Comparative analysis of creatinine and osmolality as urine normalization strategies in targeted metabolomics for the differential diagnosis of asthma and COPD.

INTRODUCTION: Urine is an ideal matrix for metabolomics investigation due to its non-invasive nature of collection and its rich metabolite content. Despite the advancements in mass spectrometry and 1H-NMR platforms in urine metabolomics, the statistical analysis of the generated data is challenged with the need to adjust for the hydration status of the person. Normalization to creatinine or osmolality values are the most adopted strategies, however, each technique has its challenges that can hinder its wider application. We have been developing targeted urine metabolomic methods to differentiate two important respiratory diseases, namely asthma and chronic obstructive pulmonary disease (COPD). OBJECTIVE: To assess whether the statistical model of separation of diseases using targeted metabolomic data would be improved by normalization to osmolality instead of creatinine. METHODS: The concentration of 32 metabolites was previously measured by two liquid chromatography-tandem mass spectrometry methods in 51 human urine samples with either asthma (n = 25) or COPD (n = 26). The data was normalized to creatinine or osmolality. Statistical analysis of the normalized values in each disease was performed using partial least square discriminant analysis (PLS-DA). Models of separation of diseases were compared. RESULTS: We found that normalization to creatinine or osmolality did not significantly change the PLS-DA models of separation (R2Q2 = 0.919, 0.705 vs R2Q2 = 0.929, 0.671, respectively). The metabolites of importance in the models remained similar for both normalization methods. CONCLUSION: Our findings suggest that targeted urine metabolomic data can be normalized for hydration using creatinine or osmolality with no significant impact on the diagnostic accuracy of the model.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry quantitative method for the cellular analysis of varying structures of gemini surfactants designed as nanomaterial drug carriers.

Diquaternary gemini surfactants have successfully been used to form lipid-based nanoparticles that are able to compact, protect, and deliver genetic materials into cells. However, what happens to the gemini surfactants after they have released their therapeutic cargo is unknown. Such knowledge is critical to assess the quality, safety, and efficacy of gemini surfactant nanoparticles. We have developed a simple and rapid liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative determination of various structures of gemini surfactants in cells. Hydrophilic interaction liquid chromatography (HILIC) was employed allowing for a short simple isocratic run of only 4min. The lower limit of detection (LLOD) was 3ng/mL. The method was valid to 18 structures of gemini surfactants belonging to two different structural families. A full method validation was performed for two lead compounds according to USFDA guidelines. The HILIC-MS/MS method was compatible with the physicochemical properties of gemini surfactants that bear a permanent positive charge with both hydrophilic and hydrophobic elements within their molecular structure. In addition, an effective liquid-liquid extraction method (98% recovery) was employed surpassing previously used extraction methods. The analysis of nanoparticle-treated cells showed an initial rise in the analyte intracellular concentration followed by a maximum and a somewhat more gradual decrease of the intracellular concentration. The observed intracellular depletion of the gemini surfactants may be attributable to their bio-transformation into metabolites and exocytosis from the host cells. Obtained cellular data showed a pattern that grants additional investigations, evaluating metabolite formation and assessing the subcellular distribution of tested compounds.

Stool calprotectin in necrotizing enterocolitis.

BACKGROUND: Calprotectin is a 36 kDa protein present in the cytoplasm of the neutrophil has antimicrobial and apoptosis inducing activities. In vitro studies have shown that calprotectin inhibits the growth of various microorganisms. Necrotizing enterocolitis (NEC) remains one of the leading causes of morbidity and mortality in neonatal intensive care units (NICU), affecting up to 5% of premature infants. Fecal calprotectin is resistant to degradation and has been proposed as a useful marker of gastrointestinal inflammation. OBJECTIVE: The objective of the present study is to evaluate fecal calprotectin concentrations in NEC. STUDY DESIGN: Fifteen neonates with a clinical diagnosis of NEC were studied; they admitted at NICU of Zagazig University Hospital. In addition, 20 age sex matched neonates fed all caloric requirement served as the control group. All neonates were subjected to history taking, clinical examination, laboratory investigations (complete blood count, C-reactive protein) and determination of stool calprotectin. RESULTS: There was a highly significant increase in fecal calprotectin in patients than control and there was a highly significant increase in its fecal level in died patients than living one. Also significant increase in fecal calprotectin level with increasing severity of NEC. CONCLUSION: Fecal calprotectin measurements could be a valuable tool for the investigation of preterm and full term infants suspected of having NEC.

AMPK and FoxO1 regulate catalase expression in hypoxic pulmonary arterial smooth muscle.

BACKGROUND: Hypoxia and reactive oxygen species (ROS) including H(2)O(2) play major roles in triggering and progression of pulmonary vascular remodeling in persistent pulmonary hypertension. Catalase (CAT), the major endogenous enzyme scavenging H(2)O(2), is regulated in a tissue- and context-specific manner. OBJECTIVE: To investigate mechanisms by which hypoxia and H(2)O(2) regulate catalase expression, and the role of AMPK-FoxO pathway, in neonatal porcine pulmonary artery smooth muscle (PASMC). DESIGN/METHODS: PASMC were grown in hypoxia (10% O(2)) or normoxia (21% O(2)) for 72 hr. We measured catalase activity and lipid peroxidation; CAT, FoxO1, and FoxO3a expression by qPCR; protein contents of CAT, FoxOs, p-AMPK, p-AKT, p-JNK, p-ERK1/2 in whole lysates, and FoxOs in nuclear extracts, by immunoblot; and FoxO-1 nuclear localization by immunocytochemistry, quantified by laser scanning cytometry. RESULTS: Hypoxia upregulated CAT transcription, content and activity, by increasing CAT transcription factors FoxO1 and FoxO3a mRNA, and promoting nuclear translocation of FoxO1. However, lipid peroxidation increased in hypoxic PASMC. Among candidate FoxO regulatory kinases, hypoxia activated AMPK, and decreased p-Akt and ERK1/2. AMPK activation increased FoxO1 (total and nuclear) and CAT, while AMPK inhibition inhibited FoxO1 and CAT, but not FoxO3a. Exogenous H(2)O(2) decreased p-AMPK and increased p-AKT in hypoxic PASMC. This decreased active FoxO1, and reduced mRNA and protein content of CAT. Hypoxic induction of CAT, AKT inhibition (LY294002), or addition of PEG-catalase partly ameliorated the H(2)O(2) -mediated loss of nuclear FoxO1. CONCLUSIONS: Hypoxia induces catalase expression, though this adaptation is insufficient to protect PASMC from hypoxia-induced lipid peroxidation. This occurs via hypoxic activation of AMPK, which promotes nuclear FoxO1 and thus catalase expression. Exogenous ROS may downregulate cellular antioxidant defenses; H(2)O(2) activates survival factor Akt, decreasing nuclear FoxO1 and thus catalase.

Enantioselectivity of mass spectrometry: challenges and promises.

With the fast growing market of pure enantiomer drugs and bioactive molecules, new chiral-selective analytical tools have been instigated including the use of mass spectrometry (MS). Even though MS is one of the best analytical tools that has efficiently been used in several pharmaceutical and biological applications, traditionally MS is considered as a "chiral-blind" technique. This limitation is due to the MS inability to differentiate between two enantiomers of a chiral molecule based merely on their masses. Several approaches have been explored to assess the potential role of MS in chiral analysis. The first approach depends on the use of MS-hyphenated techniques utilizing fast and sensitive chiral separation tools such as liquid chromatography (LC), gas chromatography (GC), and capillary electrophoresis (CE) coupled to MS detector. More recently, several alternative separation techniques have been evaluated such as supercritical fluid chromatography (SFC) and capillary electrochromatography (CEC); the latter being a hybrid technique that combines the efficiency of CE with the selectivity of LC. The second approach is based on using the MS instrument solely for the chiral recognition. This method depends on the behavioral differences between enantiomers towards a foreign molecule and the ability of MS to monitor such differences. These behavioral differences can be divided into three types: (i) differences in the enantiomeric affinity for association with the chiral selector, (ii) differences of the enantiomeric exchange rate with a foreign reagent, and (iii) differences in the complex MS dissociation behaviors of the enantiomers. Most recently, ion mobility spectrometry was introduced to qualitatively and quantitatively evaluate chiral compounds. This article provides an overview of MS role in chiral analysis by discussing MS based methodologies and presenting the challenges and promises associated with each approach.

A validated HPLC assay method for the determination of sodium alginate in pharmaceutical formulations.

A high-performance liquid chromatography-diode array detector method was developed and validated for the quantification of sodium alginate in antacid oral suspension using a phenyl stationary phase and buffer solution at pH 7.0 as a mobile phase. The method was validated for specificity, linearity, range, accuracy, precision and robustness. The method was specific for the determination of sodium alginate in the bulk drug, pharmaceutical dosage form and under stress degradation. The method was linear over the range of 600 to 1,400 µg/mL with r(2) = 0.9999, and accuracy and precision were acceptable with relative standard deviation < 2.0%. The described method is simple, specific, precise, accurate, robust and stability-indicating, and can be successfully applied for the routine analysis of sodium alginate in bulk drug and pharmaceutical dosage form.

Mechanistic action of mesenchymal stem cell injection in the treatment of chemically induced ovarian failure in rabbits.

BACKGROUND: No curative treatment is known for primary ovarian failure; however, mesenchymal stem cells (MSCs), through self-renewal and regeneration, push the trial to evaluate their role in the treatment of ovarian failure. The aim of this study was to explore the impact of MSCs on cyclophosphamide (CTX)-induced ovarian failure in rabbits and to clarify the mechanism(s) by which MSCs exert their action. METHODS: Thirty-five adult female rabbits were injected with CTX to induce ovarian failure. Five rabbits were euthanized after the last injection of CTX for histological examination. The others (30 rabbits) were further subdivided into two groups: group 1 (ovarian failure group, 15 rabbits) received no treatment; group 2 (ovarian failure and MSC recipient group, 15 rabbits) received MSCs isolated from extracted bone marrow of male rabbits. RESULTS: A decrease of follicle-stimulating hormone and an increase of estrogen and vascular endothelial growth factor (VEGF) levels in the MSC recipient group versus the ovarian failure group were found. Weak caspase-3 expression and +ve proliferating cell nuclear antigen staining after MSC injection were detected. Cytological and histological examinations showed increased follicle numbers with apparent normal structure of ovarian follicles in the MSC recipient group. Moreover, Y chromosome-containing cells from male donors were present within the ovarian tissues in group 2. CONCLUSIONS: The current study suggests that intravenous injection of MSCs into rabbits with chemotherapy-induced ovarian damage improved ovarian function. MSCs accomplish this function by direct differentiation into specific cellular phenotypes and by secretion of VEGF, which influence the regeneration of the ovary.

A validated enantioselective HPLC assay of dexibuprofen in dexibuprofen tablet formulations.

A high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed and validated for the quantitation of dexibuprofen in dexibuprofen tablets using ovomucoid chiral stationary phase (Ultron ES-OVM). The mobile phasewas composed of 0.025 M potassium phosphate dibasic (pH 4.5)-methanol-ethanol (85:10:5 v/v/v). The method was validated for specificity, linearity, range, accuracy, precision and robustness. The method was enantiomerspecific for the determination of dexibuprofen [S-(+)-isomer ibuprofen] in the presence of R-(-)-isomer ibuprofen in bulk drug, pharmaceutical dosage form and under stress degradation. The method was linear over the range 15-35 mg/mL with r² = 0.9995; accuracy and precision were acceptable with %RSD < 2.0%. The method was found to be specific, precise, accurate, robust and stability-indicating, and can be successfully applied for the routine analysis of dexibuprofen in bulk drug and pharmaceutical dosage form.

Toll-like receptor 2 and Toll-like receptor 4 polymorphisms and susceptibility to asthma and allergic rhinitis: a case-control analysis.

The aim of the study was to investigate whether polymorphisms in genes encoding Toll-like receptors (TLR2 and TLR4) may modify relative risk for development of asthma or allergic rhinitis. The results showed that the genotype and allele frequencies of the TLR2 Arg753Gln and TLR4 Asp299Gly polymorphisms were not significantly different between asthmatic children or allergic rhinitis when compared to controls (p>0.05 for each) or even when compared further with IgE level. However, it was shown that the mutant allele of TLR2 or TLR4 polymorphisms were significantly associated with the moderate-severe group compared to the mild group in both atopic asthmatics and allergic rhinitis group (p>0.001 for each). In conclusion, our study demonstrates a lack of association of TLR2 and TLR4 polymorphisms with asthma and allergic rhinitis but suggests significant association between these genetic variants and the disease severity.

Impact of DNA repair genes polymorphism (XPD and XRCC1) on the risk of breast cancer in Egyptian female patients.

The genes involved in DNA repair system play a crucial role in the protection against mutations. It has been hypothesized that functional deficiencies in highly conserved DNA repair processes resulting from polymorphic variation may increase genetic susceptibility to breast cancer (BC). The aim of the present study was to evaluate the association of genetic polymorphisms in 2 DNA repair genes, XPD (Asp312Asn) and XRCC1 (A399G), with BC susceptibility. We further investigated the potential combined effect of these DNA repair variants on BC risk. Both XPD (xeroderma pigmentosum group D) and XRCC1 (X-ray repair cross-complementing group 1) polymorphisms were characterized in 100 BC Egyptian females and 100 healthy women who had no history of any malignancy by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method and PCR with confronting two-pair primers (PCR-CTPP), using DNA from peripheral blood in a case control study. Our results revealed that the frequencies of AA genotype of XPD codon 312 polymorphism were significantly higher in the BC patients than in the normal individuals (P ≤ 0.003), and did not observe any association between the XRCC1 Arg399Gln polymorphism and risk of developing BC. Also, no association between both XPD Asp312Asn and XRCC1 A399G polymorphisms and the clinical characteristics of disease. Finally, the combination of AA(XPD) + AG(XRCC1) were significantly associated with BC risk. Our results suggested that, XPD gene is an important candidate gene for susceptibility to BC. Also, gene-gene interaction between XPD(AA) + XRCC1(AG) polymorphism may be associated with increased risk of BC in Egyptian women.

Changes in the haemocytes of Agrotis ipsilon larvae (Lepidoptera: Noctuidae) in relation to dimilin and Bacillus thuringiensis infections.

Five types of haemocytes are observed in the fourth larval instar of the black cutworm, Agrotis ipsilon: prohaemocytes (PRs), plasmatocytes (PLs), granulocytes (GRs), spherule cells (SPs) and adipohaemocytes (ADs). Infection of A. ipsilon fourth larval instar with Bacillus thuringiensis and dimilin resulted in a reduction of the total haemocyte count. Changes in the differential haemocyte population during bacterial and dimilin infections have been assessed. The PRs % decreased significantly while SPs, PLs, and GRs % increased significantly after the application of the two insecticides at 12 and 24h. Ultrastructural alternations and malformations have been observed in circulating haemocytes of A. ipsilon larvae treated with dimilin and B. thuringiensis.