Determination of trace lead in biological and water samples with dispersive liquid-liquid microextraction preconcentration

A new method for the determination of trace lead was developed by dispersive liquid–liquid microextraction preconcentration and graphite furnace atomic absorption spectrometry. In the proposed approach, 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) was used as a chelating agent, and carbon tetrachloride and ethanol were selected as extraction and dispersive solvents. Some factors influencing the extraction efficiency of lead and its subsequent determination, including extraction and dispersive solvent type and volume, pH of sample solution, concentration of the chelating agent, and extraction time, were studied and optimized. Under the optimum conditions, the enrichment factor of this method for lead was reached at 78. The detection limit for lead was 39 ng L⁻¹ (3σ), and the relative standard deviation (RSD) was 3.2% (n = 7, c = 10 ng mL⁻¹). The method was successfully applied to the determination of trace amounts of lead in human urine and water samples.     

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of a new oral thrombin inhibitor (compound I) in the blood of rats and dogs. The analyte was deproteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a mixture of acetonitrile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate of 1.0 ml/min at UV 231 nm. The retention time of compound I was approximately 9.3 min. The calibration curve was linear over the concentration range of 0.05–100 mg/l for rat blood (r²>0.9995, n=6) and dog blood (r²>0.9993, n=6). The limit of quantitation was 0.05 mg/l for both bloods using a 100-μl sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n=4) were 98.1–104.1% and 1.5–6.8% for rat blood and 95.4–105.7% and 1.4–5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8–105.3% and 3.7–12.6% for rat blood and 87.5–107.1% and 2.9–15.3% for dog blood, respectively. The absolute recoveries were 82.4–93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproducible HPLC method proved and was used successfully for the determination of compound I in the preclinical pharmacokinetics.     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

Comparison of the cytotoxic,pro-oxidant and pro-inflammatory characteristics of different oxysterols

Oxidized low-density lipoproteins play important roles in the development of atherosclerosis and contain several lipid-derived, bioactive molecules which are believed to contribute to atherogenesis. Of these, some cholesterol oxidation products, refered to as oxysterols, are suspected to favor the formation of atherosclerotic plaques involving cytotoxic, pro-oxidant and pro-inflammatory processes. Ten commonly occurring oxysterols (7α-, 7β-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, 22R-, 22S-, 25-, and 27-hydroxycholesterol) were studied for both their cytotoxicity and their ability to induce superoxide anion production (O₂^{⋅ −}) and IL-8 secretion in U937 human promonocytic leukemia cells. Cytotoxic effects (phosphatidylserine externalization, loss of mitochondrial potential, increased permeability to propidium iodide, and occurrence of cells with swollen, fragmented and/or condensed nuclei) were only identified with 7β-hydroxycholesterol, 7-ketocholesterol and cholesterol-5β,6β-epoxide, which also induce lysosomal destabilization associated or not associated with the formation of monodansylcadaverine-positive cytoplasmic structures. No relationship between oxysterol-induced cytotoxicity and HMG-CoA reductase activity was found. In addition, the highest O₂^{⋅ −} overproduction quantified with hydroethidine was identified with 7β-hydroxycholesterol, 7-ketocholesterol and cholesterol-5β,6β-epoxide, with cholesterol-5α, 6α-epoxide and 25-hydroxycholesterol. The highest capacity to simultaneously stimulate IL-8 secretion (quantified by ELISA and by using a multiplexed, particle-based flow cytometric assay) and enhance IL-8 mRNA levels (determined by RT-PCR) was observed with 7β-hydroxycholesterol and 25-hydroxycholesterol. None of the effects observed for the oxysterols were detected for cholesterol. Therefore, oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever.     

Development of an amperometric assay for phosphate ions in urine based on a chemically modified screen-printed carbon electrode

An amperometric assay for the determination of inorganic phosphate (Pi) in urine has been developed without the need for sample preparation. A screen-printed carbon electrode modified with the electrocatalyst cobalt phthalocyanine (CoPC–SPCE) and covered with a cellulose acetate membrane (CAM) serves as the sensor. The sensor detects hydrogen peroxide (H₂O₂), which is produced as a result of the oxidative decarboxylation of pyruvate, catalyzed by pyruvate oxidase (PyOd), in the presence of Pi, oxygen, and cofactors. Following optimization of solution conditions, and in the presence of a urine sample, a linear range was found to exist between the rate of current increase and phosphate concentration over the range of 2.27 × 10⁻⁵ to 1.81 × 10⁻⁴ M, and the limit of detection was found to be 4.27 × 10⁻⁶ M. The assay was applied to the determination of phosphate ions in the urine of a normal subject, and the mean concentration in unspiked urine was found to be 3.40 × 10⁻⁵ M with a coefficient of variation of 8.0% (n = 5). The mean recovery of phosphate added to urine samples was 98.7% with a coefficient of variation of 5.5% (n = 3). To the authors’ knowledge, this is the first report of an amperometric assay for Pi that incorporates a CoPC–SPCE as the sensing device.     

High-performance liquid chromatographic assay of a new HIV-1 protease inhibitor, LB71350, in the plasma of dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of LB71350 in the plasma of dogs. The analyte was deproteinized with 1.5 volumes of methanol and 0.5 volumes of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a stepwise gradient mixture of acetonitrile and 0.2% triethylamine–HCl with a flow-rate of 1 ml/min and detection at UV 245 nm. The proportion of acetonitrile was kept at 52% for the first 6 min, increased to 100% for the next 0.5 min, kept at 100% for the next 2 min, decreased to 52% for the next 0.5 min, and finally kept at 52% for the next 7 min. The retention time of LB71350 was 6.9 min. The calibration was linear over the concentration range of 0.1–100 mg/l for dog plasma (r>0.997) and the limit of quantitation was 0.1 mg/l using 0.1 ml plasma. The quality control samples were reproducible with acceptable accuracy and precision at 0.1, 1, 10 and 100 mg/l concentrations. The within-day recovery (n=5) was 90.2–93.9%, the between-day recovery (n=5) was 89.5–93.5%, and the absolute between-day recovery (n=5) was 77–81%. The within-day precision (n=5) and between-day precision (n=5) were 2.59–5.82% and 3.17–4.55%, respectively. No interferences from endogenous substances were observed. Taken together, the above HPLC assay method by deproteinization and UV detection was suitable for the determination of LB71350 in the preclinical pharmacokinetics.     

Automated determination of 5-fluorouracil and its metabolite in urine by high-performance liquid chromatography with column switching

We report a quantitative assay of 5-fluorouracil (FU) and its metabolite, 5-fluorodihydrouracil (FDHU) in human urine by used a column-switching high-performance liquid chromatographic method. The analyses were carried out using a molecular exclusion column for sample purification, and a cation-exchange column for separation. Each sample required only 40 min to analyze, and required no preparation other than filtration. Linearity was verified up to 1000 nmol/ml (r>0.993). The recovery of FU was 96–101%; recovery of FDHU was 96–105%. The imprecision (RSD) for FU (10–100 nmol/ml) was <1.5%, same-day (n=5), and <1.8%, day-to-day (n=5). The imprecision (RSD) for FDHU (10–100 nmol/ml) was <3.2%, same-day (n=5), and <4.0%, day-to-day (n=5). The detection limits were, respectively, 0.1 nmol/ml. We measured FU and FDHU in urine of seven cancer patients after oral administration of FU. The cumulative quantity ratio of the FDHU and FU (FDHU/FU) excreted in their urine within 120 min after FU administration was a constant value in all seven patients. Based on these results, we believe that our method provides a useful tool for evaluating FU metabolism.     

An amino acidic adjuvant to augment cryoinjury of MCF-7 breast cancer cells

One of the major challenges in cryosurgery is to minimize incomplete cryodestruction near the edge of the iceball. In the present study, the feasibility and effectiveness of an amino acidic adjuvant, glycine was investigated to enhance the cryodestruction of MCF-7 human breast cancer cell at mild freezing/thawing conditions via eutectic solidification. The effects of glycine addition on the phase change characteristics of NaCl–water binary mixture were investigated with a differential scanning calorimeter and cryo-macro/microscope. The results confirmed that a NaCl–glycine–water mixture has two distinct eutectic phase change events – binary eutectic solidification of water–glycine, and ternary eutectic solidification of NaCl–glycine–water. In addition, its effects on the cryoinjury of MCF-7 cells were investigated by assessing the post-thaw cellular viability after a single freezing/thawing cycle with various eutectic solidification conditions due to different glycine concentrations, end temperatures and hold times. The viability of MCF-7 cells in isotonic saline supplemented with 10% or 20% glycine without freezing/thawing remained higher than 90% (n = 9), indicating no apparent toxicity was induced by the addition of glycine. With 10% glycine supplement, the viability of the cells frozen to −8.5 °C decreased from 85.9 ± 1.8% to 38.5 ± 1.0% on the occurrence of binary eutectic solidification of glycine–water (n = 3 for each group). With 20% glycine supplement, the viability of the cells frozen to −8.5 °C showed similar trends to those with 10% supplement. However, as the end temperature was lowered to −15 °C, the viability drastically decreased from 62.5 ± 2.0% to 3.6 ± 0.7% (n = 3 for each group). The influences of eutectic kinetics such as nucleation temperature, hold time and method were less significant. These results imply that the binary eutectic solidification of water–glycine can augment the cryoinjury of MCF-7 cells, and the extent of the eutectic solidification is significant.     

Leukotriene pathway polymorphisms are associated with altered cysteinyl leukotriene production in children with acute asthma

Cysteinyl leukotrienes (cysLTs) are pro-inflammatory mediators with increasing evidence for a role in childhood acute asthma. This study examined the influence of polymorphisms in cysLT pathway genes on urinary leukotriene E₄ (uLTE₄) levels and clinical status in acute asthmatic children. Children aged 2–16 years were recruited during an asthma attack (n=205). Where possible, asthma severity scores were assigned, ALOX5AP G-336A, ALOX5 G-1708A, LTC4S A-444C and G-1072A, GPX4 C718T, and CYSTLTR1 T927C genotypes were determined and uLTE₄ was measured in acute and convalescent samples. uLTE₄ levels were higher acutely compared with convalescence (acute GM: 115.7 pg/mg creatinine; 95% CI 88.6–151.1, convalescence GM: 66.4 pg/mg creatinine; 95% CI 51.5–85.6; n=50 paired samples, p=0.003) and paired sample analysis showed genotype-specific effects with significantly increased uLTE₄ for LTC₄S-444AA (acute GM: 127.9 pg/mg creatinine; 95% CI 91.8–178.3, convalescence GM: 68.2 pg/mg creatinine; 95% CI 50.5–92.0; n=32, p=0.002), LTC₄S-1072 GG (acute GM: 126.7 pg/mg creatinine; 95% CI 95.4–168.3, convalescence GM: 78.9 pg/mg creatinine; 95% CI 59.7–104.1; n=39, p=0.019) and CYSLTR1 927 TT/T_ (acute GM: 96.8 pg/mg creatinine; 95% CI 73.8–126.9, convalescence GM: 62.4 pg/mg creatinine; 95% CI 46.8–83.3; n=28, p=0.036) but not AC/CC, GA/AA, or TC/CC/C_, respectively. When we compared the allele frequencies of the CYSLTR1 SNP between asthmatics and non-asthmatics, the 927C allele was found to be a risk allele for asthma (OR=2.13, 95% CI: 1.06–4.26, p=0.033). Genotypes were not associated with acute or convalescent uLTE₄ levels alone and neither the SNPs nor uLTE₄ correlated with acute asthma severity. Leukotriene pathway gene polymorphisms may influence the magnitude of cysLT production during an attack, yet their influence alone may not be substantial enough to alter the severity of exacerbations.     

High-performance liquid chromatographic analysis of amoxicillin in human and chinchilla plasma, middle ear fluid and whole blood

We extended the application of a sensitive high-performance liquid chromatography assay of amoxicillin developed in this laboratory for human plasma and middle ear fluid (MEF) to other sample matrices including chinchilla plasma or MEF and human and chinchilla whole blood with minor modification and validated the limit of quantitation at 0.25 μg/ml with a 50-μl sample size for human and chinchilla plasmas or MEFs. Amoxicillin and cefadroxil, the internal standard, were extracted from 50 μl of the samples with Bond Elut C₁₈ cartridges. The extract was analyzed on a Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer, pH 6.5 and 5 mM tetrabutylammonium. The within-day coefficients of variation were 2.7–9.9 (n=4) and 1.7–7.2% (n=3) for chinchilla plasma and MEF samples, respectively; 2.8–8.1% (n=3) and 2.9–4.7% (n=3) for human and chinchilla whole blood, respectively. An alternative mobile phase composition for chinchilla plasma and MEF samples reduced the analysis time significantly.     

High-performance liquid chromatographic assay of bromocriptine in plasma and eye tissue of the rabbit

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid–liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-μm Nova-Pak C₁₈ column (150×3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2–10 μg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1–50 μg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 μg/l for plasma and vitreous humour using a 1-ml sample and was 1 μg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100–102% for plasma, 91–106% for aqueous humour and 96–111% for vitreous humour. The between-day recovery (n=9) was 90–114% for plasma, 83–115% for aqueous humour and 90–105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7–7.0% and 8.1–13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.     

Concise synthesis of 4-methylumbelliferyl-penta-N-acetylchitopentaoside and its inhibition effect on chitinase

The synthesis of 4-methylumbelliferyl (UMB)-penta-N-acetylchitopentaoside 4 and its inhibition effect on chitinase are described. The fluorophore-assisted carbohydrate electrophoresis (FACE) analysis showed that the partially N-acetylated chitooligosaccharide (COS) mixture mainly contained glucosamine (GlcN) and oligomers [(GlcN)_n, n = 2–7]. The peracetylated COSs [(GlcNAc)_n, n = 1–7] were synthesized by treating the partially N-acetylated COS mixture with Ac₂O–NaOAc. The peracetylated chitopentaoside 1 was obtained by isolation of peracetylated COS mixture. The peracetylated UMB chitopentaoside 3 was synthesized by treating compound 1 with 4-methylumbelliferone and a Lewis acid (SnCl₄) catalyst. NaOMe in dry methanol was used for deacetylation of the blocked derivative, to give the target compound 4 in an overall yield of 32%. In binding chitinase assay, it indicates that compound 4 is much more stable than the corresponding penta-N-acetylchitopentaose 2.     

Corpuscular oxidative stress in desert sheep naturally deficient in copper

Oxidative stress arises when there is an imbalance between radical-generating and radical-scavenging activity; it may therefore cause an increase in oxidation products and cell damage. This study aimed to determine antioxidant status, lipid peroxidation, and their relation to anemia of grazing sheep deficient in copper (Cu). For this purpose, 39 male lambs of native (Balady) breed, aged 6–7 months and reared in El-Dakhla oasis (in the western Egyptian desert), were divided according to plasma Cu (pCu) concentration into three groups, marginally deficient (MD, pCu = 4–8 μmol/l, n = 12), functionally deficient (FD, pCu < 3 μmol/l, n = 12) and control (pCu > 9 μmol/l, n = 15). Jugular blood was sampled for determination of red blood cell count (RBC), packed cell volume (PCV), hemoglobin concentration (Hb), plasma ceruloplasmin activity (pCp), antioxidant activities of erythrocytic superoxide dismutase (eSOD), catalase (eCAT), glutathione peroxidase (eGSH-Px), and levels of erythrocytic malondialdehyde (eMDA, as a biomarker of lipid peroxidation). The Cu-deficient lambs were characterized by microcytic hypochronic anemia accompanied by decreased pCp, eSOD, eCAT and eGSH-Px activities and increased eMDA level when compared to the controls. The indices of anemia, pCp and eSOD were lower and eMDA was higher in FD compared to MD lambs. The enhanced eMDA was strongly correlated (P < 0.01) with the inhibited activity of pCu (r = −0.79), pCp (r = −0.65) and eSOD (r = −0.71) and to a lesser extent (P < 0.05) with eGSH-Px (r = −0.38) and eCAT (r = −0.41). In addition, eMDA was negatively correlated (P < 0.01) with RBC (r = −0.75), PCV (r = −0.69) and Hb (r = −0.72). This study suggests that Cu-deficient lambs incur an erythrocytic oxidative damage secondary to impaired oxidant defenses, which may be one of the mechanisms underlying Cu deficiency-induced anemia in grazing sheep.     

Quantitation of mitomycin C in human ocular tissues by high-performance liquid chromatography–photo-diode array detection

A chromatographic method, which can quantitate mitomycin C (MMC) along with two antiglaucoma drugs, is described. The separation of MMC, alphagan and timolol was performed on a reversed-phase C₁₈ column with water–methanol–trifluoroacetic acid (65:35:0.01, v/v) as the mobile phase. By monitoring at 360, 248 and 296 nm, the lower limits of detection for MMC, alphagan and timolol are, respectively, 1.0, 2.0 and 5.0 ng (injection amount) at three-time S/N ratio. The dynamic ranges of quantitation for the three drugs are, respectively, 1.0 ng–10.0 μg, 2.0 ng–10.0 μg and 5.0 ng–10.0 μg with linearity being larger than 0.9960. This method was applied to the determination of MMC levels in Tenon’s and trabeculum tissues of 10 glaucoma patients. MMC levels in these tissues, which were obtained from glaucoma filtering surgery, were determined following a multiple extraction with methanol. The recovery of MMC for a two-batch extraction was better than 91.2%. The reproducibility of measurement for the MMC levels in these tissues is 2.5–6.0% RSD for triplicate injections. The intra-day variation of retention times for the MMC peaks was less than 1.6% RSD (n=3). The inter-day variation of retention times for the MMC peaks was less than 4.8% RSD (n=3). MMC was detectable in three trabeculum tissues out of 10 cases (ranging from 0.8 to 25.5 ng/mg specimen), while MMC was detected in nine Tenon’s tissues out of 10 cases (ranging from 0.3 to 21.1 ng/mg specimen). The results obtained show that the method is sensitive and selective for the quantitation of MMC.     

Skewed birth sex ratio and premature mortality in elephants

Sex allocation theories predict equal offspring number of both sexes unless differential investment is required or some competition exists. Left undisturbed, elephants reproduce well and in approximately even numbers in the wild. We report an excess of males are born and substantial juvenile mortality occurs, perinatally, in captivity. Studbook data on captive births (CB, n = 487) and premature deaths (PD, <5 years of age; n = 164) in Asian and African elephants in Europe and North America were compared with data on Myanmar timber (Asian) elephants (CB, n = 3070; PD, n = 738). Growth in CB was found in three of the captive populations. A significant excess of male births occurred in European Asian elephants (ratio: 0.61, P = 0.044) and in births following artificial insemination (0.83, P = 0.003), and a numerical inclination in North American African elephants (0.6). While juvenile mortality in European African and Myanmar populations was 21–23%, it was almost double (40–45%) in all other captive populations. In zoo populations, 68–91% of PD were within 1 month of birth with stillbirth and infanticide being major causes. In Myanmar, 62% of juvenile deaths were at >6 months with maternal insufficient milk production, natural hazards and accidents being the main causes. European Asian and Myanmar elephants PD was biased towards males (0.71, P = 0.024 and 0.56, P < 0.001, respectively). The skewed birth sex ratio and high juvenile mortality hinder efforts to help captive populations become self-sustaining. Efforts should be invested to identify the mechanism behind these trends and seek solutions for them.     

Effect of dehydration prior to cryopreservation of large equine embryos

Cryopreservation of equine embryos > 300 μm in diameter results in low survival rates using protocols that work well for smaller equine embryos. These experiments tested the potential benefit of incorporating a dehydration step prior to standard cryopreservation procedures. Forty-six, day 7–8, grade 1, equine embryos 300–1350 μm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol, slow freeze (n = 21); (B) 10 min in 1.5 M glycerol, slow freeze (n = 15); (C) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol, followed by exposure to thaw solutions, then culture medium (n = 5); (D) transferred directly to culture medium (n = 5). Frozen embryos were thawed and subjected to a three-step cryoprotectant removal. Five embryos from each treatment were evaluated morphologically after 24 and 48 h culture (1 = excellent, 5 = degenerate/dead). All treatments had at least 4/5 embryos with a quality score  3 at these time points except treatment B (2/5 at 24 h, 1/5 at 48 h). Subsequent embryos from treatment A (n = 16) or B (n = 10) were matched in sets of two for size and treatment, thawed, and immediately transferred in pairs to 13 recipients. Only two recipient mares were pregnant; one received two 400 μm embryos from treatment A, and the other one 400 and one 415 μm embryo from treatment B. There was no advantage of incorporating a 2 min dehydration step into the cryopreservation protocol for large equine embryos.     

Genetic contribution of chemokine receptor 2 (CCR2) polymorphisms towards increased serum total IgE levels in Indian asthmatics

The chemokine (C–C motif) receptors (CCR) 2 and 5 are members of a large family of G protein-coupled receptors, playing important roles in asthma pathogenesis. Using standard sequencing techniques, a total of 15 single nucleotide and 8 insertion/deletion polymorphisms (DIPs) (5 novels) were identified in and around these two genes. None of the studied polymorphisms (N = 7, selected on the basis on linkage disequilibrium) was associated with asthma in a case (N = 315) – control (N = 337) study and showed no evidence for non-random transmission to individuals with asthma/atopy in Indian pedigrees (n = 235). However, multilocus haplotype analysis based on simulations yielded a P = 0.00005 in the case–control study and a P = 0.03 for the family-based association studies. Furthermore, rs3918356 and rs743660 polymorphisms in CCR2 were found to be associated with total serum IgE levels in both the study designs. Thus, our study supports a significant role for chemokine receptor polymorphisms in genetic susceptibility to asthma.     

Fixed-time artificial insemination in red deer (Cervus elaphus) in Argentina

Synchronization of estrous and fixed-time artificial insemination (FTAI) was conducted during the reproductive season of 2008 (March–April) in a local red deer breeding farm in Argentina. Multiparous suckling hinds (n = 38) were artificially inseminated following hormonal treatment (intravaginal sponge containing 100 mg of medroxiprogesterone acetate). At the time of sponge removal (day 12) 250 IU of eCG and 500 μg of PGF2α were given to each hind. The FTAI was performed at 48–55 h after device removal with cryopreserved semen imported from New Zealand. Rectal-transcervical AI method (similar to that in cattle) was performed and semen was deposited within the uterine body (n = 28) or the cervix (n = 10). Pregnancy was diagnosed by means of ultrasonography 44 days after FTAI. The overall pregnancy rate was 36.8% (14/38). Percentage of does that became pregnant with intrauterine seminal deposition was 42.9% (12/28) whereas pregnancy rate in the hinds with intracervical AI was 20% (2/10; P = 0.27).     

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.     

Interleukin-6 and procalcitonin in children with sepsis and septic shock

Objectives. To examine the behavior of interleukin-6 (IL-6) and procalcitonin (PCT) and verify whether they can be used to differentiate children with septic conditions. Methods. Septic children aged between 28 days and 14 years, prospectively enrolled from 01/2004 to 12/2005, were divided into sepsis (SG; n = 47) and septic shock (SSG; n = 43) groups. IL-6 and PCT were measured at admission (T0) and 12 h later (T12h). PCT results were classed as: 0.5 ng/mL = sepsis unlikely; 0.5 to <2 = sepsis possible; 2 to <10 = systemic inflammation; 10 = septic shock. Results. Ninety children were included. At T0, there was a higher frequency of SSG with higher PCT compared with SG [SSG: 30 (69.7%) > SG: 14 (29.8%); p < 0.05]. Similar results were observed at T12h. PRISM was significantly higher for SSG patients with higher PCT than SG patients. At T0, IL-6 levels were higher in SSG [SSG: 213.10 (10.85–396.70) > SG: 63.21 (0.86–409.82); p = 0.001], but not statistically different at T12h. IL-6 levels positively correlated with PRISM score in SSG patients at admission (p = 0.001; r = 0.86). Conclusion. PCT and IL-6 appear to be helpful in early assessment of pediatric sepsis, are of diagnostic value at admission, and are related to disease severity.