Principal milk components in buffalo, holstein cross, indigenous cattle and red chittagong cattle from bangladesh.

The aim of the present study was to get a total physical and chemical characterization and comparison of the principal components in Bangladeshi buffalo (B), Holstein cross (HX), Indigenous cattle (IC) and Red Chittagong Cattle (RCC) milk. Protein and casein (CN) composition and type, casein micellar size (CMS), naturally occurring peptides, free amino acids, fat, milk fat globule size (MFGS), fatty acid composition, carbohydrates, total and individual minerals were analyzed. These components are related to technological and nutritional properties of milk. Consequently, they are important for the dairy industry and in the animal feeding and breeding strategies. Considerable variation in most of the principal components of milk were observed among the animals. The milk of RCC and IC contained higher protein, CN, ß-CN, whey protein, lactose, total mineral and P. They were more or less similar in most of the all other components. The B milk was found higher in CN number, in the content of αs2-, κ-CN and α-lactalbumin, free amino acids, unsaturated fatty acids, Ca and Ca:P. The B milk was also lower in ß-lactoglobulin content and had the largest CMS and MFGS. Proportion of CN to whey protein was lower in HX milk and this milk was found higher in ß-lactoglobulin and naturally occuring peptides. Considering the results obtained including the ratio of αs1-, αs2-, ß- and κ-CN, B and RCC milk showed best data both from nutritional and technological aspects.

Quantitative determination of fifteen basic pharmaceuticals in ante- and post-mortem whole blood by high pH mobile phase reversed phase ultra high performance liquid chromatography-tandem mass spectrometry.

An ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of fifteen basic pharmaceuticals, for analysis of post- and ante-mortem whole blood samples. The following compounds were included: amitriptyline and its metabolite nortriptyline, trimipramine, mianserin, mirtazapine, citalopram, paroxetine, sertraline, and venlafaxine (all antidepressants), levomepromazine and quetiapine (antipsychotics), ketobemidone and tramadol (analgesics), alimemazine (sedative antihistamine), and metoprolol (beta-blocker). The sample pretreatment consisted of liquid-liquid extraction (LLE) using ethylacetate:n-heptane (80:20, v/v). Six deuterated analogues were used as internal standards (IS). The compounds were separated using a reversed phase C18-column (2.1mm×100mm, 1.7µm), a flow rate of 0.5mL/min, and gradient elution with 5mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode, using two transitions for the compounds and one transition for the IS. The run time of the method was 8min including equilibration time. The calibration curves had R(2) values above 0.995 for all the compounds. The intermediate precision had a relative standard deviation (RSD, %) ranging between 2.0 and 16%. Recoveries of the compounds were ≥81%. The lower limits of quantifications (LLOQs) for the compounds varied from 5.0nmol/L to 0.10µmol/L (1.3-26ng/mL) and the limits of detections (LODs) from 1.0 to 20nmol/L (0.24-5.3ng/mL). LLOQ corresponds to 0.28-5.5pg injected on column. Matrix effects (ME) were between 91 and 113% when calculated against an IS. A comparison with former confirmation LC-MS methods at the Norwegian Institute of Public Health, Division of Forensic Medicine and Drug Abuse Research (NIPH) was performed during method validation. Good correlation was seen for all compounds except sertraline, where the old LC-MS method was showing 33% higher results. The method has been running on a routine basis for more than a year, and has proven to be very robust and reliable with results for external quality samples, including sertaline, corresponding well to consensus mean or median.

Determination of benzodiazepines in ante-mortem and post-mortem whole blood by solid-supported liquid-liquid extraction and UPLC-MS/MS.

A solid-supported liquid-liquid extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of benzodiazepines commonly found in Norway, for use in cases with suspected driving impairment and autopsy cases by analysis of human whole blood samples. The following compounds were included: alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordiazepam (metabolite of diazepam), oxazepam and phenazepam. Aliquots of 500 µL whole blood were added 500 µL of borate buffer pH 11 and extracted by solid-supported liquid-liquid extraction on ChemElut(®) columns using three times 2.5 mL of methyl tert-butyl ether. Deuterated analogues were used as internal standards (IS) for all analytes, except for midazolam, phenazepam and bromazepam which had no commercially available deuterated analogues at the time the method was developed, and therefore used diazepam-d(5), flunitrazepam-d(7) and nitrazepam-d(5), respectively. The analytes were separated using UPLC with a 2.1×100 mm BEH C(18)-column, 1.7 µm particle size, and quantified by MS/MS using multiple reaction monitoring (MRM) in positive mode. Two transitions were used for the analytes and one transition for the IS. The run time of the method was 8 min including equilibration time. The concentrations of the benzodiazepines in the method span a broad range varying from the lowest concentration of 0.005 µM for flunitrazepam to the highest of 20 µM for oxazepam. The calibration curves of extracted whole blood standards were fitted by second-order calibration curves weighted 1/x, with R(2) values ranging from 0.9981 to 0.9998. The intermediate precision had a CV (%) ranging between 2 and 19%. Recoveries of the analytes were from 71 to 96%. The LLOQs for the analytes varied from 0.0006 to 0.075 µM and the LODs from 0.005 to 3.0 nM. Matrix effects were studied by post extraction addition and found to be between 95 and 104% when calculated against an internal standard. A comparison with two other LC-MS methods was performed during method validation. Good correlation was seen for all analytes. The method has been running on a routine basis for several years, and has proven to be very robust and reliable with good results for external quality samples. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs to be introduced in the Norwegian legislative system from 2012.

Retention and removal of the fish pathogenic bacterium Yersinia ruckeri in biological sand filters.

AIMS: To investigate the retention and removal of the fish pathogenic bacterium Yersinia ruckeri in biological sand filters and effects on the microbial community composition. METHODS AND RESULTS: Sand filter columns were loaded (70 mm day(-1)) with fish farm wastewater and a suspension (10(8) CFU ml(-1)) of Y. ruckeri. Bacterial numbers and protozoan numbers were determined by plate counts and epifluorescence microscopy, respectively, and microbial biomass and community composition were assessed by phospholipid fatty acids (PLFA) analysis. Concentrations of Y. ruckeri in the filter effluent decreased from 10(8) to 10(3)-10(5) CFU ml(-1) during the experiment. Numbers of Y. ruckeri in the sand decreased from 10(6) CFU g(-1) dry weight (DW) sand to 10(4) CFU g(-1) DW sand. In contrast, microbial biomass determined with plate counts and total PLFA increased during the whole experiment. Principal component analysis (PCA) revealed a change in microbial community composition with time, with the most pronounced change in surface layers and towards the end of the experiment. Protozoan numbers increased from ca 0-600 cells g(-1) DW sand, indicating the establishment of a moderate population of bacterial grazers. CONCLUSIONS: The removal of Y. ruckeri improved during the experiment. Introduction of Y. ruckeri to the sand filter columns stimulated growth of other micro-organisms, which in turn caused a shift in the microbial community composition in the sand. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the understanding of the dynamics of sand filters subjected to a high loading of a pathogenic bacterium and can therefore be used in future work were the overall aim is to provide a more reliable and efficient removal of pathogenic bacteria in biological sand filter systems.

Isolation and partial characterisation of a new antiproliferative substance from human leucocytes inhibiting growth of Candida albicans.

AIM: To purify and partially characterise a fraction from human leucocytes containing a substance cytotoxic to Candida albicans. METHODS: Leucocytes were isolated from the buffy coats of healthy blood donors. The cytotoxic factor (CF) was isolated from the soluble fraction of the cells. A cell lysate was passed through a filter with a cut off value of 3 kDa, and the filtrate was processed by anionic exchange chromatography and gel filtration. The purified CF was analysed for its chemical and biological properties. The cytotoxicity of CF was tested on C albicans grown on agar plates. RESULTS: Mass spectrometry showed a molecular mass of 2.148 kDa. CF was found in polymorphonuclear neutrophilic cells only. No amino acids were detected, and a low ultraviolet absorbance at 260 nm and resistance to nuclease indicate the absence of nucleic acids. An anthrone test was positive for carbohydrate. The substance was soluble in water. CF showed a dose related cytotoxicity in the range of 0.1-1 mg/ml. The cytotoxic effect was abrogated by zinc ions. Preliminary testing indicated that CF also had cytotoxic effects against some bacteria. CONCLUSIONS: This report describes a factor from isolated human leucocytes that is cytotoxic to C albicans. The substance contains a carbohydrate moiety, whereas no amino acids were detected. The cytotoxicity can be abrogated by zinc ions in vitro. This substance is probably part of the repertoire by which leucocytes prevent infections.

Synthesis of 3-hexuloses from 1,2:5,6-di-O-isopropylidenehexitols.

A simple, but low-yielding method for the synthesis of 3-hexuloses has been elaborated. Oxidation of 1,2:5,6-di-O-isopropylidenehexitols with bromine in the presence of barium carbonate, followed by mild-acid hydrolysis of the oxidation products gave the free hexuloses. Oxidation occurred at only one of the carbon atoms bearing free hydroxyl groups. From the D-mannitol derivative, D-arabino-3-hexulose was obtained via the di-O-isopropylidene derivative, whereas the D-glucitol derivative gave a mixture of the 1,2:5,6-di-O-isoprpylidene derivatives of L-xylo- and D-ribo-3-hexulose, separable by column chromatography. Mild-acid hydrolysis of the oxidation products afforded the free hexuloses.

Negative-ion electrospray ionisation-mass spectrometry (ESI-MS) as a tool for analysing structural heterogeneity in kappa-carrageenan oligosaccharides.

Oligosaccharides, enzymically produced from kappa-carrageenan, have been investigated by electrospray ionisation mass spectrometry (ESI-MS). The technique was used without prior derivatisation of the oligosaccharide originally obtained by size-exclusion chromatography (SEC). The structure of the oligosaccharides was mainly 4-sulphated neocarrabiose (A-G4S) with an increasing length ranging from di- to dodecasaccharides. However, in the larger oligosaccharides, structural motifs deviating from the perfect alternating A-G4S structure were detected, i.e. (A2S-G4S). Although resulting in reduced signal intensity, samples to which NaCl was added also gave rise to reliable mass spectra. Desulphation was induced at elevated cone voltages and in acidic or alkaline salt solutions.

Promoting a positive attitude in pediatric patients undergoing limb lengthening.

Pediatric patients undergoing external fixation for limb lengthening have to adjust to having the devices on their extremities for many months. This treatment requires a great deal of time and commitment from both the child and family in managing the device. Our hospital interdisciplinary limb lengthening (LLD) team discovered that having the patient and family involved is the key to getting children to resume their home routines. The team also discovered that since children's locus of control develops as they mature, the LLD process could give these patients a greater opportunity to develop an internal ("I'm in charge") rather than an external ("powerful others") locus of control. Therefore, it is essential to include the patient and family as part of the interdisciplinary team so that a positive psychologic outcome to limb lengthening can be attained.