Plasma concentrations of delta-9-tetrahydrocannabinol in dams and fetuses following acute or multiple prenatal dosing in rats

Delta-9-tetrahydrocannabinol (THC) was administered by gastric intubation to pregnant rats to study the effects of dose-level and dosing regimen on plasma concentration in dams and fetuses. Two multiple-dose groups were administered either 15 or 50 mg/kg of delta-9-THC once daily during the last two weeks of gestation. Two acute groups were administered the same dose as above but only once on the last day of gestation. Sixty min after receiving the last dose all dams and their fetuses were sacrificed by decapitation, blood collected, centrifuged and plasma removed. Quantitative measurement of delta-9-THC in plasma was carried out using GS/MS. Among the dams, plasma concentrations covaried with dose and multiple dosing produced higher concentrations than acute, especially at the high dose. Among the fetuses, plasma concentrations were approximately 10% of those found in the dams. The fetuses from the high, multiple-dose dams similarly yielded significantly higher concentrations. These findings are discussed with respect to other studies of the placental transfer of delta-9-THC and effects of postnatal developmental.     

Enhanced concentrations of relevant markers of nitric oxide formation after exercise training in patients with metabolic syndrome

Metabolic syndrome (MetS) denotes a clustering of risk factors that may affect nitric oxide (NO) bioavailability and predispose to cardiovascular diseases, which are delayed by exercise training. However, no previous study has examined how MetS affects markers of NO formation, and whether exercise training increases NO formation in MetS patients. Here, we tested these two hypotheses. We studied 48 sedentary individuals: 20 healthy controls and 28 MetS patients. Eighteen MetS patients were subjected to a 3-month exercise training (E + group), while the remaining 10 MetS patients remained sedentary (E−group). The plasma concentrations of nitrite, cGMP, and ADMA (asymmetrical dimethylarginine; an endogenous nitric oxide synthase inhibitor), and the whole blood nitrite concentrations were determined at baseline and after exercise training using an ozone-based chemiluminescence assay, and commercial enzyme immunoassays. Thiobarbituric acid reactive species (TBA-RS) were measured in the plasma to assess oxidative stress using a fluorometric method. We found that, compared with healthy subjects, patients with MetS have lower concentrations of markers of NO formation, including whole blood nitrite, plasma nitrite, and plasma cGMP, and increased oxidative stress (all P < 0.05). Exercise training increased the concentrations of whole blood nitrite and cGMP, and decreased both oxidative stress and the circulating concentrations of ADMA (both P < 0.05). These findings show clinical evidence for lower endogenous NO formation in patients with MetS, and for improvements in NO formation associated with exercise training in MetS patients.     

Concentrations of preptin,salusins and hepcidins in plasma and milk of lactating women with or without gestational diabetes mellitus

This study was undertaken to ascertain whether human milk contains preptin, salusin-alpha (salusin-α) and -beta (salusin-β) and pro-hepcidin and hepcidin-25, and whether there are relationships between plasma and milk preptin, salusin-α and -β and pro-hepcidin and hepcidin-25 concentrations in lactating mothers with and without gestational diabetes mellitus (GDM). Blood was obtained from non-lactating women (n = 12), non-diabetic lactating women (n = 12), and GDM lactating women (n = 12). Colostrum, transitional milk, and mature milk samples were collected just before suckling from healthy and GDM lactating women. Peptides concentrations were determined by ELISA and EIA. Mammary gland tissues were screened immunohistochemically for these peptides. Women with GDM had significantly higher plasma and colostum preptin concentrations than healthy lactating women during the colostral and transitional milk period. Salusin-alpha and -beta levels in milk and plasma were lower in women with GDM. Salusin-α and -β were significantly lower in both plasma and colostrums of GDM than of healthy lactating women. Women with GDM had significantly higher colostum prohepcidin and hepcidin-25 concentrations than healthy lactating women during the colostral period. Plasma prohepcidin was also higher in women with GDM than in healthy lactating women during the colostral period, but plasma prohepcidin and hepcidin-25 levels decreased during mature milk period. Transitional milk pro-hepcidin and hepcidin-25 levels in women with GDM were higher than in healthy lactating women. All these results revealed that the mammary gland produces those peptides, which were present in milk at levels correlating with plasma concentrations.     

Effects of Plant Growth Regulators on Seed Filling,Endogenous Hormone Contents and Maize Production in Semiarid Regions

In this study, we determined whether the application of uniconazole alone or combined with ethephon could enhance the seed-filling rates in maize by regulating the endogenous hormone contents. Uniconazole was applied to the foliage at concentrations of 0 (CK), 25 (U₂₅), 50 (U₅₀) and 75 (U₇₅) mg L⁻¹ at the 12-leaf stage. In addition, uniconazole was applied to the foliage at the 12-leaf stage and ethephon at 10 days after silking stage at concentrations of 0 (CK), 25 mg L⁻¹ uniconazole + 100 mg L⁻¹ ethephon (U₂₅ + E₁₀₀), 50 mg L⁻¹ uniconazole + 200 mg L⁻¹ ethephon (U₅₀ + E₂₀₀) and 75 mg L⁻¹ uniconazole + 300 mg L⁻¹ ethephon (U₇₅ + E₃₀₀). Uniconazole applied alone or in combination with ethephon significantly improved ear characters and grain yield. Uniconazole applied alone or combination with ethephon significantly improved the dry matter accumulation in seeds and seed-filling rates. Uniconazole significantly increased the abscisic acid (ABA) and zeatin (Z) + zeatin riboside (ZR) contents of seeds, but reduced the gibberellic acid (GA) contents. The application of uniconazole combined with ethephon decreased the ABA, Z + ZR and GA contents in seeds. The ABA and Z + ZR contents were significantly positively correlated, whereas the GA content was negatively correlated with the maximum seed weight, maximum seed-filling rate and mean seed-filling rate. The application of uniconazole alone significantly improved the seed-filling rates in maize by regulating the endogenous hormone contents. Thus, we conclude that uniconazole application at 50 mg L⁻¹ in the 12-leaf stage can enhance the maize production.

     

Influence of linear alkylbenzene sulfonate and ethanol on the degradation kinetics of domestic sewage in co-digestion with commercial laundry wastewater

The influence of ethanol on the degradation kinetics of linear alkyl benzene sulfonate (LAS) and organic matter was investigated using batch experiments with different initial LAS concentrations (8.3 mg L⁻¹ to 66.9 mg L⁻¹) and biomass immobilized on sand. Data were fitted with a substrate inhibition model. Concentrations of 2.4 mg LAS L⁻¹ and 18.9 mg LAS L⁻¹ (without and with ethanol) provided the maximum LAS utilization rate by the biomass (S_bm). For LAS degradation, ethanol addition favored a lower decrease in the specific substrate utilization rate (r_obs), even at the LAS concentration usually reported as inhibitory (> 14.4 mg L⁻¹). For organic matter degradation, r_obs was higher with ethanol. Higher biomass differentiation was observed at higher LAS concentrations. With ethanol, microbial selection occurred at LAS concentrations near S_bm. At higher LAS concentrations, the dominance and diversity values did not change significantly with ethanol, whereas without ethanol, their behaviors were irregular.

     

Pharmacokinetics, safety and inducible cytokine responses during a phase 1 trial of the oral histone deacetylase inhibitor ITF2357 (givinostat)

ITF2357 (givinostat) is a histone deacetylase inhibitor with antiinflammatory properties at low nanomolar concentrations. We report here a phase I safety and pharmacokinetics trial in healthy males administered 50, 100, 200, 400 or 600 mg orally. After 50 mg, mean maximal plasma concentrations reached 104 nmol/L 2 h after dosing, with a half-life of 6.9 h. After 100 mg, maximal concentration reached 199 nmol/L at 2.1 h with a half-life of 6.0 h. Repeat doses for 7 consecutive days of 50, 100 or 200 mg resulted in nearly the same kinetics. There were no serious adverse effects (AEs) and no organ toxicities. However, there was a dose-dependent but transient fall in platelets. After 7 daily doses of 50 or 100 mg, the mean decrease in platelets of 17 and 25% was not statistically significant and returned to baseline within 14 d. Blood removed from the subjects after oral dosing was cultured ex vivo with endotoxin, and the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-1Ra, interferon (IFN)-γ and IL-10 was determined. Maximal reduction in IL-1β, TNFα, IL-6 and IFNγ was observed 4 h after dosing but returned to baseline at 12 h. There was no significant reduction in IL-1Ra or IL-10. With daily dosing, the fall in cytokine production in blood cultures observed on day 7 was nearly the same as that of the first day. We conclude that dosing of 50 or 100 mg ITF2357 is safe in healthy humans and transiently but repeatedly reduces the production of proinflammatory cytokines without affecting production of antiinflammatory cytokines.     

Prognostic significance of plasma visfatin levels in patients with ischemic stroke

Plasma visfatin concentration has been enhanced in ischemic stroke. The aim of the current investigation was to test whether determination of visfatin in plasma is associated with 6-month clinical outcomes including mortality and unfavorable outcome (modified Rankin Scale score > 2) in the patients with ischemic stroke. Between July 2009 and January 2012, plasma visfatin concentrations of 186 patients and 100 healthy individuals were quantified by enzyme-linked immunosorbent assay. Plasma visfatin concentrations were higher in patients than in healthy individuals (108.5 ± 41.1 ng/mL vs. 13.8 ± 3.9 ng/mL, P < 0.001). A logistic regression analysis selected plasma visfatin concentration as an independent predictor for 6-month clinical outcomes (both P < 0.01). Using receiver operating characteristic curve analysis, plasma visfatin concentration was found to predict 6-month clinical outcomes with the high predictive performance. The predictive value of visfatin was in the range of National Institutes of Health Stroke Scale score (both P > 0.05). Combined use of visfatin and National Institutes of Health Stroke Scale score did not improve the predictive significance (both P > 0.05). Thus, visfatin may help in the prediction of long-term clinical outcomes in patients with ischemic stroke.     

Pharmacokinetics of meloxicam in rabbits after single and repeat oral dosing

We evaluated the pharmacokinetic profile of meloxicam (0.3 and 1.5 mg/kg) given as single and repeated (once daily for 5 d) oral doses to female rabbits (n = 5/group) to define the optimal dose and dosing interval for clinical use. Clinical signs, body weight, and serum chemistry parameters (sodium, potassium, chloride, total protein, urea, creatinine, glucose, alkaline phosphatase, gamma glutamyl transferase, and alanine aminotransferase) were evaluated before and 5 d after dosing to monitor safety at the 2 dose levels in both studies. Plasma samples were collected serially, and concentrations were determined by high performance liquid chromatography. After single oral dosing at 0.3 or 1.5 mg/kg, maximal plasma concentrations of meloxicam were achieved at 6 to 8 h and were 0.14 and 0.3 microg/ml, respectively. Plasma drug levels decreased rapidly to near-undetectable levels by 24 h. There was moderate interindividual variability in plasma meloxicam concentrations with less than proportional increases in peak plasma concentration and area under the concentration curve values at the higher dose after the single and repeat dosing. The elimination half-life was approximately 8 h at both dose levels, suggesting that metabolism was not saturated. Oral clearance of meloxicam is high in rabbits, indicating rapid metabolism and elimination. There was no accumulation of meloxicam when given at 0.3 or 1.5 mg/kg for 5 d, and meloxicam was rapidly eliminated after discontinuation of dosing. Rabbits may require a dose exceeding 0.3 mg/kg given once daily to achieve optimal plasma levels of meloxicam over a 24-h interval.     

The Vitamin D Dose Response in Obesity

ObjectiveThe prevalence of vitamin D inadequacy is high in obese individuals. Determining the response of serum 25-hydroxyvitamin D (25[OH]D) to vitamin D₃ supplementation in obese and nonobese individuals may lead to concurrent recommendations for optimal vitamin D intake in these populations. The objective of this study was to determine the dose response of vitamin D₃ in subjects with a body mass index ≥ 35 kg/m².MethodsRandomized, double-blind, placebo-controlled study. This study is an extension of our previous study of vitamin D dosing in healthy adults. After an assessment of baseline 25(OH)D levels, participants were randomized to a vitamin D supplementation arm (100 μg daily if baseline 25[OH]D was < 50 nmol/L, or 50 μg daily if baseline 25[OH]D was ≥ 50 nmol/L) or placebo arm. Subjects with baseline 25(OH)D level ≥ 80 nmol/L were excluded from the study. Two months following randomization, a repeat 25(OH)D measurement was done.ResultsFinal analysis included 25 subjects (14 placebo, 11 active). At 2 months, serum 25(OH)D concentration increased to a mean of 75 nmol/L in the active group. Mean slope (i.e., vitamin D₃ response), defined as 25(OH) D change/baseline dose, was 0.398 nmol/L/μg/day.ConclusionThe dose response of vitamin D₃ (slope) in obese subjects was significantly lower (P < .03) at 0.398 nmol/L/μg/day compared to the slope in the previous study of healthy subjects (0.66 nmol/L/μg/day). These results suggest that obese individuals may require 40% higher vitamin D intake than nonobese individuals to attain the same serum 25(OH)D concentration. (Endocr Pract. 2014;20:1258-1264)     

Decreased levels of the gelsolin plasma isoform in patients with rheumatoid arthritis

Introduction  

Gelsolin is an intracellular actin-binding protein involved in cell shape changes, cell motility, and apoptosis. An extracellular gelsolin isoform, plasma gelsolin circulates in the blood of healthy individuals at a concentration of 200 ± 50 mg/L and has been suggested to be a key component of an extracellular actin-scavenging system during tissue damage. Levels of plasma gelsolin decrease during acute injury and inflammation, and administration of recombinant plasma gelsolin to animals improves outcomes following sepsis or burn injuries. In the present study, we investigated plasma gelsolin in patients with rheumatoid arthritis.     

Biotic conversion of sulphate to sulphide and abiotic conversion of sulphide to sulphur in a microbial fuel cell using cobalt oxide octahedrons as cathode catalyst

Varying chemical oxygen demand (COD) and sulphate concentrations in substrate were used to determine reaction kinetics and mass balance of organic matter and sulphate transformation in a microbial fuel cell (MFC). MFC with anodic chamber volume of 1 L, fed with wastewater having COD of 500 mg/L and sulphate of 200 mg/L, could harvest power of 54.4 mW/m², at a Coulombic efficiency of 14%, with respective COD and sulphate removals of 90 and 95%. Sulphide concentration, even up to 1500 mg/L, did not inhibit anodic biochemical reactions, due to instantaneous abiotic oxidation to sulphur, at high inlet sulphate. Experiments on abiotic oxidation of sulphide to sulphur revealed maximum oxidation taking place at an anodic potential of −200 mV. More than 99% sulphate removal could be achieved in a MFC with inlet COD/sulphate of 0.75, giving around 1.33 kg/m³ day COD removal. Bioelectrochemical conversion of sulphate facilitating sulphur recovery in a MFC makes it an interesting pollution abatement technique.

     

Lapatinib Plasma and Tumor Concentrations and Effects on HER Receptor Phosphorylation in Tumor

Purpose

The paradigm shift in cancer treatment from cytotoxic drugs to tumor targeted therapies poses new challenges, including optimization of dose and schedule based on a biologically effective dose, rather than the historical maximum tolerated dose. Optimal dosing is currently determined using concentrations of tyrosine kinase inhibitors in plasma as a surrogate for tumor concentrations. To examine this plasma-tumor relationship, we explored the association between lapatinib levels in tumor and plasma in mice and humans, and those effects on phosphorylation of human epidermal growth factor receptors (HER) in human tumors.

Experimental Design

Mice bearing BT474 HER2+ human breast cancer xenografts were dosed once or twice daily (BID) with lapatinib. Drug concentrations were measured in blood, tumor, liver, and kidney. In a randomized phase I clinical trial, 28 treatment-naïve female patients with early stage HER2+ breast cancer received lapatinib 1000 or 1500 mg once daily (QD) or 500 mg BID before evaluating steady-state lapatinib levels in plasma and tumor.

Results

In mice, lapatinib levels were 4-fold higher in tumor than blood with a 4-fold longer half-life. Tumor concentrations exceeded the in vitro IC₉₀ (~ 900 nM or 500 ng/mL) for inhibition of HER2 phosphorylation throughout the 12-hour dosing interval. In patients, tumor levels were 6- and 10-fold higher with QD and BID dosing, respectively, compared to plasma trough levels. The relationship between tumor and plasma concentration was complex, indicating multiple determinants. HER receptor phosphorylation varied depending upon lapatinib tumor concentrations, suggestive of changes in the repertoire of HER homo- and heterodimers.

Conclusion

Plasma lapatinib concentrations underestimated tumor drug levels, suggesting that optimal dosing should be focused on the site of action to avoid to inappropriate dose escalation. Larger clinical trials are required to determine optimal dose and schedule to achieve tumor concentrations that maximally inhibit HER receptors.

Trial Registration

Clinical Trial Registration: NCT00359190     

Tissue Distribution of Anidulafungin in Neonatal Rats

Anidulafungin, an echinocandin, is currently approved for treatment of fungal infections in adults. There is a high unmet medical need for treatment of fungal infections in neonatal patients, who may be at higher risk of infections involving bone, brain, and heart tissues. This in vivo preclinical study investigated anidulafungin distribution in plasma, bone, brain, and heart tissues in neonatal rats. Postnatal day (PND) 4 and PND 8 Fischer (F344/DuCrl) rats were dosed subcutaneously once with anidulafungin (10 mg/kg) or once daily for 5 days (PND 4–8). Plasma and tissue samples were collected and anidulafungin levels were measured by liquid chromatography‐tandem mass spectrometry. The mean plasma C_max and AUC_0‐24 values were consistent with single‐dose plasma pharmacokinetics (dose normalized) reported previously for adult rats. Observed bone concentrations were similar to plasma concentrations regardless of dosing duration, with bone‐to‐plasma concentration ratios of approximately 1.0. Heart concentrations were higher than plasma, with heart to plasma concentration ratios of 1.3‐ to 1.8‐fold. Brain concentrations were low after single dose, with brain‐to‐plasma concentration ratio of approximately 0.23, but increased to approximately 0.71 after 5 days of dosing. Tissue concentrations were nearly identical after single‐dose administration in both PND 4 and PND 8 animals, indicating that anidulafungin does not appear to differentially distribute in this period in neonatal rats. In conclusion, anidulafungin distributes to bone, brain, and heart tissues of neonatal rats; such results are supportive of further investigation of efficacy against infections involving bone, brain, and heart tissues.     

High-dose vitamin C supplementation increases skeletal muscle vitamin C concentration and SVCT2 transporter expression but does not alter redox status in healthy males

Antioxidant vitamin C (VC) supplementation is of potential clinical benefit to individuals with skeletal muscle oxidative stress. However, there is a paucity of data reporting on the bioavailability of high-dose oral VC in human skeletal muscle. We aimed to establish the time course of accumulation of VC in skeletal muscle and plasma during high-dose VC supplementation in healthy individuals. Concurrently we investigated the effects of VC supplementation on expression levels of the key skeletal muscle VC transporter sodium-dependent vitamin C transporter 2 (SVCT2) and intramuscular redox and mitochondrial measures. Eight healthy males completed a randomized placebo-controlled, crossover trial involving supplementation with ascorbic acid (2×500 mg/day) over 42 days. Participants underwent muscle and blood sampling on days 0, 1, 7, and 42 during each treatment. VC supplementation significantly increased skeletal muscle VC concentration after 7 days, which was maintained at 42 days (VC 3.0±0.2 (mean±SEM) to 3.9±0.4 mg/100 g wet weight (ww) versus placebo 3.1±0.3 to 2.9±0.2 mg/100 g ww, p=0.001). Plasma VC increased after 1 day, which was maintained at 42 days (VC 61.0±6.1 to 111.5±10.4 µmol/L versus placebo 60.7±5.3 to 59.2±4.8 µmol/L, p<0.001). VC supplementation significantly increased skeletal muscle SVCT2 protein expression (main treatment effect p=0.006) but did not alter skeletal muscle redox measures or citrate synthase activity. A main finding of our study was that 7 days of high-dose VC supplementation was required to significantly increase skeletal muscle vitamin C concentration in healthy males. Our findings implicate regular high-dose vitamin C supplementation as a means to safely increase skeletal muscle vitamin C concentration without impairing intramuscular ascorbic acid transport, antioxidant concentrations, or citrate synthase activity.     

Elevated Fructosamine Levelsin Acquired Immunodeficiency Syndrome

ObjectiveTo investigate whether the mechanism of increased glycation in acquired immunodeficiency syndrome (AIDS) is due to an alteration in a circulatory plasma enhancer.MethodsWe assessed glycation of serum protein and hemoglobin in patients with AIDS without altered carbohydrate metabolism. Fasting concentrations of glucose, ethanol, vitamin E, fructosamine, hemoglobin, hemoglobin A1c (A1C), and partial pressure of alveolar oxygen (Pao₂) were determined in 50 men with AIDS and in 25 age-matched healthy men in whom normal glucose tolerance was established by oral glucose tolerance tests.ResultsFasting serum glucose was not significantly different between the men with AIDS (87 ± 4 mg/dL) and the healthy male volunteers (84 ± 6 mg/dL); however, A1C (6.9 ± 0.2%) and serum fructosamine levels (288 ± 15 μmol/L) were significantly higher (P < .01) in the patients with AIDS than in the normal subjects (A1C, 5.6 ± 0.1%; fructosamine, 204 ± 14 μmol/L). Moreover, both A1C and fructosamine concentrations were significantly higher (P < .01) in the patients with AIDS than in the normal subjects divided into subgroups on the basis of fasting plasma glucose concentrations (70 to 79 mg/dL, 80 to 89 mg/dL, and 90 to 99 mg/dL). None of the study participants had anemia (hemoglobin < 12 g/dL) or hypoxia (Pao₂ < 95 mm Hg), and serum ethanol was undetectable. Furthermore, vitamin E concentrations were not significantly different between the patients with AIDS (25 ± 3 mg/L) and the normal subjects (22 ± 4 mg/L).ConclusionOn the basis of this study, glycation of some circulating proteins appears to be enhanced in AIDS and may be induced by an undetermined plasma enhancer, inasmuch as known circulating factors promoting glycation were absent. (Endocr Pract. 2008;14:686-690)     

Pharmacological and Host Considerations Surrounding Dose Selection and Duration of Therapy with Echinocandins

Caspofungin, micafungin and anidulafungin are antifungal drugs with excellent safety profiles. Dosing regimens and treatment durations must be appropriate for optimal patient outcomes. Overall, factors that affect dosing of all three drugs are similar. Drug-specific properties, including in vitro concentration-dependent antifungal activity, activity against fungal biofilms, and pharmacokinetic and pharmacodynamic parameters influence dose selection and duration of therapy. Dosing strategies that provide “unbound” plasma drug concentrations exceeding the minimum inhibitory concentration (or minimum effective concentration) of the fungus are essential. Patient weight, age and illness severity are also important considerations for adequate exposure to drug: individuals >66 kg, pediatric patients and the critically-ill clear drug at higher rates although drug product information guidelines do not recommend for these populations to receive doses higher than those currently used. Clinical studies of treatment of, and prophylaxis against, Candida and Aspergillus infection indicate that currently recommended dosing regimens are adequate in most instances.     

Modeling of growth kinetics for <Emphasis Type="Italic">Pseudomonas putida</Emphasis> during toluene degradation

Glucose has been often used as a secondary substrate to enhance the degradation of primary substrate as well as the increase of biomass, especially for the inhibitory range of substrate concentration. In this study, we investigated the effect of glucose concentration on growth kinetics of Pseudomonas putida during toluene degradation for a wide concentration range (60–250 mg/l). Batch microcosm studies were conducted in order to monitor bacterial growth for three different initial concentrations (2, 5, 10 mg/ml) of glucose for a given toluene concentration. Modeling of growth kinetics was also performed for each growth curve of glucose dose using both Monod and Haldane kinetics. Batch studies revealed that bacterial growth showed a distinct inhibitory phase above some limit (∼170 mg/l) for the lowest (2 mg/ml) glucose dose, but the degree of inhibition decreased as the glucose dose increased, leading to three different growth patterns. The bacterial growth followed each of the modified Wayman and Tseng, Wayman and Tseng, and Luong model as the glucose dose increased from 2 to 10 mg/ml. This indicates that glucose has a prominent influence on bacterial growth during toluene degradation and that different kinetics should be adopted for each broth condition.     

Simple GC-FID method development and validation for determination of α-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of α-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of α-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. α-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min^− 1. Calibration curves were linear over the concentration range 1–30 μg ml^− 1 (for standard solutions and solutions without endogenous α-tocopherol in plasma) and 5–34 μg ml^− 1 (for solutions with endogenous α-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of α-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 μg.ml^− 1 and 0.30 μg.ml^− 1, respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of α-tocopherol. The endogenous α-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.     

Interaction between tobramycin and CSA-13 on clinical isolates of Pseudomonas aeruginosa in a model of young and mature biofilms

The bactericidal activity of a cholic acid antimicrobial derivative, CSA-13, was tested against eight strains of Pseudomonas aeruginosa (both reference and clinical strains) and compared with the response to tobramycin. In planktonic cultures, the minimal inhibitory and minimal bactericidal concentrations of CSA-13 and tobramycin were in the 1–25 mg/L range except for one mucoid clinical strain which was much less sensitive to tobramycin (minimal bactericidal concentration, 65–125 mg/L). In young (24 h) biofilms, the sensitivity to CSA-13 was reduced (half-maximal concentration CSA-13 averaged 88 mg/L) and varied among the eight strains. The sensitivity to tobramycin was also very variable among the strains and some were fully resistant to the aminoglycoside. The combination of tobramycin with CSA-13 was synergistic in five strains. Only one strain showed antagonism between the two drugs at low concentrations of CSA-13. One reference and five clinical strains were tested in mature (12 days) biofilms. The effect of CSA-13 was delayed, some strains requiring 9 days exposure to the drug to observe a bactericidal effect. All the strains were tolerant to tobramycin but the addition of CSA-13 with tobramycin was synergistic in three strains. CSA-13 permeabilized the outer membrane of the bacteria (half-maximal concentration, 4.4 mg/L). At concentrations higher than 20 mg/L, it also permeabilized the plasma membrane of human umbilical vein endothelial cells. In conclusion, CSA-13 has bactericidal activity against P. aeruginosa even in mature biofilms and cationic steroid antibiotics can thus be considered as potential candidates for the treatment of chronic pulmonary infections of patients with cystic fibrosis. Considering its interaction with the plasma membrane of eukaryotic cells, less toxic derivatives of CSA-13 should be developed.     

Vitamin D-Binding Protein in Healthy Pre- and Postmenopausal Women: Relationship with Estradiol Concentrations

Objective: To examine the relationship between endogenous serum estradiol and vitamin D–binding protein (DBP) and total, free, and bioavailable 25-hydroxyvitamin D (25OHD) concentrations in pre- and postmenopausal women.Methods: In 165 healthy women (ages, 26 to 75 years) not taking any form of exogenous estrogen, the serum concentrations of estradiol, 25OHD, DBP, parathyroid hormone, and albumin were measured. Free and bioavailable 25OHD (free + albumin-bound) levels were calculated from total 25OHD, DBP, and serum albumin levels.Results: Premenopausal women had higher serum 25OHD (31.5 ± 7.9 ng/mL), DBP (45.3 ± 6.2 mg/dL), and estradiol (52.8 ± 35.0 pg/mL) levels than postmenopausal women (26.5 ± 4.9 ng/mL, 41.7 ± 5.7 mg/dL, and 12.9 ± 4.9 pg/mL), respectively. In addition, the calculated free and bioavailable 25OHD levels were higher in prethan postmenopausal women (P<.05). Serum estradiol correlated with DBP (r = 0.22; P<.01) and total 25OHD (r = 0.27; P<.01). In multivariate regression models (with or without serum 25OHD), estradiol was independently associated with DBP (P<.05).Conclusion: Lower estradiol level is one of the factors that contribute to lower DBP levels in older women. Our data indicate that besides well-known factors such as age, gender, and race, serum estradiol concentrations are also a physiologic predictor of DBP concentration.Abbreviations: 25OHD = 25-hydroxyvitamin D BMI = body mass index CV = coefficient of variation DBP = vitamin D–binding protein PTH = parathyroid hormone SHBG = sex hormone–binding globulin