Using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS), a highly sensitive analytical method was proposed for the determination of five phenylarsonic acids. It was shown that CM-α-CD-Fe3O4 could extract the five phenylarsonic acids in complex chicken tissue samples with high extraction efficiency. Under the optimal conditions, a high enrichment factor, ranging from 349 to 606 fold, was obtained. The limits of detection (LODs)  were in the range of 0.05–0.11 µg/kg for the five phenylarsonic acids. The proposed method was applied for the determination of five target phenylarsonic acids in chicken muscle and liver samples. Recoveries for the spiked samples with 0.2 µg/kg, 2.0 µg/kg and 20 µg/kg of each phenylarsonic acids were in the range of 77.2%–110.2%, with a relative standard deviation (RSD) of less than 12.5%.Carboxymethyl α-cyclodextrin immobilized Fe First, they can be easily isolated from solutions by applying an external magnetic field 3O4 nanoparticles have been demonstrated to have higher affinity toward arsenic element than other nano-materials, resulting in higher arsenic extraction efficiency. Fe3O4 magnetic nanoparticles have been used for removing inorganic arsenic ions in water sample. For example, Fe3O4 nanoparticles dispersed in chelating resins or coated with adequate chelating agents have been used for the removal of a wide range of metal ions from wastewater, overall displaying higher adsorption capacity than traditional materials 3O4 nanoparticles have been successfully applied for the removal of methylene blue and copper ions After feeding, nearly all phenylarsonic acids are excreted unchanged to the environment through the disposal of poultry litter, so the residues in animal tissues are very low 3O4 magnetic nanoparticles was not as good as inorganic arsenic compounds. It was then decided to use CM-α-CD to couple on the surface of Fe3O4 providing CD cavities to fit the benzene rings in the structures of phenylarsonic acid compounds. In addition, hydrogen bonding and electrostatic interactions between hydroxyl/carboxyl groups of modified CM-α-CD and amino/nitro of phenyl arsenic acids were also increased. The adsorption properties of modified Fe3O4 magnetic nanoparticles to phenylarsonic acids were studied, and the interactions between nanoparticles and phenylarsonic acids were examined. The synthesized materials were successfully applied in the sample clean-up and pre-concentration of phenylarsonic acids in chicken muscle and liver samples, which were subsequently separated and detected by UPLC-MS/MS.In this work, a highly sensitive determination method was established to monitor five phenylarsonic acids in chicken tissues. In our first attempt, it was found that the adsorption of phenylarsonic acids by FeICP-MS  was used to study the adsorption properties of synthesized material for phenylarsonic acids. The optimum operation parameters of ICP-MS were selected by tuning. The power was 1550W, the flow rates of cooling air, auxiliary air and carrier were 15.0 L/min, 1.0 L/min and 1.12 L/min, respectively. The sample rate of ascension by using peristaltic pump was set as 1.0 mL/min. The integration time for arsenic was 0.3 s/isotope. The operating parameters of UPLC-MS/MS  were as follows: capillary voltage = 2.8 kv, desolvation temperature = 450°C, desolvation gas flow rate = 600 L/Hr. The mobile phase was a mixture of acetonitrile (solvent A) and water containing 0.1% formic acid (solvent B) at a flow rate of 0.3 mL/min. All chromatographic separations were carried out in linear gradient mode as follows. In the first minute, solvent A was maintained at 98% (v/v). Solvent B quickly dropped to 30% from 1 to 3 min, followed by dramatic increase back to 98% from 3 to 5 min. MS parameters of UPLC-MS/MS are showed in The closed microwave digestion system  was used to digest samples for the determination of the total arsenic in the crude samples. The homogenizer  was applied to sample pretreatment and rocking hammock bed was from Zhicheng, Shanghai, China.Five phenylarsonic acids (98%) were obtained from the Chinese Academy of Agricultural Sciences. The standard stock solutions were prepared by dissolving each arsenic species in pure water at an arsenic concentration equivalent to that of 1 mg/mL phenylarsonic acids and stored at 4°C in the dark.2⋅4H2O and FeCl3⋅6H2O  were purchased from Tianjin Guangfu Fine Chemical Research Institute. Sodium chloroacetate (98%) was bought from Alfa Aesar. α-cyclodextrin (98%) was purchased from Beijing Dilang Biochemical Technology Co., Ltd., China. Other reagents including methanol, ethanol, acetone, toluene, formic acid and sodium hydroxide were of analytical reagent grade and all bought from Beijing Chemical Plant. The water used throughout the experiment was purified using a Milli-Q water purification system .Reagents for preparing magnetic nanoparticles: FeClCM-α-CD was prepared according to the following procedures. α-CD (3.55 mmoL) and NaOH (90.2 mmoL) were first dissolved into 20 ml water. The solution was then heated at 90°C for 5 min, followed by the addition of 74.6 mmoL sodium chloroacetate. The solution was heated for 3 h at 90°C under stirring. Once cooled to room temperature, the pH of the solution was adjusted to 6–7 by hydrochloric acid. The nearly neutral solution obtained was then poured into about 500 mL methanol. CM-α-CD was precipitated out as white solids, which was filtered and washed with methanol for a few times and then dried under vacuum for 3 d at 50°C and 0.085 MPa. The melting point of the CM-α-CD product was about 245.5°C as determined by micro melting point apparatus.3O4 magnetic nanoparticles were prepared according to the conventional co-precipitation method 2⋅4H2O and 5.2 g FeCl3⋅6H2O was added into a 500 mL conical flask containing 200 ml 0.05 M HCl. After dissolution of the solids, 250 ml 0.75 M NaOH solution was poured into the flask under a blanket of N2. The mixture was stirred for another 2 h at 80°C. The Fe3O4 nanoparticles were then obtained in the form of black precipitates, which were separated with a magnet and washed subsequently by water (3 times) and ethanol (twice). It should be noted that both the HCl and NaOH solutions were degassed by a sonicator for 20 min before use.Fe3O4 nanoparticles prepared in the previous step were added into 60 ml PBS buffer solution (pH = 6.6) containing 1.6 g of CM-α-CD. The suspension was sonicated for 3 min and then stirred for 3 h at 80°C. After cooling to room temperature, the nanoparticles were washed several times by PBS buffer solution to remove excess CM-α-CD. The CM-α-CD modified Fe3O4 nanoparticles were then dried at 80°C in a vacuum oven.The Fe3O4 nanoparticles were mixed with 8 mL standard solution of phenylarsenic acid with a given concentration. The centrifuge tube was placed on a rocking hammock bed at a rate of 270 rpm. After equilibrating for 30 min, the magnetic nanoparticles were separated from the solution with external magnetic field. The nanoparticles were rinsed twice with ethanol and dried in N2. To desorb target compounds, 1.0 mL pure water was added to the nanoparticles followed by equilibration for 10 min. The aqueous solution containing target compounds was filtered through a 0.22 µm Poly (ether sulfones) (PES) syringe filter and analyzed by ICP-MS. Standard phenylarsenic acid solutions of other concentrations were analyzed in the same way.In a 10 mL centrifuge tube, 5 mg CM-α-CD-Fe3O4 nanoparticles were mixed with 8 mL standard solution of phenylarsenic acid with a given concentration. A number of centrifuge tubes were prepared in this manner for a given concentration of standard. The centrifuge tubes were placed on an orbital shaker at a rate of 270 r/min. At different time points, one tube was removed and the magnetic nanoparticles contained in the tube were separated from the solution with external magnetic field. The following washing, desorption and ICP-MS analysis procedures were the same as those in the static adsorption step. Standard phenylarsenic acid solutions of other concentrations were analyzed in the same way.In a 10 mL centrifuge tube, 5 mg CM-α-CD-Fe3O4 nanoparticles for 10 min. Then the magnetic nanoparticles which adsorbed target analytes were separated under external magnetic field, the analytes adsorbed on the nanoparticles were then desorbed with 2 mL deionized water. The aqueous solution containing target compounds was filtered through a 0.22 µm PES syringe filter and analyzed by UPLC-MS-MS.Chicken tissues including meat and liver were bought from a supermarket in Beijing. Chicken meat and chicken liver samples were pulverized and freeze-dried for 24 h. The freeze-dried sample was homogenized by grinding and frozen until analysis. In a 50 mL centrifuge tube, 5.0 g freeze-dried chicken tissue sample and 10 mL ethanol were added. The extraction was repeated twice, each lasting 30 min. The combined extracts were equilibrated with 5 mg CM-α-CD-FeThe total amount of arsenic in chicken tissue samples was determined by microwave digestion ICP-MS according to reference 3O4 nanoparticles were varied in six dosages of 0.8, 1.6, 2.4, 3.2, and 4.8 g to obtain 450 mL of Fe3O4 suspension as described in section 2.3.2. The adsorption was carried out by equilibrating 5 mg CM-α-CD-Fe3O4 in 8 mL ROX standard solution at a concentration of 1 µg/L. The adsorption efficiency was then determined by ICP-MS. As shown in 3O4. The amount of adsorbed ROX is linear over a large concentration range of ROX. To verify the important role of CM-α-CD in the adsorption of phenylarsonic acid, the adsorption efficiencies of modified and unmodified Fe3O4 nanoparticles for p-NPAA were compared. As shown in For optimal adsorption efficiency, the amounts of CM-α-CD for the modification of Fe3O4 and CM-α-CD-Fe3O4 nanoparticles are shown in (a) and (b), respectively. Unmodified Fe3O4 nanoparticles, approximately 10 nm in diameter, trend to aggregate because of size effect of Fe3O4 nanoparticles. On the other hand, CM-α-CD-Fe3O4 nanoparticles shown in (b) are much better dispersed in an aqueous solution with diameters of about 5 nm, because the Fe3O4 nanoparticles were modified with CM-α-CD and the surface of Fe3O4 nanoparticles was protected by CM-α-CD. It can be observed in (b) that the composite of modified nanoparticles was more compacted and displayed roughly spherical shapes.The transmission electron microscope (TEM), fourier transform infrared spectrometry (FTIR) and thermo gravimetric analyzer (TGA) were used to characterized the magnetic nanoparticles. The TEM images of Fe3O4, CM-α-CD and CM-α-CD- Fe3O4 nanoparticles respectively were scanned . According to Y. P. Wu 3O4 can be calculated in 3O4, R is the mean radius of CM-α-CD-Fe3O4 (5.0 nm based on the TEM results), ρ is the density of the nanoparticle (4.8 g/cm3), NA is Avogadro's number, WCD and WFe3O4 are the weight losses of the CM-α-CD-Fe3O4 and Fe3O4 respectively, MCD is the molar mass of CM-α-CD immobilized on CM-α-CD-Fe3O4. The calculated number of CM-α-CD molecules immobilized on each CM-α-CD-Fe3O4 is about 70.The amount of CM-α-CD grafted on the surface of FeThe solvent for phenylarsonic acid solutions is an important factor affecting the adsorption efficiency. Five candidate solvents, i.e., water, methanol, ethanol, acetone and toluene, were compared . The adsThe adsorption saturation curve is shown in 3O4 could be estimated from the saturation of adsorption. Since one phenylarsonic acid molecule was supposed to fit one CD cavity, there should be approximately equal amount of CD and phenylarsonic acid. Thus, the amount of CM-α-CD might be calculated through its molecular mass of 1029 according to The amount of CM-α-CD coated on the surface of Fe3O4 as well as enrichment factors were investigated. Ultimately, the enrichment factors of p-APAA, AAPAA, ROX, p-HPAA and p-NPAA were 414, 426, 581, 606 and 349  at the concentration of 0.1 µg/L. Enrichment efficiency was 69.8%-121.2% and experimental results are shown in The lowest concentrations of phenylarsonic acids that could be absorbed by CM-α-CD-FeThe water was selected as desorption solvent. The adsorption efficiency was optimized by varying the adsorption time in the range of 1–40 min with all other parameters held constant. The adsorption efficiency increased with the adsorption time from 1 to 30 min followed by a plateau. The rate of adsorption was so high that 5 min was enough to adsorb the target compounds. Similarly, desorption efficiency was optimized by varying desorption time in the range of 1–10 min. The desorption efficiency increased with desorption time till 10 min, at which point it plateaued. Therefore, 10 min was selected as desorption time and desorption efficiency was achieved above 75%.2+, Cu2+, K+, Zn2+, Ba2+, Fe3+, Mg2+, Ca2+, Pb2+, Cr3+, Cd2+, and Sn2+ at concentrations of 500, 5000, and 50000 µg/L for each inorganic ions respectively, interference of inorganic ions on the adsorption of phenylarsonic acids was studied. No significant interferences were observed under the optimum conditions described above in the presence of inorganic ions as high as 50000 µg/L. The influence of four organic analogues including phenol, benzoic acid, p-hydroxbenzoic and m-hydroxbenzoic acid at concentrations of 50, 250 and 500 µg/L for each organic analogues respectively on the adsorption of target arsenic species was investigated. As shown in 3O4 and amino groups of p-APAA and AAPAA was very strong but there was hardly any hydrogen bonding between the hydroxyl and nitro of p-NPAA. In any case, phenylarsonic acid could form inclusion complex with α-CD and therefore the arsenate, hydroxyl or amino group were forced closer to the magnetic particles, leading to stronger interactions between the modified material and phenylarsonic acids. Both selectivity and adsorption efficiency were improved compared to that of Fe3O4 nanoparticles without modification.By fixing the concentration of each phenylarsonic acids at 50 µg/L and 12 coexisting inorganic ions including MnA mixture of five arsenic compound standards, each at a concentration of 2.0 µg/L, was successfully separated and analyzed by UPLC-MS/MS in less than 3 min on a C18 column. Under the optimal separation conditions, baseline separation was achieved for every arsenic compound. The multi-reaction monitoring (MRM) chromatograms of arsenic compounds are shown in To extract phenylarsonic acids from chicken tissue samples, different extraction methods , extraction time  and extraction solvents  were studied. The optimal recovery was above 75% with the ultrasonic extraction method and toluene as the extraction solvent. The proper extraction time was found to be 30 min and extraction should be conducted twice.2 ranging from 0.9951 to 1.0000. The repeatability study was performed for each of the phenylarsonic acids under the optimal conditions. The LOD of each phenylarsonic acid was estimated by analyzing blank samples spiked at 0.2 µg/kg of each target analytes and they were determined as the lowest concentrations of the analyte for which signal-to-noise ratios were 3 respectively. The resultant repeatabilities expressed as RSD varied from 0.85% to 4.49%. These results show that the method has a high sensitivity and good repeatability.The developed method was validated by determining the linearity and LOD of arsenic species listed in The phenylarsonic acid in tissue was stored with the prototype compound. So the sample preparation of real tissue samples is same as the spiked samples 3O4, five phenylarsonic acids were spiked individually in each sample at 0.2 µg/kg and determined by ICP-MS. Recoveries were in the range of 81.5%–119.2% which agreed with the UPLC-MS/MS results. This shows that the CM-α-CD-Fe3O4 nanoparticles have high selectivity for phenylarsonic acid and can be used for sample clean-up. Combining CM-α-CD-Fe3O4 nanoparticles clean-up procedure with UPLC-MS-MS technique would potentially result in practical application in the analysis of trace phenyl arsenic acids.In the samples of chicken meat and liver, 0.2, 2 and 20 µg/kg of each phenylarsonic acids were spiked, respectively. The recoveries of five phenylarsonic acids in chicken tissue samples, as shown in 3O4 nanoparticles were synthesized to selectively extract and enrich phenylarsonic acids. CM-α-CD- Fe3O4 nanoparticles exhibited excellent selectivity and adsorption efficiency for five phenylarsonic acids because of the size selectivity of α-CD and the affinity of Fe3O4 to arsenic. In the sub ppb level of phenylarsonic acids, the enrichment factor was higher than 400 and the extraction efficiency higher than 70%. Coupled with UPLC-MS/MS, a fast, selective and convenient analytical method for the determination of phenylarsonic acid was developed. Comparing with published method 3O4 nanoparticles for phenylarsonic acids in complex sample matrix.In this work, CM-α-CD-FeFigure S13O4 magnetic nanoparticles (a), CM-α-CD (b) and CM-α- CD- Fe3O4 (c).FTIR spectra of Fe(TIF)Click here for additional data file.


Additionally, mRNA content and protein expression of the transporter OATP1A2 were increased up to ninefold with 1,25(OH)2D3. We investigated whether these in vitro findings can be confirmed in humans in vivo.In Caco-2 cells, folate uptake via the proton-coupled folate transporter (PCFT) increases significantly by a 3-day treatment with 1,25-dihydroxyvitamin D2D3 orally. One hundred twenty milligrams fexofenadine, an OATP1A2 substrate, was taken in 1 day before the first folic acid intake, and again on the ninth day of 1,25(OH)2D3 intake. Duodenal biopsies were taken for transporter mRNA assessments once before and once on the ninth or tenth day of the vitamin D3 course. Serum folic acid and fexofenadine concentrations were quantified with a chemiluminescence immunoassay and LC-MS/MS, respectively. Pharmacokinetics were compared between periods with standard bioequivalence approaches.Ten healthy volunteers (six women) received 5 mg folic acid orally once before and once together with the last intake of a 10-day course of 0.5 μg 1,25(OH)0-2h, which mainly reflects absorption, was 0.403 and 0.414 mg/L·h before and after the vitamin D3 course (geometric mean ratio (GMR), 1.027; 90 % confidence interval (90 % CI), 0.788–1.340), the geometric mean fexofenadine AUC0-2h was 1.932 and 2.761 mg/L·h, respectively . PCFT- and OATP1A2-mRNA expressions in duodenal biopsies were essentially unchanged.While geometric mean folic acid AUC2D3 in humans in vivo. This study underlines the importance of confirming in vitro findings in vivo in humans.No significant changes in folic acid and fexofenadine absorption were observed after a 10-day course of 1,25(OH) As a heterodimer with the retinoid-X-receptor α (RXR), VDR regulates the transcription of numerous genes [2D3 biodegradation, tryptophan hydroxylase 2 in the brain [2D3 in the functional regulation of these proteins usually comes from in vitro or animal experiments, and the relevance in humans is unknown.The active form of vitamin D, 1,25-dihydroxyvitamin Dhe brain . HoweverSLC46A1) and, to a lesser extent, by the reduced folate carrier , which has a lower affinity for folic acid [The water-soluble vitamin folic acid is the precursor for the production of the metabolically active folates, which are essential for the synthesis of nucleic acids in human cells. Naturally occurring folates in food are conjugated to a polyglutamyl chain which is removed by the enzyme folate conjugase in the mucosal cells before the folate monoglutamates can be absorbed in the duodenum and jejunum  mainly blic acid .Tissues with a high turnover rate like bone marrow, lymphatic tissue, hair follicles, and tumor cells are most susceptible to folate deficiencies . During 2D3 [PCFT is an electrogenic transporter located in the apical enterocyte membrane of the proximal part of the small intestine and works best at low pH values , 9. Muta2D3 .SLCO1A2) and OATP2B1 (gene code SLCO2B1), are expressed in several tissues, notably at the blood brain barrier, in cholangiocytes, in the apical domain of tubular cells of the distal nephron, and at the apical membrane of small intestinal epithelial cells. Although the expression level of OATP1A2 in human intestine is still controversial [2, etc. On the other hand, apical and basolateral intestinal efflux transporters like P-glycoprotein  and multidrug-resistance-related protein 3 , respectively, also exert influences on the absorption of drugs like fexofenadine [2D3 [2D3 on OATP2B1. Studies in vitro suggest that intestinal P-gp is upregulated by 1,25(OH)2D3 [There are numerous other intestinal transporters for nutrients, ions, water, peptides, and also xenobiotics. Two important representatives of the organic anion transporting polypeptide family, OATP1A2 2D3 , 16, whi5(OH)2D3 , 17.2D3. However, the extent to which these in vitro findings are relevant in vivo in humans is unknown. The aim of this study, therefore, was to investigate the influence of a sufficiently long 1,25(OH)2D3 administration on the pharmacokinetics of folic acid and fexofenadine as marker substrates of PCFT and OATP1A2 activities, respectively, in healthy human volunteers. To investigate the link between pharmacokinetics and transporter expression and activities, we additionally quantified mRNA of PCFT, OATP1A2, OATP2B1, P-gp, and MRP3 in duodenal biopsies.These in vitro findings suggest that intestinal folate uptake and uptake of OATP1A2 substrates can be increased by 1,25(OH)2, and were in a good healthy condition, as determined by physical examination and laboratory assessment conducted prior to the study. The intake of concomitant medication was not allowed, except oral contraceptives in women. Participants were instructed not to take in any nutrition supplement, especially folic acid or food fortified with folic acid.The study was performed as a prospective single arm, single center, pharmacokinetic study in 10 healthy volunteers at the Department of Clinical Pharmacology and Toxicology, University Hospital of Zurich, Switzerland. Subjects were eligible if they were between 18 and 65 years of age, had a body mass index between 18 and 30 kg/mThe study was approved by the Ethics Committee of the Canton of Zurich, Switzerland (KEK-ZH-no. 2012–0051), and by the Swiss National Health authority, Swissmedic. The clinicaltrials.gov identifier is NCT01856348. Written informed consent was obtained from each volunteer before any study related procedures were carried out. The study was carried out in accordance with the Declaration of Helsinki, amendment of Seoul, South Korea, 2008, and all pertinent guidelines and laws in force in Switzerland.On study day 1, 120 mg fexofenadine , and on study day 2, 5 mg folic acid  was given to the participants orally together with 200 mL light sparkling water. An esophago-gastro-duodenoscopy with biopsies from the duodenum to assess PCFT, OATP1A2, OATP2B1, P-gp, and MRP3 mRNA expression was performed on study day 1 or 2, depending on the availability of the endoscopy unit.2D3  daily in the morning at home and to fill in a diary for treatment adherence, adverse event reporting, and nutrition assessment. On study days 11 and 12, a second dose of 120 mg fexofenadine and 5 mg folic acid, respectively, was given together with the daily dose of 1,25(OH)2D3, and a second endoscopy was carried out.During the following 7 days (study days 3 to 10), participants were requested to take in 0.5 μg 1,25(OH)All drug administrations on study days 1, 2, 11, and 12 were supervised by study personnel, and a mouth check was carried out. Before the study drug intakes on days 1, 2, 11, and 12, participants fasted for at least 1 h. Subjects consumed a light breakfast after the 2-h blood sampling and a lunch at least 6 h after study drug intakes on the pharmacokinetics days.2D3 serum concentrations were obtained on study days 1, 2, 11, and 12.Serial blood samples to assess pharmacokinetics of fexofenadine  and folic acid  were obtained on study days 1 (fexofenadine), 2 (folic acid), 11 (fexofenadine), and 12 (folic acid). Blood samples for the quantification of pre-dose 1,25(OH)2D3 in serum were measured at the Institute of Clinical Chemistry of the University Hospital Zürich, Switzerland, and fexofenadine was analyzed by XenoGesis Ltd, Nottingham, UK.Blood samples were drawn from an indwelling catheter in the cubital vein and centrifuged at 4 °C and 3000 rpm for 10 min. The resulting serum was frozen at −80 °C until analysis. Concentrations of folic acid and 1,25(OH)Folic acid was quantified in serum using a chemiluminescence immunoassay on the Access 2 immunoanalyzer . This test is standardized to the World Health Organization International Standard 03/178.In this assay, folic acid in the sample competes with a conjugate of folic acid and alkaline phosphatase for binding sites on a folic acid-binding protein. After immobilization of the resulting complex on a solid phase and removal of unbound material, addition of a chemiluminescent substrate induces a light signal that is inversely proportional to the concentration of folic acid in the sample.The detection limit of this test is <1 μg/L and the limit of quantification <2 μg/L as indicated by the manufacturer. Inter- and intra-assay imprecision has been found to be lower than 10 % for concentrations >3.6 μg/L. Since the dynamic measurement range of this test is between 1.0 and 24.8 μg/L, many of the samples had to be diluted to allow measurement of folic acid concentrations higher than 24.8 μg/L. As recommended by the manufacturer, the Access Folate Calibrator S0 (zero) was used for the dilutions.Fexofenadine (FEX) and fexofenadine-d6 (FEX-d6) were purchased from Sequoia Research Products Ltd.  and Santa Cruz Biotechnology , respectively. HPLC grade methanol and formic acid were from Fisher Scientific . Water for HPLC was purified on a Milli Q system .FEX serum concentrations were assayed by LC-MS/MS using FEX-d6 as internal standard. FEX and FEX-d6 stock solutions were prepared at a concentration of 1 mg/mL in DMSO and stored at −20 °C. Independent weighings were used for standards and quality controls (QC). Working solutions were prepared by dilution of stock solutions in methanol. Standards and QCs were prepared by spiking 10 μL methanolic working solution into 50 μL blank human serum and adding 40 μL methanol plus 150 μL methanol containing 100 nM FEX-d6 as internal standard. Serum concentration curves were prepared at 2, 5, 10, 25, 50, 100, 250, 500, 1000, and 2000 ng/mL. QC samples were prepared in the same way containing 16, 160, and 1600 ng/mL FEX.g, 4 °C for 20 min, and supernatants were transferred for analysis by LC-MS/MS.To each 50 μL serum sample, 50 μL methanol and 150 μL methanol containing 100 nM FEX-d6 as internal standard were added. Blank serum samples were prepared in the same way. All samples, standards, and QCs were vortex mixed and kept at −20 °C for a minimum of 2 h to allow complete protein precipitation. Samples were then centrifuged at 2500×v/v formic acid as mobile phases. Flow rate was 0.5 mL/min, and the column temperature was 50 °C. A solvent gradient was used: solvent B increased from 5 % in the beginning to 95 % at 1.5 min, rose to 99 % from 1.55 till 2 min, returned to 5 % at 2.05 min, and remained stable until the next injection started.Five microliters of each sample was analyzed in MRM mode on a Waters Premier XE tandem quadrupole mass spectrometer using electrospray ionization and a Waters Acquity UPLC system . High performance liquid chromatography was performed on a ACE Excel 2 C18-AR, 50 × 2.1 mm column  using water (A) and methanol (B) both containing 0.1 % m/z 502.2 → 171.1 and 508.2 → 177.1 for FEX and FEX-d6, respectively. Dwell time was 50 ms, cone voltage was 46 V, and collision energy was 34 eV for both analytes. Peak area ratios of FEX and FEX-d6 were used for quantification. The lower and upper limits of quantification were 2 and 2000 ng/mL, respectively.MS/MS transitions were 2D3 from the serum sample using a monoclonal antibody, the concentration of this parameter was determined with a commercially available 1,25(OH)2D3 assay on an immunoanalyzer  using a competitive immunoassay method. 1,25(OH)2D3 from the serum sample competed with a 1,25(OH)2D3-acridinium conjugate for binding to a biotinylated anti-1,25(OH)2D3-antibody. After immobilization and washing of the resulting antigen-antibody-complex, light signal induced by a chemiluminescence reaction was measured.After manual immunoextraction of 1,25(OH)2D3 concentrations could be quantified in all samples without further dilution. Intra-assay imprecision has been found to be lower than 10 % at concentrations of 37.9 and 82.3 ng/L , inter-assay precision was 12.6 % at 34.9 ng/L and 11.9 % at a 1,25(OH)2D3 concentration of 81.9 ng/L.Limit of detection of this test was 6.5 ng/L and limit of quantification, defined in accordance with the manufacturer’s recommendations as the concentration which can be measured with an inter-assay coefficient of variation not higher than 20 %, was 12.2 ng/L. Measurement range of this test was between 6.5 and 210 ng/L; thus, 1,25(OH)2D3 administration. All mRNA quantifications were done in triplicate.Duodenal biopsies were immediately snap-frozen in liquid nitrogen and stored at −80 °C. Using the “Nucleospin miRNA” kit , frozen biopsies were immersed in the appropriate buffer and homogenized with a 1.5-mL pestle , and RNA was extracted following the manufacturer’s instructions. Extracted RNA was subsequently analyzed by real-time PCR (RT-PCR) using TaqMan probes , and results were normalized to the enterocyte-specific housekeeping gene villin. Biopsies were identified according to subject (number) and whether they were collected before or after 1,25(OH)Cmax) and the time to reach Cmax (tmax) was directly taken from the plasma concentration-time data. The terminal elimination half-life (t1/2) was calculated by dividing ln(2) by the terminal elimination rate constant λz, which was estimated by linear regression of ln-concentration data of the terminal part of the concentration-time curve. The area under the concentration-time curve (AUC) was calculated using the log-linear trapezoidal rule, either from the time of administration to a specified time point thereafter, or to the last quantifiable time point (AUC0-last). To obtain the AUC from administration extrapolated to infinity (AUC0-∞), the estimated AUClast-∞, which was calculated as Clast / λz, was added to AUC0-last. The parameters Cmax and AUC0-2 were selected for the characterization of the absorption phase of fexofenadine and folic acid.A non-compartmental pharmacokinetic approach was chosen. The maximum serum concentration after drug intake ndations ), and ifund 25 % , and thee 28.4 % ), a samp2D3 intake, an ANOVA-based relative bioequivalence assessment was carried out. The pharmacokinetic parameters of fexofenadine and folic acid before the start of intake of 1,25(OH)2D3 were taken as reference for these calculations. Point estimates of the geometric means ratios of the pharmacokinetic parameters of fexofenadine and folic acid during 1,25(OH)2D3 intake over the pharmacokinetic parameters of fexofenadine and folic acid before the 1,25(OH)2D3 dosing period were calculated alongside with their corresponding 90 % confidence intervals. An influence of 1,25(OH)2D3 administration on the pharmacokinetics of fexofenadine, or folic acid, was accepted as absent if the 90 % confidence intervals of the point estimates fell entirely within the bioequivalence zone of 0.8–1.25.For the comparison of pharmacokinetics of fexofenadine and folic acid, respectively, before and after 10 days of 1,25(OH)2, all non-smokers) were enrolled and completed the study. All adverse events were mild and transient. There was no statistically significant change in serum 1,25(OH)2D3 concentrations before and on the 11th and 12th study day: the geometric mean 1,25(OH)2D3 concentrations (geometric coefficient of variation) were as follows: day 1, 18.7 ng/mL (49 %); day 2, 19.1 ng/mL (48 %); day 11, 20.9 ng/mL (44 %); and day 12, 20.5 ng/mL (41 %).A total of 10 healthy volunteers 2D3 are shown in Fig. 2D3 intake (geometric means ratio (GMR), 89.66; 90 % confidence interval (90 % CI), 65.87–122.03). For OATP2B1, the geometric mean mRNA contents were 1.149 before and 1.146 after the 10-day course , and for PCFT, values were 0.997 before and 0.996 after the 1,25(OH)2D3 intake . For the efflux transporter P-gp, the geometric mean mRNA contents were 1.860 before and 1.608 after the 1,25(OH)2D3 intake , and corresponding values for MRP3 were 2.988 and 2.724 .The villin-normalized mRNA contents of PCFT, OATP1A2, and OATP2B1 in the duodenal biopsies taken before and at the end of a 10-day course of 1,25(OH)2D3 on folate uptake via PCFT could be confirmed in vivo in humans, as well as on the transport capacity of OATP1A2 and OATP2B1, because inducing effects on PCFT and OATP1A2 have been demonstrated in cell culture experiments [2D3 has an effect on folate uptake and on PCFT expression in healthy human volunteers. Although the mean results for fexofenadine favored an increase in fexofenadine uptake of about 30–40 % after 10 days of 1,25(OH)2D3, these results were not statistically significant and the intestinal mRNA expression of OATP1A2 and OATP2B1 was not changed.This study aimed at clarifying whether an inducing effect of 1,25(OH)eriments . We coul2D3 to a VDR-RXR heterodimer, which then binds to the PCFT promoter and roughly doubles PCFT expression and folate uptake in Caco-2 cells in vitro [2D3 on folic acid absorption in vivo in humans. These results underline the importance of verifying in vitro findings by in vivo studies, although the explanations for the observed discrepancies are not obvious. It can be speculated that the 1,25(OH)2D3 concentration changes in the enterocytes 2D3 doses used) were too low to lead to a quantifiable effect on PCFT expression and activity. In vitro, a 3-day treatment with only 25 nM 1,25(OH)2D3 (corresponding to approx. 10 μg/L) was sufficient to show a significant PCFT mRNA increase in Caco-2 cells, but more pronounced effects were seen with 100–200 μg/L 1,25(OH)2D3 [2D3 serum concentrations are tightly controlled in the human body. It may be possible that higher doses would have led to a quantifiable effect. However, since higher doses of 1,25(OH)2D3 may confer an undue risk for prolonged hypercalcemia and related adverse effects, we did not select higher doses for the present study.Two intestinal uptake mechanisms exist for folates in humans. While the PCFT is the most relevant transport mechanism, the RFC does not seem to play an important role in folate uptake in the intestines . As expein vitro . Howeverin vitro . In our 5(OH)2D3 . Trough 2D3. OATP1A2 has been shown to be upregulated in vitro up to ninefold by a 3-day treatment of Caco-2 cells with a large dose of 500 nM (approx. 200 μg/L) 1,25(OH)2D3 for 24 h [2D3, and that this effect diminished with time [2D3 on MRP3 led to contradictory results [2D3, and since we did not observe any relevant changes in the duodenal mRNA of the four transporters involved in fexofenadine pharmacokinetics, there is no mechanistic explanation which would support the non-significant trend towards slightly higher mean fexofenadine exposure after a 10-day treatment with 1,25(OH)2D3 observed in the present study in healthy volunteers. Taken together, a 10-day course of 1,25(OH)2D3 does not lead to major changes in fexofenadine pharmacokinetics and in duodenal mRNA of OATP1A2, OATP2B1, P-gp, and MRP3.Most drugs which are absorbed from the intestine via drug transporters are not specific substrates for a single transporter. This is also the case with fexofenadine, which is taken up from the gut lumen by OATP1A2 and OATP2B1 , and is ith time . In vitr results , 17. The2D3 concentrations were at the lower range of normal, but who did not have manifest vitamin D deficiency. This may have prevented an effect of 1,25(OH)2D3 on folic acid pharmacokinetics becoming quantifiable. Second, a 10-day course of 1,25(OH)2D3 may have been too short to lead to substantial effects, although the induction was statistically significant in cell culture already after a 3-day treatment. Higher 1,25(OH)2D3 doses may have led to a visible effect on folic acid absorption and a statistically significant effect on fexofenadine pharmacokinetics. Last, since the variability in fexofenadine pharmacokinetics was higher than expected, a larger number of participants would have been necessary to unequivocally answer the question whether fexofenadine transport is significantly affected by 1,25(OH)2D3 treatment.Some limitations of this study have to be considered. First, we investigated healthy volunteers whose 1,25(OH)2D3 does neither affect folic acid uptake nor fexofenadine pharmacokinetics in a significant manner. The results of this study underscore the importance of investigating whether in vitro findings can be confirmed in vivo.In summary, we show that a 10-day course of usual doses of 1,25(OH)


To induce peroxisomal proliferator-activated receptor α (PPARα) expression and increase milk fat utilization in pigs at birth, the effect of maternal feeding of the PPARα agonist, clofibrate (2-(4-chlorophenoxy)-2-methyl-propanoic acid, ethyl ester), on fatty acid oxidation was examined at full-term delivery (0 h) and 24 h after delivery in this study. Each group of pigs (n = 10) was delivered from pregnant sows fed a commercial diet with or without 0.8% clofibrate for the last 7 d of gestation. Blood samples were collected from the utero-ovarian artery of the sows and the umbilical cords of the pigs as they were removed from the sows by C-section on day 113 of gestation.P < 0.01) 14C-accumulation in CO2 and acid soluble products 2.9-fold from [1-14C]-oleic acid and 1.6-fold from [1-14C]-lignoceric acid respectively. Correspondingly, clofibrate increased fetal hepatic carnitine palmitoyltransferase (CPT) and acyl-CoA oxidase (ACO) activities by 36% and 42% over controls (P < 0.036). The mRNA abundance of CPT I was 20-fold higher in pigs exposed to clofibrate (P < 0.0001) but no differences were detected for ACO and PPARα mRNA between the two groups.HPLC analysis identified that clofibric acid was present in the plasma of the clofibrate-fed sow (~4.2 μg/mL) and its offspring (~1.5 μg/mL). Furthermore, the maternal-fed clofibrate had no impact on the liver weight of the pigs at 0 h and 24 h, but hepatic fatty acid oxidation examined in fresh homogenates showed that clofibrate increased (These data demonstrate that dietary clofibrate is absorbed by the sow, crosses the placental membrane, and enters fetal circulation to induce hepatic fatty acid oxidation by increasing the CPT and ACO activities of the newborn. High postnatal mortality has been recognized as a critical problem by the modern swine industry since the 1990’s. Although genetic improvement has increased the number of pigs born alive per litter, the death rate of newborn pigs during the postnatal period has increased slightly in recent years, from 13.2% in 2006 . In addi14C]-palmitate by 60% and 186%, respectively, in addition to the increased CPT I and ACO specific activities [Hepatic fatty acid β-oxidation pathways are controlled mainly by the key enzymes carnitine palmitoyltransferase I (CPT I) in mitochondria and acyl-CoA oxidase (ACO) in peroxisomes. The activities of these key enzymes are transcriptionally regulated by the peroxisomal proliferator-activated receptor α . This ctivities . Moreovetivities  as obserin vivo fatty acid oxidation in neonatal pigs [Recently we have demonstrated that supplementation of clofibrate could improve tal pigs . Howevertal pigs . Maternatal pigs . The reltal pigs . Furthertal pigs . These f14C]-fatty acid substrates.To test our hypothesis, we investigated the effect of maternal supplementation of PPARα agonist clofibrate on the development of oxidative capacity of newborn pigs at birth and 24 h after birth. We confirmed that PPARα agonist clofibrate transfers across porcine placental tissues by measuring maternal and fetal plasma clofibrate concentrations and increases hepatic oxidative capacity by measuring hepatic fatty acid oxidation in the newborns using -fatty acids. Homogenate protein was determined using the biuret method -oleic acid  and [1-14C]-lignoceric acid  as substrates. The measurements followed the same procedure as described previously by Lin et al. [14C]-C18:1 (4.2 MBq/mmol) or [1-14C]-C24:0 (1 MBq/mmol) and terminated by adding 0.5 mL HCLO4. The 14CO2 collected in ethanolamine and the 14C-acid soluble products (ASP) processed from the acid-killed medium were quantified using liquid scintillation counter . The rate of total β-oxidation and the rate of peroxisomal and mitochondrial β-oxidation were calculated as described by Yu et al. -oleic acid (C18:1) and [1-14C]-lignoceric acid (C24:0) were purchased from American Radiolabeled Chemicals, Inc. . 2-(4-Chlorophenoxy)-2-methylpropionic acid ethyl ester, 2-(4-chlorophenoxy)-2-methylpropionic acid, 4-chlorophenylacetic acid, peroxidase and all other chemicals were obtained from Sigma-Aldrich .Clofibrate was purchased from Cayman Chemical Company . [1-P ≤ 0.05.Data from fatty acid oxidation measurements were analyzed according to a split-plot design with the main plots in a completely randomized design using the SAS GLM procedure. The clofibrate effect was assigned to the main plot and pig postnatal age and fatty acid chain length effects were treated as the subplots. Least squares means ± SEM for variables are presented in tables and figures. Data from enzyme activity and mRNA enrichment assays were analyzed as a 2 × 2 factorial design using the GLM procedure. Least square means for treatment (clofibrate vs. control) and the postnatal age (0 vs. 24 h) effects were calculated. Differences between least squares means were determined using a Tukey test and considered significant when Chromatograms from HPLC analysis confirmed that clofibric acid was present in the plasma of both the clofibrate-fed sow and their newborn pigs Figure . In addiP < 0.0001), but the stimulatory effects of clofibrate and postnatal age for C18:1 oxidation were different from C24:0 oxidation. The 14C accumulations for C18:1 on average in CO2, ASP and CO2 + ASP (μmol/h/g protein) measured at 0 h and 24 h were 1.9, 2.2, and 2.1 fold higher, respectively, from clofibrate exposed pigs  than control pigs . Similarly, the 14C accumulations in CO2, ASP and CO2 + ASP (μmol/h/g protein) for C18:1 from both clofibrate treatment and control were on average 331%, 49% and 71% greater respectively in the livers of 24 h old pigs  than the pigs at delivery . In contrast with C18:1, the 14C accumulation for C24:0 on average in CO2 + ASP (μmol/h/g protein) measured at 0 h and 24 h was 1.8 fold higher from clofibrate exposed pigs (3.32) than control pigs (1.81), but no differences were detected in the 14C accumulation in CO2 and ASP , but there was no postnatal age effect on the 14C accumulations in ASP and CO2 + ASP .Results from total fatty acid oxidation in vitro Figure  showed t2 and ASP . The 14C-accumulations in CO2, ASP and CO2 + ASP (μmol/h/g protein) in the livers of the pigs from sow received clofibrate  were on average 2.1-fold higher than that from controls . Similarly, the 14C-accumulations in CO2, ASP and CO2 + ASP also were increased on average by 3.1, 0.49 and 0.71 fold respectively in the pigs at 24 h after delivery  as compared to the pigs at birth . However, the stimulatory effect by clofibrate on the 14C-accumulations in ASP and CO2 + ASP tended to attenuate with the increase in postnatal age. There was no effect of age on the 14C accumulation in ASP in the pigs exposed to clofibrate . In contrast to C18:1, the mitochondrial oxidative flux of C24:0 to ASP and CO2 + ASP (μmol/h/g protein) remained similar in the livers of the pigs exposed to clofibrate  and the controls , although the flux to CO2 tended to be higher in the liver from the clofibrate-exposed pigs than control pigs. There was no significant change in the oxidative flux of C24:0 as the postnatal age increased (P > 0.05).The mitochondrial oxidative flux of C18:1 to COP Figure , A & B a14C-accumulation in CO2  was detected in peroxisomes  from C18:1was on average 2 fold greater from clofibrate-treated  than control  pigs, and 56% higher from the 24 h-old pigs than that from pigs at delivery . In comparison to C18:1, clofibrate increased the 14C accumulation from C24:0 peroxisomal oxidation in CO2 in the liver of pigs at delivery but decreased in the liver of 24 h-old pigs. Thus there was no significant difference (P = 0.93) in the average flux to CO2 (μmol/h/g protein) between clofibrate-treated (0.15) and control pigs (0.14). There were no effects of clofibrate on the 14C accumulation in ASP and CO2 + ASP from C24:0 in pigs at delivery, but the 14C accumulations was 84% greater from clofibrate-treated (4.0) than control in 24 h-old pigs (2.17). Postpartum age also had no influence on C24:0 oxidation in pigs at delivery (P > 0.05), but the 14C accumulation in ASP and CO2 + ASP was 94% higher from the clofibrate-treated pigs measured at 24 h (4.0) than at delivery (2.06).No substantial P > 0.05). However, the peroxisomal proportion of total fatty acid oxidation was on average 2.3 fold higher from C24:0 (85.5) than C18:1 (37.7%).Clofibrate and postnatal age had no effect on the distribution of oxidative flux C18:1 and C24:0 between mitochondria and peroxisome . There was no change in the activities with postnatal age (P = 0.2). The total and inhibited enzyme activity (μmol/h/g protein) were 31.2, 52.4 on average for the pigs at delivery and 39.3, 64 on average for the 24 h-old pigs. Hepatic ACO activity was increased by 2.3 fold in the clofibrate-exposed pigs .Hepatic CPT specific activity Figure A measureP < 0.0001), but no differences were detected for the relative mRNA abundance of either ACO or PPARα . The percentage of C24:0 oxidation in peroxisomes was more than 80% of the total oxidation. Clofibrate had no effect on the relative oxidative capacity of peroxisomes although the efficacy of clofibrate was increased by postpartum age. This result suggests that the very long-chain fatty acid is catabolized exclusively in peroxisomes. In contrast with C24:0, high14C accumulations in CO2, ASP and CO2 + ASP from C18:1 were observed in both mitochondria and peroxisomes, suggesting that C18:1can be oxidized initially in both of the organelles. Similar results were observed also using C18:1 and erucic acid as substrates in our previously study [Peroxisomal fatty acid oxidation catalyzes chain-length shortening of monounsaturated fatty acid and saturated very long-chain fatty acid. Evidence indicated that C24:0 could only be oxidized initially in peroxisomes after activation by the acyl-CoA sythetase enzyme in endoplasmic reticulum and peroxisomes in brain and liver ,42. Peroly study .In conclusion, clofibrate from maternal oral feeding during gestation is absorbed and hydrolyzed to clofibric acid, which can cross the porcine placenta and enter the fetal circulation system. Fetuses that were exposed to clofibric acid via maternal-placental transfer had a higher hepatic capacity to oxidize fatty acid at birth as compared to control fetuses. The high fatty acid oxidative capacity resulted from an increase in key enzyme activities induced by clofibric acid via activation of PPARα and its target genes. The promoted fatty acid oxidation by activation of PPARα and its target genes was attenuated in fasted newborns with postpartum age. Postpartum age also increased fatty acid oxidation. The increase was associated with development and was not influenced by clofibrate supplementation. Results from this study suggest that clofibrate could be examined as an agent to induce precocious development of ACO and possibly improve milk fat oxidation and pig survivability.


Here, we describe the pharmacokinetics of an accidental single oral overdose of 2,000,000 IU of vitamin D3 in two elderly nursing home patients and discuss safety issues.Administration of intermittent high doses of vitamin D3 concentrations showed a prompt increase with the highest peak area already hours after the dose, followed by a rapid decrease to undetectable levels after day 14. Peak blood 25(OH)D3 concentrations were observed 8 days after intake ). Remarkably, plasma calcium levels increased only slightly up to 2.68 and 2.73 mmol/L, respectively (ref: 2.20–2.65 mmol/L) between 1 and 14 days after intake, whereas phosphate and creatinine levels remained within the reference range. No adverse clinical symptoms were noted.Two patients, a Caucasian 90-year old man and a 95-year old woman, were monitored from 1 h up to 3 months after intake for clinical as well as biochemical signs of vitamin D intoxication. Blood vitamin D3 does not cause clinically apparent toxicity requiring hospitalization, with only slightly elevated plasma calcium levels in the first 2 weeks. Toxicity in the long term cannot be excluded as annual doses of 500,000 IU of vitamin D3 for several years have shown an increase in the risk of fractures. This means that plasma calcium levels may not be a sensitive measure of vitamin D toxicity in the long term in the case of a single high overdose. To prevent a similar error in the future, the use of multiple-dose bottles need to be replaced by smaller single-unit dose formulations.A single massive oral dose of 2,000,000 IU of vitamin D Plasma calcium and urinary calcium excretion both are recognized markers of vitamin D toxicity [Vitamin D deficiency is a highly prevalent condition, present in approximately 30–50% of the general population , and mayractures , similarractures . On the  placebo . In clintoxicity , 500,000toxicity , 540,000toxicity , or 600,toxicity ,13.3 in two elderly nursing home patients and discuss safety issues from single massive oral doses of vitamin D3.Here, we describe the pharmacokinetics of an accidental overdosing by 2,000,000 IU vitamin D3 doses of 100,000 IU three times a year as a lyophilized solution . Two nursing home patients each received an accidental single overdose of 2,000,000 IU vitamin D from a whole bottle of a concentrated vitamin D3 solution . The pharmacy department was surprised by the order for 25 new bottles immediately after the dose, as the two 40-mL bottles would have lasted for half of a year of treatment for the patients at the ward. It then became clear that an overdose had been given. For safety reasons, we immediately started biochemical and clinical monitoring of both patients. Plasma calcium, phosphate, creatinine, and 25(OH)D levels were measured from 1 h after dosing up to 106 days (case 1) and 71 days (case 2) , were performed using liquid chromatography-tandem mass spectrometry as previously described [1/21/2[a = analytical variation, and CVi = intra-individual variation. For calcium, CVa and CVi were 1.5% and 1.9%, respectively [Measurements of plasma 25(OH)D concentrations, as well as semi-quantitative measurement of vitamin Described . Plasma +CVa2)1/2, where Zectively .3 peak area showed a prompt increase with the highest peak area already at the second measurement, 5.5 h after dosing, followed by a rapid decrease to undetectable levels at day 14. Plasma 25(OH)D3 concentrations also were already markedly increased 5.5 h after dosing, but rose more slowly than vitamin D3, reaching a maximum of 527 nmol/L at day 8. Over the following 3-month period, 25(OH)D3 concentrations decreased by 50% over an approximately 50-day period. At the end of the 106-day follow-up period, the patient’s 25(OH)D3 concentration was 113 nmol/L and fell within the laboratory reference range (50–200 nmol/L). Plasma phosphate and creatinine levels showed a modest increase within the reference range  equation >60 mL/min), and an absence of significant pathology. He did not report any clinical signs or symptoms of vitamin D toxicity. His plasma calcium was 2.52 mmol/L 1 h after dosing and showed a non-significant increase from day 1 to day 8, with a maximum of 2.68 mmol/L at day 8. After 8 days, plasma calcium levels fell to within the reference range (2.2–2.65 mmol/L) D3 concentrations also were already markedly increased 1 day after dosing, but rose more slowly than vitamin D3, reaching a maximum of 422 nmol/L at day 8. At the end of the 71-day follow-up period, the 25(OH)D3 concentration was 233 nmol/L, with an approximate 50% decrease from the highest level being still above the upper normal limit of 200 nmol/L. Plasma phosphate and creatinine levels both showed a modest increase within the reference range  and an absence of significant pathology. She did not report any clinical signs or symptoms of vitamin D toxicity. Her plasma calcium was 2.46 mmol/L 1 h after dosing and showed a biologically significant increase from day 1 to day 11 with a maximum of 2.73 mmol/L at the second day. After 11 days, plasma calcium levels dropped to within the reference range of 2.2–2.65 mmol/L D3 levels peak on days 1 and 7, respectively [3 concentrations fell rapidly, being close to baseline by day 7, with a much slower fall of the 25(OH)D3 concentrations. Plasma calcium levels did not rise at any time, and no subject experienced hypercalcemia at any of the measured time points [3 concentrations in our nursing home residents (59 nmol/L in case 1 and 77 nmol/L in case 2) at the first measurement (1 h post-dosing) can be explained as the result of previous osteoporosis prophylaxis treatments with 100,000 IU of oral vitamin D3. However, we cannot exclude the possibility that the plasma 25(OH)D concentrations were already increased 1 h after the high dose of vitamin D3, implicating that the baseline 25(OH)D values in both cases actually may have been lower.Pharmacokinetic studies in human volunteers given a single large oral dose of 100,000 IU of vitamin Dectively ,18. The e points . The pha3 and serum levels of 25(OH)D3 up to 500 nmol/L, well above naturally occurring levels of 25(OH)D3[3 are not seen by a physician until they are admitted to the emergency department with clinical signs of intoxication, such as life-threatening dehydration, after a prolonged period of oral intake of high-dose vitamin D, either from contaminated food components [The toxicity of vitamin D in daily administration has been studied extensively. The present view is that hypercalcemia is the hazard criterion for vitamin D . Thus, v 25(OH)D3. Most pamponents ,22 or sumponents ,24.3 following a single high oral dose of vitamin D3. In studies using 300,000 IU [3, no adverse effects were noted and plasma calcium levels or urinary calcium excretion did not change, or only slightly increased for a short period of time, with all cases remaining within the physiological range. In contrast to these reassuring data, Sanders et al. [3 for 3–5 years in older women compared with placebo. Falls and fractures occurred especially within the first 3 months following the annual dose of vitamin D3. In that study, plasma calcium levels and urinary calcium excretion were not measured; however, bearing in mind the former data, it is unlikely that hypercalcemia occurred in the study. These unexpected findings led Rossini et al. [3. A single dose of 600,000 IU of vitamin D3 increased serum C-terminal telopeptide of type 1 collagen and cross-linked N-telopeptide of type 1 collagen significantly at day 1, attained a peak increment greater than 50% at day 3, and subsequently decreased almost back to baseline values at day 90, without statistically significant changes in plasma calcium [Much less is known about the toxicity of vitamin D0,000 IU , 540,0000,000 IU , and 6000,000 IU ,13 of ors et al.  found a i et al.  to inves calcium . A possi3, such as measurement of urinary calcium excretion and plasma levels of 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D, PTH, and bone turnover markers. Because of the nature of this study, baseline concentrations (prior to dosing) of biochemical parameters such as plasma vitamin D3, 25(OH)D, and calcium were lacking.The present study displays some limitations. We lacked the opportunity for studying other relevant parameters in the risk assessment for vitamin D3 did not result in immediate clinical and biochemical toxicity requiring hospitalization, with only slightly elevated plasma calcium levels in the first 2 weeks. However, toxicity in the long term cannot be excluded as an annual dose of 500,000 IU of vitamin D3 for several years has shown an increased risk of fractures. This may implicate that markers other than plasma calcium measurement, such as bone turnover markers, are needed to assess the safety of high doses of vitamin D in the long term. Clearly, the safety of high-dose vitamin D supplementation warrants further study. To prevent a similar error in the future, the use of multiple-dose bottles were replaced by smaller single-unit dose formulations of 25.000 IU/mL. In general, we believe that available formulations of vitamin D3 should not exceed 100,000 IU, as long as the safety of high doses of vitamin D in the long term has not been established.In our two patients, the massive single overdose of 2,000,000 IU of vitamin DWritten informed consent for publication of their clinical details was obtained from the relatives of both patients. A copy of the written consent is available for review by the Editor of this journal.a: analytical variation; CVi: intra-individual variation; RCV: reference change value.25(OH)D: 25-hydroxyvitamin D; CVThe authors declare that they have no competing interests.MD was the treating physician of the patients reported. JvdO carried out the biochemical measurements, evaluated the test results and drafted the manuscript. HF and HV evaluated the test results and helped to draft the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/15/57/prepub


FP gene family and color diversity. Examining gene duplication events and subsequent evolution may improve our understanding of FP gene family diversity.Reef-building corals (Scleractinia) exhibit various colors, of which fluorescent proteins (FPs) are a major determinant. Gene duplication is considered a major mechanism in the generation of the FP gene family from one individual of Montipora sp., which we named monGFP (GFP gene from Montipora sp.). This gene family consists of at least four genes that produce at least six different cDNA sequences. The sequences were categorized into two types based on the length of cDNA; this difference is attributed to alternative splicing. Although the amino acid sequences were different, the emission spectra of the monGFP variants were nearly identical (518–521 nm). In addition to this gene family, we isolated ten paralogous AdiFP10 (Adi-Fluorescent protein-10 gene from Acropora digitifera) sequences from cDNA of two Acropora species, A. digitifera and A. tenuis. Based on our phylogenetic analysis, five sequences from A. digitifera and four sequences from A. tenuis appeared to be in a different cluster from AdiFP10, suggesting a new FP gene cluster. The FP sequences were likely to have been generated independently in each species or generated by gene duplications in the ancestral lineage of Acropora, followed by extensive gene conversion within each species.We isolated a novel FP genes during the evolutionary history of Montipora and Acropora species. Our analyses of monGFP indicate that FPs translated from different splicing variants and gene copies have evolved without changes in the function of fluorescence, and gene copies have been evolved under purifying selection. On the other hand, AdiFP10 paralogs and other RFP genes in Acropora species may have diversified their functions. Identification of conserved and divergent modes of evolution after the duplication of FP genes may reflect variation in the biological roles of different FPs.Our results clarify a part of the diversification process of The online version of this article (doi:10.1186/s40851-015-0020-5) contains supplementary material, which is available to authorized users. FP gene [FP genes represents the evolution of colors in corals [In tropical shallow oceans, reef-building corals (Scleractinia) exhibit various colors. Fluorescent proteins (FPs) are a major color determinant in corals –3; indeeFP gene . FPs areFP gene , and thin corals .Aequorea victoria (synonym A. aequorea) [FP genes have been cloned from anthozoa [in vivo imaging techniques [The initial discovery and determination of the structure of FP was first studied in the crystal jelly, equorea) , 8. Thisequorea) . Since tanthozoa , marine anthozoa , and deuanthozoa –14. The chniques .FP genes, it is also classified as an FP gene family member [FP genes pre-dates the divergence of coral families, and may have occurred as early as the Jurassic period [A variety of FPs has been detected in anthozoan species, with emission spectra in the visible light range. FPs are classified into four groups based on the color of light detected: cyan (CFP), green (GFP), yellow (YFP), and red (RFP) , 17. Siny member . The oric period . During In addition to fluorescent emission, many roles of FPs have been proposed; they are thought to be essential for viability, and may have photoprotective and antioxidant roles , 18–20. FP genes, gene duplication is considered to be a major mechanism of generating FP gene family and color diversity [FP genes have been isolated from the both genome and transcriptome of Acropora digitifera [FP genes [amilFP597 genes are present in head-to-tail tandem arrangements in the Acropora millepora genome [FP gene sequences showed that FP gene duplication and the subsequent acquisition of various fluorescent colors has occurred multiple times, indicating repeated evolution of FP colors [amilFP597 is correlated with red pigment concentration [FP genes is important for understanding FP gene family diversity.Gene duplication is a major source of genetic variation and can lead to neofunctionalization of the genes , 22. Neogitifera , 25. Gena genome . The phyP colors . In addintration , indicatFP transcripts has not been reported, to our knowledge.Alternative splicing, in which mature mRNAs are generated from a single pre-mRNA transcript, is another mechanism that may generate variation of protein functions. Alternative splicing increases protein diversity because the mature mRNAs sometimes encode proteins with subtly different or opposing functional domains . HoweverFP gene family, we analyzed gene duplication events and alternative splicing of FP genes in Scleractinia. We isolated novel GFP genes from Montipora sp., and identified a role of both gene duplication and alternative splicing in FP diversification. In addition to the GFP family, novel paralogs of an RFP gene were also identified in Acropora species. Our results shed new light on FP gene diversification during the evolutionary history of corals.In this study, to evaluate the genetic diversification of the Montipora sp. was acquired via an aquarium trade. The genus was predicted based on the sequence of the mitochondrial COI region , and subjected to a phylogenetic analysis  with 1,Genomic DNA was extracted from adult coral tissues of each species using the DNeasy Blood & Tissue Kit . Total RNA was extracted from adult tissues and larvae of each species using the RNeasy Mini Kit (Qiagen).Montipora sp.. An FP partial cDNA was cloned by RT-PCR using degenerate primers RFP_F2 (5’-ATGGAAGGGTSTGTCGATGG-3’) and RFP_R (5’-TKTTCTACCAGTYGCCATTTCTG-3’), and full-length cDNAs were isolated from the RNA of Montipora sp. by 5’ rapid amplification of cDNA ends (RACE) and 3’ RACE using the nested primers M5_RACE_F1 (5’-CAGTTCCACAGTACTTACAAGGCAAAGAC-3’), M5_RACE_R1 (5’-CATCAGGGCATGAGTTCTTGAAGTAGTC-3’), M5_RACE_F2 (5’-CGAAAGAGATGCCAGACTTCCACTTC-3’), and M5_RACE_R2 (5’-CAACTATGCCTCTGGGATATTTAGTGATG-3’), and a GeneRacer Kit . PCR was performed using the GeneAmp PCR System 9700 . All positions of primers are described in Fig. S2. The PCR reaction condition for partial cDNA was a denaturation step for 3 min at 94 °C, followed by 30 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C. The 5’ and 3’ RACE procedures were performed with two rounds of nested PCR using M5_RACE_F1 and M5_RACE_R1 for the first round, and M5_RACE_F2 and M5_RACE_R2 for the second round. The first and second PCRs were performed under the same condition, consisting of a denaturation step for 3 min at 94 °C, followed by 30 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 65 °C, and extension for 3 min at 72 °C. The novel FP cDNA was amplified using M5_F1 (5’-ATGCAAGCCAACAAATGCGCAA-3’) and M5_R1 (5’-CAAGGAGCTTGCAGATGCAGAAG-3’) primers and cDNA of Montipora sp. as a template. The PCR condition for the FP cDNA consisted of a denaturation for 3 min at 94 °C, followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C. The PCR product was purified for direct sequencing or cloned into the T-Vector pMD20 vector (Takara), and the sequences were determined using the Applied Biosystems Automated 3130 Sequencer.cDNAs were synthesized using a PrimeScript II 1st Strand cDNA Synthesis Kit  from total RNA of FP gene was amplified using M5_TC_LF (5’-GCAAACCGTGTGCTTGGATTCAC-3’) and M5_TC_LR (5’-CTTCCCACCAGTTGCCATTTCTG-3’) primers from the genomic DNA of Montipora sp. The PCR condition for the genomic FP gene sequence was denaturation for 3 min at 94 °C, followed by 30 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 65 °C, and extension for 10 min at 72 °C. The sequences of the whole intron 1, both ends of the other introns, and all exons were determined from the PCR product using M5eF1 (5’-CAACAAATGCGCAAAKAAGGCAAAC-3’), M5eR1 (5’- ACAGATCGCTTGATGCCTCAGAAG-3’), M5eR1_2 (5’- GGAATTTCAGGTCCATTTTGC-3’), M5eF2 (5’-TTGTATTACTTCTGAGGCATCAAG-3’), M5_RACE_R2 (5’-CAACTATGCCTCTGGGATATTTAGTGATG-3’), M5eF3 (5’-GGTTGACTATTTCAAGAACTCATG-3’), M5eR2 (5’- CATCACAGGTCCATTAGCAGGAAAG-3’), M5_TC_LR, and M5_GT_LR as sequencing primers. Partial intron 1 sequences between exon 1 and the additional 105 bp sequence (see results and discussion) were amplified using primers M5eF1 and M5eR1 from the genomic DNA of Montipora sp.. PCR products were cloned into T-Vector pMD20 and the sequences were determined using M5eF1, M5eR1, and M5i1_2_4F (5’-CGTCAAGATCCAGATTAACCTGT-3’) for all intron 1 sequences; M5i1L770 (5’-TGCTGAGACGGAAAATCTCAATGCT-3’) and M5i1-770R (5’-AGTCTGTTCTTAATAGCATTGAGA-3’) for i1_1250 ; M5i1-1300R (5’-TAGATCTCAGGTTACGTAATTCTGA-3’) and M5i1_3R (5’-CGACAGCTGACGTGCGATCTAAC-3’) for i1_1800; M5i1_2R (5’-AGTTAAGACATACGACAATAACAAAACGCA-3’), M5i1L1700 (5’- TTAGCTCGTAGTGCCGTGGAAAGTTAG-3’), and M5i1-1700R (5’-AGTCACGCTCCATTAACTACGT-3’) for i1_2100; and M5i1_1R (5’-AAAGACATTATTAGATCTGTTACGTAATTT-3’), M5i1-2400 F (5’-TCTCAGTATAAAAGGTCCAAAGTC-3’), and M5i1-2400R (5’-TTAAAGAACTTAGTGTTCACTAG-3’) for il_2800. The rest of intron 1 sequences (downstream part) were amplified from the genomic DNA of Montipora sp., using the primer pairs of M5eR1_2 and specific to each intron type M5i1-2400 F, M5i1-1700 F, and M5i1-770. The rest of the intron 1 sequences were determined from the PCR product using M5i1_2_4F and M5eR1. The whole intron 1 sequences were amplified from the genomic DNA of Montipora sp., using a primer pair of M5eF1 and M5eR1_2. DNA fragments of each intron type from exon 1 through to intron 1, and intron 1 through to exon 3 were amplified from the genomic DNA of Montipora sp., using the primer pairs of M5F1 and specific to each intron type M5i1-2400R, M5i1-1700R, and M5i1-770R for exon 1, and those of M5_RACE_R2 and specific to each intron type M5i1-2400 F, M5i1L1700, and M5i1L770 for exons 2 and 3. The sequences of intron 2 and partial sequences of exon 1 and exon 3 were determined using the M5i1-2400R, M5i1-1700R, M5i1-770R, and M5i2LF (5′-CGAATAGCTCAAGTTGAACTCAAGCGA-3′) primers.The novel A. digitifera and A. tenuis. A set of primers AdiFP10_F2 (5’-TACGGAAACAGGGTCTTCACTG-3’) and AdiFP10_R1 (5’-TGTTCTGCCAGTTGCCATTTCTG-3’) were designed based on AdiFP10 (accession number: BR000970). Using these primers and cDNA of A. digitifera and A. tenuis as templates, FP cDNAs were amplified. PCR products were cloned and the sequences were determined. The sequences were aligned using Genetyx (ver. 16.0.0). In order to remove PCR errors, cDNA sequences appeared at least two times were used for phylogenetic analysis.Complementary DNAs were synthesized using a PrimeScript II 1st Strand cDNA Synthesis Kit (Takara) from total RNA of FP gene was aligned with FP genes from other Scleractinia species by using Genetyx (ver. 16.0.0) and ClustalW [The coding sequence of the novel ClustalW , and subClustalW ) with 1,ClustalW . The nucFP cDNAs were cloned into a pCold I expression vector (Takara), and then transformed into the BL 21 Escherichia coli strain (Takara). Each clone was grown in 30 ml of LB medium overnight, and the recombinant protein was extracted by sonication and centrifugation. The S2 sequence in the expression vector was constructed from the S3 sequence by in vitro mutagenesis. In vitro mutagenesis of FP was performed by extension of DNA synthesis of overlapped oligo-nucleotides using PCR [FP genes from Montipora sp., we designed a set of primers to amplify the RFP gene, as we expected that the RFP gene might be highly expressed in our reddish-colored specimen. We designed a set of degenerated primers (RFP_F2 and RFP_R) based on amilRFP  and meffRFP  genes. We cloned and sequenced a PCR product amplified using these primers and cDNA of Montipora sp. as a template. We confirmed a single FP-like sequence showing high similarity (>80 %) with an FP nucleotide sequence  and protein sequences . This indicated that the newly isolated sequence was an FP family gene, which we tentatively named monFP . To isolate the full-length coding region of monFP, we performed 5’ and 3’ RACE. Similar to known FP genes in other species, the coding region of monFP was 723 bp. We constructed a neighbor-joining phylogenetic tree using nucleotide sequences of the monFP together with other FP genes available from Acropora digitifera (AdiFP1-AdiFP10), Acropora millepora (amilRFP), Montipora millepora, (mmilCFP), Montipora effloresce , and Psammocora sp. (psamCFP). The monFP gene formed a monophyletic group with AdiFP10, amilRFP, meffGFP, meffRFP, psamCFP, meffCFP, and mmilCFP , this group contained two different fluorescent color classes. One of the three amino acids  responsible for chromophore-formation of FPs was leucine in two GFPs, but aspartic acid in the other three RFPs. This leucine was unique to GFPs and the site may be responsible for the difference in the fluorescent color, representing the wavelength of emission light of monGFP.As a first step to isolate era AdiFP-AdiFP10,as a GFP , 17 and monGFP from cDNA of Montipora sp. by PCR, we designed primers (M5_F1 and M5_R1) at both ends of the monGFP sequence. The PCR products showed a band with expected size (short type) and an unexpected one (long type) in the electrophoresis image shown in Fig. amilRFP and meffRFP . The sequence of intron 1 of this PCR product was 1,950 bp, shown in Fig. i1_1800. Next, we designed primers specific to the i1_1250, i1_2100, and i1_2700 intron 1 sequences, and amplified the sequences from the genomic DNA between intron 1 and exon 3 using the primers specific to intron 1 and M5_RACE_R2. We obtained four products with nearly the same length (4 kb) and deduced the length of the sequences of intron 2 for each gene , but could not obtain amplified products. The introns 3 and/or 4 of these genes might be too long to be amplified by using PCR. We then determined the sequences of exon 1 to exon 3 of each monGFP gene, except the sequences of exon 1 from i1_2100 and i1_2700 genes. We could not determine these exon 1 sequences by direct sequencing, probably due to variations of short indels in intron 1 of PCR products. In the sequences of exon 1 to exon 3, the variation observed in each cDNA sequence was confirmed. Based on the exons 1 and 3 sequence types, only one pair, S2 and L2 variants with exon1 (TT) exon3 (AGG) matched with i1_1800 gene with exon1 (TT) exon3 (AGG)  exon3 (GTA) did not match with other variants  matched with i1_2100 gene with exon3 (GTA)  Fig.  and 4b. nts Fig.  and 4b, TA) Fig.  and 4b. TA) Fig.  and 4b. monGFP genes have evolved under purifying selection.To examine the functions of different genes and splice variants, we generated monGFP proteins from variants L1, L2, S1, and S2 to measure fluorescence. As shown in Fig. monGFP genes thus arises. One possible role is in increasing the expression of proteins with the same function. In A. millepora, the copy number variation of amilFP597 with a particular promoter type is correlated with red pigment concentration. The higher expression of amilFP597 has a photoprotective role to reduce photodamage to zooxanthellae under acute light [Mef2D gene [The question of the biological role of gene duplication and alternative splicing of te light . The othte light  if each f2D gene .monGFP forms a monophyletic clade with the other RFP and GFP genes. To examine whether genes in other species in this clade have undergone gene duplication, we designed a set of primers (AdiFP10_F2 and AdiFP10_R1) based on the AdiFP10 sequence to identify other FP genes expressed in A. digitifera and A. tenuis. We extracted RNA and synthesized cDNA separately from a single adult and large samples (over 100) of larvae from each species. We determined 97 sequences in total from the PCR products and isolated six and four distinct sequences from A. digitifera and A. tenuis, respectively. We constructed a phylogenetic tree using the isolated sequences, together with monGFP genes, AdiFP10, and two RFP genes, using AdiFP1 as an outgroup  were isolated from a single adult individual of A. digitifera. Since A. digitifera is diploid [Da2_12, clustered with the amilRFP gene, and the others formed a monophyletic group (Fig. Acropora species, and additional duplication occurred in the A. digitifera lineage, if no gene conversion is assumed in this species. In A. tenuis, three sequences were obtained from larvae cDNA, and we are not sure that whether the sequence variation was due to gene duplication events or allelic variation. These results suggest that there are at least three paralogs of AdiFP10 in Acropora species.As shown in Fig. oup Fig. . In thisade Fig. . Moreove diploid , we estiFP genes in Scleractinia occurred by gene duplication events and alternative splicing. We found at least four monGFP genes in the genome of Montipora sp., each with a similar function in terms of emitted fluorescence. Regarding the wavelength of fluorescence, monGFP genes have evolved without change in function even though the amino acid sequences of the first exon in different splicing variants are completely different. The similar function of duplicated monGFP may be attained through both purifying selection and homogenization by gene conversion, although the evidence supporting gene conversion in monGFP remains unclear. One of possible biological causes for the homogenization might be a requirement of increasing amount of the protein. Although the conserved evolution of the function of FPs is not well studied, this mode of evolution may be an important aspect with regard to FPs. When corals regulate the expression level of FPs to adapt or acclimatize to different environments, such as various light stresses [FP genes may be advantageous due to their ability to greatly change FP expression levels. On the other hand, gene duplication events and the acquisition of new functions has also been suggested in the FP family [AdiFP10 paralogs have diversified in Acropora species (Fig. amilRFP and meffRFP possess distinct fluorescent spectra, with emission peaks at 593 nm and 576 nm, respectively [AdiFP10 paralogs and other RFP genes in Acropora species may have diversified with respect to their functions, i.e., fluorescence. Thus, these FP genes are consistent with divergent evolution, as suggested previously [FP genes are consistent with various modes of evolution. These differences might reflect variation in the biological roles of different FPs. Further detailed evolutionary analysis of FP genes may reveal the roles of FPs in live corals.In this study, we showed that diversification of stresses , the funP family . AdiFP10ies Fig. . Althougectively , suggesteviously . Our res


However, the role of S100B in an inflammatory situation is not clear. The aim of the study was to determine whether S100B is likely to influence inflammation through its effect on macrophages. A murine macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages were used for in vitro studies and a model of retinal inflammatory disease in which pathogenesis is highly dependent on macrophage infiltration, Experimental Autoimmune Uveoretinitis, for in vitro study. Experimental Autoimmune Uveoretinitis is a model for the human disease posterior endogenous uveoretinitis, a potentially blinding condition, with an autoimmune aetiology, that mainly affects the working age group. To date the involvement of S100B in autoimmune uveoretinitis has not been investigated. Real-time PCR array analysis on RAW 246.7 cells indicated up-regulation of gene expression for various cytokines/chemokines in response to S100B, IL-1β and CCL22 in particular and this was confirmed by real-time PCR. In addition flow cytometry and ELISA confirmed up-regulation of protein production in response to S100B for pro-IL-1β and CCL22 respectively. This was the case for both RAW 264.7 cells and bone marrow derived macrophages. Induction of EAU with retinal antigen in mice in which S100B had been deleted resulted in a significantly reduced level of disease compared to wild-type mice, as determined by topical endoscopic fundus imaging and histology grading. Macrophage infiltration was also significantly reduced in S100B deleted mice. Real-time PCR analysis indicated that this was associated with reduction in CCL22 and IL-1β in retinas from S100B knock-out mice. In conclusion S100B augments the inflammatory response in uveoretinitis and this is likely to be, at least in part, via a direct effect on macrophages.S100B is a Ca Intraceactivity . S100B cactivity . Howeveractivity . S100B mactivity ,6.S100B has been shown to be involved in neurodegeneration and brain injury  with eleIn vitro studies on microglia cultured from murine BV-2 microglial cell lines have suggested that excessive production of S100B by astrocytes might lead to production of TNF-α, IL-1β, NO and COX-2 by microglia and subsequent enhanced inflammation [There is evidence to suggest that inflammation may be enhanced in these conditions by the action of S100B on macrophages/microglia. ammation ,13. S100ammation . Howeverin vitro, on both a macrophage cell line (RAW 264.7) and on primary macrophages, and also in vivo, in a model of retinal inflammatory disease in which pathogenesis is highly dependent on macrophage infiltration . S100B has long been known to be present within the eye particularly associated with Mϋller cells [The aim of the study was to clarify the involvement of S100B in inflammation and determine whether its influence is likely to be via macrophages. We have therefore examined its effects er cells –17 and ier cells . RAGE haer cells .In vitro studies showed that S100B had direct effects on macrophages, enhancing CCL22 and IL-1β expression in particular. EAU disease severity was shown to be reduced in mice in which S100B was deleted and this was related to a reduction in IL-1β and CCL22 expression in the retina. This suggests that S100B may play an active role in enhancing inflammation via its action on macrophages.S100B knockout (S100B KO) mice on a C57BL/6 background were supplied by the Medical Research Facility, University of Aberdeen. S100B KO mice were originally established and obtained from the RIKEN Brain Science Institute, Japan [et al., 2002 [Animal studies were conducted under a project licence granted by the UK Home Office according to the Animals Scientific Procedures Act 1986. The project was also subject to the University of Aberdeen’s Ethical Review Process and was approved by its Animal Welfare and Ethical Review Body, in accordance with the University Code of Practice for Research Involving the Use of Animals. C57BL/6 wild type (WT) control mice and e, Japan . A breed2. Primary macrophages were also cultured from bone marrow (BMDM) of C57BL/6 mice, aged between 8 and 12 weeks. Tibias and femurs were taken and excess tissue removed before sterilizing in 70% ethanol and rinsing in PBS. Bone marrow was flushed out using DMEM/F12 and cells were grown in 6 well plates in DMEM/F12, plus 10% FCS and 1% penicillin and streptomycin, containing 15% L929 conditioned DMEM. After 6 days of culture the cells were characterised as bone marrow derived macrophages by flow cytometry showing high expression of F4/80 , CD11b  and low expression of GR1 LY6C  and FLT3 . When the RAW 264.7 cells reached 80% confluence or on day 6 of primary bone marrow culture, where cells were also approximately 80% confluent, the media was replaced with serum-free media overnight before addition of bovine S100B protein .To determine the effect of S100B on macrophages, a murine macrophage cell line RAW 264.7  was cultured in DMEM plus 10% FCS and 1% penicillin and streptomycin at 37°C 5% COThe S100B preparation was filtered (0.22 μm) and underwent endotoxin removal treatment using endotoxin removal beads according to the manufacturer’s protocol . LAL endotoxin test  confirmed that the endotoxin levels in the S100B protein preparation were minimal at < 6 pg/ml following this treatment. S100B was heated to 95°C for 15 min and used as a control to confirm no endotoxin contamination.To determine whether 2 μM S100B was influencing cell viability, morphology was checked and an acid phosphatase cell viability assay done. RAW 264.7 or BMDM were treated with S100B for 24 h. Cells were imaged using a Widefield Ziess Observer microscope  and an A1R confocal microscope . For acid phosphatase assay, cells were washed in PBS and incubated with 100 μl of substrate, sodium acetate buffer containing 5 mM p-nitrophenyl phosphate substrate . One substrate tablet was dissolved in 2.4 ml sodium acetate buffer (0.1 M sodium acetate and 0.1% Triton). Cells were incubated for 2 h at 37°C. After the incubation period the reaction was stopped by adding 25 μl of 0.1 M sodium hydroxide. The plate was read at 405 nm in a microplate reader .RNA was extracted from RAW 264.7 and BMDM cells using TRIzol reagent  following manufacturer’s instructions. RNA was quantified  and cDNA generated using superscript II  according to manufacturer’s instructions with 2.0 μg total RNA in each reaction and oligo-(dT) priming. The reverse transcription was performed at 42°C for 50 min and 95°C for 10 min. PCR was in a 25 μl total volume reaction mixture containing 12.5 μl Go Taq DNA polymerase reaction buffer , 9.0 μl nuclease free water, 0.4 μM forward primer, 0.4 μM reverse primer and 1.5 μl cDNA template. RAGE primers were forward primer, 5’-CAGCATCAGGGTCACAGAAA-3’, and reverse primer, 5’-CTGGTTGGAGAAGGAAGTGC-3’. β-Actin was used as a reference gene, using forward primer, 5’-TGTGATGGTGGGAATGGGTCA-3’, and reverse primer, 5’-TTTGATGTCACGCACGATTTCC-3’. Primers were designed to be intron spanning using the NCBI and primer 3 software. The PCR program consisted of, 1 cycle of denaturation at 95°C for 3 min followed by 30 cycles of 95°C for 30 s, 63°C for 1 min and 72°C for 5 min before a final extension at 72°C for 5 min. The amplified products were analysed by gel electrophoresis on a 1.8% agarose gel containing ethidium bromide. PCR products were visualised under UV light. The resulting cDNA product was visualised at approximately 350 bp for RAGE and 500 bp for β-Actin.To confirm RAGE protein expression, western blot was carried out on BMDM. BMDM were collected, washed with PBS, centrifuged and re-suspended with 100 μl of 1% NP-40 PBS lysis buffer with the addition of protease inhibitor  and vortexed and placed on ice for 10 min before a further vortex for 20 s. The samples were then centrifuged at 162 g for 15 min and supernatant removed and stored at -80°C. Protein concentration for the supernatant was determined using BCA protein assay . Approximately 60 μg in LDS sample buffer without reducing agents  was applied to a non-reduced 12.5% PAGE gel. Mouse lung tissue was homogenised by passing the tissue through a 25G needle and syringe in 1ml NP-40 PBS lysis buffer with protease inhibitor and then treated as for the BMDM and used as a positive control. To identify protein band size, 8 μl MagicMark XP Western Protein Standard (Life Technologies) was also loaded onto the gel.Proteins were transferred to a PVDF membrane and blocked in 5% non-fat dry milk  in 0.1% Tween in PBS for 1 h followed by incubation with 2 μg/ml of Rat-Anti-Mouse RAGE monoclonal antibody  for 1 h at room temperature. The membrane was then washed 3 x 5 min with 0.1% Tween PBS and incubated with a HRP-conjugated goat anti-rat secondary antibody  for 1 h at room temperature. The membrane was washed again in PBS Tween as previously with an additional 15 min wash. Protein bands were visualised by enhanced chemiluminesence detection system  by mixing 1 ml ECL reagents 1 and 2 for 1 min and reading on a luminescent image analyser .To compare pro-inflammatory cytokine and chemokine gene expression in RAW 264.7 cells cultured for 6 h with or without 2 μM S100B, RNA was extracted using an RNeasy Micro kit  according to manufacturer’s instructions. RNA quality was checked using an Agilent RNA 6000 Nano kit  and only RNA samples showing clear 18S and 28S peaks, with the 28S:18S ratio higher than 2 were accepted for analysis . RNA was2 first strand kit . The cDNA was then used in a 96 well RT2 Profiler PCR Array for inflammatory cytokines and chemokines  all following manufacturer instructions. A Light Cycler 480 (Roche) was used for the PCR analysis. Cycle threshold values (CT) were converted into fold change using Qiagen data analysis software. The RT2 Profiler PCR Array for inflammatory cytokines and chemokines has been shown to be a robust method for this type of comparison and to give a good indication of the cytokines and chemokines which warrant further study at the protein level [From the total RNA extracted, 1 μg was used in cDNA synthesis, using RTin level ,23.CCL22 and IL-1β as responding to S100B, quantitative real-time PCR was done and the time and dose response to S100B determined. RNA was extracted and quantified and cDNA generated as described above. Real-time PCR was set up with 5 μl SYBR Green 2x concentration master mix (Roche), 1 μl forward primer (5 μM), 1 μl reverse primer (5 μM) and 3 μl cDNA and analysed using a Light Cycler 480 (Roche). Primer efficiency was determined by preparation of a standard curve (10 fold) using pooled cDNA, and primers with an efficiency of 1.85–2 were accepted for use. The primer sequences used were designed by Roche universal probe library to be intron spanning and were for CCL22, forward 5’-TCTTGCTGTGGCAATTCAGA-3’ and reverse 5’-gagggtgacggatgtagtcc-3’; and for IL-1β, forward 5’-agttgacggaccccaaaag-3’ and reverse 5’-agctggatgctctcatcagg-3’. Cycle threshold values were normalised to GAPDH gene expression using GAPDH primers, forward 5’-gggttcctataaatacggactgc-3’ and reverse 5’- ccattttgtctacgggacga-3’. The PCR program was 95°C for 5 min followed by 40 cycles of 95°C for 10 s, 60°C for 10 s and 72°C for 10 s. Results are expressed as relative fold change as calculated by the delta delta CT method [To confirm the PCR array results from RAW 264.7 cells, which identified T method .To confirm that S100B increases CCL22 production by RAW 264.7 macrophages or BMDM, supernatant was collected after 24 h incubation with or without S100B, centrifuged at 167 g for 5 min at 4°C and stored at -80°C. Supernatants were analysed for CCL22 using a DuoSet ELISA kit (R&D Systems) as recommended by the manufacturer.For studies on IL-1β pro-form production, RAW 264.7 cells or BMDM were treated with 2 μM S100B and 5 μg/ml Brefeldin A for 4 h . Cells were harvested in 2% FCS in PBS for flow cytometric analysis of intracellular pro-IL-1β. Cells were incubated on ice for 20 min with Fc block  before surface staining with PerCP-CY5.5 rat monoclonal anti-mouse CD11b . Cells were then fixed and permeabilised in BD BioSciences fixative and permeabilisation buffer, following manufacturer guidelines. PE-conjugated rat anti-mouse IL-1β pro-form  was added and incubated at room temperature for 30 min in the dark. Cell suspensions were then washed twice in permeabilisation buffer, mixing before each wash, with a final wash in FACS buffer (2% v/v FCS in PBS containing 2% w/v sodium azide). Cells were re-suspended in 300 μl FACS buffer before data acquisition on the LSR II flow cytometer (BD BioSciences). Analysis of FACS data was carried out using FlowJo Software, .1−20; GPTHLFQPSLVLDMAKVLLD; New England Peptide LLC, MA, USA) emulsified with Complete Freunds Adjuvant  containing additional 2.5 mg/ml Mycobacterium tuberculosis . An additional 100 μl, 10 μg/ml Bordetella pertussis toxin was administered by intraperitoneal injection. Mice were sacrificed by CO2 asphyxiation and cardiac puncture and eyes collected.EAU was induced using a standard method . Both S1et al.,2008 [et al., 2008 [Disease progression was followed using Topical Endoscope Fundal Imaging (TEFI) at day 15, day 21 and day 24 post peptide immunization (pi) to compare S100B KO and C57BL/6 WT mice. Mice were anaesthetised, pupils dilated using 1% (w/v) tropicamide and 2.5% (w/v) phenylephrine hydrochloride , and Viscotear liquid gel  was applied to each eye to provide good endoscope contact to cornea and avoid eyes drying. Fundus images obtained were clinically graded on a scale 0–4 by scoring changes in retinal infiltrate and lesions, retinal vessels and clarity of the optic disc according to Xu al.,2008 . At apprl., 2008 . Section2 Fab polyclonal anti-mouse IgG (Serotec) was applied for 30 min before incubation with anti-mouse S100B primary antibody  at a 1 in 500 dilution in 1% normal swine serum for 1 h at room temperature followed by washing in PBS. Sections were then incubated with the secondary antibody, 1 in 200 dilution, for 1 h before washing in PBS. ABC elite peroxidase  was applied for 30 min at room temperature, slides washed in PBS as previously, then the Nova Red substrate (SK4800) was applied. The substrate reaction was stopped after 30 s by washing in water. Sections were counterstained with haematoxylin before being dehydrated and mounted in DPX (Sigma).To examine the expression of S100B in the retina, immunohistochemistry was carried out using a Nova Red peroxidase substrate kit prepared as per manufacturer instructions . Sections were fixed in 100% ethanol for 5 min before being rehydrated in PBS and blocked with 10% normal rabbit serum for 30 min. F(ab’)2) in 3 sections at 3 spaced intervals through the eye. The average count for the sections was calculated for each eye.Sections were stained for macrophages with MOMA-2 antibody  using a 1 in 25 dilution. The secondary antibody was used at a 1 in 100 dilution . In control staining, primary antibody was replaced with tris-buffered saline. Sections were masked and total stained and unstained infiltrating cells present across the retinal layers, or within the rod outer segments were counted using a graticule to count cells in an exact area  value, , was calculated using the second derivative max, and allowed fold change calculations to be made. Each array provided quality control checks for PCR reproducibility, reverse transcription and genomic DNA contamination. Five reference genes were available to select the optimum gene normalisation. All samples were normalised to GAPDH and CT values greater than 35 were not reported. RNA samples from RAW 264.7 cells treated with or without S100B and meeting PCR array quality control standards were reverse transcribed, analysed using the PCR array and normalised to GAPDH. The PCR array showed an overall up-regulation of pro-inflammatory mediators in S100B treated compared to untreated cells  in response to incubation of RAW 264.7 cells with 2 μM S100B for 9 h (P = 0.0170) after 6 h incubation at this concentration. IL-1β showed a significant increase in RAW 264.7 cells compared to untreated cells with 1 μM and 2 μM S100B  and 1 μM S100B (P = 0.0078) compared to untreated control cultures .To confirm that S100B up-regulated transcription of IL-1β results in protein translation, flow cytometry was used to detect pro-IL-1β after treatment of RAW 264.7 cells and BMDM with 2 μM S100B for 4 h. Cells were gated using CD11b expression . Comparein vivo we used a murine model Experimental Autoimmune Uveoretinitis (EAU), in which retinal pathogenesis is highly dependent on macrophage infiltration and activation [To investigate the relevance of the pro-inflammatory effect of S100B on macrophages tivation ,30. To dtivation . An incr1−20. Progression of disease was followed using TEFI at day 15 and day 21 pi (P<0.05)  . Inflamm(P<0.05) .P = <0.05).Eyes were collected at day 24 pi for histological evaluation. Histology sections were graded according to cell infiltration and confirmed TEFI observations showing significantly reduced inflammatory infiltrate in the S100B KO mice compared to WT  confirmed reduction of CCL22 and IL-1β in retinas where S100B had been deleted.Thus we have shown for the first time that S100B is an important component in the development of retinal inflammation. It is likely to be present at levels that may have a direct action on macrophages, the key effector cell in uveoretinitis, up-regulating chemokine/chemokine receptor and cytokine expression, CCL22 and IL-1β in particular. Recently it has been shown that S100B can increase migration of BMDMs  and this