Development of pectin microparticles by using ionotropic gelation with chlorhexidine as cross-linking agent.

Having previously highlighted the gelation of pectin with chlorhexidine (CX), pectinate microparticles were prepared here by vibrational prilling using CX, not only as an active ingredient encapsulated but also as a cross-linking agent. CX amount required for pectin gelation was smaller than usual dications (Ca2+, Zn2+) used as cross-linking agent for pectin ionotropic gelation: CX seemed to bind more easily to pectin chains that could be explained by its large molecular size. Three batches of CX microparticles with different mean size were prepared. Whatever the droplet mean diameter, similar particle characteristics in terms of encapsulation efficiency, CX encapsulation yield and drug release were observed. The encapsulation efficiency was about 5.5%, the CX encapsulation yield was approximately 44% and the maximal amount of CX released after 6 h was about 7%. Finally, zinc diacetate was added to the formulation as a competitive pectin cross-linking agent in order to limit CX binding to pectin and to improve CX release. The influence of CX and Zn2+ concentrations on the particles properties was studied by the means of a Doehlert design. Results showed the interest of such a mixture since the competition between both cations led to more or less structured and large microparticles, some of them having promoted the quantity of CX released.

Acute aquatic toxicity of organic solvents modeled by QSARs.

To limit in vivo experiments, the use of quantitative structure-activity relationships (QSARs) is advocated by REACH regulation to predict the required fish, invertebrate, and algae EC50 for chemical registration. The aim of this work was to develop reliable QSARs in order to model both invertebrate and algae EC50 for organic solvents, regardless of the mechanism of toxic action involved. EC50 represents the concentration producing the 50 % immobilization of invertebrates or the 50 % growth inhibition of algae. The dataset was composed of 122 organic solvents chemically heterogeneous which were characterized by their invertebrate and/or algae EC50. These solvents were described by physico-chemical descriptors and quantum theoretical parameters calculated via density functional theory. QSAR models were developed by multiple linear regression using the ordinary least squares method and descriptor selection was performed by the Kubinyi function. Invertebrate EC50 was well-described with LogP, dielectric constant, surface tension, and minimal atomic Mulliken charges while algae EC50 of organic solvents (except amines) was predicted with LogP and LUMO energy. To evaluate robustness and predictive performance of the QSARs developed, several strategies have been used to select solvent training sets (random, EC50-based selection and a space-filling design) and both internal and external validations were performed.

Bone-muscle unit activity, salivary steroid hormones profile, and physical effort over a 3-week stage race.

Muscle traction and bone metabolism are functionally linked and co-regulated by a series of factors. Although a role for steroid hormones was hypothesized, a clear definition of the bone-muscle interconnection still lacks. To investigate this relationship, we studied bone metabolism, muscle activity, and salivary steroid hormones profile in relation with the physical effort across a cycling stage race, a model of effort in absence of load. Nine pro-cyclists were recruited; body weight and power output/energy expenditure were recorded. Diet was kept constant. Saliva was collected at days -1, 4, 8, 12, 14, 19, and 23; blood and urine were collected at days -1, 12, and 23. Salivary steroid hormones [cortisol, dehydroepiandrosterone (DHEA), testosterone, and estradiol], serum lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and creatine kinase (CK) activities, plasma sclerostin, and urinary calcium and phosphorous were measured. Cortisol remained constant, testosterone decreased at day 4, and estradiol and DHEA firstly increased and then returned to basal levels. Hormone concentrations were not correlated with plasma volume shifts. LDH, CK, AST, sclerostin, and urinary calcium and phosphorous increased. DHEA and estradiol correlated with the physical effort and the bone-muscular markers. A relationship between muscle activity, in absence of load, and bone resorption emerged under a putative regulation by DHEA and estradiol.

Modifications of biochemical parameters related to protein metabolism and renal function in male soccer players after a match.

AIM: The impact of a soccer match on parameters related to protein catabolism and renal function was evaluated in male players. METHODS: Blood was collected before and immediately after a 90 minutes soccer match from 19 athletes of two first division teams in Brazil. Red blood cells (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), ammonia, uric acid, urea and creatinine were analyzed. The modification of plasma volume was calculated, and biochemical values were corrected for this change. Urea/creatinine ratio and equations to estimate the glomerular filtration rate (eGFR) were used to assess kidney function. RESULTS: Plasma volume decreased from pre- to post-match. Post-match values higher than the pre-match ones were observed for RBC, Hb and Ht, as a consequence of plasma volume decrease. An increase in ammonia and creatinine concentrations post-match in comparison with pre-match values was registered, without changes in uric acid and urea levels. A reduction in urea/creatinine ratio and in eGFR was observed post-match, suggesting a decrease of renal function. CONCLUSION: A soccer match induced alterations in parameters linked to renal function and protein metabolism in male athletes. Particular attention should be paid in the monitoring of the ammonia concentration as an indicator of metabolic activity and energy requirement during prolonged exercise.

Salivary steroid hormone response to whole-body cryotherapy in elite rugby players.

Saliva represents a low stress, not-invasively collected matrix that allows steroid hormone monitoring in athletes by reflecting type, intensity and duration of exercise. Whole body cryotherapy (WBC) consists of short whole-body exposures to extremely cold air (-110° to -140°C) which, despite being initially used to treat inflammatory diseases, is currently acquiring increasing popularity in sports medicine. Cryostimulation practice is now widely accepted as an effective treatment to accelerate muscle recovery in rugby players. The aim of this work was to study the changes of steroid hormones in saliva of rugby players after both 2 and 14 consecutive WBC sessions, in order to investigate the effects of the treatment on their salivary steroid hormonal profile. Twenty-five professional rugby players, belonging to the Italian National Team, underwent a 7-day cryotherapy protocol consisting of 2 daily sessions. Saliva samples were taken in the morning prior to the start of the WBC, in the evening after the end of the second WBC, and in the morning of the day after the last WBC session. The samples were analyzed for cortisol, DHEA, testosterone and estradiol using competitive enzyme-linked immunosorbent assays. Cortisol and DHEA showed a reduction already after the 2 WBC sessions of the first day; after 14 consecutive WBC sessions cortisol, DHEA, and estradiol levels decreased, while testosterone increased as did the testosterone to cortisol ratio. These results were confirmed by the fact that the majority of subjects showed variations exceeding the critical difference (CD). In conclusion, we found that WBC acutely affects the salivary steroid hormone profile, and the results are evident already after only one twice-daily session. Most significantly, after one-week of consecutive twice-daily WBC sessions, all the hormones were modified. This is the first experimental report that links changes in the hormonal asset to WBC.

Reciprocal regulation of calcium-/phosphate-regulating hormones in cyclists during the Giro d'Italia 3-week stage race.

Calcium and phosphate are essential for cell functions, and their serum concentrations result from the balance between intestinal absorption, bony storage, and urinary excretion. Fibroblast growth factor 23 (FGF23), expressed by osteocytes and osteoblasts, acts in the kidney, leading to hypophosphatemia and low 1,25-dihydroxycholecalciferol synthesis, but suppresses parathyroid function. The aim of this study was to explore the effects of a high-energy demanding cycling race on this bone-kidney-parathyroid axis. We studied nine cyclists during the 2011 Giro d'Italia stage race. Pre-analytical and analytical phases followed academic and anti-doping recommendations. Serum parathyroid hormone (PTH), 25(OH)D, total calcium, inorganic phosphorus, and plasma FGF23 were measured on days -1, 12, and 22 and corrected for changes in plasma volume. Dietary calcium and phosphorus, anthropometric parameters (height, weight, and body mass index) and indexes of metabolic effort (net energy expenditure, power output) were recorded. Dietary calcium and phosphorus intakes were kept at the same levels throughout the race. Twenty-five (OH)D, PTH, and calcium concentrations remained stable. FGF23 increased 50% with a positive correlation with the indexes of metabolic effort and, consequently, phosphorous decreased, although only in the first half. The strong metabolic effort acts on the bone-kidney-parathyroid system, and the rise in FGF23 plasma concentration might be aimed at maintaining calcium and phosphorus homeostasis.

Quantitative structure-activity relationship to predict acute fish toxicity of organic solvents.

REACH regulation requires ecotoxicological data to characterize industrial chemicals. To limit in vivo testing, Quantitative Structure-Activity Relationships (QSARs) are advocated to predict toxicity of a molecule. In this context, the topic of this work was to develop a reliable QSAR explaining the experimental acute toxicity of organic solvents for fish trophic level. Toxicity was expressed as log(LC50), the concentration in mmol.L(-1) producing the 50% death of fish. The 141 chemically heterogeneous solvents of the dataset were described by physico-chemical descriptors and quantum theoretical parameters calculated via Density Functional Theory. The best subsets of solvent descriptors for LC50 prediction were chosen both through the Kubinyi function associated with Enhanced Replacement Method and a stepwise forward multiple linear regressions. The 4-parameters selected in the model were the octanol-water partition coefficient, LUMO energy, dielectric constant and surface tension. The predictive power and robustness of the QSAR developed were assessed by internal and external validations. Several techniques for training sets selection were evaluated: a random selection, a LC50-based selection, a balanced selection in terms of toxic and non-toxic solvents, a solvent profile-based selection with a space filling technique and a D-optimality onions-based selection. A comparison with fish LC50 predicted by ECOSAR model validated for neutral organics confirmed the interest of the QSAR developed for the prediction of organic solvent aquatic toxicity regardless of the mechanism of toxic action involved.

Sclerostin concentrations in athletes: role of load and gender.

Bone mass is the net product of formation and resorption, which are closely regulated by the equilibrium between endogenous/exogenous factors. Sclerostin inhibits the Wnt canonical signaling and is considered an anti-anabolic factor. We compared sclerostin serum concentrations between genders in athletes belonging to different sport disciplines, characterized by a different weight-bearing, and in their sedentary counterparts in order to study the possible link between bone metabolism in athletes and its peripheral concentration. We also compared sclerostin levels with bone alkaline phosphatase activity, a marker of bone formation. Sixty-one elite athletes, belonging to weight-bearing (15 male rugby players, 11 male enduro racers, 8 female basketball players), high-impact (6 male tennis players, 8 female ice skaters), non weight-bearing sports (13 male cyclists) and 16 sedentary controls were enrolled. Higher levels of sclerostin were found in females. Sclerostin was higher in weight-bearing than in non-weight-bearing disciplines in males. Significant inverse age-related correlation was found. Higher bone alkaline phosphatase activity was observed in females. The young adult elite athlete represents a peculiar physiologic model for studying sclerostin behavior: the applied load increased the marker concentrations, testifying a high bone turnover rate; however, a gender effect is evident.

Mucoadhesion evaluation of polysaccharide gels for vaginal application by using rheological and indentation measurements.

The influence of hyaluronic acid (HA) and fructooligosaccharides (FOS) addition on low methyl pectin (LMP) gelation has been investigated in order to produce adhesive gel-based microparticles suitable for the development of a vaginal delivery system of pro- and prebiotics. First, dynamic rheological measurements were performed on LMP/Ca(2+) gels with or without FOS and HA in presence or not of porcine stomach mucins. This rheological method is known to translate the interactions between polymer and mucins and then simulate the polymer bioadhesion potential. Nevertheless, as this method is disputed, in vitro and ex vivo indentation test measurements were also achieved in order to correlate the results obtained. Despite some different results, the overall tendency indicates that addition of HA and FOS enhanced the mucoadhesive properties of LMP gels. Moreover, gel-based microparticles obtained according to an emulsification/gelation method and composed by LMP 3% (w/v), FOS 5% (w/v) and HA 0.5% (w/v) displayed a mucoadhesive potential adapted to vaginal delivery system.

Improvement of an encapsulation process for the preparation of pro- and prebiotics-loaded bioadhesive microparticles by using experimental design.

The purpose of this study was to design a new vaginal bioadhesive delivery system based on pectinate-hyaluronic acid microparticles for probiotics and prebiotics encapsulation. Probiotic strains and prebiotic were selected for their abilities to restore vaginal ecosystem. Microparticles were produced by emulsification/gelation method using calcium as cross-linking agent. In the first step, preliminary experiments were conducted to study the influence of the main formulation and process parameters on the size distribution of unloaded microparticles. Rheological measurements were also performed to investigate the bioadhesive properties of the gels used to obtain the final microparticles. Afterwards an experimental design was performed to determine the operating conditions suitable to obtain bioadhesive microparticles containing probiotics and prebiotics. Experimental design allowed us to define two important parameters during the microencapsulation process: the stirring rate during the emulsification step and the pectin concentration. The final microparticles had a mean diameter of 137µm and allowed a complete release of probiotic strains after 16h in a simulated vaginal fluid at +37°C.

Determination of poly(epsilon-caprolactone) solubility parameters: application to solvent substitution in a microencapsulation process.

The evolution of regulation on chemical substances (i.e. REACH regulation) calls for the progressive substitution of toxic chemicals in formulations when suitable alternatives have been identified. In this context, the method of Hansen solubility parameters was applied to identify an alternative solvent less toxic than methylene chloride used in a microencapsulation process. During the process based on a multiple emulsion (W/O/W) with solvent evaporation/extraction method, the solvent has to dissolve a polymer, poly(epsilon-caprolactone) (PCL), which forms a polymeric matrix encapsulating or entrapping a therapeutic protein as the solvent is extracted. Therefore the three partial solubility parameters of PCL have been determined by a group contribution method, swelling experiments and turbidimetric titration. The results obtained allowed us to find a solvent, anisole, able to solubilize PCL and to form a multiple emulsion with aqueous solutions. A feasibility test was conducted under standard operating conditions and allowed the production of PCL microspheres.

New separation method for organic and inorganic selenium compounds based on anion exchange chromatography followed by inductively coupled plasma mass spectrometry.

We describe a new method for separating the organic and inorganic selenocompounds methaneseleninic acid, selenite, selenate, methylselenocysteine, selenocystine as well as both selenomethionine and its oxidized form. The separation is performed on a Hamilton PRP-X100 column. According to the literature, the oxidized form of selenomethionine-which is easily formed-is eluted close to the dead volume when this column is used. The choice of parahydroxybenzoic acid as mobile phase enabled us to elute all of these species after this oxidized form, resulting in better identification and quantification. The factors determining separation (eluent concentration, pH, gradient) were optimized via an experimental design. Application of the method to yeast and commercial tablets showed that the principal Se compound present was selenomethionine, which was also present in its oxidized form.

Optimization of the coupling of high-temperature liquid chromatography and flame ionization detection application to the separations of alcohols.

The feasibility of coupling high-temperature liquid chromatography (HTLC) to flame ionization detection (FID) has been studied. FID parameter values (hydrogen flow-rate, air flow-rate and FID temperature), typically set in gas chromatography are rarely suitable for liquid chromatography. Best values depend obviously on the water flow rate which is defined depending on both column temperature and column internal diameter. The FID parameters were optimized according to the water flow-rate by means of an experimental design. The potential of the method is shown with some alcohol separations and the value of increasing column temperature while reducing the column diameter is highlighted.

Motor evoked potential monitoring for spinal cord and brain stem surgery.

Intraoperative Neurophysiology (ION) has established itself as one of the means by which modern neurosurgery can improve surgical results while minimizing morbidity. The advent of motor evoked potential (MEP) monitoring represents a landmark in this recent progress. ION consists of monitoring (the continuous "on-line" assessment of the functional integrity of neural pathways) and mapping (the functional identification and preservation of anatomically ambiguous nervous tissue) techniques. In this chapter we have attempted to critically review the evolution of MEP use during monitoring and mapping techniques for neurosurgical procedures in the brainstem and the spinal cord, providing the neurophysiological theoretical background and practical aspects of clinical applications. According to the experience from our and other groups involved in ION, we suggest the following: 1) ION is mandatory whenever neurological complications are expected as predicted by a known pathophysiological mechanism. It is therefore advisable to perform ION when dealing with brain stem and intramedullary spinal cord lesions. 2) MEP monitoring after transcranial electrical stimulation is today a feasible and reliable technique for use under general anesthesia. MEP monitoring is the most appropriate technique to assess the functional integrity of descending motor pathways in the brainstem and, foremost, in the spinal cord. 3) Mapping of the corticospinal tract at the level of the cerebral peduncle as well as mapping of the VII, IX-X and XII cranial nerve motor nuclei on the floor of the fourth ventricle is of great value with which to identify "safe entry zones" into the brainstem. 4) Other techniques, although safe and feasible, still lack rigorous validation in terms of prognostic value and correlation with the postoperative neurological outcome. These techniques include mapping of the corticospinal tract within the spinal cord and monitoring of the corticobulbar tracts. These techniques, however, are expected to open new perspectives in the near future.

Brain surgery in motor areas: the invaluable assistance of intraoperative neurophysiological monitoring.

AIM: Surgery for tumors in the central and precentral region, as much as for insular tumors, places at risk the functional integrity of the motor cortex and the subcortical motor pathways. These procedures may therefore benefit from the assistance of intraoperative neurophysiological monitoring (INM). INM consists of "mapping" and true "monitoring" (the continuous "on-line" assessment of the functional integrity of neural pathways) techniques. In spite of the large interest in mapping techniques, monitoring techniques have received less attention. We describe our experience with intraoperative neurophysiological mapping and monitoring of motor tracts during surgery for brain gliomas in or near motor areas, in order to support the feasibility and reliability of monitoring as an essential adjunct to mapping during surgery in these areas. METHODS: Between September 2000 and January 2002, 51 patients were surgically treated for brain gliomas located in the precentral gyrus (45.1%), the postcentral gyrus (23.5%), anterior to the precentral gyrus (15.6%), or in the insula (15.6%). INM of the motor system consisted of monitoring muscle motor evoked potentials (mMEPs) recorded via needle electrodes inserted into the controlateral upper and lower extremity muscles and elicited by transcranial multipulse electrical stimulation (TES). Once the dura was open and the central sulcus was identified using the phase reversal technique, mMEPs were elicited by direct stimulation of the motor cortex (DCS). Motor mapping was performed with a monopolar electrode using the same stimulation parameters as used for monitoring except for much lower intensity (up to 20 mA). RESULTS: Ninety-eight percent of the patients exhibited recordable baseline mMEPs. The success rate of the phase reversal technique was 95.8%. Eight patients presented disappearance of mMEPs during tumor removal. Using corrective measures, all intraoperative changes in mMEPs were reversed in time to prevent an irreversible complete injury to the motor system and no patient lost mMEPs at the end of the operation. At discharge, 66% of the patients remained at their preoperative status, 4% improved, and 24% had a mild worsening as compared to the preoperative status assessed using the Medical Research Council scale; 6% of the patients presented a moderate to severe supplementary motor area syndrome. CONCLUSION: Monitoring techniques significantly implement the reliability and effectiveness of INM since these provide: 1) continuous "on-line" assessment of the functional integrity of motor pathways with higher chance to early detect a progressive mechanical or vascular injury to the neural tissue, as compared to mapping techniques; 2) lower risk to induce intraoperative seizures and strong muscular twitches as compared to the single pulse mapping technique; 3) possibility to monitor motor pathways using TES also when there is no direct access to the motor cortex.

Hydrophobic mechanochemical treatment of metallic surfaces wettability measurements as a means of assessing homogeneity.

Hydrophobicity, lubrication and anticorrosion properties of steel substrates have been obtained by a deposition of thin film (i.e. by mechanochemical treatment) at room conditions. Stearic acid and paraffin were chosen as reactive molecules. Different abrasive powders were selected to generate active sites on the treated surfaces for adsorption of the reactive molecules and then, the results were compared. The surfaces were analyzed by reflection-absorption infrared spectroscopy (RAIRS). The results emphasize that, a thick layer of mixed stearic acid/paraffin was deposited onto the metallic surface after the treatment. After hexane rinsing we could only detect a very thin layer of oriented stearic acid molecules chemically adsorbed onto the metallic surface and which engages strong interactions with it. Whereas, RAIRS only provides molecular analysis, the XPS technique was complementary for discriminating the different surfaces. It was possible to show differences in thickness as well as in coverage according to the size and shape of abrasive particles. Furthermore, we could conclude that deposit layer is not uniform. Defects were always present and were dependent on abrasive powders used. Then wettability was assessed as a way to test the homogeneity of thin films generated by the mechanochemical treatment. In agreement with theoretical data, receding contact angle was very dependent on the defects in the deposited film. If holes or aggregates were increased in the deposit layer, the receding contact angle was decreased while advancing contact angles and equilibrium contact angles remained constant. A very important point for technological applications was that the homogeneity of the deposited film was governed by abrasive powder involved in mechanochemical treatment and contact angle values were a direct measurement of the homogeneity of surfaces generated by mechanochemical treatment.

Experimental designs to investigate capillary electrophoresis-electrospray ionization-mass spectrometry enantioseparation with the partial-filling technique.

An experimental design approach is described to evaluate the main electrophoretic parameters involved in the enantioseparation of pharmaceuticals by capillary electrophoresis (CE) coupled to electrospray ionization-mass spectrometry (ESI-MS). For all experiments, the partial-filling technique was applied to avoid the chiral selector entering in the mass spectrometer ion source with a negative effect on the electrospray performance. To carry out enantioseparation, a volatile buffer constituted of 20 mM ammonium acetate at pH 4.0, and a polyvinyl alcohol-coated capillary were used. Methadone was employed as the model compound and three different cyclodextrins (CDs), namely sulfobutyl ether-beta-CD, carboxymethylated-beta-CD and hydroxypropyl-beta-CD, were selected in order to study the countercurrent process. Two different experimental designs were chosen: (i) a full-factorial design to examine the effects and significance of the investigated factors, and (ii) a central composite face-centered design to establish the mathematical model of the selected responses in function of experimental factors. The chiral selector concentration, percentage of the capillary filled with the chiral selector, and drying gas nebulization pressure were three relevant factors taken into consideration. For each CD, the methadone enantiomeric resolution, apparent selectivity, and migration time of the second enantiomer were established as responses. The latter were systematically related to experimental parameters with the help of multiple linear regression. It is noteworthy that the behaviour was different in function of the chiral selector charge. Results revealed that the nebulization pressure involved in the electrospray process and the CD concentration had a significant effect on the enantiomeric resolution, while the effect of the separation zone length was less pronounced. Finally, response surfaces were drawn from the mathematical model and experimental conditions were selected to allow a robust determination of methadone enantiomers by CE-MS.

Electrophysiologic monitoring in neurointensive care.

Cumulative evidence of potential benefits of electroencephalography (EEG) and evoked potentials in the management of patients with acute cerebral damage has been confirmed. Continuous EEG monitoring is the best method for detecting nonconvulsive seizures and is strongly recommended for the treatment of status epilepticus. Continuously displayed, validated quantitative EEG may facilitate early detection of secondary cerebral insults and may play a decision-making role in the management of patients with head injury, stroke, or subarachnoid hemorrhage. Long-latency auditory evoked potentials and cognitive components constitute a new field of interest for the progress of comatose patients. Motor evoked potentials may become clinically important both in acutely injured and elective postoperative patients. In the neurointensive care units adequate techniques can be selected to answer targeted clinical questions. The efficacy can be improved by implementing educational projects based on ad hoc training of nurses and neurointensive care specialists.

Experimental design in supercritical fluid extraction of cocaine from coca leaves.

An optimisation procedure for the supercritical fluid extraction (SFE) of cocaine from the leaves of Erythroxylum coca var. coca was investigated by means of experimental design. After preliminary experiments where the SFE rate-controlling mechanism was determined, a central composite design was applied to evaluate interactions between selected SFE factors such as pressure, temperature, nature and percentage of the polar modifier, as well as to optimise these factors. Predicted and experimental contents of cocaine were compared and robustness of the extraction method estimated by drawing response surfaces. The analysis of cocaine in crude extracts was carried out by capillary GC equipped with a flame ionisation detector (GC-FID), as well as by capillary GC coupled with a mass spectrometer (GC-MS) for peak identification.

Acid-Base Approach to Latex Particles Containing Specific Groups Based on Wettability Measurements.

Polar properties of latex particles containing specific groups with various surface charge densities were examined from contact angle measurements using "old" polar/dispersive models. It was determined that the Fowkes approach is quite sufficient for classifying our functionalized polystyrene latices. The pertinence to our systems of the acid-base approach, which has a more pronounced chemical background, was then investigated, and the strong dependence of the acid-base parameters (gamma plus sign in circle, gammaTheta) on the acid-base scale chosen for the reference liquid (water) was shown. Finally, using the receding angle values revealed the outermost surface buried moieties in the latex particles, which were not seen in the advancing angle measurements. Copyright 1998 Academic Press.