Choline kinase inhibition in rheumatoid arthritis.

OBJECTIVES: Little is known about targeting the metabolome in non-cancer conditions. Choline kinase (ChoKα), an essential enzyme for phosphatidylcholine biosynthesis, is required for cell proliferation and has been implicated in cancer invasiveness. Aggressive behaviour of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) led us to evaluate whether this metabolic pathway could play a role in RA FLS function and joint damage. METHODS: Choline metabolic profile of FLS cells was determined by (1)H magnetic resonance spectroscopy ((1)HMRS) under conditions of ChoKα inhibition. FLS function was evaluated using the ChoKα inhibitor MN58b (IC50=4.2 µM). For arthritis experiments, mice were injected with K/BxN sera. MN58b (3 mg/kg) was injected daily intraperitoneal beginning on day 0 or day 4 after serum administration. RESULTS: The enzyme is expressed in synovial tissue and in cultured RA FLS. Tumour necrosis factor (TNF) and platelet-derived growth factor (PDGF) stimulation increased ChoKα expression and levels of phosphocholine in FLS measured by Western Blot (WB) and metabolomic studies of choline-containing compounds in cultured RA FLS extracts respectively, suggesting activation of this pathway in RA synovial environment. A ChoKα inhibitor also suppressed the behaviour of cultured FLS, including cell migration and resistance to apoptosis, which might contribute to cartilage destruction in RA. In a passive K/BxN arthritis model, pharmacologic ChoKα inhibition significantly decreased arthritis in pretreatment protocols as well as in established disease. CONCLUSIONS: These data suggest that ChoKα inhibition could be an effective strategy in inflammatory arthritis. It also suggests that targeting the metabolome can be a new treatment strategy in non-cancer conditions.

Determination of the purity of valine by isocratic liquid chromatography coupled with charged aerosol detection (CAD).

A liquid chromatography coupled with charged aerosol detection (LC-CAD) procedure; capable of separating and quantifying the most common impurities of valine at levels as low as 0.05 per cent (m/m), has been developed. The procedure is simple (isocratic), rapid, linear, sensitive and repeatable. It employs a widely available and inexpensive stationary phase (C18).

Determination of water content by capillary gas chromatography coupled with thermal conductivity detection.

This article presents some experience obtained by applying capillary gas chromatography coupled with thermal conductivity detection (GC/TCD) to the determination of water in substances for pharmaceutical use. This technique represents a useful, orthogonal tool complementary to water determination methods based on volumetric or coulometric titration. It can also represent an alternative technique when such titrations are not applicable. This article presents the preliminary results obtained in a number of case studies where a GC/TCD procedure was applied in comparison with pharmacopoeial methods to substances with different water contents.

An interlaboratory study on the suitability of a gradient LC-UV method as a compendial method for the determination of erythromycin and its related substances.

A liquid chromatographic (LC) method for the analysis of erythromycin and related substances has been adapted from an isocratic method developed by Chepkwony et al. (2001). The suitability of the method for general application as a compendial (pharmacopoeia) method has been assessed by means of an interlaboratory (collaborative) study. The method involves LC separation on a XTerra C18 column kept at 65 degrees C and UV detection at 210 nm. Five laboratories, located in Europe and the United States (US), participated in the study. Four erythromycin samples were tested. The main components (erythromycin A (EA), erythromycin B (EB), erythromycin C (EC)) and the impurities were determined. The analysis of variance was carried out on the results of the five laboratories to evaluate the between-laboratory consistencies and the laboratory-sample interaction. The estimates for the repeatability and reproducibility of the method, expressed as relative standard deviation (RSD) of the result of the determination of EA, were calculated to be 0.8% and 1.4% respectively. It is concluded that the method examined is a good replacement for the methods currently described in the European Pharmacopoeia (Ph. Eur.) and the United States Pharmacopoeia (USP), especially for its enhanced selectivity.

An unusual bilateral areolar dermatitis.

This article describes an uncommon clinical manifestation of contact dermatitis due to dyed tissues, showing bilateral plaque lesions on the mammary areolas. The clinical history, histological and patch testing examinations confirmed the diagnosis. The possible triggering factors related with this kind of contact dermatitis and possible differential diagnosis are briefly discussed.

Biogas production and valorization by means of a two-step biological process.

The scope of this research work was to investigate biogas production and purification by a two-step bench-scale biological system, consisting of fed-batch pulse-feeding anaerobic digestion of mixed sludge, followed by methane enrichment of biogas by the use of the cyanobacterium Arthrospiraplatensis. The composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5-76.0% and 13.2-19.5%, respectively. Biogas yield reached a maximum value (about 0.4 m(3)(biogas)/kg COD(i)) at 50 days-retention time and then gradually decreased with a decrease in the retention time. Biogas CO(2) was then used as a carbon source for A. platensis cultivation either under batch or fed-batch conditions. The mean cell productivity of fed-batch cultivation was about 15% higher than that observed during the last batch phase (0.035+/-0.006 g(DM)/L/d), likely due to the occurrence of some shading effect under batch growth conditions. The data of carbon dioxide removal from biogas revealed the existence of a linear relationship between the rates of A. platensis growth and carbon dioxide removal from biogas and allowed calculating carbon utilization efficiency for biomass production of almost 95%.

Cadmium biosorption on Spirulina platensis biomass.

Dry biomass of Spirulina platensis re-hydrated for 48 h was employed as a biosorbent in tests of cadmium(II) removal from water. Various concentrations of biomass (from 1 to 4 g l(-1)) and metal (from 100 to 800 mg l(-1)) were tested. Low biomass levels (X(o)2 g l(-1)) ensured metal removal up to 98% only at Cd(0)= 100 and 200 mg l(-1), while X(o)2.0 g l(-1) were needed at Cd(0)=400 mg l(-1) to achieve satisfactory results. Whereas X(o)=4.0 g l(-1) was effective to remove up to Cd(0)=500 mg l(-1), a further increase in metal concentration (Cd(0)=600 and 800 mg l(-1)) led to progressive worsening of the system performance. At a given biomass levels, the kinetics of the process was better at low Cd(2+) concentrations, while, raising the adsorbent level from 1.0 to 2.0 g l(-1) and then to 4.0 g l(-1), the rate constant of biosorption increased by about one order of magnitude in both cases and the adsorption capacity of the system progressively decreased from 357 to 149 mg g(-1).

The evolution of the pharmacopoeial test for heavy metals.

This article describes some recent improvements to simplify the test for heavy metals, avoiding loss of analytes and increasing sensitivity.

Inter-laboratory reproducibility of the LC-ELSD method for the control of composition of sesame oil recently introduced in the Ph. Eur.

Evaporative light-scattering detection (ELSD) is an increasingly important technology for the control of substances for pharmaceutical use. A LC-ELSD method has recently been introduced in the Ph. Eur. monograph for sesame oil, refined to control the composition of triglycerides. A collaborative study was run to assess the reproducibility of the new method and to confirm that it was suitable for pharmacopoeial use.

Copper removal by dry and re-hydrated biomass of Spirulina platensis.

Dried and re-hydrated biomass of Spirulina platensis was employed as a sorbent in tests of copper removal from water. Biomass re-hydrated for 24 h before use exhibited a shorter adsorption time as well as an increased percentage removal when compared with simply dried biomass. The combined effects of the concentrations of re-hydrated biomass (from 1.0 to 4.0 g l-1) and copper (from 0.1 to 0.4 g l-1) were then investigated. Copper was almost entirely removed (91% removal) at relatively high biomass levels (X0>or=2.0 gDM l-1), while 1.0 gDM l-1 removed only 81% of copper present initially, suggesting a situation of excess metal with respect to the adsorption capacity of biomass. Additional tests performed with biomass re-hydrated for variable time demonstrated that no less than 48 h of this treatment are needed to ensure a satisfactory copper removal, while no significant improvement was detected using biomass re-hydrated for longer times.

Ammonium and urea removal by Spirulina platensis.

Different concentrations either of ammonium chloride or urea were used in batch and fed-batch cultivations of Spirulina platensis to evaluate the possibility of substituting nitrate by cheaper reduced nitrogen sources in wastewaters biotreatment. The maximum nitrogen concentration able to sustain the batch growth of this microalga without inhibition was 1.7 mM in both cases. Ammonium chloride was limiting for the growth at lower concentrations, whereas inhibition took place at higher levels. This inhibition effect was less marked with urea, likely because the enzymatic hydrolysis of this compound by urease controlled the ammonia transfer into the cell. Fed-batch experiments carried out by pulse-feeding either ammonium or urea proved that the use of these compounds as nitrogen sources can sustain the long term-cultivation of S. platensis, provided that the conditions for their feeding are accurately optimized.

Evaporative light scattering detection (ELSD): a tool for improved quality control of drug substances.

Thanks to the recent technological advancements, evaporative light-scattering detection (ELSD) is regarded as a valuable alternative to UV detection for liquid chromatographic analysis of substances that do not contain a chromophore. In the field of substances for pharmaceutical use, LC-ELSD appears to be suitable for aminoglycosides, most of which (for ex. gentamicin) are presently controlled in the Ph. Eur. by pulsed amperometric detection. Other substances (ex sugars, triglycerides) presently employing refractometrric detection, could be conveniently analysed by LC-ELSD. ELS detection is regarded as robust and relatively simple, although not particularly sensitive. A key feature of ELSD is that - unlike refractometry - it can operate in gradient mode, thus allowing application of more selective liquid chromatographic methods. ELSD can also be used to set up MS-compatible methods, as the mobile phase constraints are essentially the same. Due to all the above, ELSD is becoming increasingly used in pharmacopoeial methods.

A liquid chromatographic method using a reversed-phase hybrid stationary phase to control potential impurities of imipramine hydrochloride.

A reversed-phase liquid chromatographic method has been developed for the detection and control of impurities of imipramine hydrochloride at a level of 0.2 microg/ml (corresponding to 0.02 per cent in the procedure described). This method is included in a proposed revision of the monograph (Pharmeuropa 18.4) and replaces the thin-layer chromatographic method currently employed.

The control of impurities in chlortalidone using a reversed-phase stationary phase.

A reversed-phased liquid chromatographic method for the control of impurities of nine impurities in Chlortalidone drug substance is presented. The method is sufficiently sensitive to quantify down to the level of 0.0015 per cent equivalent to a concentration of 15 microg/ml.

The control of impurities in amitriptyline hydrochloride using a reversed-phase hybrid stationary phase.

An isocratic reversed-phase liquid chromatographic method is described using a hybrid column for the control of synthetic impurities potentially found in amitriptyline hydrochloride. The method has been validated and is capable of controlling impurities to 0.1 per cent.

Nitrate and phosphate removal by Spirulina platensis.

The cyanobacterium Spirulina platensis was used to verify the possibility of employing microalgal biomass to reduce the contents of nitrate and phosphate in wastewaters. Batch tests were carried out in 0.5 dm3 Erlenmeyer flasks under conditions of light limitation (40 micromol quanta m(-2) s(-1)) at a starting biomass level of 0.50 g/dm3 and varying temperature in the range 23-40 degrees C. In this way, the best temperature for the growth of this microalga (30 degrees C) was determined and the related thermodynamic parameters were estimated. All removed nitrate was used for biomass growth (biotic removal), whereas phosphate appeared to be removed mainly by chemical precipitation (abiotic removal). The best results in terms of specific and volumetric growth rates (mu=0.044 day(-1), Qx=33.2 mg dm(-3) day(-1)) as well as volumetric rate and final yield of nitrogen removal (Q(N-NO3-)=3.26 mg dm(-3) day(-1), Y(N-NO3-)=0.739) were obtained at 30 degrees C, whereas phosphorus was more effectively removed at a lower temperature. In order to simulate full-scale studies, batch tests of nitrate and phosphate removal were also performed in 5.0 dm3 vessels (mini-ponds) at the optimum temperature (30 degrees C) but increasing the photon fluence rate to 80 micromol quanta m(-2) s(-1) and varying the initial biomass concentration from 0.25 to 0.86 g/dm3. These additional tests demonstrated that an increase in the inoculum level up to 0.75 g/dm3 enhanced both NO3- and PO4(3-) removal, confirming a strict dependence of these processes on biomass activity. In addition, the larger surface area of the ponds and the higher light intensity improved removal yields and kinetics compared to the flasks, particularly concerning phosphorus removal (mu=0.032-0.050 day(-1), Qx=34.7-42.4 mg dm(-3) day(-1), Q(N-NO3-)=3.24-4.06 mg dm(-3) day(-1), Y(N-NO3-)=0.750-0.879, Q(P-PO4(3-))=0.312-0.623 mg dm(-3) day(-1), and Y(P-PO4(3-))=0.224-0.440).

The control of nitrilotriacetic acid in edetic acid and its salts by liquid chromatography.

Two liquid chromatographic methods have been developed and validated for the determination of nitrilotriacetic acid (NTA) in edetic acid (EDTA) and its salts. In one method NTA and EDTA are separated on a polymer column without pretreatment, and are detected amperometrically using a glassy-carbon electrode. In the other method NTA and EDTA are complexed with ferric ions and the complexes separated on a porous graphite carbon stationary phase with ultraviolet detection at 215 nm. Both methods were sufficiently selected and sensitive to allow the control of NTA (0.1% m/m) in sample of EDTA and its salts.

Bioleaching of zinc and aluminium from industrial waste sludges by means of Thiobacillus ferrooxidans.

Biological solubilisation of heavy metals contained in two different kinds of industrial wastes was performed in batches employing a strain of Thiobacillus ferroxidans. The wastes tested were: a dust coming from the iron-manganese alloy production in an electric furnace (sludge 1) and a sludge coming from a process treatment plant of aluminium anodic oxidation (sludge 2). The experimental results pointed out the ability of the used strain to maintain the environment, that initially has a pH about 8, at strongly acid conditions (pH 2.5-3.5), producing sulphuric acid that is the chemical agent responsible for the metals solubilisation. At wastes initial concentration of 1%, the percentage of solubilised metals was 76 and 78% for the wastes 1 and 2, respectively, but the lag phase was considerably longer for sludge 2 than for sludge 1, indicating a different affinity of microorganisms for the solid phase. Increasing the initial slurry concentration, the percentage of removed metal reached 72-73% for the sludge 1, while in case of sludge 2, the total amount of solubilized metal progressively decreased. Two kinetic models are proposed to describe the trends of metals solubilization curves.

Treatment of effluent containing micropollutants by means of activated carbon.

Different amounts of granular activated carbon (GAC) have been tested for the removal of aliphatic and aromatic micropollutants contained in a liquid stream coming from an industrial plant. Tests have been carried out in a JAR-Test apparatus, using plugged flasks, in order to eliminate the oxygen influence on the adsorption process and to obtain information for studying the process in a pilot plant. The removal of aliphatic compounds resulted better than aromatic ones, probably because these substances are enveloped by water molecules which make adsorption on the GAC surface easier; in contrast, aromatic compounds show a lower affinity for the GAC, owing to their steric conformation. The good results obtained confirm that the proposed system is applicable to the examined effluent, even when the concentration of the pollutant load varies. In the latest part of this work, a plan for the construction of a full-scale plant to treat the examined wastewater has been developed.