A GC-EI-MS/MS Method for the Determination of 15 Polybrominated Diphenyl Ethers (PBDEs) in Fish and Shellfish Tissues

This paper describes the development and validation of an analytical method to determine 15 polybrominated diphenyl ethers (PBDEs) in fish and shellfish tissues with gas chromatography coupled to triple quadrupole mass spectrometry using electron impact source (GC-EI-MS/MS). A QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe)-like procedure was applied; the extract was cleaned up on an acidic Extrelut-NT3 column connected on top of a silica cartridge and further purified with gel permeation chromatography (GPC). The SIM acquisition proved to yield better results than traditional MRM approach for octa-, nona-, and deca-brominated congeners. The validation study was carried out in inter-laboratory conditions. The average recoveries were in the range 66–118%, with relative standard deviations generally lower than or equal to 22%. Quality assurance and quality control practices were thoroughly described and highlighted. These long and tedious practices are fundamental to obtain reliable quantitative data during food monitoring programs and, therefore, to support daily intake assessments. Finally, the procedure was applied to the analysis of marine fish samples collected in local markets. Mackerel and mullet were the most contaminated species.     

Effects of plasma total ammonia content and pH on urea excretion in Nile tilapia

Nile tilapia (Oreochromis niloticus) were infused with ammonium salts, acid, and base to investigate the effects of changes in arterial plasma total ammonia content (Tamm) and pH (pHa) on plasma urea-nitrogen (urea-N) levels and urea-N excretory fluxes (Jurea-N). The tilapia did not possess a functional hepatic ornithine urea-cycle (no significant carbamyl phosphate synthetase III activity). Infused substances were dissolved in a saline vehicle and injected twice (5 mL kg-1), the first infusion to "prime" the animal and promote a more marked response to the second infusion, given 2.5 h later. The results reported are those of the second infusion. Infusion of 200 mM NH4Cl increased Tamm, reduced pHa, and increased plasma urea-N and Jurea-N. Two hundred mM NH4HCO3 increased Tamm and arterial plasma total CO2 content (TaCO2), reduced pHa, and increased Jurea-N. Fifty mM HCl reduced pHa but had no effects on urea dynamics. Fifty mM NaOH increased pHa, plasma urea-N levels, and Jurea-N. Two hundred mM NaHCO3 increased pHa, TaCO2, plasma urea-N levels, and Jurea-N. Infusion of the saline vehicle was without effect. The results indicate that ammonia loading and plasma alkalosis both stimulate urea excretion in uricolytic fish. The responses to hyperammonemia or alkalosis were not modified when combined with elevated plasma bicarbonate levels.     

Transferable, plasmid-mediated vanB-type glycopeptide resistance in Enterococcus faecium

An approximately 60-kb transferable, vanB-carrying plasmid has been identified in a clinical Enterococcus faecium strain. A similar plasmid has been observed in an unrelated E. faecium strain, suggesting that plasmid transfer of vanB operons occurs in nature and plays a role in the dissemination of VanB-type resistance among strains of E. faecium.     

Repetitive axonal stimulation of the same single motor unit: a longitudinal tracking study

We elicited three single motor unit action potentials (S-MUAPs) via multiple point stimulation and subjected them to repetitive stimulation (RS) in 3 healthy subjects. We tracked each S-MUAP and its RS trains over two separate sessions as well as the compound motor action potential (CMAP) RS trains obtained from the same muscle following whole nerve stimulation. Repetitive axonal stimulation yields consistent results when carefully performed with minimal variation in each S-MUAP RS train from session to session, providing previously unobtainable data regarding neuromuscular junction function in the same single motor unit over time. In this small, normative sample, no significant correlation between S-MUAP and CMAP RS responses was observed.     

Dual microcolumn immunoaffinity liquid chromatography: an analytical application to human plasma proteins

Dual-column microcolumn immunoaffinity chromatography was accomplished by coupling immunoaffinity (IAC) and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis columns. The IAC support was prepared by hydrophobically adsorbing the appropriate antibody onto an octyl silica-based stationary phase contained in a 250-microns-l.d. fused-silica microcolumn. Once the antigen was captured, the antigen-subtracted human plasma could be sent to the RP-HPLC microcolumn for plasma profiling. The antigen was quantitated by desorbing it from the IAC column and transferring it to the RP-HPLC system. Human plasma profiles were generated after removal of albumin, and transferrin and alpha 1-antitrypsin were quantitated with nanogram sensitivity. All analyses were accomplished with less than 1 microL of human plasma.     

Treatment of cutout of a lag screw of a dynamic hip screw in an intertrochanteric fracture

Sixteen consecutive patients with cutout of a lag screw of a dynamic hip screw fixation in an intertrochanteric fracture were treated with reinsertion of a lag screw, bone cement supplementation in the neck-trochanter, and subtrochanteric valgus osteotomy. Postoperatively, patients were permitted to ambulate with protected weight-bearing. Fourteen patients were followed-up for at least 1 year (median 2 years; range 1-3 years), and all had a solid union. The union period took a median of 5 months, with a range of 3-7 months. Usually, union of an intertrochanteric fracture was faster than that of subtrochanteric osteotomy (P < 0.01). There were no complications of wound infection, loss of reduction, cutout of a lag screw, or osteonecrosis of the femoral head. From clinical and theoretical considerations, we conclude that despite cutout of a lag screw of a dynamic hip screw fixation being difficult to treat, out technique still can provide an excellent outcome. Therefore, we strongly recommend its wide use.     

The efficacy of oral appliances in the treatment of persistent sleep apnea after uvulopalatopharyngoplasty

Twenty-four patients who failed uvulopalatopharyngoplasty (UPPP) for obstructive sleep apnea (OSA) had an adjustable oral (Herbst) appliance made to treat the persistent apnea. Six patients discontinued the device prior to sleep evaluation. Eighteen patients had polysomnographic evaluations at baseline, post-UPPP, and with the Herbst appliance in place. The apnea-hypopnea index baseline (AHI) and arterial oxygen saturation (SaO2) nadir were 42.3+/-6.1 and 83.6+/-1.8%, respectively. There was no significant change in either parameter with surgery. With the oral appliance, the AHI fell to 15.3+/-4.4 (p < or = 0.01) and the SaO2 nadir increased to 87.9+/-1.2% (p < or = 0.05). Ten of the patients had control of the OSA with the Herbst appliance with a fall in the AHI to < 10. There were, in addition, two partial responders as defined by an AHI of <20 and a >50% fall in AHI compared with baseline and post-UPPP values. All but one of the responders and partial responders had complete resolution of subjective symptoms of daytime sleepiness with the appliance. An adjustable oral appliance appears to be an effective mode of therapy to control OSA after an unsuccessful UPPP.     

TIMP-1 contact sites and perturbations of stromelysin 1 mapped by NMR and a paramagnetic surface probe

Surfaces of the 173 residue catalytic domain of human matrix metalloproteinase 3 (MMP-3(DeltaC)) affected by binding of the N-terminal, 126 residue inhibitory domain of human TIMP-1 (N-TIMP-1) have been investigated using an amide-directed, NMR-based approach. The interface was mapped by a novel method that compares amide proton line broadening by paramagnetic Gd-EDTA in the presence and absence of the binding partner. The results are consistent with the X-ray model of the complex of MMP-3(DeltaC) with TIMP-1 (Gomis-Rüth et al. (1997) Nature 389, 77-81). Residues Tyr155, Asn162, Val163, Leu164, His166, Ala167, Ala169, and Phe210 of MMP-3(DeltaC) are protected from broadening by the Gd-EDTA probe by binding to N-TIMP-1. N-TIMP-1-induced exposure of backbone amides of Asp238, Asn240, Gly241, and Ser244 of helix C of MMP-3(DeltaC) to Gd-EDTA confirms that the displacement of the N-terminus of MMP-3(DeltaC) occurs not only in the crystal but also in solution. These results validate comparative paramagnetic surface probing as a means of mapping protein-protein interfaces. Novel N-TIMP-1-dependent changes in hydrogen bonding near the active site of MMP-3(DeltaC) are reported. N-TIMP-1 binding causes the amide of Tyr223 of MMP-3(DeltaC) bound by N-TIMP-1 to exchange with water rapidly, implying a lack of the hydrogen bond observed in the crystal structure. The backbone amide proton of Asn162 becomes protected from rapid exchange upon forming a complex with N-TIMP-1 and could form a hydrogen bond to N-TIMP-1. N-TIMP-1 binding dramatically increases the rate of amide hydrogen exchange of Asp177 of the fifth beta strand of MMP-3(DeltaC), disrupting its otherwise stable hydrogen bond.     

Screening calcaneal ultrasound and risk factors for osteoporosis among lesbians and heterosexual women

We compared calcaneal ultrasound measurements (speed of sound [SOS], broadband ultrasound attenuation [BUA], and stiffness index [SI]) of lesbian and heterosexual women to examine the medical and lifestyle risk factors for osteoporosis in each group. This was an exploratory, community-based, cross-sectional study. Subjects were mailed food frequency, health, and physical activity questionnaires. Weight, height, and calcaneal ultrasound measurements were taken at one office visit. In communities in southern and eastern Maine, 71 lesbians and 77 heterosexual women between the ages of 30 and 50 with regular menses and in good general health were the subjects. Statistical analysis used t-tests and chi-square tests to evaluate differences between study groups. Linear regression models were used to evaluate risk factors for low calcaneal ultrasound measurements. There were no differences between the lesbian and heterosexual women in age, body mass index (BMI), exercise, calcium intake, alcohol use, or calcaneal ultrasound measurements. There was a positive association between BUA and both BMI and alcohol consumption (p < 0.01). Antidepressant use significantly reduced SOS and SI (p < 0.05). There were no differences in calcaneal ultrasound measurements between lesbian and heterosexual women. BMI was strongly and positively associated with BUA. Antidepressant use in both populations was associated with a significant reduction in calcaneal bone mass. Studies are needed to define the relationship of depression and its treatment to bone mineral density and the future risk of osteoporosis.     

Functional maturation of the primate fetal adrenal in vivo: 3. Specific zonal localization and developmental regulation of CYP21A2 (P450c21) and CYP11B1/CYP11B2 (P450c11/aldosterone synthase) lead to integrated concept of zonal and temporal steroid biosynthesis

Previous studies in the primate fetal adrenal gland have indicated that the gland is comprised of three functional zones: 1) the inner fetal zone (FZ), which has the enzymes necessary for dehydroepiandrosterone sulfate (DHEAS) production beginning early in gestation; 2) the transitional zone (TZ), which possesses enzymes necessary for cortisol production; and 3) the outer, definitive zone (DZ), which appears to function as a reservoir of progenitor cells that may populate the remainder of the gland and does not acquire a steroidogenic phenotype with the capacity to produce mineralocorticoids until near term. The enzymes CYP21A2 (P450 21 hydroxylase, or P450c21), CYP11B1 (11beta hydroxylase or P450c11) and CYP11B2 (aldosterone synthase) are necessary for glucocorticoid and mineralocorticoid synthesis but have not been localized previously in an ontogenic manner in the primate fetal adrenal gland. Therefore, we used immunocytochemistry (ICC) to assess specific zonal localization and developmental regulation of CYP21A2 and CYP11B1/CYP11B2 in the human (13-24 weeks' gestation) and rhesus monkey (109 d-term) fetal adrenal gland. In the fetal rhesus, ICC was performed with and without metyrapone administration to the fetus to assess the effects of endogenously increased fetal ACTH. In the human fetal adrenal, CYP21A2 immunoreactivity (IR) was present in only a few isolated cells in the DZ but was detectable in almost all cells in the TZ and FZ. In the fetal rhesus, CYP21A2-IR was present in cells throughout the DZ and TZ and, to a lesser degree, in the FZ. Staining intensity increased with advancing gestational age and was up-regulated in the DZ and TZ, but not the FZ, of the metyrapone-treated fetuses. In the human fetal adrenal gland, CYP11B1/CYP11B2-IR was absent in the DZ but present in the TZ and FZ. In the fetal rhesus monkey adrenal, CYP11B1/CYP11B2-IR was present in all cells of the TZ and FZ but was absent from the DZ until near term. After metyrapone, CYP11B1/CYP11B2-IR was induced in the DZ and was up-regulated in the TZ and FZ. Taken together, these data indicate that in the primate fetal adrenal gland, the FZ has the capacity to synthesize DHEA and DHEAS beginning early in development, the TZ has the capacity to synthesize cortisol after midgestation, and the DZ has the capacity to synthesize mineralocorticoids, but not until near term. The spatial localization of steroid metabolizing enzymes and steroid products in the human and rhesus monkey fetal adrenal suggests analogies of the three functional zones of the fetus (DZ, TZ, and FZ) to their adult counterparts (zona glomerulosa, zona fasciculata, and zona reticularis) and their steroid products (mineralocorticoids, glucocorticoids and androgens, respectively), although the reason for the presence of CYP11B1/CYP11B2- and CYP21A2-IR in the FZ remains to be elucidated.