Radioimmunoassay of Bovine,Ovine and Porcine Luteinizing Hormone with a Monoclonal Antibody and a Human Tracer

A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human 125 ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH and oLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 µg/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar.     

Dose-response of female llamas to ovulation-inducing factor from seminal plasma

The present study was designed to determine if the dose of purified ovulation-inducing factor (OIF) from llama seminal plasma required to provoke an ovulatory response is physiologically relevant in terms of the proportion present in a normal ejaculate and to test the hypothesis that corpus luteum (CL) form and function are affected by OIF in a dose-dependent manner. Female llamas were assigned randomly to five groups (n = 10 per group) and given a single i.m. dose of 500, 250, 125, or 60 μg of purified OIF (representative of the amount present in 1/25th to 1/200th of a normal ejaculate) or 1 ml of PBS (control). Ovulation and CL development were monitored by transrectal ultrasonography. Blood samples were taken to measure plasma progesterone concentrations and to determine changes in plasma concentrations of luteinizing hormone (LH). The high dose of OIF (500 μg) was associated with the highest incidence of ovulation (P < 0.05), the greatest maximum CL diameter (P < 0.05), and the largest day-to-day profiles of CL diameter (P < 0.05) and plasma progesterone concentrations (P < 0.01). A rise in plasma LH concentration was apparent in all llamas that ovulated and was most rapid and highest in the high-dose group (P < 0.01). The low dose of OIF (60 μg) was minimally effective for induction of ovulation and the least luteotrophic, as evidenced by the smallest maximum CL diameter and the smallest day-to-day profiles for CL diameter and plasma concentrations of progesterone and LH. Responses were intermediate for the middle-dose groups (125 and 250 μg). We conclude that OIF from llama seminal plasma has a dose-dependent effect on ovulation rate and CL form and function in llamas and that the biological effect of OIF is evident at physiologically relevant doses (i.e., as little as 1/100th of that present in an ejaculate).     

GnRH dose reduction decreases pituitary LH release and ovulatory response but does not affect corpus luteum (CL) development and function in llamas

Gonadotrophin releasing hormone (GnRH) is commonly used in llamas to induce ovulation; however, the consequence of reduced doses of GnRH on luteinizing hormone (LH) release, ovulatory response, and subsequent corpus luteum (CL) development and function have apparently not been investigated. Hence, we examined the effect of gradual reduction of gonadorelin acetate (GnRH) dosage on pituitary LH release, ovulatory response, CL development, and plasma progesterone concentrations in llamas. Non-pregnant, non-lactating adult llamas were examined once daily by transrectal ultrasonography, and those with a follicle ≥8 mm in diameter that had grown for three consecutive days were randomly assigned to receive 50 (GnRH50, n = 23), 25 (GnRH25, n = 29), 12.5 (GnRH12.5, n = 29), or 6.25 μg (GnRH6.25, n = 29) of GnRH, or 0.5 mL of PBS (Control group, n = 16) im. In a subset (7 or 8 animals/group), intense blood sampling was done to measure LH concentrations. All females were examined by ultrasonography every 12 h from treatment (Day 0) to Day 2 to determinate ovulation, and thereafter on alternate days until Day 16 to evaluate CL development (9-13 animals/group). Also, blood samples for progesterone determination were taken (9 or 10 animals/group) on alternate days from Days 0-16. Ovulatory response (%) was highest (P < 0.05) in the GnRH50 (82.6), intermediate in the GnRH25 (72.3) and GnRH12.5 (75.9) groups, and lowest in the GnRH6.25 group (48.3). No ovulations were detected in the Control group. Mean peak LH concentrations (ng/mL) were highest (P < 0.05) for GnRH50 (6.2), intermediate for GnRH25 (4.4) and GnRH12.5 (2.9), and lowest for GnRH6.25 (2.2) groups. In addition, based on regression analysis, llamas with an LH peak <4 ng/mL were less likely to ovulate. Llamas given 50 μg of GnRH released more (P < 0.05) pituitary LH and had an LH surge of longer duration than those given 25, 12.5, or 6.25 μg. However, in those that ovulated, neither GnRH treatment nor treatment by time interaction affected (P > 0.05) CL diameter or plasma progesterone concentrations. In summary, reducing the dose of GnRH gradually decreased the magnitude of the preovulatory LH surge and ovulatory response; however, subsequent CL development and plasma progesterone concentrations were not affected.     

Normal or induced secretory patterns of luteinising hormone and follicle-stimulating hormone in anoestrous gonadotrophin-releasing hormone-immunised and cyclic control heifers

The objective was to determine the effect of gonadotrophin-releasing hormone (GnRH), GnRH analogue (GnRH-A) or oestradiol administration on luteinising hormone (LH) and follicle-stimulating hormone (FSH) release in GnRH-immunised anoestrous and control cyclic heifers. Thirty-two heifers (477 ± 7.1 kg) were immunised against either human serum albumin (HSA; controls; n = 8), or a HSAGnRH conjugate. On day 70 after primary immunisation, control heifers (n = 4 per treatment; day 3 of cycle) received either (a) 2.5 μg GnRH or (b) 2.5 μg of GnRH-A (Buserelin®) and GnRH-immunised heifers (blocked by GnRH antibody titre; n = 6 per treatment) received either (c) saline, (d) 2.5 μg GnRH, (e) 25 μg GnRH or (f) 2.5 μg GnRH-A, intravenously. On day 105, 1 mg oestradiol was injected (intramuscularly) into control (n = 6) and GnRH-immunised anoestrous heifers with either low (13.4 ± 1.9% binding at 1:640; n = 6) or high GnRH antibody titres (33.4 ± 4.8% binding; n = 6). Data were analysed by ANOVA. Mean plasma LH and FSH concentrations on day 69 were higher (P < 0.05) in control than in GnRH-immunised heifers (3.1 ± 0.16 vs. 2.5 ± 0.12 ng LH ml⁻¹ and 22.5 ± 0.73 vs. 17.1 ± 0.64 ng FSH ml⁻¹, respectively). The number of LH pulses was higher (P < 0.05) in control than in GnRH-immunised heifers on day 69 (3.4 ± 0.45 and 1.0 ± 0.26 pulses per 6 h, respectively). On day 70, 2.5 μg GnRH increased (P < 0.05) LH concentrations in control but not in GnRH-immunised heifers, while both 25 μg GnRH and 2.5 μg GnRH-A increased (P < 0.05) LH concentrations in GnRH-immunised heifers, and 2.5 μg GnRH-A increased LH in controls. FSH was increased (P < 0.05) in GnRH-immunised heifers following 25 μg GnRH and 2.5 μg GnRH-A. Oestradiol challenge increased (P < 0.05) LH concentrations during the 13–24 h period after challenge with a greater (P < 0.05) increase in control than in GnRH-immunised heifers. FSH concentrations were decreased (P < 0.05) for at least 30 h after oestradiol challenge. In conclusion, GnRH immunisation decreased LH pulsatility and mean LH and FSH concentrations. GnRH antibodies neutralised low doses of GnRH (2.5 μg), but not high doses of GnRH (25 μg) and GnRH-A (2.5 μg). GnRH immunisation decreased the rise in LH concentrations following oestradiol challenge.     

Ovarian estradiol modulates the stimulatory effect of ovulation-inducing factor (OIF) on pituitary LH secretion in llamas

This study was designed to: 1) characterize the effect of ovulation-inducing factor (OIF) on pituitary LH secretion in ovariectomized (OVX) llamas; and 2) determine the effect of OIF on LH secretion in OVX llamas pretreated with estradiol-17β (E-17β) or estradiol benzoate (EB). In Experiment 1, intact and OVX llamas (n = 5 or 6 per group) were assigned to a two by two factorial design: 1) Intact llamas treated with 1 mL of phosphate buffered saline (PBS); 2) Intact llamas treated with 1 mg of purified OIF; 3) OVX llamas treated with 1 mL of PBS; or 4) OVX llamas treated with 1 mg of purified OIF. In Experiment 2, intact and OVX llamas (n = 5 or 6 per group) were randomly assigned to the following groups: 1) Intact llamas treated with 1 mg of purified OIF; 2) OVX llamas treated with 1.0 mL of PBS; 3) OVX llamas treated with 1.0 mg of purified OIF; 4) OVX llamas primed with E-17β, followed by 1.0 mg of purified OIF. Experiment 3 was similar as described for Experiment 2, except that priming was done with EB. In Experiment 1, animal category by treatment and animal category by treatment by time interactions tended (P = 0.08) to affect LH concentration. The effect of OIF on LH released was partly restored (P < 0.05), to the values observed for the intact OIF-treated females, when OVX llamas were primed with E-17β or BE (Experiments 2 and 3). We concluded that peripheral estradiol concentrations in llamas partially modulates the effect of OIF on pituitary LH secretion; however, other ovarian factor(s) could also participate in this modulatory action.     

Detecting TSH-receptor antibodies with the recombinant TBII assay: technical and clinical evaluation.

We evaluated the technical robustness of the new commercial TBII assay using human recombinant TSH-R, and describe its use for the clinician in the routine laboratory. The human recombinant TSH-R assay (DYNOtest TRAK human) was compared to a conventional TBII assay (TSH-REZAK). Specificity was adjusted at 99.1% for both assays by ROC plot analysis including 113 healthy individuals. Sensitivity in 115 patients with active Graves' Disease (GD) was 98.2% for the DYNOtest TRAK human compared to 68.4% for the TSH-REZAK (p<0.0001). Comparison of the ROC-calculated cut off confirmed the recommended cut-off for the DYNOtest TRAK human, since 11% inhibition of tracer equals 1 IU/L, which is recommended as the grey zone. At the recommended cut-off (2 IU/L, 22% inhibition), the sensitivity is still 93.9% with 100% specificity. The ROC plot-derived cut-off of the TSH-REZAK (4.4%, 2 to 10 U/L) is below the grey zone of 10-15 U/L. At the recommended cut off of 15 U/L, the sensitivity is 43.0% with a specificity of 100%. Both assays showed a good correlation (r = 0.82, p < 0.0001); however, assay comparison revealed a constant bias in favour of the DYNOtest TRAK human. Applying the ROC plot-derived cut-off of 11 % inhibition (1 IU/L) for the DYNOtest TRAK human, we found 15 of 50 patients with autoimmune thyroiditis (AIT) and 6 of 23 patients with goitre (all < 1.5 IU/L). These patients would have been missed using the recommended 2 IU/L. The difference in sensitivity between the DYNOtest TRAK human and the TSH-REZAK was highly significant in the GD group, but not in other groups, indicating that the DYNOtest TRAK human has a higher sensitivity for GD without compromising specificity. In summary, the proposed high sensitivity of the new TBII assay using human recombinant TSH-R could be confirmed with the commercial product. This method offers a clear advantage over conventional TBII assays to confirm or exclude the diagnosis of GD. The recommended cut-off is very stringent, and until we have more information on the clinical relevance of low-level TBII between 1 and 1.5 IU/L, those patients should be monitored for the development of autoimmune thyroid disease.     

Ovarian follicular wave synchronization and pregnancy rate after fixed-time natural mating in llamas

The study was designed to compare the efficacy of treatments intended to induce follicular wave synchronization among llamas (Experiment 1), and to determine the effect of these treatments on pregnancy rates after fixed-time natural mating (Experiment 2). In Experiment 1, llamas were treated with: (1) saline (control, n=20); (2) estradiol and progesterone (E/P, n=20); (3) LH (LH, n=20); or (4) transvaginal ultrasound-guided follicle ablation (FA, n=20). The ovarian response was monitored daily by transrectal ultrasonography. The intervals from treatment to follicular wave emergence and to the day on which the new dominant follicle reached ≥7 mm, respectively, did not differ between the LH (2.1±0.3 days and 5.2±0.5 days, respectively) and FA groups (2.3±0.3 days and 5.0±0.5 days), but both were shorter (P<0.05) and less variable (P<0.01) than in the control group (5.5±1.0 days and 8.4±2.0 days), while the E/P group (4.5±0.8 days and 7.7±0.5 days) was intermediate. In Experiment 2, llamas at unknown stages of follicular development were assigned randomly to control, E/P, and LH groups (n=30 per group). A single, fixed-time natural mating was permitted 10–12 days after treatment. Ovulation rates did not differ among groups (control, 93%; E/P, 90%; LH, 90%; P=0.99), but the pregnancy rate was higher (P<0.05) for synchronized llamas (LH and E/P groups combined, 41/54) than for non-synchronized llamas (control group, 15/28). In conclusion, LH and FA treatments were most effective for inducing follicular wave synchronization, while E/P treatment was intermediate. Synchronization treatments did not influence ovulation rate subsequent to fixed-time natural mating, but a higher pregnancy rate in synchronized than non-synchronized llamas warrants critical evaluation of the effects of follicular status on the developmental competence of the contained oocyte.     

Iodine status of Turkish pregnant women and their offspring: A national cross-sectional survey

BackgroundThis national cross-sectional survey aimed to assess the iodine status in pregnant women and their offspring, and also to demonstrate regional differences by measuring urinary iodine concentration (UIC). For each woman and her newborn a questionnaire was prepared with basic facts as age, parity number or birth weight and additional information regarding thyroid diseases, use of iodized salt in the household, extra iodine supplementation during pregnancy, education level and wage income.MethodsThe target population represented 1444 pregnant women who gave birth between January 1 st, 2018 and 2019, and their offspring. Iodine deficiency for pregnant women and their offspring were defined as urine iodine level <150 μg/L and <100 μg/L, respectively. Results are given as median (25th–75th percentile).ResultsThe median UIC in the group of pregnant woman was 94 (52–153) μg/L. Within the sample of 1444 pregnant women, UIC indicative of mild iodine deficiency (100−149 μg/L) was present in 21 % (n = 306), moderate deficiency (50−99 μg/L) in 30 % (n = 430), and severe deficiency (<50 μg/L) in 23 % (n = 337). This study showed a prevalence of 74 % of iodine deficiency in Turkish pregnant woman. The median UIC in the group of offspring was 96 (41−191) μg/L. Within the new-borns, UIC indicative of mild iodine deficiency (50−99 μg/L) was present in 22 % (n = 323), moderate deficiency (20−49 μg/L) in 15 % (n = 222), and severe deficiency (<20 μg/L) in 13 % (n = 192). This survey showed a prevalence of 51 % of iodine deficiency in Turkish new-borns. Pregnant women with lower socioeconomic and education level, lower access to household iodized salt, lower rates of exposure to povidone-iodine containing skin disinfectant, higher parity and higher iodine deficiency had higher rates of iodine deficiency in their offspring. Regional differences were observed both in mothers and their offspring concerning their iodine status.ConclusionsOur findings suggest that iodine deficiency is still an important public health problem in Turkey. More drastic measures should be taken to decrease these important iodine deficiencies, both in pregnant women and in their offspring.     

Pituitary response to repeated copulation and/or gonadotropin-releasing hormone administration in llamas and alpacas.

The response of the pituitary gland and ovary to repeated copulatory periods and/or gonadotropin-releasing hormone (GnRH, i.v. 1000 micrograms) administration was determined in llamas and alpacas. Eighty adult females (41 llamas and 39 alpacas with ovulatory follicles) were divided into three general groups for each species as follows: copulation (one or two copulations at either 6- or 24-h intervals) GnRH treatment (one or two treatments at either 6- or 24-h intervals), and combined treatment (copulation followed by GnRH treatment, or GnRH followed by copulation at either 6- or 24-h intervals). An additional control (nontreated) group was composed of 4 llamas and 4 alpacas. The first copulation or treatment with GnRH provoked LH release sufficient to cause ovulation in most of the females (alpacas, 89%; llamas, 92%); urinary pregnanediol glucuronide values, used to verify ovulation, were significantly elevated 48 h after copulation and/or GnRH treatment. A second stimulus, copulation or GnRH, provoked no LH response with concentrations similar to those in nontreated controls and in females not ovulating. Llamas and alpacas thus were refractory to a second copulatory or GnRH stimulus with regard to LH release for up to 24 h following an initial ovulatory release of LH.     

The activity of ferulic and gallic acids in biofilm prevention and control of pathogenic bacteria

The activity of two phenolic acids, gallic acid (GA) and ferulic acid (FA) at 1000 μg ml(-1), was evaluated on the prevention and control of biofilms formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. In addition, the effect of the two phenolic acids was tested on planktonic cell susceptibility, bacterial motility and adhesion. Biofilm prevention and control were tested using a microtiter plate assay and the effect of the phenolic acids was assessed on biofilm mass (crystal violet staining) and on the quantification of metabolic activity (alamar blue assay). The minimum bactericidal concentration for P. aeruginosa was 500 μg ml(-1) (for both phenolic acids), whilst for E. coli it was 2500 μg ml(-1) (FA) and 5000 μg ml(-1) (GA), for L. monocytogenes it was >5000 μg ml(-1) (for both phenolic acids), and for S. aureus it was 5000 μg ml(-1) (FA) and >5000 μg ml(-1) (GA). GA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. FA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. Colony spreading of S. aureus was completely inhibited by FA. The interference of GA and FA with bacterial adhesion was evaluated by the determination of the free energy of adhesion. Adhesion was less favorable when the bacteria were exposed to GA (P. aeruginosa, S. aureus and L. monocytogenes) and FA (P. aeruginosa and S. aureus). Both phenolics had preventive action on biofilm formation and showed a higher potential to reduce the mass of biofilms formed by the Gram-negative bacteria. GA and FA promoted reductions in biofilm activity >70% for all the biofilms tested. The two phenolic acids demonstrated the potential to inhibit bacterial motility and to prevent and control biofilms of four important human pathogenic bacteria. This study also emphasizes the potential of phytochemicals as an emergent source of biofilm control products.     

Sensitivity of Certain Porcine and Bovine Mycoplasmas to Antimicrobial Agents in a Liquid Medium Test Compared to a Disc Assay

The sensitivity of some porcine and bovine mycoplasmas to potent antimicrobial agents was examined. Minimal inhibitory concentration (MIC) values were estimated for M. hyosynoviae, M. hyopneumoniae, M. dispar and M. bovis against enrofloxacin, lincomycin, tetracycline, tiamulin and tylosin, in a liquid medium test and in a disc assay. All 6 examined strains of each species and the respective type strains were significantly inhibited. The greatest sensitivity was noted for tiamulin against strains of M. hyosynoviae with a final MIC50 broth value of 0.025 µg ml−1 and disc value of 0.03 µg per disc. Enrofloxacin was found very potent against M. hyopneumoniae with a final MIC50 of 0.025 µg ml−1 and 0.1 µg per disc, and for M. dispar with 0.05 µg ml−1 and 0.03 µg per disc. Most disc assay estimates in ug per disc were similar to or moderately greater than corresponding final broth figures in µg ml−1. It may be possible to convert observed disc assay values into representative final broth MIC values for use in the clinic.     

Surfactant induces ROS-mediated cell membrane permeabilization for the enhancement of mannatide production

A surfactant was a substance that had an important influence on the excretion of intracellular substances. In this work, it was found that cetyltrimethylammonium bromide (CTAB) inhibited cell viability but increased the mannatide production by optimizing the addition time. Results revealed that CTAB changed cell surface properties (cell surface hydrophobicity and Zeta potential was increased from 3% to 14% and −14.5 mV to −10.2 mV, respectively) and permeabilized cell membrane (intercellular ATP content was decreased from 28.599 μg/g to 9.737 μg/g while extracellular ATP content was increased from 33.051 μg/g to 82.809 μg/g; the concentrations of K⁺ and Ca²⁺ were increased to 3.9 mg/L and 2.1 mg/L, respectively; membrane potential was formed). Moreover, the images of scanning electron micrographs indicated distinct morphological changes and disruption on the surface of the cells. Further pyridinium iodides staining showed CTAB could induce cell apoptosis from 4.24% to 31% with increasing the relative intracellular reactive oxygen species (ROS) from 0.11% to 7.31%. It is the most noteworthy that the addition of CTAB increased the mannatide production to 1.46 g/L, 98.6% higher than that of untreated cells. Consequently, the utilization of CTAB for the preparation of mannatide provide theoretical foundation for the further large-scale production.     

A rapid radioimmunoassay for determining plasma concentrations of LH in dogs

A heterologous dog LH radioimmunoassay was modified to provide accurate results for LH concentrations in blood plasma of dogs within 3-4 h. This assay utilizes radioiodinated ovine LH (LER-1056-C2), antiserum against ovine LH (GDN-15) at a final dilution of 1:48,000 and dog LH (LER-1685-1) as standard. A 60-min incubation, including a 30-min delay in the addition of tracer, was carried out at 37 degrees C. The free and antibody-bound hormone were separated by addition of a Micro Sepharose bead suspension containing anti-gamma-globulin, followed by incubation at room temperature for 30 min. The minimum detectable concentration in this assay, calculated from the precision profile, was 1.5 micrograms/l. The amount of dog LH needed to cause 50% reduction of the initial binding was 1.57 +/- 0.13 ng/tube (15.7 micrograms/l for 100-microliters samples). Daily blood samples were collected in heparinized tubes from the cephalic vein of 5 pointer and 7 beagle bitches from the onset of pro-oestrus until 3-4 days after either the last mating or artificial insemination with frozen semen or until metoestrus. Samples were assayed for LH content by the short and normal incubations as well as for progesterone and oestradiol-17 beta content. In all bitches plasma concentrations of progesterone increased rapidly within 1 week after the LH peak which indicates that they had ovulated. Comparison of the short (1.5 h) with the normal (24 h) incubation system resulted in a regression equation: y = 1.0 + 0.7 x (r = 0.95, n = 153 samples).(ABSTRACT TRUNCATED AT 250 WORDS)     

9-fluorenylmethyl chloroformate as a fluorescence-labeling reagent for derivatization of carboxylic acid moiety of sodium valproate using liquid chromatography/tandem mass spectrometry for binding characterization: a human pharmacokinetic study

In High Performance Liquid Chromatographic (HPLC) determination of chemicals with acidic functions, different labeling agents are used to improve sensitivity of the assay. 9-Fluorenylmethyl chloroformate (FMOC-Cl), on the other hand, is a suitable labeling agent, which reacts with both primary and secondary amines and less readily with hydroxyl groups in alkaline conditions. However, the reagent has not been applied in labeling of chemicals with acidic function yet. In this study which is the first report on application of FMOC-Cl in derivatization and analysis of a drug with acidic function, valproic acid (VPA), one of a series of fatty carboxylic acids with anticonvulsant activity, was derivatized using the reagent and quantified in serum samples by HPLC with fluorescence detection. In addition, to document the reaction between the labeling agent and carboxylic acid moiety of the drug, we developed a liquid chromatography-tandem MS/MS (LC-MS/MS) method. Following liquid-liquid extraction, derivatization of the drug and an internal standard was achieved in alkaline medium. The elute was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The present method is more sensitive comparing with other published HPLC procedures for analysis of VPA. The assay is sensitive enough to measure drug levels obtained in human single dose studies with a limit of quantification of 0.01 μg/mL. Also the method is linear over the concentrations range of 0.01-32 μg/mL of VPA in human serum using 100 μL serum sample and 5 μL injection. The coefficient variation values of both inter and intra day analysis were less than 12% and the percentage error was less than 4%. The method performance was studied and the validated procedure applied in a randomized cross-over bioequivalence study of two different VPA preparations in 24 healthy volunteers.     

Growth and nitrogen removal characteristics of Halomonas sp. B01 under high salinity

At present, the nitrogen (N) removal efficiency of the microbial treatment in the high-salinity nitrogenous wastewaters is relatively low. Study on the N removal behavior and properties of moderately halophilic bacteria Halomonas under high salinity is of great significance for the microbial treatment of high-salinity nitrogenous wastewater. The response mechanism of Halomonas sp. B01 to high osmotic pressure stress was investigated by measuring the compatible solute ectoine concentration and superoxide dismutase (SOD) activity. The salt tolerance during growth and N removal of the strain was evaluated by measuring the activities of growth-related and N removal–related enzymes and the mRNA expression abundance of ammonia monooxygenase-encoding gene (amoA). The process of simultaneous heterotrophic nitrification and aerobic denitrification (SND) under high salinity was described by measuring the concentration of inorganic N. Halomonas sp. B01 synthesized ectoine under NaCl stress, and the intracellular ectoine concentration increased with increased NaCl concentration in the growth medium. When the NaCl concentration of the medium reached 120 g L−1, the malondialdehyde concentration and SOD activity were significantly increased to 576.1 μg mg−1 and 1.7 U mg−1, respectively. The growth-related and N removal–related enzymes of the strain were active or most active in medium with 30–60 g L−1 NaCl. The amoA of the strain cultured in medium with 60 g L−1 NaCl had the highest mRNA expression abundance. In the N removal medium containing 60 g L−1 NaCl and 2121 mg L−1 NH4+-N, SND by Halomonas sp. B01 was performed over 96 h and the N removal rate reached 98.8%. In addition to the protective mechanism of synthetic compatible solutes, Halomonas sp. B01 had the repair mechanism of SOD for lipid peroxidation. The growth-related and N removal–related enzymes of the strain were most active at a certain salt concentration; amoA also had the highest mRNA expression abundance under high salinity. Halomonas sp. B01 could efficiently perform N removal by SND under high salinity.     

Ovulation-inducing factor in the seminal plasma of alpacas and llamas

Studies were conducted to document the existence of an ovulation-inducing factor in the seminal plasma of alpacas (experiment 1) and llamas (experiment 2) and to determine if the effect is mediated via the pituitary (experiment 3). In experiment 1, female alpacas (n = 14 per group) were given alpaca seminal plasma or saline intramuscularly or by intrauterine infusion. Only alpacas that were given seminal plasma i.m. ovulated (13/ 14, 93%; P < 0.01). In experiment 2, ovulation was detected in 9/10 (90%) llamas at a mean of 29.3 +/- 0.7 h after seminal plasma treatment. Plasma progesterone concentrations were maximal by Day 9 and were at nadir by Day 12 posttreatment. In experiment 3, female llamas were given llama seminal plasma, GnRH, or saline i.m., and ovulation was detected in 6/6, 5/ 6, and 0/6 llamas, respectively (P < 0.001). Treatment was followed by a surge (P < 0.01) in plasma LH concentration beginning 15 min and 75 min after treatment with GnRH and seminal plasma, respectively. Plasma LH remained elevated longer in the seminal plasma group (P < 0.05) and had not yet declined to pretreatment levels after 8 h. Compared with the GnRH group, corpus luteum tended to grow longer and to a greater diameter (P = 0.1) and plasma progesterone concentration was twice as high in the seminal plasma group (P < 0.01). Results document the existence of a potent factor in the seminal plasma of alpacas and llamas that elicited a surge in circulating concentrations of LH and induced an ovulatory and luteotropic response.     

Association of lead and cadmium exposure with kidney stone incidence: A study on the non-occupational population in Nandan of China

BackgroundEnvironmental lead (Pb) and cadmium (Cd) pollution has been considered a risk factor in the etiology of kidney stones. However, the association between Pb and Cd exposure and kidney stone incidence has yet to be determined.ObjectivesThis study aimed to determine a possible the association between kidney stones with Pb and Cd exposure (alone or combined) in a non-occupational population.MethodsPb and Cd contaminations in soil-plant system were determined by flame atomic absorption spectrophotometry. Health risk assessment of dietary Pb or Cd intake from rice and vegetables were calculated. Kidney stones were diagnosed with urinary tract ultrasonography. Urinary cadmium (UCd) and blood lead (BPb) levels were determined by graphite-furnace atomic absorption spectrometry. Multivariate logistic regression models were constructed.ResultsThe hazard indexes (HI) of Pb and Cd were 7.91 and 7.31. The odds ratio (OR) was 2.83 (95 %CI:1.38−5.77) in males with high BPb (BPb ≥ 100 μg/L), compared with those with low BPb (BPb＜100 μg/L). Compared to those with low BPb and low UCd (BPb＜100 μg/L and UCd＜2 μg/g creatinine), the ORs were 2.58 (95 % CI:1.17−5.70) and 3.43 (95 % CI:1.21−9.16) in females and males with high BPb and high UCd (BPb ≥100 μg/L and UCd ≥2 μg/g creatinine), respectively. The OR was 3.16 (95 % CI:1.26−7.88) in males with high BPb and low UCd (BPb ≥ 100 μg/L and UCd ＜2 μg/g creatinine), compared to those with low BPb and low UCd.ConclusionsKidney stones incidence was increased by high Pb exposure in males, and by Pb and Cd co-exposure in males and females.     

Liquid chromatographic–mass spectrometric determination of the novel,recently identified thioTEPA metabolite,thioTEPA-mercapturate,in urine

An assay for the quantitative determination of the mercapturic acid conjugate of N,N′,N″-triethylenethiophosphoramide (thioTEPA-mercapturate) in human urine has been developed. ThioTEPA-mercapturate, a recently identified metabolite of the alkylating anticancer agent thioTEPA, was analyzed using LC–MS and with direct sample injection. Sulphadiazine was used as internal standard. Linearity was accomplished in the therapeutic relevant range of 1–25 μg/ml; recovery was 84% and both accuracy and precision were less than 20% for the lower limit of quantification (1.0 μg/ml) and less than 10% for the other concentration levels. The stability of thioTEPA-mercapturate proved to be satisfactory over a period of 2 months, when kept at −80°C. ThioTEPA-mercapturate urine concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples.     

Determination of dexamethasone and dexamethasone sodium phosphate in human plasma and cochlear perilymph by liquid chromatography/tandem mass spectrometry

A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of dexamethasone (Dex) and dexamethasone sodium phosphate (Dex SP) in plasma and human cochlear perilymph. After proteins were precipitated with a mixture of acetonitrile and methanol, Dex, Dex SP and flumethasone, the internal standard, were resolved on a C18 column using gradient elution of 5 mM ammonium acetate and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.5-500 μg/L (r>0.99), bias was <±10%, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.5 μg/L for both Dex and Dex SP. The assay has been used successfully in a clinical pharmacokinetics study of Dex and Dex SP in cochlear perilymph and plasma.     

Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 μg/mL, while total EPA and DHA concentration range was 0.5-250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.