Breast muscle and plasma metabolomics profile of broiler chickens exposed to chronic heat stress conditions.

Understanding the variations of muscle and plasma metabolites in response to high environmental temperature can provide important information on the molecular mechanisms related to body energy homeostasis in heat-stressed broiler chickens. In this study, we investigated the effect of chronic heat stress conditions on the breast muscle (Pectoralis major) and plasma metabolomics profile of broiler chickens by means of an innovative, high-throughput analytical approach such as the proton nuclear magnetic resonance (1H NMR) spectrometry. A total of 300 Ross 308 male chicks were split into two experimental groups and raised in either thermoneutral conditions for the entire rearing cycle (0-41 days) (TNT group; six replicates of 25 birds/each) or exposed to chronic heat stress conditions (30 °C for 24 h/day) from 35 to 41 days (CHS group; six replicates of 25 birds/each). At processing (41 days), plasma and breast muscle samples were obtained from 12 birds/experimental group and then subjected to 1H NMR analysis. The reduction of BW and feed intake as well as the increase in rectal temperature and heterophil: lymphocyte ratio confirmed that our experimental model was able to stimulate a thermal stress response without significantly affecting mortality. The 1H NMR analysis revealed that a total of 26 and 19 molecules, mostly related to energy and protein metabolism as well as antioxidant response, showed significantly different concentrations respectively in the breast muscle and plasma in response to the thermal challenge. In conclusion, the results obtained in this study indicated that chronic heat stress significantly modulates the breast muscle and plasma metabolome in fast-growing broiler chickens, allowing to delineate potential metabolic changes that can have important implications in terms of body energy homeostasis, growth performance and product quality.

Fatty acid profile, oxidative status, and content of volatile organic compounds in raw and cooked meat of different chicken strains.

Chicken meat is rich in unsaturated fatty acids. Therefore, it is more susceptible to lipid oxidation and production of volatile organic compounds (VOC). In this study, we evaluated the fatty acids, antioxidants, and VOC profiles of raw and cooked meat samples derived from 4 strains of chicken differing in their growth rates, which were as follows: slow-growing (SG, Leghorn), medium-growing (MG, Hubbard and Naked Neck), and fast-growing (FG, Ross). The VOC profile of meat was measured using proton-transfer reaction-mass spectrometry (PTR-MS). The VOC were identified using PTR-time of flight-MS (PTR-ToF-MS). The data were analyzed using both univariate and multivariate models. Twenty main VOC were identified, which were classified into the following chemical categories: aldehydes, alkadienes, alkenes, furans, amides, alcohols, and other compounds. Our results revealed that the chicken genotype and the method of cooking strongly influenced the VOC profile of the meat. Identifying the relationships between these traits allowed us to highlight the trade-off of the main substrates such as n-3 and n-6 polyunsaturated fatty acids (PUFA), protective substances (antioxidants), and degradation products (VOC) of the poultry meat produced during cooking. The extent of VOC production and n-3 loss was found to be higher for the SG genotype. Reduction of n-6 was higher in MG, whereas small losses in antioxidants and PUFA were observed in the FG genotype, consequently, resulting in the lowest production of VOC. The SG and MG are genotypes more active from a kinetic point of view respect to the FG ones. For this reason, in the FG genotypes, the antioxidants are less involved in the oxidative stress induced by the movement; thus, they were available to protect the lipid of the meat during the cooking process. These results suggested that the use of SG and MG genotypes requires a specific dietary protocol (i.e., increasing the antioxidants content) to counteract the lipid oxidations in all the phases: in vivo, postmortem, and during/after cooking.

Performance and egg quality of laying hens fed flaxseed: highlights on n-3 fatty acids, cholesterol, lignans and isoflavones.

Flaxseed is a rich source of α-linolenic acid and phytoestrogens, mainly lignans, whose metabolites (enterodiol and enterolactone) can affect estrogen functions. The present study evaluated the influence of dietary flaxseed supplementation on reproductive performance and egg characteristics (fatty acids, cholesterol, lignans and isoflavones) of 40 Hy-Line hens (20/group) fed for 23 weeks a control diet or the same diet supplemented with 10% of extruded flaxseed. The flaxseed diet had approximately three times the content of lignans (2608.54 ng/g) as the control diet, mainly secoisolariciresinol diglucoside (1534.24 v. 494.72 ng/g). When compared with the control group, hens fed flaxseed showed a similar deposition rate (72.0% v. 73.9%) and egg yield. Furthermore, there was no effect of flaxseed on the main chemical composition of the egg and on its cholesterol content. Estradiol was higher in the plasma of the control group (1419.00 v. 1077.01 pg/ml) probably due to the effect of flaxseed on phytoestrogen metabolites. The plasma lignans were higher in hens fed flaxseed, whereas isoflavones were lower, mainly due to the lower equol value (50.52 v. 71.01 ng/ml). A similar trend was shown in eggs: the flaxseed group had higher level of enterodiol and enterolactone, whereas the equol was lower (198.31 v. 142.02 ng/g yolk). Secoisolariciresinol was the main lignan in eggs of the flaxseed group and its concentration was three times higher then control eggs. Flaxseed also improved the n-3 long-chain polyunsaturated fatty acids of eggs (3.25 v. 0.92 mg/g egg), mainly DHA, however, its oxidative status (thiobarbituric reactive substances) was negatively affected. In conclusion, 10% dietary flaxseed did not affect the productive performance of hens or the yolk cholesterol concentration, whereas the lignans and n-3 polyunsaturated fatty acid content of eggs improved. Further details on the competition between the different dietary phytoestrogens and their metabolites (estrogen, equol, enterodiol and enterolactone) should be investigated.

Ceftibuten stability to active-site serine and metallo-beta-lactamases.

Ceftibuten is an oral third-generation cephalosporin active against a wide range of bacteria and shows an improved stability to hydrolysis by several beta-lactamases because of the carboxyethilidine moiety at position 7 of the ss-acyl side chain. The kinetic interactions between ceftibuten and active-site serine and metallo-ss-lactamases were investigated. The activity of several TEM-derived extended spectrum beta-lactamases (ESbetaLs) against ceftibuten, cefotaxime and ceftazidime was compared using K(m), K(cat) and K(cat)/K(m). Ceftibuten behaved as a poor substrate for class A and B beta-lactamases compared with cefotaxime. The chromosomal class C beta-lactamase from Enterobacter cloacae 908R gave a high K(cat) value (21 s(-1)), whereas there was poor activity with enzymes from Acinetobacter baumannii and Morganella morganii and ceftibuten. Ceftibuten resists hydrolysis in the presence of typical respiratory or urogenital-tract pathogens producing beta-lactamases.

Excretion and metabolism of propionyl-L-carnitine in the isolated perfused rat kidney.

Propionyl-L-carnitine (PLC) is an ester of L-carnitine (LC) under evaluation for the treatment of cardiovascular disorders. The renal disposition of PLC was studied in the isolated perfused rat kidney with deuterium-labeled derivative (PLC-CD3). Kidneys of male Sprague-Dawley rats were perfused at initial PLC-CD3 concentrations of 10 (n = 4) and 200 microM (n = 5). High-performance liquid chromatography/mass spectrometry was used to quantify PLC-CD3, deuterated L-carnitine (LC-CD3) and acetyl-L-carnitine (ALC-CD3) in perfusate and urine. PLC-CD3 in perfusate decreased in a monoexponential manner with a half-life of 90 +/- 24 min (S.D.) (10 microM) and 94 +/- 11 min (200 microM). The renal excretory clearance of PLC-CD3 was significantly lower (P < .05, unpaired t test) at an initial concentration of 10 microM (45 +/- 23 microl/min) than at 200 microM (85 +/- 28 microl/min), but in both cases it was substantially less than the glomerular filtration rate, which indicates extensive tubular reabsorption. The renal excretory clearance of PLC-CD3 represented less than 6% of the total clearance, which suggests that metabolism is the major renal elimination route for this compound. The appearance in perfusate and urine of LC-CD3 and ALC-CD3 provided additional evidence for a metabolic role of the kidney. The apparent renal excretory clearance values for these metabolites were always significantly higher than the values obtained for the corresponding endogenous compounds, which suggests that LC-CD3 and ALC-CD3, as formed metabolites, underwent passive or carrier-mediated movement directly into urine.

Determination of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine in human plasma by high-performance liquid chromatography after pre-column derivatization with 1-aminoanthracene.

A new sensitive high-performance liquid chromatographic procedure for the determination of L-carnitine (LC), acetyl-L-carnitine (ALC) and propionyl-L-carnitine (PLC) in human plasma has been developed. Precolumn derivatization with 1-aminoanthracene (1AA), performed in phosphate buffer in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as catalyst, is involved. The fluorescent derivatives were isocratically separated on a reversed-phase column (C18). The eluate was monitored with a fluorimetric detector set at 248 nm (excitation wavelength) and 418 nm (emission wavelength). Because of the presence of endogenous carnitines, the validation was performed using dialyzed plasma. The identity of the derivatized compounds was assessed by mass spectrometry and the purity of the chromatographic peaks was confirmed by HPLC-tandem mass spectrometry. The limits of quantitation were 5 nmol/ml for LC, 1 nmol/ml for ALC and 0.25 nmol/ml for PLC. The recovery of the extraction procedure was in the range 82.6%-95.4% for all 3 compounds. Good linearity (R approximately 0.99) was observed within the calibration ranges studied: 5-160 nmol/ml for LC, 1-32 nmol/ml for ALC and 0.25-8 nmol/ml for PLC. Precision was in the range 0.3-16.8% and accuracy was always lower than 10.6%.

Disposition of L-carnitine and acetyl-L-carnitine in the isolated perfused rat kidney.

The isolated perfused rat kidney was used to investigate the regulation, specificity and concentration-dependence of the renal tubular disposition of L-carnitine (LC) and its ester, acetyl-L-carnitine (ALC). Tritiated markers were used to study the renal disposition of LC and ALC and HPLC was used to purify 3H-LC and 3H-ALC before radiochemical analysis. At perfusate concentrations comparable to those found in plasma in vivo (50 microM for LC and 5 microM for ALC), the renal clearance of both analogues was substantially less than GFR (P < .05) which, in view of their negligible binding to perfusate proteins, is indicative of extensive reabsorption. During the first 20 min of perfusion, the percent tubular reabsorption (%TR) of LC and ALC was 94 +/- (SD) 2.6% and 97 +/- 0.6%, respectively. The extent of 3H-ALC and 3H-LC enrichment of perfusate in experiments with 3H-LC and 3H-ALC, respectively, provided evidence for the capability of the rat kidney to acetylate LC and deacetylate ALC. In addition, a portion of renally generated 3H-ALC and 3H-LC was found to undergo leakage into renal tubules and escape subsequent reabsorption. It was also found that the %TR of both compounds decreased substantially when the perfusate concentration was increased above endogenous levels; each compound was capable of decreasing the %TR of the other; and trimethylamine-N-oxide, a metabolite of LC, had no significant effect on the renal handling of the carnitine derivatives.

Determination of pentoxifylline and its metabolites in human plasma by high-performance liquid chromatography with solid-phase extraction.

A simple and reliable method for the determination of pentoxifylline and its main metabolites in human plasma has been developed using high-performance liquid chromatography. After selective solid-phase extraction, pentoxifylline, its metabolites and an internal standard, 7-(2'-chloroethyl)theophylline, were separated on a 5-micron LiChrospher 100 RP-18 column using water-dioxan-acetonitrile (87:6.5:6.5, v/v/v) acidified with acetic acid (0.5%, v/v) as the mobile phase. The analytes were detected at 275 nm. The lowest detectable concentration for all analytes was 25 ng/ml; the recovery was 85%. The assay has been successfully applied to analysis of these compounds in human plasma after administration of an oral dose of 400 mg of pentoxifylline to healthy volunteers.

Determination of the tricyclic compound adosupine and its three metabolites in plasma and brain of rat using high-performance liquid chromatography.

An analytical method for the detection in biological samples of the novel tricyclic compound adosupine (10-acetoamido-5-methyl-5,6-dihydro-11H-dibenzo[b,e]azepin-6 ,11-dione), which is capable of influencing various forms of urinary bladder hyperreflexia has been developed using high-performance liquid chromatography with UV detection. Liquid-liquid extraction was used to isolate the parent compound, three metabolites and an analogue (added as internal standard) from plasma and brain of rat. Adosupine was well separated from its three metabolites with 0.01 M disodium hydrogenphosphate-acetonitrile-methanol-nonylamine (59.986:38:2:0.014) at pH 4.5 as mobile phase using a C18 reversed-phase column. The standard curves were linear in the range 50-5000 ng/ml (or ng/g) for adosupine and metabolites in both plasma and brain. The between- and within-assay variations for high and low concentrations of the parent compound and the three metabolites were 8.2-14%. In the range 50-5000 ng/ml (or ng/g) the accuracy of the method was satisfactory, with the relative error always lower than 10%. Analytical recoveries of added adosupine and the three metabolites were higher than 82%. The method has been applied successfully, to investigate the pharmacokinetics of the drug and its distribution in the central nervous system of rats.

Pharmacokinetic investigation of ST 789 in rats and mice.

Pharmacokinetics of ST 789 were investigated in rats and mice after oral, intravenous, subcutaneous and intramuscular routes. A HPLC method validated for pharmacokinetic studies allowed the Authors to assay ST 789 concentration in plasma, urine and tissues. ST 789 interacted poorly with albumin and plasma proteins. Blood-to-plasma concentration ratio proved to range on average from 1.3 to 2.0 in both in vivo and in vitro studies. Plasma concentration-time behaviour after i.v. injection fitted according to the open three-compartment model; after subcutaneous and intramuscular routes two phases were observed and after oral route the absorption and one elimination phases were detected. Pharmacokinetics of ST 789 proved to vary linearly with the dose administered. Cumulative urinary excretion after parenteral administration ranged on average 60-80% and cumulative biliary excretion was 9.47% of the dose given. Oral administration allowed only 2.5% of the drug given to be excreted in urine, this leading to conclude that this drug is poorly absorbed through the intestine wall. After oral administration ST 789 produced relatively high concentration in lungs and lymphatic tissues, this leading to hypothesize a lymphatic component in its enteral absorption.

In vivo phytochrome-mediated perception of reflected light signals.

The spectrophotometric assay of phytochrome in vivo in etiolated plant material was used to determine the effects of changes in reflected light on the state of the photoreceptor in etiolated seedlings exposed simultaneously to direct and reflected light. Changes in reflected light that were small in terms of the total (direct + reflected) radiation incident on the seedlings produced detectable changes in the state of phytochrome in vivo. The contribution of reflected light to the state of phytochrome in vertical organs was greater than expected from its low contribution to total incident light. These data from laboratory studies complement and are consistent with results of field studies on the effects of light reflected from neighboring vegetation on plant growth under natural radiation conditions.

High-performance liquid chromatographic evaluation of Med 15 and its metabolites Med 5 and tolmetin in rat plasma.

A simple and reliable high-performance liquid chromatographic method is described for the quantitative analysis of the new non-steroidal anti-inflammatory agent Med 15 and its metabolites Med 5 and tolmetin in rat plasma. After selective extraction the three analytes and an internal standard (p-phenyl-phenol) were separated on a reversed-phase Ultrasphere 5 micron column using potassium dihydrogenphosphate (0.05 M)-acetonitrile (52:48) (pH 4.7) as the mobile phase. The analytes were detected at 313 nm; the sensitivity of the method proved to be 0.05 microgram/ml for all three compounds. The method has been applied to investigate Med 15 pharmacokinetics in rats.

Cryptochrome, phytochrome, and anthocyanin production.

Anthocyanin production in cabbage (Brassica oleracea L.) and tomato (Lycopersicon esculentum Mill.) seedlings exposed to prolonged irradiations was studied under conditions that allowed discrimination, within certain limits, between the contribution of cryptochrome and phytochrome in the photoregulation of the response. The results of the study provide confirming evidence for the involvement of cryptochrome and direct evidence for a significant contribution of cryptochrome to the fluence rate dependence of the response to blue. The results provide some preliminary, direct indication for an interaction between cryptochrome and phytochrome in the photoregulation of anthocyanin production in seedlings exposed to the prolonged irradiations required for a high level of expression of the response. The type and degree of interaction between the two photoreceptors vary significantly, depending on the species and experimental conditions.

Potential antidepressant activity and enhancement of serotonin uptake of a new dibenzothiadiazepine derivative.

A molecule, 6-methyl-6,11-dihydro-11-[(N,N-dimethylamino) acetyl]dibenzo[c,fl-[1,2,5]thiadiazepine 5,5-dioxide, (IM/P/3/4, CAS 128377-70-8), was identified in a screening program, which had the scope of finding compounds with antidepressive potential without the common sideeffects of existing antidepressive medication. IM/P/3/4 was found active a) in antagonizing apomorphine (16 mg/kg) and reserpine-induced hypothermia in mice; b) in potentiating yohimbine-induced lethality in mice; c) in reducing immobility of rats forced to swim and of mice suspended by the tail. IM/P/3/4 does not affect a) apomorphine-induced stereotypy; b) amphetamine-induced hypermotility; c) haloperidol-induced catalepsy and water-induced grooming and d) does not induce stereotypy or alter motor activity. The compound also a) reduced the beating of rat right heart atria only at a concentration of 3 x 10-4 mol/l; b) had weak anticholinergic activity; c) antagonized electroshock-induced convulsions and d) prevented indometacin-induced duodenal ulcers. IM/P/3/4 does not have good affinity for noradrenergic, serotonergic, dopaminergic, histaminergic or muscarinic receptors and does not displace imipramine, desipramine and mianserine from their binding sites. IM/P/3/4 increases 5-hydroxyindolacetic acid content and 3H-serotonin uptake in the hypothalamus. The present results suggest that IM/P/3/4 is a potential antidepressant with reduced side effects and with a mechanism of action which is different from that of other antidepressants.

Effect of enantiomers of propranolol on desipramine-induced anti-immobility in the forced swimming test in the rat.

The effect of d- and l-enantiomers of propranolol on desipramine-induced anti-immobility effects and on brain desipramine levels was studied in the rat. Intraperitoneal propranolol and desipramine were administered three times, 25, 6, 2 and 24, 5, 1 h respectively, before the test. It was found that l-propranolol but not d-propranolol, at the same doses (2.5 and 5 mg/kg), antagonized 20 mg/kg desipramine without altering desipramine brain levels. It is suggested that blockade of beta-adrenergic receptors rather than membrane-stabilizing or pharmacokinetic effects is responsible for the antagonism of propranolol toward desipramine.

Phytochrome-mediated detection of changes in reflected light.

Measurements of phytochrome photoequilibria and photoconversion rates in vivo, in seedlings of Cucurbita pepo L. exposed to light in growth chambers, indicate that significant changes in the state of phytochrome can be brought about by changes in the quality and quantity of the light reflected from the walls of the growth chambers. The changes in reflected light, although large, were small in terms of the total radiation (direct light from the lamps plus wall-reflected light) to which the seedlings were exposed. The conditions used were approximate simulations of direct and reflected sunlight conditions in the natural environment. Keeping in mind the limitations imposed by the approximation of the simulations, the results from this study are consistent with the hypothesis that, in the natural environment, a plant might be capable of detecting the presence of nearby plants, before being shaded by them, through the phytochrome-mediated perception of changes in reflected light.