while no significant difference was observed with a co-administration of GAL.

Differential Changes in Expression of Stress- and Metabolic-Related Neuropeptides in the Rat Hypothalamus during Morphine Dependence and Withdrawal

Chronic morphine treatment and naloxone precipitated morphine withdrawal activates stress-related brain circuit and results in significant changes in food intake, body weight gain and energy metabolism. The present study aimed to reveal hypothalamic mechanisms underlying these effects. Adult male rats were made dependent on morphine by subcutaneous implantation of constant release drug pellets. Pair feeding revealed significantly smaller weight loss of morphine treated rats compared to placebo implanted animals whose food consumption was limited to that eaten by morphine implanted pairs. These results suggest reduced energy expenditure of morphine-treated animals. Chronic morphine exposure or pair feeding did not significantly affect hypothalamic expression of selected stress- and metabolic related neuropeptides - corticotropin-releasing hormone (CRH), urocortin 2 (UCN2) and proopiomelanocortin (POMC) compared to placebo implanted and pair fed animals. Naloxone precipitated morphine withdrawal resulted in a dramatic weight loss starting as early as 15–30 min after naloxone injection and increased adrenocorticotrophic hormone, prolactin and corticosterone plasma levels in morphine dependent rats. Using real-time quantitative PCR to monitor the time course of relative expression of neuropeptide mRNAs in the hypothalamus we found elevated CRH and UCN2 mRNA and dramatically reduced POMC expression. Neuropeptide Y (NPY) and arginine vasopressin (AVP) mRNA levels were transiently increased during opiate withdrawal. These data highlight that morphine withdrawal differentially affects expression of stress- and metabolic-related neuropeptides in the rat hypothalamus, while relative mRNA levels of these neuropeptides remain unchanged either in rats chronically treated with morphine or in their pair-fed controls.

AB0761 Synthesis and evaluation of the novel folate receptor ligand [18f]fluoro-peg-folate for macrophage targeting in a rat model of arthritis

BackgroundDetection of (subclinical) synovitis is relevant for both early diagnosis and monitoring of therapy of rheumatoid arthritis (RA). Previously, the potential of imaging of (sub)clinical arthritis was demonstrated by targeting...

Determination of cefdinir levels in rat plasma and urine by high‐performance liquid chromatography–tandem mass spectrometry: application to pharmacokinetics after oral and intravenous administration of cefdinir

A selective and sensitive liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination of cefdinir in rat plasma and urine. Following a simple protein precipitation using methanol, chromatographic separation was achieved with a run time of 10 min using a Synergi 4 µ polar-RP 80A column (150 × 2.0 mm, 4 µm) with a mobile phase consisting of 0.1% formic acid in water and methanol (65:35, v/v) at a flow rate of 0.2 mL/min. The protonated precursor and product ion transitions for cefdinir (m/z 396.1 → 227.2) and cefadroxil, an internal standard (m/z 364.2 → 208.0) were monitored in the multiple reaction monitoring in positive ion mode. The calibration curves for plasma and urine were linear over the concentration range 10-10,000 ng/mL. The lower limit of quantification was 10 ng/mL. All accuracy values were between 95.1 and 113.0% and the intra- and inter-day precisions were <13.0% relative standard deviation. The stability under various conditions in rat plasma and urine was also found to be acceptable at three concentrations. The developed method was applied successfully to the pharmacokinetic study of cefdinir after oral and intravenous administration.

Stress in the Adult Rat Exacerbates Muscle Pain Induced by Early-Life Stress

Early-life stress and exposure to stressful stimuli play a major role in the development of chronic widespread pain in adults. However, how they interact in chronic pain syndromes remains unclear. Dams and neonatal litters were submitted to a restriction of nesting material (neonatal limited bedding [NLB]) for 1 week. As adults, these rats were exposed to a painless sound stress protocol. The involvement of sympathoadrenal catecholamines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) in nociception was evaluated through behavioral and enzyme-linked immunosorbent assays, surgical interventions, and intrathecal antisense treatments. Adult NLB rats exhibited mild muscle hyperalgesia, which was markedly aggravated by sound stress (peaking 15 days after exposure). Adrenal medullectomy did not modify hyperalgesia in NLB rats but prevented its aggravation by sound stress. Sustained administration of epinephrine to NLB rats mimicked sound stress effect. Intrathecal treatment with antisense directed to IL-6 receptor subunit gp130 (gp130), but not to tumor necrosis factor receptor type 1 (TNFR1), inhibited hyperalgesia in NLB rats. However, antisense against either gp130 or TNFR1 inhibited sound stress-induced enhancement of hyperalgesia. Compared with control rats, NLB rats exhibit increased plasma levels of IL-6 but decreased levels of TNFα, whereas sound stress increases IL-6 plasma levels in control rats but not in NLB rats. Early-life stress induces a persistent elevation of IL-6, hyperalgesia, and susceptibility to chronic muscle pain, which is unveiled by exposure to stress in adults. This probably depends on an interaction between adrenal catecholamines and proinflammatory cytokines acting at muscle nociceptor level.

253 REMOTE ISCHAEMIC PRECONDITIONING IS MEDIATED VIA THE SDF 1Α/CXCR4 SIGNALLING AXIS

Background Ischaemic preconditioning is one of the most potent experimental modalities known to decrease infarct size following ischaemia-reperfusion (IR) injury. Much interest has been stimulated by the phenomenon of Remote Ischaemic Preconditioning (RIPC) which occurs when a preconditioning stimulus is applied to a limb remote from the heart to stimulate cardioprotection. The protection is believed to be mediated by an unidentified humoral factor between 3.5 and 30 kDa. Stromal cell-derived factor-1α (SDF-1α or CXCL12) is a chemokine of 10 kDa that is induced by hypoxia and recruits stem cells. However it also exerts a direct and acute cardioprotection via its receptor, CXCR4. Aims We hypothesized that SDF-1α is a potential mediator of RIPC-induced protection. Methods and Results Rats were anaesthetized and subjected to RIPC, consisting of 3 cycles of 5 minutes ischaemia by application of a tourniquet around the hind limb followed by 5 minutes of reperfusion. SDF-1α levels in rat plasma were measured using an ELISA kit. There was a 50% increase (p Conclusion RIPC increases circulating levels of SDF-1α, which acts via the CXCR4 receptors in the heart to attenuate IR injury. These results suggest that this chemokine might be an essential mediator of RIPC.

Highly-Sensitive Detection of Free Advanced Glycation End-Products by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry with 2,4,6-Trinitrobenzene Sulfonate Derivatization

The common derivatization method of primary amino groups by 2,4,6-trinitrobenzene sulfonate (TNBS) was applied to the detection of free adducts of advanced glycation end-products (AGEs), in combination with liquid chromatography-tandem mass spectrometry-multiple reaction monitoring (LC-MS/MS-MRM). The proposed TNBS-MS method provided a surprisingly significant improvement of the detection of AGE-free adducts (e.g., by a factor of >1000 for methylglyoxal-derived hydroimidazolone) with a detection limit of 1.0 nM (10 fmol/injection volume), which was due to the high ionization efficiency of the derived trinitrophenol moiety and its hydrophobicity. With the aid of stable-isotope-labeled internal standard (MG-H1-d3), the convenient TNBS method that allowed the derivation of AGE-free adducts bearing amino groups under mild reaction conditions (30 mM, pH 8.5, 30 min, 30 °C) also permitted successive detection and quantification of five typical AGE-free adducts at nanomolar levels in rat plasma (50 μL) with high reproducibility (2-9% of RSD) and recovery (93-113%), using LC-MS/MS-MRM.

Validation of an LC–MS bioanalytical method for quantification of phytate levels in rat, dog and human plasma

Myo-inositol hexakisphosphate (phytate, IP6) is a naturally occuring compound whose determination in biological matrices is chanllenging. Several benefitial properties have been attributed to IP6 in parallel with the development of suitable analytical methodologies for its analytical determination in urine and some tissues. However, there is a lack of appropriate tools for its determination in plasma samples. In this paper, a direct, sensitive and selective bioanalytical method for the determination of IP6 based on LC-MS is presented. It is the first method published to quantify IP6 in plasma matrices directly through its molecular weight, being consequently a highly specific methodology. The method has been validated in rat, dog and human plasma, according to the acceptance criteria laid down in the FDA guidance Bioanalytical Method Validation. Accuracy and precision were not greater than 15% at medium and high concentrations and not greater than 20% at the LLOQ concentration. The mean absolute recovery obtained ranged from 78.74 to 102.44%, 62.10 to 87.21% and 61.61 to 86.99% for rat, dog and human plasma respectively. The LLOQ was 500ngmL(-1) due to the presence of endogenous IP6 in blank plasma samples and the limit of detection was within the range 30-80ngmL(-1).

The effect of shifting food‐intake period on rat body weight and plasma levels of glucose, leptin, melatonin, cholesterol and triglyceride

The suprachiasmatic nucleus of the hypothalamus receives photic information from the retina and contribute to the control of body functions and hormonal secretion in a rhythmic manner through day/night cycle. We hypothesized that disturbance of circadian rhythm by shifting food‐intake period may reflect on body metabolism‐related blood parameters and hence body weight. Hence, we investigated the effect of repeated shifting food intake (over a period of six weeks) on daily changes of rat body weight, food/water intake, feces output and plasma levels of glucose, leptin, melatonin, cholesterol and triglyceride. Two groups of male WKY rats weighing 150–180g were used. The first was subjected to a constant day/night of 12:12h of light/dark cycle (fixed light/day cycle throughout the experimental period; Fixed Group); whereas the second group was subjected to an alternating (every two weeks) pattern of light/dark‐dark/light cycle of 12:12h each (Shifted Group). The plasma glucose, triglycerides, leptin and melatonin levels as well as feces output, food intake showed significant differences between Shifted and Fixed Groups. However, body weight and other measured plasma parameters were not significantly different. It is concluded that rats may need more frequent shifting of food‐intake period to manifest more conclusive effects.