Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or = 99% purity, a yield of 25.0 ± 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60°C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37°C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.

Plasma and testicular testosterone levels, volume density and number of Leydig cells and spermatogenic efficiency of rabbits

Plasma and tissue testosterone concentrations were determined by radioimmunoassay in 12 eight-month-old sexually mature New Zealand White rabbits and evaluated for possible associations with spermatogenic efficiency as well as with volume density and number of Leydig cells. Testicular tissue was processed histologically and histometry was performed in order to quantify germ cells, Sertoli cells and Leydig cells. Spermatogenic efficiency, reported as the ratios among germ cells (spermatogonia, primary spermatocytes and round spermatids) and by the ratio of germ cells to Sertoli cells, was not associated with testosterone levels. However, Leydig cell parameters such as number of Leydig cells per gram of testis, total number of Leydig cells per testis and percent cell volume of Leydig cell nuclei were correlated significantly with testosterone levels. The statistically significant correlation (r = 0.82, P<0.05) observed between testosterone levels and the number of Leydig cells per gram of testis suggests that, in the rabbit, the latter parameter can serve as a criterion for monitoring testosterone levels in this species under normal conditions.

Effect of perimuscular injection of Bothrops jararacussu venom on plasma creatine kinase levels in mice: influence of dose and volume

The effect of dose and volume of a perimuscular injection of Bothrops jararacussu venom on myonecrosis of skeletal muscle was studied in mice. An increase of the venom dose (0.25 to 2.0 µg/g) at a given volume (50 µl) resulted in an increase in plasma creatine kinase (CK) levels 2 h after injection. Plasma CK activity increased from the basal level of 129.27 ± 11.83 (N = 20) to 2392.80 ± 709.43 IU/l (N = 4) for the 1.0 µg/g dose. Histological analysis of extensor digitorum longus muscle 4 h after injection showed lesion of peripheral muscle fibers, disorganization of the bundles or the complete degeneration of muscle fibers. These lesions were more extensive when higher doses were injected. Furthermore, an increase in volume (12.5 to 100 µl) by dilution of a given dose (0.5 µg/g) also increased plasma CK levels from 482.31 ± 122.79 to 919.07 ± 133.33 IU/l (N = 4), respectively. These results indicate that care should be taken to standardize volumes and sites of venom injections.

The role of complement in the modulation by fluid-phase IgG of the production of reactive oxygen species by polymorphonuclear leukocytes stimulated with IgG immune complexes

The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMN) can be induced by immune complexes and is an important component of phagocytosis in the killing of microorganisms, but can also be involved in inflammatory reactions when immune complexes are deposited in tissues. We have observed that fluid-phase IgG can inhibit the generation of ROS by rabbit PMN stimulated with precipitated immune complexes of IgG (ICIgG) in a dose-dependent manner, acting as a modulatory factor in the range of physiological IgG concentrations. This inhibitory effect is compatible with the known affinity (Kd) of monomeric IgG for the receptors involved (FcRII and FcRIII). The presence of complement components in the immune complexes results in a higher stimulation of ROS production. In this case, however, there is no inhibition by fluid-phase IgG. The effect of complement is strongly dependent on the presence of divalent cations (Ca2+ or Mg2+) in the medium, whereas the stimulation of ICIgG (without complement) does not depend on these cations. We have obtained some evidence indicating that iC3b should be the component involved in the effect of complement through interaction with the CR3 receptor. The absence of the inhibitory effect of fluid-phase IgG in ROS production when complement is present in the immune complex shows that complement may be important in vivo not only in the production of chemotactic factors for PMN, but also in the next phase of the process, i.e., the generation of ROS.

Comparison of the effects of lactated Ringer solution with and without hydroxyethyl starch fluid resuscitation on gut edema during severe splanchnic ischemia

The type of fluid used during resuscitation may have an important impact on tissue edema. We evaluated the impact of two different regimens of fluid resuscitation on hemodynamics and on lung and intestinal edema during splanchnic hypoperfusion in rabbits. The study included 16 female New Zealand rabbits (2.9 to 3.3 kg body weight, aged 8 to 12 months) with splanchnic ischemia induced by ligation of the superior mesenteric artery. The animals were randomized into two experimental groups: group I (N = 9) received 12 mL·kg-1·h-1 lactated Ringer solution and 20 mL/kg 6% hydroxyethyl starch solution; group II (N = 7) received 36 mL·kg-1·h-1 lactated Ringer solution and 20 mL/kg 0.9% saline. A segment from the ileum was isolated to be perfused. A tonometric catheter was placed in a second gut segment. Superior mesenteric artery (Q SMA) and aortic (Qaorta) flows were measured using ultrasonic flow probes. After 4 h of fluid resuscitation, tissue specimens were immediately removed for estimations of gut and lung edema. There were no differences in global and regional perfusion variables, lung wet-to-dry weight ratios and oxygenation indices between groups. Gut wet-to-dry weight ratio was significantly lower in the crystalloid/colloid-treated group (4.9 ± 1.5) than in the crystalloid-treated group (7.3 ± 2.4) (P < 0.05). In this model of intestinal ischemia, fluid resuscitation with crystalloids caused more gut edema than a combination of crystalloids and colloids.

The effect of epidural and general anesthesia on newborn rectal temperature at elective cesarean section

Both epidural and general anesthesia can impair thermoregulatory mechanisms during surgery. However, there is lack of information about the effects of different methods of anesthesia on newborn temperature. The purpose of this study was to determine whether there are differences in newborn rectal temperature related to type of anesthesia. Sixty-three pregnant women were randomly assigned to receive general or epidural anesthesia. Maternal core temperature was measured three times with a rectal probe just before anesthesia, at the beginning of surgery and at delivery. In addition, umbilical vein blood was sampled for pH. The rectal temperatures of the babies were recorded immediately after delivery, and Apgar scores were determined 1, 5, and 10 min after birth. The duration of anesthesia and the volume of intravenous fluid given during the procedure (833 ± 144 vs 420 ± 215 mL) were significantly higher in the epidural group than in the general anesthesia group (P < 0.0001). Maternal rectal temperatures were not different in both groups at all measurements. In contrast, newborn rectal temperatures were lower in the epidural anesthesia group than in the general anesthesia group (37.4 ± 0.3 vs 37.6 ± 0.3°C; P < 0.05) immediately after birth. Furthermore, the umbilical vein pH value (7.31 ± 0.05 vs 7.33 ± 0.01; P < 0.05) and Apgar scores at the 1st-min measurement (8.0 ± 0.9 vs 8.5 ± 0.7; P < 0.05) were lower in the epidural anesthesia group than in the general anesthesia group. Since epidural anesthesia requires more iv fluid infusion and a longer time for cesarean section, it involves a risk of a mild temperature reduction for the baby which, however, did not reach the limits of hypothermia.

Brain tissue segmentation using q-entropy in multiple sclerosis magnetic resonance images

The loss of brain volume has been used as a marker of tissue destruction and can be used as an index of the progression of neurodegenerative diseases, such as multiple sclerosis. In the present study, we tested a new method for tissue segmentation based on pixel intensity threshold using generalized Tsallis entropy to determine a statistical segmentation parameter for each single class of brain tissue. We compared the performance of this method using a range of different q parameters and found a different optimal q parameter for white matter, gray matter, and cerebrospinal fluid. Our results support the conclusion that the differences in structural correlations and scale invariant similarities present in each tissue class can be accessed by generalized Tsallis entropy, obtaining the intensity limits for these tissue class separations. In order to test this method, we used it for analysis of brain magnetic resonance images of 43 patients and 10 healthy controls matched for gender and age. The values found for the entropic q index were 0.2 for cerebrospinal fluid, 0.1 for white matter and 1.5 for gray matter. With this algorithm, we could detect an annual loss of 0.98% for the patients, in agreement with literature data. Thus, we can conclude that the entropy of Tsallis adds advantages to the process of automatic target segmentation of tissue classes, which had not been demonstrated previously.

Increased levels of glutamate in the central nervous system are associated with behavioral symptoms in experimental malaria

Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection. This condition has been associated with cognitive, behavioral and motor dysfunctions, seizures and coma. The underlying mechanisms of CM are incompletely understood. Glutamate and other metabolites such as lactate have been implicated in its pathogenesis. In the present study, we investigated the involvement of glutamate in the behavioral symptoms of CM. Seventeen female C57BL/6 mice (20-25 g) aged 6-8 weeks were infected with P. berghei ANKA by the intraperitoneal route using a standardized inoculation of 10(6) parasitized red blood cells suspended in 0.2 mL PBS. Control animals (N = 17) received the same volume of PBS. Behavioral and neurological symptoms were analyzed by the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. Glutamate release was measured in the cerebral cortex and cerebrospinal fluid of infected and control mice by fluorimetric assay. All functional categories of the SHIRPA battery were significantly altered in the infected mice at 6 days post-infection (dpi) (P ≤ 0.05). In parallel to CM symptoms, we found a significant increase in glutamate levels in the cerebral cortex (mean ± SEM; control: 11.62 ± 0.90 nmol/mg protein; infected at 3 dpi: 10.36 ± 1.17 nmol/mg protein; infected at 6 dpi: 26.65 ± 0.73 nmol/mg protein; with EGTA, control: 5.60 ± 1.92 nmol/mg protein; infected at 3 dpi: 6.24 ± 1.87 nmol/mg protein; infected at 6 dpi: 14.14 ± 0.84 nmol/mg protein) and in the cerebrospinal fluid (control: 128 ± 51.23 pmol/mg protein; infected: 301.4 ± 22.52 pmol/mg protein) of infected mice (P ≤ 0.05). These findings suggest a role of glutamate in the central nervous system dysfunction found in CM.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

Comparison of capillary electrophoresis and high performance liquid chromatography methods for caffeine determination in decaffeinated coffee

Decaffeinated coffee accounts for 10 percent of coffee sales in the world; it is preferred by consumers that do not wish or are sensitive to caffeine effects. This article presents an analytical comparison of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) methods for residual caffeine quantification in decaffeinated coffee in terms of validation parameters, costs, analysis time, composition and treatment of the residues generated, and caffeine quantification in 20 commercial samples. Both methods showed suitable validation parameters. Caffeine content did not differ statistically in the two different methods of analysis. The main advantage of the high performance liquid chromatography (HPLC) method was the 42-fold lower detection limit. Nevertheless, the capillary electrophoresis (CE) detection limit was 115-fold lower than the allowable limit by the Brazilian law. The capillary electrophoresis (CE) analyses were 30% faster, the reagent costs were 76.5-fold, and the volume of the residues generated was 33-fold lower. Therefore, the capillary electrophoresis (CE) method proved to be a valuable analytical tool for this type of analysis.

Characterization of a hydrolyzed oil obtained from fish waste for nutraceutical application

The fish industry generates high volume of waste from fish oil that can have the extraction of its lipids used as nutraceuticals and foods. The objective of this study was to produce unsaturated fatty acids from industrialized fish oil by means of a differentiated hydrolysis process. The samples used were crude fish oil obtained from Campestre industry and characterized through physical-chemical parameters, according to AOCS: acidity, peroxide, saponification, iodine and percentage of free fatty acids and also obtained the fatty acid profile through derivatization method for gas chromatography. The results obtained for the oleochemical indices for refined oil were similar to the data found on the literature. The content of polyunsaturated fatty acids (PUFA) was found of 32,78%, with 9,12% of docosahexaenoic (DHA) and 10,36% of eicosapentaenoic (EPA), regarding monounsaturated fatty acids (MUFA) content was of 30,59% in the hydrolyzed fish oil in relation to refined (20,06%). Thus, it can be concluded that the hydrolysis process used for oils from fish-waste was satisfactory on the production of absolute yield of lipids in the process and significant preservation on the percentages of EPA and DHA, interesting on the production of nutraceuticals and nutrition of aquatic animals, including shrimp in captivity.

Development of a rapid method for the determination of antibiotic residues in honey using UPLC-ESI-MS/MS

Abstract An accurate, reliable and fast multianalyte/multiclass ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the simultaneous analysis of 23 pharmaceuticals, belonging to different classes amphenicols, sulfonamides, tetracyclines, in honey samples. The method developed consists of ultrasonic extraction followed by UPLC–ESI–MS/MS with electrospray ionization in both positive mode and negative mode. The influence of the extraction solvents and mobile phase composition on the sensitivity of the method, and the optimum conditions for sample weight and extraction temperature in terms of analyte recovery were extensively studied. The identification of antibiotics is fulfilled by simultaneous use of chromatographic separation using an Acquity BEH C18 (100 mm x 2.1 mm, 1.7 µm) analytical column with a gradient elution of mobile phases and tandem mass spectrometry with an electrospray ionization. Finally, the method developed was applied to the determination of target analytes in honey samples obtained from the local markets and several beekeepers in Muğla, Turkey. Ultrasonic-extraction of pharmaceuticals from honey samples is a well-established technique by UPLC–ESI–MS/MS, the uniqueness of this study lies in the simultaneous determination of a remarkable number of compounds belonging to 23 drug at the sub-nanogram per kilogram level.