Determination of enalapril maleate and atenolol in their pharmaceutical products and in biological fluids by flow‐injection chemiluminescence

A chemiluminescent method using flow injection (FI) was investigated for rapid and sensitive determination of enalapril maleate and atenolol, which are used in the treatment of hypertension. The method is based on the sensitizing effect of these drugs on the Ce(IV)–sulfite reaction. The different experimental parameters affecting the chemiluminescence (CL) intensity were carefully studied and incorporated into the procedure. The method permitted the determination of 0.01–3.0 µg mL^?1 of enalapril maleate in bulk form with correlation coefficient r = 0.99993, lower limit of detection (LOD) 0.0025 µg mL^?1 (S/N = 2) and lower limit of quantitation (LOQ) 0.01 µg mL^?1. The linearity range of atenolol in bulk form was 0.01–2.0 µg mL^?1 (r = 0.99989) with LOD of 0.0003 µg mL^?1 (S/N = 2) and LOQ of 0.01 µg mL^?1. In biological fluids the linearity range of enalapril maleate was 0.1–2.0 µg mL^?1 in both urine and serum, and for atenolol the linearity range was 0.1–1.0 µg mL^?1 in both urine and serum. The method was also applied to the determination of the drugs in their pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Post‐translational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells

Chinese hamster ovary cells (CHO) are routinely used in industry to produce recombinant therapeutic proteins and a number of studies have reported increased recombinant mRNA levels at temperatures <37°C. Surprisingly, the effect of reduced temperature on mRNA translation in CHO cells has not been investigated despite this process being highly responsive to environmental stresses. The relationship between low temperature culturing of CHO cells and mRNA translation was therefore investigated using labeling studies and dual luciferase reporter gene technology. Global protein synthetic capacity was not greatly affected at 32°C but was diminished at lower temperatures. The expression of both cap‐dependent and cap‐independent (IRES driven) mRNA translated luciferase reporter gene activity was highest at 32°C on a per cell basis and this was partially accounted for by increased mRNA levels. Importantly, post‐translational events appear to proceed with higher fidelity and accuracy at 32 than 37°C resulting in increased yield of active protein as opposed to an increase in total polypeptide synthesis. Therefore at 32°C recombinant cap‐dependent mRNA translation appears sufficient to maintain recombinant protein yields on a per cell basis and this is associated with improved post‐translational processing. Biotechnol. Bioeng. 2010;105: 215–220. © 2009 Wiley Periodicals, Inc.     

T‐RFLP reveals high β‐Proteobacteria diversity in microbial fuel cells enriched with domestic wastewater

Aims: To assess the biodiversity of a large number of microbial fuel cell (MFC) anodes from a variety of MFC designs, all enriched with domestic wastewater, using a molecular fingerprinting method. Methods and Results: We optimized a protocol allowing the rapid characterization of MFC communities using terminal restriction fragment length polymorphism (T‐RFLP) with two different sets of primers and a varying number of restriction enzymes. This protocol was further validated by direct comparison with bacterial clone libraries. Twenty‐one MFC anodes were analysed by T‐RFLP. We also provided a statistical comparison with other bacterial communities from environments sharing common features. Conclusions: Bacterial communities were dominated by β‐Proteobacteria, mostly belonging to the Burkholderiales order, that are known to play an active role in the cycle of metals such as iron and manganese. This property may allow them to properly pass electrons to the anode of an MFC. Significance and Impact of the Study: Unlike other groups, β‐Proteobacteria have seldom been acknowledged as potentially efficient electrochemically active bacteria (EAB) in MFCs. Yet, they are plentiful in natural environments like biocorrosion biofilms and acid mine drainages that consequently show some potential for MFC enrichment.     

Age‐based life history parameters and status assessments of by‐catch species (Lethrinus borbonicus,Lethrinus microdon,Pomacanthus maculosus and Scolopsis taeniatus) in the southern Arabian Gulf

Life history and demographic parameters for Lethrinus borbonicus, Lethrinus microdon, Pomacanthus maculosus and Scolopsis taeniatus in the southern Arabian Gulf were estimated using a combination of size frequency, biological and size‐at‐age data. Defined structural increments consisting of alternating translucent and opaque bands in transverse sections of sagittal otoliths were validated as annuli. The maximum age estimates ranged from 5 years for Scolopsis taeniatus to 36 years for Pomacanthus maculosus. The size‐at‐age relationships were highly asymptotic in form with the majority of growth being achieved early in life. There were significant differences in the growth characteristics between sexes for Pomacanthus maculosus, with males approaching a larger asymptotic size at a faster rate than females. With the exception of Scolopsis taeniatus, the mean age at which fish became vulnerable to capture was lower than the mean age at first sexual maturity. The stocks of L. microdon, P. maculosus and S. taeniatus were exploited within sustainable limits, conversely, L. borbonicus was found to be overexploited and recruitment overfishing may have occurred as the relative spawner biomass per recruit was below 30% of the unexploited state.     

Carboxylic Acids as Biomarkers of Biomphalaria alexandrina Snails Infected with Schistosoma mansoni

Biomphalaria alexandrina snails play an indispensable role in transmission of schistosomiasis. Infection rates in field populations of snails are routinely determined by cercarial shedding neglecting prepatent snail infections, because of lack of a suitable method for diagnosis. The present study aimed at separation and quantification of oxalic, malic, acetic, pyruvic, and fumaric acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to test the potentiality of these acids to be used as diagnostic and therapeutic biomarkers. The assay was done in both hemolymph and digestive gland-gonad complex (DGG) samples in a total of 300 B. alexandrina snails. All of the studied acids in both the hemolymph and tissue samples except for the fumaric acid in hemolymph appeared to be good diagnostic biomarkers as they provide not only a good discrimination between the infected snails from the control but also between the studied stages of infection from each other. The most sensitive discriminating acid was malic acid in hemolymph samples as it showed the highest F-ratio. Using the Z-score, malic acid was found to be a good potential therapeutic biomarker in the prepatency stage, oxalic acid and acetic acid in the stage of patency, and malic acid and acetic acid at 2 weeks after patency. Quantification of carboxylic acids, using HPLC strategy, was fast, easy, and accurate in prediction of infected and uninfected snails and possibly to detect the stage of infection. It seems also useful for detection of the most suitable acids to be used as drug targets.     

Blood sphingolipidomics in healthy humans: impact of sample collection methodology

We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C₁₄-C₂₆ N-acyl part) of “normal” sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (C_n-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C₁₆-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C₁₆-LacCer (10.0 µM) and the abundant HexCer was C₂₄-HexCer (2.5 µM). The abundant Cer, C₂₄-Cer (4.0 µM), was not influenced by fasting; however, levels of C₁₆-C₂₀ Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL₃ was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood.     

No evidence of altered alveolar macrophage polarization,but reduced expression of TLR2, in bronchoalveolar lavage cells in sarcoidosis

Background

Sarcoidosis is a granulomatous inflammatory disease, possibly of infectious aetiology. We aimed to investigate whether the degree of functional polarization of alveolar macrophages (AMs), or Toll-like receptor (TLR) expression, is associated with sarcoidosis or with distinct clinical manifestations of this disease.

Methods

Total BAL cells (cultured four or 24 h in medium, or stimulated 24 h with LPS) from 14 patients and six healthy subjects, sorted AMs from 22 patients (Löfgren''s syndrome n = 11) and 11 healthy subjects, and sorted CD4+ T cells from 26 patients (Löfgren''s syndrome n = 13) and seven healthy subjects, were included. Using real-time PCR, the relative gene expression of IL-10, IL-12p35, IL-12p40, IL-23p19, CCR2, CCR7, iNOS, CXCL10, CXCL11, CXCL16, CCL18, CCL20, CD80, and CD86, and innate immune receptors TLR2, TLR4, and TLR9, was quantified in sorted AMs, and for selected genes in total BAL cells, while IL-17A was quantified in T cells.

Results

We did not find evidence of a difference with regard to alveolar macrophage M1/M2 polarization between sarcoidosis patients and healthy controls. TLR2 gene expression was significantly lower in sorted AMs from patients, particular in Löfgren''s patients. CCL18 gene expression in AMs was significantly higher in patients compared to controls. Additionally, the IL-17A expression was lower in Löfgren''s patients'' CD4+ T cells.

Conclusions

Overall, there was no evidence for alveolar macrophage polarization in sarcoidosis. However, there was a reduced TLR2 mRNA expression in patients with Löfgren''s syndrome, which may be of relevance for macrophage interactions with a postulated sarcoidosis pathogen, and for the characteristics of the ensuing T cell response.     

Development and Evaluation of Hydrophilic Colloid Matrix of Famotidine Tablets

The objective of the present study was to develop a once-daily sustained-release (SR) matrix tablet of famotidine. Nine different formulations (F1–F9) were prepared by direct compression method using Avicel PH101 as filler/binder in the range of 41–27% in F1–F3, 18–22% in F4–F7, and 16–18% in F8–F9 and hydroxypropyl methylcellulose (4,000 cps) as hydrophilic matrix was used in F1–F3 from 19% to 30%, around 40% in F4–F7, and 42–45% in F8–F9. Talc and Aerosil were added in the ratio of 0.7–1.2%. The tablets were subjected to various physical parameters including weight variation test, hardness, thickness, diameter, friability, and in vitro release studies. Assay was also performed according to the USP 30 NF 25 procedure. The results of the physical parameters and assay were found to be within the acceptable range. In vitro dissolution results indicated that formulation F4–F7, having around 40% of rate control polymer, produced a SR pattern throughout 24 h. F1–F3 showed drug release at a faster rate, while F8–F9 released much slower, i.e., <80% in 24 h. Model-dependent and model-independent methods were used for data analysis and the best results were observed for F4 in zero order (r ² = 0.984) and F6 in Korsmeyer and Higuchi (r ² = 0.992 and 0.988). The parameter n indicated anomalous diffusion, while β in Weibull showed a parabolic curve with higher initial slope. The f ₂ similarity test was performed taking F4 as a reference formulation. Only the F5–F7 formulations were similar to the reference formulation F4. The mean dissolution time was around 10 h for the successful formulation.