Chlorella vulgaris, this study investigated bioaugmentation with a cellulolytic and hydrogenogenic bacterium, Clostridium thermocellum, at different inoculum ratios as a possible method to improve CH4 and H2 production of microalgae.The recalcitrant cell walls of microalgae may limit their digestibility for bioenergy production. Considering that cellulose contributes to the cell wall recalcitrance of the microalgae C. thermocellum, as a result of enhanced cell disruption and excess hydrogen production. Furthermore, addition of C. thermocellum enhanced the bacterial diversity and quantities, leading to higher fermentation efficiency. A two-step process of addition of C. thermocellum first and methanogenic sludge subsequently could recover both hydrogen and methane, with a 9.4% increase in bioenergy yield, when compared with the one-step process of simultaneous addition of C. thermocellum and methanogenic sludge. The fluorescence peaks of excitation-emission matrix spectra associated with chlorophyll can serve as biomarkers for algal cell degradation.Methane production was found to increase by 17?~?24% with the addition of C. thermocellum improved the degradation of C. vulgaris biomass, producing higher levels of methane and hydrogen. The two-step process, with methanogenic inoculum added after the hydrogen production reached saturation, was found to be an energy-efficiency method for hydrogen and methane production.Bioaugmentation with  Microalgae have enormous potential as a source for biofuel and bioenergy production due to their high photosynthetic efficiencies, high growth rates, and characteristics of not requiring external organic carbon supply. Anaerobic digestion of algal biomass to biogas containing methane or hydrogen is one of the most energy-efficient and environmentally beneficial technologies. The proChlorella sp. and Scenedesmus sp. is known to contain recalcitrant cellulose[Chlorella vulgaris biomass was degraded during methanogenic fermentation. Various mechanical , physical (ultrasonication), thermal, and chemical  pretreatment methods have been investigated to improve the digestion efficiency[Rhizoclonium biomass prior to anaerobic digestion. The researchers observed that the enzymatic pretreatment led to greater methane conversions than the mechanical methods, and that the action of cellulase resulted in maximum methane yield, when compared with that of other enzymes. However, enzymes are usually only effective at the initial stage after addition and become inactive soon afterwards. Comparatively, living bacteria can continuously hydrolyze the materials through growth and proliferation. Nevertheless, appropriate bacterial species should be carefully selected to be effective for microalgae hydrolysis and be compatible with subsequent or synchronous anaerobic digestion.Anaerobic digestion could be carried out on microalgal residues after lipid extraction-6 or direllulose, which cellulose,10. Lakaellulose found thficiency,8,12-14.ficiency,15,16. Bficiency. Enzymatficiency found thficiency reportedC. vulgaris, this study investigated bioaugmentation with a thermophilic, anaerobic, cellulolytic, and hydrogenogenic bacterium, Clostridium thermocellum, which is also available from cellulose-fed anaerobic digester[C. vulgaris biomass to enhance the efficiency of methane and hydrogen production. To our best knowledge, the present study is the first report on improving C. vulgaris degradation by bioaugmentation using C. thermocellum.Considering that cellulose contributes to the cell wall recalcitrance in the microalgae digester, as a poC. vulgaris without C. thermocellum in Series 1 was 318 ml/g VS. There was a clear difference in methane production after addition of C. thermocellum. The highest methane yields for the inoculum ratios of 1%, 5%, and 10% of C. thermocellum were 376, 388, and 403 ml/g VS, respectively. Correspondingly, the maximum methane production rate was found to increase from 23.11 to 33.14 ml/g VS/day, and the lag time was noted to increase from 0.83 to 3.61 days when the inoculum ratio of C. thermocellum was increased from 0% to 10%. However, when the concentration of algal biomass was increased to 3 g VS/L in Series 2, the highest methane yield and maximum methane production rate were much lower than those noted for the corresponding treatment in Series 1 with the same inoculum ratio of 5% of C. thermocellum , in which a significant level of isovalerate was detected   method to resolve the EEM signals of the unknown samples from those of any overlapping and uncalibrated interferents. The core consistencies were 100%, 100%, 98.9%, 98.2%, 74.5%, 64.5%, and 7% for component numbers ranging from 1 to 7, respectively. Therefore, the optimal number of components was set to 4 for this model. The EEM contours of Components 1–4 are shown in Figure C. vulgaris and C. thermocellum culture medium, the following could be observed: Component 4 with the highest fluorescence was assigned to the distinct fluorophores from C. vulgaris; Component 1 was primarily contributed by C. thermocellum and partly by C. vulgaris; Component 3 was assigned to the soluble microbial products (SMP) generated from anaerobic digestion of the inoculated granular sludge; and Component 2 with low fluorescence intensity was the systematic background of buffer solutions and instruments.Algal cells contain specific fluorescent biochemical components (e.g. chlorophyll), and therefore exhibit different excitation-emission matrix (EEM) profiles. In the  Figure a with fi a and b,21. On tC. thermocellum significantly increased with the C. thermocellum inoculum ratio, suggesting that the bacteria kept growing on the substrate of C. vulgaris during the initial 7 days. Component 3 representing SMP also had higher scores at higher C. thermocellum inoculum ratio, implying that the growth of methanogenic inoculum was promoted by the addition of C. thermocellum. The scores of Component 4 representing microalgal fluorophores increased quickly on the first day and then decreased to a low value from Day 5 onwards, indicating rapid breakage and hydrolysis of the microalgal cell wall in 24 h, resulting in the release of intracellular fluorophores. The released fluorophores were instantly fermented into low-molecular intermediates in 5 days, and the fermentation rate was higher in treatments with 5% and 10% C. thermocellum.The scores of each PARAFAC component in different samples of Series 1 are presented in Figure C. vulgaris cells were observed under transmission electron microscopy (TEM). Samples taken from the treatment with 10% (v/v) C. thermocellum at 0, 7, and 15 days are shown in Figure C. vulgaris degradation, but the degradation rates were slightly lower.The H values of the treatments with C. thermocellum were significantly higher than those of the treatment without C. thermocellum during the initial 7 days and the H value of the treatment with 5% inoculum ratio was the highest. In solid phase, the highest H values were found for treatment with 10% C. thermocellum, followed by those for treatment with 5% inoculum ratio. However, the H values of treatments with 1% and 0% C. thermocellum inoculum ratios were quite similar, and much lower than those of treatments with 10% and 5% inoculum ratios.Shannon diversity index was used to analyze the ARISA profiles  were grown photoautotrophically in Liquid Bold’s Basal Medium (BBM)[2 (5% CO2 and 95% 0.45-μm filtered air) sparging. Light was provided by 8000–10000 lux LED lights . After 7 days of incubation, the microalgal biomass was harvested and concentrated by centrifugation at 3600?×?g for 15 min. The solid concentrate was subjected to anaerobic digestion. The concentrated algal biomass contained 12.9% (on wet weight basis) of total solid (TS), 93.5% (on dry weight basis) of volatile solid (VS), and 58, 11, and 14% (on dry weight basis) of proteins, lipids, and sugars, respectively.The microalgae um (BBM) with 0.1C. thermocellum (strain DSM2360) was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. Fresh cultures were maintained by routinely transferring 5% (v/v) inoculum into fresh medium containing 5 g/L of absorbent cotton. Other compounds contained in the fresh medium included (per liter of distilled water): KH2PO4, 0.50 g; K2HPO4?·?3H2O, 1.00 g; urea, 2.00 g; MgCl2?·?6H2O, 0.50 g; CaCl2?·?2H2O, 0.05 g; FeSO4?·?7H2O, 1.25 mg; morpholinopropane sulfonic acid, 10.00 g; resazurin, 1.00 mg; yeast extract, 6.00 g; glucose, 5.00 g; cysteine-HCl?·?H2O, 1.00 g. C. thermocellum was freshly harvested after 4 days of incubation when no more hydrogen production was detected.Granular sludge, cultivated in a laboratory-scale (3.5 L) anaerobic sequenced batch reactor (ASBR), was added as the methanogenic inoculum. The ASBR was operated at 55°C, and glucose and acetate  were utilized as the feedstock at an organic loading rate of 2 g COD/(L-day). The methanogenic sludge was taken after being acclimated for more than 50 days and rinsed with anaerobic preheated (55°C) buffer solution to remove the residual carbon. The buffer solution was the same as that used in the subsequent batch experiments. The TS of the granular sludge was 11.1% (w/w) and the VS was 77.8% (w/w) of the TS.C. thermocellum(1) Series 1: One-step methane production with different inoculum ratios of C. thermocellum was added at different inoculum ratios: 0%, 1%, 5%, and 10% (v/v). A control with only granular sludge and without microalgae was prepared to measure the endogenous activity of the sludge itself. Another control with granular sludge and culture medium containing 5% (v/v) of C. thermocellum was set up to determine the methane production potential of the culture medium. The bottles were filled up to 500 ml with buffer solution and flushed with nitrogen for 2 min to maintain anaerobic conditions. The composition of the buffer solution was as follows (per L): 1.0 g of NH4Cl, 0.4 g of K2HPO4?·?3H2O, 0.2 g of MgCl2?·?6H2O, 0.08 g of CaCl2?·?2H2O, 10 ml of trace element solution, and 10 ml of stock vitamin solution. The stock trace element and vitamin solutions were prepared according to Chen et al.[Batch experiments were conducted at 55°C with 2 g VS/L of the algal biomass and 3 g VS/L of methanogenic sludge. The culture medium containing n et al..(2) Series 2: Two-step co-production of hydrogen and methaneC. thermocellum containing culture medium at 55°C for 7 days in 500-ml buffer solution, as mentioned earlier, for hydrogen production. Subsequently, 2 g VS/L of methanogenic sludge was added to produce methane. As a reference, a one-step experiment with the same amount of algal biomass, granular sludge, and 5% (v/v) of C. thermocellum containing culture medium was set up. Furthermore, a control with granular sludge and 5% (v/v) of C. thermocellum containing culture medium, similar to that used in Series 1, was also included in this series. Two different inoculum to substrate ratios  were introduced to investigate the influence of inoculum to substrate ratio. All the experiments were carried out in duplicate and the results were expressed as means.In the two-step experiment, 3 g VS/L of algal biomass was first incubated with 5% (v/v) of , and carbon dioxide in the biogas were analyzed using a gas chromatograph  equipped with a thermal conductivity detector (TCD). The gas volumes were corrected to standard temperature and pressure conditions (STP: 0°C and 1013 kPa). Methane production from the culture medium and methanogenic sludge was deducted in the reported data. Gompertz modeling (Eq.1) was used according to Lü et al.[P, Rmax, and λ) were determined.Gas production was measured by manometric methods. The pressure in the headspace of the serum bottles was measured by a Testo 512 pressure meter . The concentrations of hydrogen, methaneü et al. to fit tM(t) is the cumulative methane production (ml/g VS added) at time t (days), P is the highest methane yield (ml/g VS), Rmax is the maximum methane production rate (ml/g VS/day), and λ is the lag phase (days). Lag phase refers to the initial adaptive phase, during which methane production remains relatively constant prior to rapid growth.g for 10 min. Subsequently, the supernatants were collected and analyzed for pH, volatile fatty acids (VFAs), alcohols, dissolved organic carbon (DOC), total inorganic carbon (TIC), dissolved nitrogen (DN), and three-dimensional fluorescent intensity. The pH was tested with a pHS-2 F Digital Meter. The DOC, TIC, and DN were analyzed on a TOC-VCPH Analyzer . The concentrations of VFAs  and alcohols in the supernatant were determined using an Agilent 6890 N gas chromatography (GC) system equipped with a flame ionization detector (FID). The fluorescence excitation-emission matrixes (EEM) were recorded for the supernatant in a 10-mm quartz cuvette in a Varian Cary Eclipse fluorometer . The emission was scanned from 220 to 750 nm at 2-nm intervals and 10-nm bandwidth, while the excitation was produced with a Xenon flash lamp in 10-nm bandwidth at 10-nm intervals from 200 to 700 nm. The EEM signals were processed and subjected to parallel factor analysis (PARAFAC), as described in the study by Lu et al.[The liquid samples were centrifuged at 16,000?×?u et al..C. vulgaris were observed using transmission electron microscopy (TEM). Samples were prepared according to the procedure developed by Yamamoto et al.[The cells of o et al., and exaC. vulgaris were stained successively by FITC for proteins, Con A for α-polysaccharides and calcofour white for β-polysaccharides according to Chen et al.[The cells of n et al.. The samH value was calculated using the software PAlaeontological STatistics (PAST) version 2.17b, according to the procedure proposed by Hammer et al.[Both the liquid samples and granules corresponding to different sampling dates were used for DNA extraction. The total DNA was extracted from the pellets using PowerSoil DNA isolation kit , according to the manufacturer’s protocol. The fingerprint technique of Automated Ribosomal Intergenic Spacer Analysis (ARISA) was used to monitor the microbial dynamics. The extracted DNA was amplified using primers 1389 F and 71R for archaea, and primers ITSF and ITSReub for bacteria, respectively. Polymerase chain reaction (PCR) and ARISA of the PCR product were carried out according to the method described by Qu et al.. Shannonr et al..VFA: Volatile fatty acid; EEM: Excitation-emission matrix; PARAFAC: Parallel factor analysis; SMP: Soluble microbial products; TEM: Transmission electron microscopy; ARISA: Automated ribosomal intergenic spacer analysis; PCR: Polymerase chain reaction; DOC: Dissolved organic carbon; TIC: Total inorganic carbon; DN: Dissolved nitrogen; TS: Total solid; VS: Volatile solid.The authors declare that they have no competing interests.FL conceived the study, participated in microbial community analyses, parallel factor analyses, data interpretation and thoroughly reviewed the manuscript. JJ carried out the microalgae and biomass production and harvesting, anaerobic cultivations and all related analyses, microbial community analyses, data interpretation, and drafting and completion of the manuscript. LS participated in the design of the study and data interpretation, and thoroughly reviewed the manuscript. PH participated in the design of the study and data interpretation, and thoroughly reviewed the manuscript. All authors read and approved the final manuscript.Figure S2 ARISA profile of bacteria in solid phase. The marks (e.g. “12”) above represent the time of incubation, for example, “12” means the day 12 in the incubation. The profile marked with 0 represents the profile of the seed sludge, which was used in all the reactors at day 0. Figure S3 ARISA profile of methanogens in liquid phase. The marks (e.g. “23”) above represent the time of incubation, for example, “23” means the day 23 in the incubation. Figure S4 ARISA profile of methanogens in solid phase. The marks (e.g. “12”) above represent the time of incubation, for example, “12” means the day 12 in the incubation. The profile marked with 0 represents the profile of the seed sludge, which was used in all the reactors at day 0. Figure S5 The spectral microscope images of stained Chlorella vulgaris. a phase contrast photograph, b combined image of individual images in c-e, c spectral microscope image of α-polysaccharides (con A), d spectral microscope image of β-polysaccharides , e spectral microscope image of protein (FITC). Figure S6 The spectral microscope images of stained Chlorella vulgaris cells and cell walls. a phase contrast photograph, b spectral microscope image of β-polysaccharides .ARISA profile of bacteria in liquid phase. The marks (e.g. “23”) above represent the time of incubation, for example, “23” means the day 23 in the incubation. Click here for file


Using the high-pressure cryocooling method, the high-resolution X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. This is the first ultra-high-resolution structure obtained from a high-pressure cryocooled crystal. et al., 2005Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim  Large crystals with dimensions of up to 0.6 mm were obtained after one week.The expression and purification of bovine H-protein were performed as described previously , because the ambient-pressure data were collected from a single crystal with dimensions of 200–300 mm (Higashiura et al., 2010C2 with unit-cell dimensions of a = 84.73, b = 41.40, c = 43.28 Å, β = 91.57°. The cryocooled crystal at ambient pressure also belonged to space group C2 with unit-cell dimensions of a = 84.41, b = 41.25, c = 43.05 Å, β = 91.18° (Higashiura et al., 2010et al., 2010et al., 2010et al. (2010−3. The hydrogen atoms bound to atoms with multiple conformations, partial occupancies, waters and glycerol molecules were excluded from the count. Because we observed electron density from a considerable number of hydrogen atoms, we believe that we succeeded in high-quality data collection and structural determination from a high-pressure cryocooled crystal.To obtain a complete data set the data collections were carried out at two different conditions, and the overall  al. 2010. The thret al., 2010α atoms is 0.12 Å, and the r.m.s. deviation for all atoms was 0.61 Å. Differences were mainly observed in side-chains on the molecular surface and in multiple conformations. In the high-pressure cryocooling structure, 196 of 300 water molecules were in common with water molecules in the ambient-pressure structure, and the r.m.s. deviation was 0.48 Å. Of the common water molecules, 141 of 196 interacted with protein atoms, and the r.m.s. deviation was 0.44 Å. The r.m.s. deviation of the 55 remaining water molecules was 0.56 Å. Non-common water molecules were distributed uniformly in crystals.We previously reported the rigid packing of bovine H-protein crystal (Higashiura We observed no significant differences in structural comparisons at ultra-high resolution, and there seemed to be no artificial structural variation resulting from the high-pressure cryocooling. Thus, the versatility of this technique was confirmed by the ultra-high-resolution structural comparison.3wdnPDB reference:


The Daniel Fast is a vegan diet that prohibits the consumption of animal products, refined foods, white flour, preservatives, additives, sweeteners, flavorings, caffeine, and alcohol. Following this dietary plan for 21 days has been demonstrated to improve blood pressure, LDL-C, and certain markers of oxidative stress, but it has also been shown to lower HDL-C. Krill oil supplementation has been shown to increase HDL-C.We investigated the effects of following a Daniel Fast dietary plan with either krill oil supplementation (2 g/day) or placebo supplementation  for 21 days. The subjects in this study (12 men and 27 women) were heterogeneous with respect to body mass index (BMI) , blood lipids (normolipidemic and hyperlipidemic), blood glucose , and blood pressure (normotensive and hypertensive).p > 0.05), and so the data from the krill oil group and the placebo group were collapsed and analyzed to examine the effects of following a 21-day Daniel Fast. Significant reductions were observed in LDL-C (100.6 ± 4.3 mg/dL vs. 80.0 ± 3.7 mg/dL), the LDL:HDL ratio (2.0 ± 0.1 vs. 1.7 ± 0.1), fasting blood glucose (101.4 ± 7.5 mg/dL vs. 91.7 ± 3.4 mg/dL), fasting blood insulin (7.92 ± 0.80 μU/mL vs. 5.76 ± 0.59 μU/mL), homeostasis model assessment of insulin resistance (HOMA-IR) (2.06 ± 0.30 vs. 1.40 ± 0.21), systolic BP (110.7 ± 2.2 mm Hg vs. 105.5 ± 1.7 mm Hg), and body weight (74.1 ± 2.4 kg vs. 71.5 ± 2.3 kg) .Krill oil supplementation had no effect on any outcome measure (all Following a Daniel Fast dietary plan improves a variety of cardiometabolic parameters in a wide range of individuals in as little as 21 days, and these improvements are unaffected by krill oil supplementation.Clinicaltrial.govNCT01378767 Type 2 diabetes and cardiovascular disease are two epidemics that will grow more prevalent in the near future ,2. DietaA recently investigated “Daniel Fast”  was shown to lower blood pressure, total cholesterol, and low density lipoprotein cholesterol (LDL-C) . Other iThe Daniel Fast has also been shown to lower high density lipoprotein cholesterol (HDL-C), although the reduction in this cholesterol was outpaced by a greater reduction in LDL-C . Nonethe2. Key exclusion criteria were smoking, contraindication to krill oil or coconut oil (placebo) supplementation, pregnancy or desire to become pregnant, and unwillingness to follow a vegan diet. As these exclusion criteria were included in the recruitment flyers, outside of self-exclusion, none of the individuals that expressed interest in the study had to be excluded from participating. Sample size for this study was calculated based on the changes in total cholesterol and LDL-C that were observed in our previous investigation of a 21-day Daniel Fast [2), 9 were classified as overweight (BMI 25–29.9 kg/m2), and 8 were classified as obese (BMI ≥ 30 kg/m2). Thirty-three subjects were classified as exercise-trained, which was defined as performing ≥ 2 h/week of combined anaerobic and aerobic exercise of moderate to high intensity. On average, subjects performed 2.1 ± 0.2 h/week of anaerobic exercise for 6.8 ± 1.1 years and 4.7 ± 0.4 h/week of aerobic exercise for 8.3 ± 1.0 years. Six subjects had elevated BP (≥ 140/90 mm Hg), 7 subjects had elevated total cholesterol (≥ 200 mg/dL), and 2 subjects were type 2 diabetics. The following medication usages likely affected some of the outcome measurements in this study: Two subjects were taking hormone replacements, 2 were taking hypothyroid medication, 1 was taking an anti-inflammatory, 1 was taking insulin, 1 was taking hyperthyroid medication, and 1 was taking a stimulant/appetite suppressant. Failure to exclude individuals taking these medications during the screening process can be considered a limitation of this study. Subjects were instructed to not consume antioxidant supplements from 2 weeks prior to beginning the dietary plan until end of study. Prior to participation, each subject was informed of all procedures, potential risks, and benefits associated with the study in both verbal and written form in accordance with the approved procedures of the University Institutional Review Board for Human Subjects Research (protocol H11-14). Subjects provided written informed consent prior to being enrolled in the study.Subjects were recruited from the Memphis area through the use of flyers placed on and around the University of Memphis campus. Eligibility was assessed via questionnaire. Our prior investigation of a 21-day Daniel Fast indicated that changes in outcome measures in response to the dietary plan were unaffected by gender, baseline body weight, or baseline exercise habits . Therefoiel Fast . Forty-oThe dietary intervention consisted of only consuming foods that comply with a Daniel Fast for 21 days. The 21-day duration was chosen to maximize generalizability – when motivated by religious reasons, most individuals follow a Daniel Fast dietary plan for 21 days, although 10- and 40-day Daniel Fasts have also been observed – and also to allow for a comparison against the findings from our previous study of the Daniel Fast . No mealThe outcome variables described below were measured immediately before (day 1) and after following the Daniel Fast dietary plan (day 22). All data collection occurred between 05:00 and 11:00 hours while subjects were in a 12-hour post-absorptive state.Subjects were randomly assigned to consume one of the following for the duration of the Daniel Fast: Twenty-one subjects (7 men and 14 women) were assigned to orally consume krill oil capsules , and 20 subjects (7 men and 13 women) were assigned to orally consume placebo capsules . Specifically, men and women were alternately assigned to the krill oil group and the placebo group as they were enrolled in the study. Both the subjects and the investigators were blind to treatment via the use of coded bottles. A recent investigation examining the safety and tolerability of krill oil supplementation at 2 g/day for 4 weeks indicated that “krill oil was generally well tolerated and did not show evidence of any adverse influence on safety parameters” . Both ofSubjects were seated in a chair with a BP cuff placed on their left arm. After a 10-minute period of quiet rest, two technicians measured heart rate by palpating the radial artery for 60 seconds. Blood pressure was then measured via auscultation using a dual-earpiece stethoscope that allowed for two technicians to listen simultaneously. Duplicate measures were obtained for both heart rate and BP, and the average of all measures was used in data analysis. If values deviated by more than 5 beats/minute for heart rate or 5 mm Hg for BP, an additional measure was taken.Subjects’ height was measured using a stadiometer, and bodyweight was measured using a calibrated medical scale. Waist and hip circumference measurements were obtained using a tension-regulated measuring tape; subjects wore athletic shorts for these measurements. Fat mass, fat-free mass, total body fat percentage, and trunk-specific body fat percentage were determined via dual energy x-ray absorptiometry (Hologic QDR-4500 W) using a 4-min fan array.Blood samples that were collected in tubes containing EDTA were immediately separated via centrifugation at 1500 g for 15 minutes at 4°C for collection of plasma. Blood samples that were collected in tubes containing no additives were allowed to clot at room temperature for 30 minutes and then separated by centrifugation at 1500 g for 15 minutes at 4°C for collection of serum. A portion of blood samples were analyzed within 1 day of collection as follows: A complete blood count was performed using an automated cell counter (Coulter LH750). A comprehensive metabolic panel was obtained using automated procedures (Roche/Hitachi Modular). A lipid panel was obtained using enzymatic procedures (Roche/Hitachi Modular). C-reactive protein was measured using a high-sensitivity, particle-enhanced turbidimetric immunoassay (Roche Integra 800). Insulin was measured using an immuno-chemiluminescent assay procedure (Roche Modular E170). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as: fasting glucose (mg/dL) × fasting insulin (μU/mL)/405 .The remaining blood samples were immediately stored in multiple aliquots at −70°C until analysis for the following: Malondialdehyde was analyzed in plasma following the procedures of Jentzsch et al.  using reAll subjects were instructed to maintain their normal diet until they began the Daniel Fast. During the 7 days immediately prior to beginning the Daniel Fast, subjects recorded all food and beverages consumed. Subjects also recorded all food and beverages consumed during the final 7 days of the Daniel Fast. These dietary records were analyzed using Food Processor SQL, version 9.9 . Subjects were instructed to maintain their normal physical activity and exercise habits during the entire study period with one exception: Subjects were instructed to refrain from strenuous exercise during the 48 hours immediately preceding the 2 assessment days. Subjects were also instructed to refrain from alcohol consumption during the Daniel Fast (as detailed in the dietary guidelines) and also during the 48 hours that preceded day 1.At the conclusion of the intervention period (day 22), subjects completed a questionnaire pertaining to their experience with the Daniel Fast. Using a scale of 1–10 , subjects rated their feeling of physical health and vitality, their mental health , and their level of satiety while following the dietary plan. Subjects also rated their compliance to the Daniel Fast using a scale of 0–100 .p ≤ 0.05.All outcome measures were analyzed using a 2 (condition) × 2 (pre/post intervention) ANOVA. However, data for both conditions were subsequently collapsed and analyzed using a paired t-test. Analyses were performed using JMP statistical software . Statistical significance was set at p > 0.05). Based on these analyses, data for all variables were collapsed, analyzed using a paired t-test, and are presented as pre and post intervention.Thirty-nine subjects successfully completed the study. Compliances to the krill oil and placebo supplementations were 92.4 ± 1.7% and 92.8 ± 2.2%, respectively. No condition (krill oil or placebo) or interaction effects were noted for any primary or secondary outcome measure , while the decrease in diastolic BP was not statistically significant . Body weight (74.1 ± 2.4 kg vs. 71.5 ± 2.3 kg), fat mass (21.9 ± 1.5 kg vs. 20.8 ± 1.5 kg), and fat-free mass (52.2 ± 2.0 kg vs. 50.8 ± 1.9) each decreased .Blood pressure and anthropometric data are presented in Table p < 0.01). No changes were observed in serum triglycerides or VLDL (both p > 0.05). Blood glucose (101.4 ± 7.5 mg/dL vs. 91.7 ± 3.4 mg/dL), blood insulin (7.92 ± 0.80 mg/dL vs. 5.76 ± 0.59 mg/dL), and HOMA-IR (2.06 ± 0.30 vs. 1.40 ± 0.21) each decreased . Resistin (6.06 ± 0.31 ng/mL vs. 6.72 ± 0.32 ng/mL) and NOx (19.26 ± 2.56 μmol/L vs. 28.49 ± 3.53 μmol/L) increased (both p < 0.05). No changes were observed in adiponectin, malondialdehyde, hydrogen peroxide, Trolox Equivalent Antioxidant Capacity, or C-reactive protein .Cardiometabolic-, oxidative stress-, and inflammatory data are presented in Table p < 0.05). Fiber intake increased .Dietary data are presented in Table We sought to determine if krill oil supplementation could maintain HDL-C in individuals following a Daniel Fast dietary plan for 21 days. This was not the case, as HDL-C decreased similarly regardless of whether krill oil or a placebo was consumed. Krill oil supplementation also did not affect any other outcome measure. Our findings therefore strongly contradict the work of Bunea et al. , who repFollowing a Daniel Fast dietary plan resulted in clinically significant reductions in LDL-C, the LDL:HDL ratio, blood glucose, blood insulin, HOMA-IR, systolic BP, and body weight. This study demonstrates that meaningful improvements to cardiovascular and glycemic parameters can be obtained in response to dietary changes in only 21 days.Plant-based diets have consistently been shown to reduce LDL-C . The maiThe reductions in blood glucose, blood insulin, and HOMA-IR in the present study were not observed in our previous investigation of the Daniel Fast . SmallerThe reduction in systolic BP may have been mediated by an increase in nitric oxide, a signaling molecule that promotes vasodilation . SupportThere were some limitations associated with this study. First, the study lacked a control group of subjects maintaining their usual diet during the 21-day intervention phase. Many prior investigations have demonstrated that our measured outcome variables experience minimal to no change in 21 days in the absence of modification in dietary habits, exercise habits, or medication usage. Nonetheless, such changes are possible, and our failure to include a control group clouds our ability to determine how much of the changes in our measured outcome variables were due to dietary changes. Second, as mentioned above, some subjects were enrolled in the study despite using medications that may have affected outcome measures. Finally, the inclusion criteria were broad, making it difficult to generalize the results of this investigation.2), LDL-C (100.6 mg/dL), blood glucose (101.4 mg/dL), and systolic BP (110.7 mm Hg) did not differ greatly from healthy norms . Insofar as healthy individuals have less “room for improvement”, this suggests that individuals with less-desirable values for these variables could potentially derive greater clinical benefit from this dietary plan. As mentioned above, this was observed when the results of this study were compared to those of our previous investigation of the Daniel Fast [A key strength of this study is that the observed changes were of sizeable magnitudes despite occurring in individuals whose average baseline values for BMI . None of the authors have a financial interest in the krill oil used in this study or in NOW Foods. There are no known or perceived conflicts of interest for any author.The authors’ contributions were as follows: RJB designed the experiment, performed the laboratory analyses, analyzed the data, assisted with the preparation of the manuscript, and has primary responsibility for the final content. JFT, MMK, and RJA conducted the clinical trial. JFT also wrote the manuscript. All authors read and approved the final manuscript.


We suggest that these acid dissociation constants are related to the carboxyl, enol, and amino groups.The acid dissociation constants of potential bioactive fused ring thiohydantoin-pyrrolidine compounds were determined by potentiometric titration in 20% (v/v) ethanol-water mixed at 25 ± 0.1°C, at an ionic background of 0.1 mol/L of NaCl using the HYPERQUAD computer program. Proton affinities of potential donor atoms of the ligands were calculated by AM1 and PM3 semiempiric methods. We found, potentiometrically, three different acid dissociation constants for  Acid dissociation constants are important physicochemical parameters, which can provide critical information about drug properties such as solubility, lipophilicity, acidity –3, transThiohydantoins , 12 are In the previous work , we have−3 mol/L in 20% (v/v) ethanol-water, 0.025 mol/L NaOH, and 0.1 mol/L HCl (J. T. Baker) and 1.0 mol/L NaCl (Riedel-de Haën) stock solution were prepared. Deionized water with a resistance of 18.2 MΩ·cm was obtained with an aquaMAX-Ultra water purification system (Young Lin Inst.).All reagents having analytical grade were used without further purification. Sodium hydroxide (Merck), potassium hydrogen phthalate (Fluka), and sodium tetraborate (Fluka) were dried at 110°C before use and 0.05 mol/kg potassium hydrogen phthalate and 0.01 mol/kg sodium tetraborate solutions were prepared for calibration of the electrode systems. Solution of ligands 1.10pH-Metric titrations were performed using the Molspin pH meter with an Orion 8102BNUWP ROSS ultracombination pH electrode. The temperature in the double-wall glass titration vessel was kept at 25.0 ± 0.1°C and controlled using a thermostat . During the titration, the vessel solution was stirred. The combination pH electrode was calibrated by the buffer solutions of pH 4.005  and 9.018 (sodium tetraborate) at 25.0 (±0.1)°C according to the instructions of the Molspin Manual . An autoDuring the titration, nitrogen (99.9%) was purged through the titration vessel. Acidity and stability constants were computed using the HYPERQUAD computer program .The potentiometric titrations were carried out using Molspin pH meter with an Orion 8102BNUWP ROSS ultracombined electrode. The titration temperature was adjusted at 25.0 ± 0.1°C and controlled by a thermostat . The double-wall glass titration vessel was used and the titration solution was magnetically stirred. Before and after each titration, the double-wall glass titration vessel was rinsed with distilled water and dried with a piece of tissue. The titration vessel was capped by a lid, which contained three holes for the electrode, glass tubing for nitrogen purging, and plastic tubing for alkali from the burette. Air bubbles were excluded while dropping with alkali solution. The syringe was rinsed with deionized water and at least three times with the alkali before filling with the alkali solution. The acid dissociation constants were determined by titrating 50.00 mL of ligands with standardized NaOH solution and three titrations were performed for each ligand. HYPERQUAD computer program was used for the calculation of acidity and stability constants. Standard deviations quoted only refer to random errors. 130 of the titration data were obtained for each experiment. Volume increment of the NaOH solution was 0.03 mL. The pKw value as, defined for the aqueous system was obtained as 13.98 at the ionic strength employed.1a–g), prepared according to the literature method [a values for each compound, except for 1g which had four acid dissociation constants due to the presence of the pyridine group. For the thiohydantoin-pyrrolidine fused ring systems (1a–g), pKa1, pKa2, and pKa3 values were found in a range of 3.21 ± 0.02–3.80 ± 0.05, 8.13 ± 0.01–8.66 ± 0.02, and 10.87 ± 0.01–12.70 ± 0.05, respectively. pKa1 values (a range of 3.21 ± 0.02–3.80 ± 0.05) arise from hydrolysis of the methyl ester in acidic aqueous solution. In acidic solution, the weak nucleophile water attacks carbonyl carbon atom, having protonated oxygen atom; then carboxylic acid derivatives and alcohol groups are formed [2a–g) are formed, after hydrolysis of methyl ester groups of thiohydantoin-pyrrolidine compounds (1a–g) (2a–f) may have four acidic centers as carboxyl, enthiol, enol, and amino group. As a result of theoretical and experimental calculations, we suggest that pKa1 value is related to the carboxyl group; pKa2 and pKa3 values are related to enol and amino group, respectively.Acid dissociation constants of the thiohydantoin-pyrrolidine compounds containing methyl ester group as a substituent  . Despite3, LH2, and LH were observed during titration processes. The deprotonation equilibrium is as seen in the following equations (charges are omitted for simplicity):n) are given asAcid dissociation constants were potentiometrically obtained from several series of independent measurements. Three deprotonated species that formulated as LH2a–g) and the distribution curves of species H in 20% (v/v) ethanol-water mixed are shown in All species have broad protonation space between pH 2 and 12. When pH increases, the protonated ligand losses protons and it is converted to the other forms. The titration curves of the ligands (Acid dissociation constants of ligands were computed from data which were obtained by potentiometric titrations using HYPERQUAD computer program. These acid dissociation constants are given in Hf) and the total energies (TE) of the ligands and monoprotonated species were calculated by semiempirical AM1 and PM3 methods. In addition, the proton affinity of each nitrogen, oxygen, and sulphur atom (PA) in the ligands was found using formation heats in the following equation and given in Hf°  (B) is the formation heat of B molecule, ΔHf° (BH+) is the formation heat of BH+ molecule, and 367.2 is the formation heat of H+ [Theoretical calculations were made in order to examine the structure of the species and to determine protonation order of nitrogen, oxygen, and sulphur atoms in the ligands. The formation heats , protonation orders of atoms in the ligand are 3S, 4N, 1O, and 5O, but dissociation orders of atoms in the H are 5O, 1O, 4N, and 3S. There might be differences in these orders due to forming intermolecular hydrogen bonding and forming acid thiol and enol group because of tautomerism in the ligand. It is assumed that high affinity causes the protonation of the sulfur and oxygen atoms on ligand and increases the mobility of its π-electrons. Consequently, sulfur and oxygen atoms of thiohydantoin begin to be hydrolyzed at appreciable rates in the solutions more alkaline than pH 9 [Proton affinity gives information about protonation order. Since the nitrogen, oxygen, and sulphur atoms having the highest PA are 2S in ligand, in other ways, 2S is more basic than other nitrogen and oxygen groups. Thus, the first protonated atom is 2S in this ligand. According to the calculated results  has got four ionizable groups as thiol, enol, amino, and carboxyl in ligands. In other ways, the potential coordinating sites are sulphur atom of the thiol group, nitrogen atoms of the amino group, and oxygen atoms of the carboxyl group and hydroxyl group. But, due to intermolecular hydrogen bonding between the neighbouring nitrogen atoms and the basic nature of the sulphur atom, the dissociation of the thiol group takes place at a very high pH level. Thus acid dissociation constant of thiol group was not determined in our experimental conditions.According to the electronic delocalisation, which is enhanced upon deprotonation, the ligand is very versatile, because thione and ketone forms were easily transformed into thiol and enol forms depending on pH , 33. Thea values for 1a–f were calculated. In addition, theoretical calculations were carried out by AM1 and PM3 semiempiric methods.In conclusion, the acid dissociation constants of thiohydantoin-pyrrolidine fused derivatives, which constitute important information for future studies in this area, were determined potentiometrically and the results were interpreted with the HYPERQUAD computer program. The determination of the acid dissociation constants was performed in 20% (v/v) ethanol-water hydroorganic solvent at 25 ± 0.1°C, at an ionic background of 0.1 mol/L of NaCl. Under these conditions, three different pK


Vernonia condensata Baker (Asteraceae). Dried and powdered leaves were exhaustively extracted with ethanol by static maceration followed by partition to obtain the hexane, dichloromethane, ethyl acetate, and butanol fractions. Total phenols and flavonoids contents were determined through spectrophotometry and flavonoids were identified by HPLC-DAD system. The antioxidant activity was assessed by DPPH radical scavenging activity, TLC-bioautography, reducing power of Fe+3, phosphomolybdenum, and TBA assays. The total phenolic content and total flavonoids ranged from 0.19 to 23.11 g/100 g and from 0.13 to 4.10 g/100 g, respectively. The flavonoids apigenin and luteolin were identified in the ethyl acetate fraction. The IC50 of DPPH assay varied from 4.28 to 75.10 µg/mL and TLC-bioautography detected the antioxidant compounds. The reducing power of Fe+3 was 19.98 to 336.48 μg/mL, while the reaction with phosphomolybdenum ranged from 13.54% to 32.63% and 56.02% to 135.00% considering ascorbic acid and rutin as reference, respectively. At 30 mg/mL, the ethanolic extract and fractions revealed significant effect against lipid peroxidation. All these data sustain that V. condensata is an important and promising source of bioactive substances with antioxidant activity.The present study evaluated the antioxidant potential of  After 60 min at room temperature, the absorbance was recorded at 518 nm. The experiment was performed in triplicate. Rutin was used as standard control. IC50 values denote the concentration (μg/mL) of sample, which is required to scavenge 50% of DPPH free radicals.DPPH was used for determination of free radical-scavenging activity . DiffereμL of each sample  (10 mg/mL) were applied on thin layer chromatography (TLC) (7 cm × 2 cm) of silica gel 60 F254 which was eluted with dichloromethane : methanol (5% or 15%) in duplicate  [The reducing power of iron was determined using a serial dilution of the samples  to molybdenum (V) which occurred in the presence of antioxidant substances with consequent formation of a green complex between phosphate/molybdenum (V) in acidic pH, which is spectrophotometrically determined at 695 nm . The antThe TBA test determines the presence of malonaldehyde and other substances from lipid peroxidation , formingμg/mL). The samples were oxidized with Folin-Ciocalteu reagent and the reaction was neutralized with sodium carbonate. The absorbance of the resulting blue color was measured at 765 nm after 60 min. The analyses were performed in triplicate and results were expressed as gram of gallic acid equivalent.The total phenolic content was determined by Folin-Ciocalteu method  using gaμg/mL) and results were expressed as gram of rutin equivalent.Aluminum chloride colorimetric method was used for total flavonoid determination  using ruμm particle size. Mobile phase was composed by solvents A (water pH adjusted to 4.0 with H3PO4) and B (acetonitrile). The elution conditions applied were 0–30 min, 20% B isocratic. The mobile phase was returned to the original composition over the course of 30 min and additional 5 minutes were allowed for the column to re-equilibrate before injection of the next sample. The sample volume was 50 μL at a concentration of 1 mg/mL, the flow rate of 0.6 mL/min, and the temperature was maintained at 25°C during the analysis. Detection was performed simultaneously at 210, 230, 254, 280, and 330 nm. Two pure standards, luteolin and apigenin, previously identified in Vernonia [HPLC analysis was performed using an Agilent Technologies 1200 Series, with a PDA detector and an automatic injector. The column employed was a Zorbax SB-18; 250 × 4.6 mm, 5 Vernonia , were usP < 0.05 was considered significant.Data were expressed as mean ± S.E.M. Statistical significance was determined by one-way analysis of variance followed by the Tukey test. V. condensata. After separation on TLC plates, the components of EE, HF, DF, EF, and BF with radical scavenging activity were sprayed with DPPH reagent followed by UV and visible light detection. The presence of yellowish bands on the purple background color was considered as antioxidants. In addition, TLC plates with the same samples were also revealed with specific reagents and phenolic, flavonoids, and terpenoids compounds were detected. 50 values were statistically different (P < 0.05) that ranged from 18.44 ± 0.54 to 147.14 ± 0.40 μg/mL  to Fe (+2) with IC50 of 19.98 ± 0.42 μg/mL.Initially, TLC-bioautography method was performed to screen the antioxidant capacity of 40 μg/mL . HF and ectively . In addiV. condensata was found to decrease in this order: HF < EE < BF < EF < DF  was most pronounced (P < 0.05) than EE (94.87 ± 0.09%) and BF (105.00 ± 0.04%). The antioxidant activity with ascorbic acid also decreased at the same order as rutin: HF < EE < BF < EF < DF  during 5 days. In the last day, the MDA concentration declined in the homogenate treated with EE, HF, DF, EF, and BF at concentrations of 7.5, 15, and 30 mg/mL . EF was V. condensata, the total phenolic varied from 0.19 to 23.11 g/100 g and flavonoid ranged from 0.13 to 4.10 g/100 g  and luteolin (Rt = 8.86). The chromatogram and the respective UV spectra are shown in Figures  and luteAntioxidants are radical scavengers' substances that delay or inhibit oxidative damage by blocking the oxidizing chain reactions. These compounds prevent cell and tissue damages and can neutralize free radicals by donating electrons, ending the carbon-stealing reaction. In body and at low concentration, these molecules markedly delay or prevent the oxidation of an oxidizable substrate –3. ConseV. condensata contains phytochemicals that are capable of donating hydrogen to a free radical in order to remove the odd electron which is responsible for radical's reactivity.Phenolic compounds and other natural products have been implicated in the ability to donate electrons to DPPH that turns from purple to yellow in solution and this reaction can be monitored by spectrophotometry at 518 nm . This as+3 to Fe+2 monitored by absorbance measurement at 700 nm [+3 to Fe+2, demonstrating a reducing potential of V. condensata. This data confirmed the previous findings observed by DPPH method described in details above. The ability to reduce Fe+3 may be attributed to the hydrogen donation from the phenolic compounds [The reducing power of iron is used to measure the reductive ability of antioxidant through the transformation of Fet 700 nm . This ret 700 nm . EE, HF,ompounds , which iompounds . In addiompounds .The obtained results through phosphomolybdenum method demonstrated that DF, EF, and BF exhibited the highest antioxidant capacity for phosphomolybdate reduction. The results suggested that the strong antioxidant activity of extracts might be due to the presence of phenolics compounds present in the extract . MoreoveV. condensata reduced the production of MDA (MDA, a major degradation product of lipid hydroperoxides, is more attractive as a marker for assessing the extent of lipid peroxidation , 43. Thin of MDA . HoweverV. condensata leaves was reported in the literature. EF showed the highest total phenolic and flavonoid contents. Phenolic constituents, among them the flavonoids, have been investigated for antioxidant [Vernonia, which may represent an important chemotaxonomic marker [It was the first time that the quantification of the total phenolic and flavonoid contents of EE, HF, DF, EF, and BF of ioxidant , antimicioxidant , antinocioxidant , 48. In c marker .Vernonia species, studies with V. amygdalina showed that the content of total phenols varied in accordance with references as follow: 0.20 to 0.27 g/100 g [Vernonia blumeoides, the content of total phenols ranged from 0.014 to 0.41 g/100 g in ethanol, chloroform, ethyl acetate, and butanol extracts [V. condensata is an excellent source of phenolic substances (Vernonia's species [Considering the phenolic constituents present in  g/100 g , from 0. g/100 g , and 0.2 g/100 g . In Vernextracts . In solvbstances . Based o species , 52.V. amygdalina, the total flavonoid content, another important phenolic parameter, was 0.22 g/100 g [V. condensata is a promising source of this compound. As the quantification of total phenols, ethyl acetate produced a higher yield of flavonoids and this solvent could be the most appropriate to extract substances of this class of special metabolite in Vernonia genus.In  g/100 g  or 0.041 g/100 g  dependinV. condensata. Two flavonoids, apigenin [Vernonia. However, besides flavonoids, other phenolic compounds such as condensed and hydrolysable tannins, not identified in the present study, could also contribute with the antioxidative effectivity of V. condensata [In view of the chemical studies on the bioactive components of this genus, among the identified constituents, flavonoids that exhibit antioxidant activity are highlighted , 33, 48.apigenin  and luteapigenin , were sendensata .Evidence of natural products with antioxidant activity has been described in the literature and this property seems to add up to that of the endogenous antioxidant system . In the V. condensata leaves can be stated in the occurrence of substances that present donor of electrons and protons capable of neutralizing free radicals and make them more stable products plus the ability to reduce oxidative intermediates of lipid peroxidation processes. The total phenolic and flavonoid contents were also determined to quantify and identify antioxidant constituents and EF showed the highest values established. Finally, the chemical characterization revealed the presence of apigenin and luteolin as previously reported in other species of the genus Vernonia. All this data sustain that V. condensata is an important and promising source of bioactive substances with antioxidant activity.Based on the results obtained in the present study, the presence of antioxidant effect of the ethanol extract and fractions obtained from


To evaluate the on-going scale-up of HIV programs, we assessed trends in patient characteristics at enrolment and ART initiation over 7 years of implementation.Data were from Optimal Models, a prospective open cohort study of HIV-infected (HIV+) adults (≥15 years) and children (<15 years) enrolled from January 2005 to December 2011 at 44 HIV clinics in 3 regions of mainland Tanzania  and Zanzibar. Comparative statistics for trends in characteristics of patients enrolled in 2005–2007, 2008–2009 and 2010–2011 were examined.Overall 62,801 HIV + patients were enrolled: 58,102(92.5%) adults, ; 4,699(7.5%) children.Among adults, pregnant women enrolment increased: 6.8%, 2005–2007; 12.1%, 2008–2009; 17.2%, 2010–2011; as did entry into care from prevention of mother-to-child HIV transmission (PMTCT) programs: 6.6%, 2005–2007; 9.5%, 2008–2009; 12.6%, 2010–2011. WHO stage IV at enrolment declined: 27.1%, 2005–2007; 20.2%, 2008–2009; 11.1% 2010–2011. Of the 42.5% and 29.5% with CD4+ data at enrolment and ART initiation respectively, median CD4+ count increased: 210 cells/μL, 2005–2007; 262 cells/μL, 2008–2009; 266 cells/μL 2010–2011; but median CD4+ at ART initiation did not change . Stavudine initiation declined: 84.9%, 2005–2007; 43.1%, 2008–2009; 19.7%, 2010–2011.Among children, median age (years) at enrolment decreased from 6.1(IQR:2.7-10.0) in 2005–2007 to 4.8(IQR:1.9-8.6) in 2008–2009, and 4.1(IQR:1.5-8.1) in 2010–2011 and children <24 months increased from 18.5% to 26.1% and 31.5% respectively. Entry from PMTCT was 7.0%, 2005–2007; 10.7%, 2008–2009; 15.0%, 2010–2011. WHO stage IV at enrolment declined from 22.9%, 2005–2007, to 18.3%, 2008–2009 to 13.9%, 2010–2011. Proportion initiating stavudine was 39.8% 2005–2007; 39.5%, 2008–2009; 26.1%, 2010–2011. Median age at ART initiation also declined significantly.Over time, the proportion of pregnant women and of adults and children enrolled from PMTCT programs increased. There was a decline in adults and children with advanced HIV disease at enrolment and initiation of stavudine. Pediatric age at enrolment and ART initiation declined. Results suggest HIV program maturation from an emergency response. In the United Republic of Tanzania (Tanzania), HIV prevalence among adults aged 15–49 years was last estimated at 5.7% in 2008/2009 . ART covThe scale-up of ART services and the large number of patients receiving ART are important successes in Tanzania. An evaluation conducted in the early years of program implementation revealed that ART services had been introduced at 210 facilities within 3 years, but there was limited male and pediatric enrolment . Like maICAP at Columbia University with funding from the United States President’s Emergency Plan for AIDS Relief (PEPFAR) provided programmatic, health facility and health systems support to Tanzania in the establishment of HIV care and treatment clinics (CTCs). ICAP’s support in Tanzania began in 2004 and was regionalized to Kagera, Kigoma (north-west Tanzania), Pwani , and the island of Zanzibar. In 2007–2008, HIV prevalence among adults aged 15–49 years in these regions was estimated at 5.3% in Pwani, 3.4% in Kagera, 0.9% in Kigoma and 0.6% in Zanzibar. By December 2011, a total of 127 CTCs had been supported in these regions and a total of 89,780 HIV-infected patients enrolled into HIV care . ICAP suWhen patients attend HIV care and treatment clinics, demographic and clinical characteristics and treatment details e.g. age, gender, source of referral, WHO staging, CD4+ cell count (CD4+), ART status and regimen are recorded on their patient medical card. During the study observation period, ART initiation was according to national guidelines which aligned with the 2003 and 2006 WHO ART initiation guidelines ,11. The Health facility and program characteristics were captured annually using a site assessment tool ,13. DataThe patient record data were de-identified and anonymized, then combined across health facilities to create an analytic dataset. The analysis (using SPSS 18) explored trends in patient characteristics among adults (≥15 years) and children (<15 years) enrolled from January 2005 to December 2011. Seven patients were missing age data and excluded from the analysis. WHO stage and CD4+ at enrolment and ART initiation, respectively, were based on records within < 90 days before and < 30 days after date of enrolment/ART initiation with priority given to data before and closest to the enrolment/ART initiation date. ART eligibility was defined using the 2006 WHO guidelines . DescripOptimal Models involves analysis of de-identified routinely collected data and received non-human subjects research determination from Columbia University Institutional Review Board and the Centers for Disease Control. The study was also approved by Institutional Review Boards in mainland Tanzania and Zanzibar.A total of 62,801 patients, 58,102 (92.5%) adults and 4,699 (7.5%) children, were enrolled at the 44 clinics, with peak enrolment in 2009  . The percentage of adults enrolled with WHO clinical stage IV declined from 27.1% in 2005–2007 to 20.2% in 2008–2009 and 11.1% in 2010–2011 (p < 0.0001). This decline was statistically significant among males as well as non-pregnant and pregnant women Figure . There wOverall, 52.8% were female and median age at enrolment was 4.1 years (IQR 1.9-8.8), and at ART initiation 5.6 years (IQR 2.2-9.7). Main points of entry into care were VCT (44.4%) and PMTCT (11.2%); and 10.8% of children transferred in from other clinics Table . The majAlthough absolute numbers of children increased, there were no changes in the proportion of children over time: 7.4%, 2005–2007; 7.8%, 2008–2009; 7.4%, 2010–2011. The children’s median age at enrolment decreased from 6.1 yrs (IQR 2.7-10.0) in 2005–2007 to 4.8 yrs (IQR 1.9-8.6) in 2008–2009, and 4.1 yrs (IQR 1.5-8.1) in 2010–2011 (p < 0.0001). Children aged <24 months at enrolment increased from 18.5% in 2005–2007 to 26.1% in 2008–2009 and 31.5% in 2010–2011, p < 0.0001, . Among the 2,318 children ≥5 years, there was no statistically significant change in documentation of CD4+ at enrolment, and documentation of CD4+ at ART initiation declined over time , these data from sites with electronic databases represent 43% of the adult population estimated to be HIV+ by December 2009, implying that a significant proportion of the population in these regions had accessed HIV services . ObserveOver time, PMTCT programs had an increasing contribution to adults and children entering HIV care. This is further evidenced by the significant increase in the proportion of women pregnant at enrolment and may be an indication that the scale up of PMTCT programs is having a positive impact on linkage into care and treatment. Though small, the proportion of women pregnant at ART initiation also increased over time. The largest contributor to adults and children entering HIV care was VCT, particularly after 2007. This could reflect the success of the National Voluntary HIV Testing Campaign conducted in 2007/2008 . The popThere have been significant improvements in WHO stage documentation over time, but CD4+ documentation remains low, even among the most recently enrolled adults and children. As a result, ART eligibility at enrolment could not be determined for at least 1 in five patients in the study population (with the exception of the most recently enrolled adults). Considering the liberal window attached to the enrolment visit in our analysis (<90 days before and <30 days after date of enrolment), this is a concern. CD4+ testing is an essential complement to WHO staging in accurately diagnosing HIV disease progression ,20, but Despite the limitations with CD4+ testing, a large number of patients initiated ART, and it is possible that due to limited resources, CD4+ testing was deliberately omitted where patients had advanced HIV disease. According to Tanzania and WHO guidelines used during the study period ,6,8,11, We observed a modest increase in median CD4+ at enrolment over time comparable to other sub-Saharan Africa HIV programs ,27, and There was a decline in stavudine use in the initial ART regimen, particularly significant among adults. This is in line with national and international guidelines that have advocated for the phasing out of this drug due to toxicities ,14. It iOne in three adults enrolled were male, and this did not change significantly over time. Evaluations conducted in other regions of Tanzania and Africa ,31,32 haThe strengths of Optimal Models lie in the large sample of HIV-infected adults and children, and a variety of sites represented from multiple regions of Tanzania over a long observation period. The data are collected from ‘real-world’ settings: clinics and facilities where patients received care, giving insight to grass roots activities. This analysis serves as a baseline, and additional analyses evaluating critical implementation issues are planned. A limitation of this analysis was inadequate data to determine which clinics transferred-in patients were coming from, possibly overestimating the overall number enrolled if patients transferred within the 44 study sites. However, the impact of this overestimation is likely minimal since patients could potentially transfer-in from over 900 other clinics in Tanzania and beyond. Further review of the data indicated that transferring out of the study clinics did not proportionally increase over time: it declined from 17.9% to 13.1% to 5.9% in the respective calendar year groups.This analysis did not control for site-level effects when testing for trends, however the entire patient population at the study sites was used. Whereas the trends are indicative of observations at the 44 study sites, the data cannot be considered as applying to all clinics in Tanzania more generally. Another limitation with this analysis pertains to data quality barriers generally associated with routine program data -43. For Over the early 7 years of HIV care and treatment program implementation in Tanzania, there was an increase in enrolment of pregnant women and adults and children enrolled from PMTCT programs. The proportion of adults and children with advanced HIV disease at enrolment declined, as did the stavudine use in the initial ART regimen. Median pediatric age at enrolment and ART initiation also declined. A large number of adults and children were enrolled into HIV care and initiated ART, demonstrating successful expansion of HIV services in the country. However, many patients were missing documentation of CD4+ at enrolment and ART initiation, an aspect that will require focus in further program enhancements.This study was based on de-identified routinely collected data. There was no interaction with patients for study purposes. The protocol received non-human subjects research designation and consent waiver under 45 CFR46. As such, individual consent was not sought for participating individuals.The authors declare that they have no competing interests.HNB, BK, GA, AK, AAA, GR, RM, WR, EJA contributed to the conception of the analysis idea; HNB, BK, GA, AK, AAA, WR, ML, RM, contributed to data collection and cleaning; ML merged and coded the data; ML, GA, AAA advised on the analysis; HNB conducted the analyses and wrote the manuscript; BK, GA, AK, AAA, WR, GR, ML, RM, EJA, reviewed and contributed to the manuscript, EJA gave overall technical oversight for the analytic process and manuscript writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/1016/prepub


Red yeast rice (RYR) can reduce cholesterol through its active component, lovastatin. This study was to investigate the pharmacokinetic properties of lovastatin in RYR products and potential RYR-drug interactions. Extracts of three registered RYR products  were more effective than pure lovastatin in inhibiting the activities of cytochrome P450 enzymes and P-glycoprotein. Among CYP450 enzymes, RYR showed the highest inhibition on CYP1A2 and CYP2C19, with comparable inhibitory potencies to the corresponding typical inhibitors. In healthy volunteers taking the RYR product LipoCol Forte, the pharmacokinetic properties of lovastatin and lovastatin acid were linear in the dose range of 1 to 4 capsules taken as a single dose and no significant accumulation was observed after multiple dosing. Concomitant use of one LipoCol Forte capsule with nifedipine did not change the pharmacokinetics of nifedipine. Yet, concomitant use of gemfibrozil with LipoCol Forte resulted in a significant increase in the plasma concentration of lovastatin acid. These findings suggest that the use of RYR products may not have effects on the pharmacokinetics of concomitant comedications despite their effects to inhibit the activities of CYP450 enzymes and P-gp, whereas gemfibrozil affects the pharmacokinetics of lovastatin acid when used concomitantly with RYR products. Monascus purpureus, has been used as a medicinal food supplement to improve blood circulation by decreasing cholesterol levels [Cardiovascular disease remains the leading cause of morbidity and mortality worldwide and hyperlipidemia is a major contributing factor to the development of cardiovascular disease . Red yeal levels –7. The al levels , also knl levels , 3, 9. Il levels , 10–12, Statins are a class of drugs that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an enzyme that plays a central role in the production of cholesterol in the liver . LovastaAlthough RYR products have been demonstrated to be well tolerated , 16, 17,β-hydroxytestosterone, furafylline, ticlopidine, sulfaphenazole, quinidine, and ketoconazole were from Sigma-Aldrich . Bupropion, hydroxybupropion, S-mephenytoin, S-4-hydroxymephenytoin, and hydroxytolbutamide were from Toronto Research Chemicals Inc. . Testosterone was from ACROS Organic . Human liver microsomes (UltraPool HLM 150) were purchased from BD Biosciences . Other reagents and solvents were obtained from standard sources and were of the highest quality available.LipoCol Forte was purchased from NatureWise Biotech & Medicals Corporation, Taiwan, Xuezhikang from Peking University WBL Biotech Co., Ltd., China, and Cholestin from Nu Skin Pharmanex, USA. Nifedipine capsule  was purchased from Bayer AG, Germany. Gemfibrozil capsule (Lopid) was purchased from Pfizer Inc., USA. Pure lovastatin was obtained from Merck KGaA . Phenacetin, acetaminophen, tolbutamide, dextromethorphan, dextrorphan, 6μm, Biotic Chemical Co., Ltd., Taiwan) with elution with a 70/30 v/v mixture of acetonitrile and 0.2% formic acid at a flow rate of 1.0 mL/min. Lovastatin was detected at 238 nm. For in vitro studies, the residue was dissolved in the appropriate assay buffer containing 1% methanol.The ingredients of the RYR products were weighed and 600 mg extracted with 10 mL of methanol at room temperature. Following centrifugation , the supernatants were collected and evaporated to dryness under nitrogen. The analytical condition was performed according to the methods described previously with minor modifications . To measβ-hydroxytestosterone/testosterone ratio was measured for CYP3A4, the 4′-hydroxymephenytoin/mephenytoin ratio for CYP2C19, the hydroxybupropion/bupropion ratio for CYP2B6, the hydroxytolbutamide/tolbutamide ratio for CYP2C9, the dextrorphan/dextromethorphan ratio for CYP2D6, and the acetaminophen/phenacetin ratio for CYP1A2. RYR product extracts or pure lovastatin at the same molar dose of lovastatin or CYP450 enzyme inhibitors  were used to inhibit the metabolism of typical substrates of CYP450 enzymes. To measure inhibition of CYP enzyme activity, the reaction mixture contained 20 μL of human liver microsomes (5 mg protein/mL in 500 mM potassium phosphate), 20 μL of an NADPH-generating system , 20 μL of 250 μM marker substrates, and 0.5–25 μM lovastatin (RYR product extracts or pure lovastatin) or 0.001–50 μM specific inhibitor in a total volume of 100 μL. After incubation at 37°C for 10 min (for dextromethorphan or testosterone), 20 min (for phenacetin or bupropion), 30 min (for tolbutamide), or 60 min (for S-mephenytoin), the reaction was terminated by addition of 50 μL of acetonitrile and centrifugation at 9,000 g at 4°C for 15 min. An aliquot of 10 μL of the supernatant was injected onto the LC/MS/MS system   and the percentage of metabolic activities at I by nonlinear regression analysis using Scientist v2.01 .The inhibition assay was performed according to the methods described previously with minor modifications , 24. TheMA, USA) . The IC54 cells/cm2) (from The Netherlands Cancer Institute) were cultured in polystyrene Nunclon multidishes under an atmosphere of 5% CO2/95% air. The minimal essential medium (MEM)  contained 10% fetal bovine serum , 5.5 mM L-glutamine, 1.5 mg/mL of sodium bicarbonate, and 1 mM sodium pyruvate , and 100 unit/mL of penicillin and 0.1 mg/mL of streptomycin (both from Invitrogen). After 4 days, the cells were washed with ECF buffer  and preincubated in ECF buffer for 30 min at 37°C, then digoxin uptake was initiated by addition of 40 nM 3H-digoxin  in ECF buffer with or without extracts of RYR products containing 25 μM lovastatin, 25 μM pure lovastatin, or 25 μM PSC833  and the mixture incubated at 37°C for 60 min, then the reaction was stopped by aspirating the supernatant and adding 500 μL of ice-cold ECF buffer, then the cells were solubilized overnight at room temperature in 1% Triton X-100 and radioactivity was measured by liquid scintillation counter using an external standard method for quench correction. The percentage of inhibition of P-gp activity and the IC50 values for P-gp inhibition were determined by the methods described by Rautio et al. [MDCK-MDR1 cells (2.5 × 105 cells/cm2) were seeded into 30 mm Millicell inserts  and cultured according to the conditions described previously [μM lovastatin or the equivalent concentration of RYR product extract in the apical compartment, then after 30, 60, or 120 min incubation, a 250 μL sample was collected from the basolateral compartment. At the end of the 120 min incubation, the cells were extracted with 1 mL of methanol by sonication for 30 min at room temperature and centrifuged at 14,000 g for 10 min at 4°C and the supernatants evaporated to dryness under nitrogen. The residues were reconstituted in 250 μL of mobile phase and injected into the HPLC system described in the previous section (see extraction procedure for RYR products). Caco-2 cells  and were performed in accordance with the Declaration of Helsinki and according to good clinical practice guidelines and any relevant local laws, regulations, and guidelines. The subjects were 20 to 30 years old and in good health as determined by past medical history, physical examination, electrocardiogram, chest X-ray, laboratory tests, and urinalysis. Premenopausal women were required to have a negative pregnancy test. All subjects had to sign an informed consent form before being included in the study. The major exclusion criteria included clinical significant abnormalities detected on physical examination with the potential to alter the absorption or elimination of the study drugs or that could constitute a risk to the subject when taking the study drugs. In addition, subjects were excluded if they had taken any prescription or over-the-counter medications within 14 days before administration of the study drugs. All studies were approved by an institutional review board/ethics committee before initiation. The studies were registered at n = 14) received one, two, or four LipoCol Forte capsules in the fed state in periods one to three, respectively, with a minimum of a 5-day washout period between each treatment. A high fat breakfast  was provided 30 min before dosing. All doses were administered with 240 mL of water. Blood samples were collected prior to drug administration and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 h after taking the capsules. In the multiple dose study, the volunteers (n = 14) received one LipoCol Forte capsule in the fed state twice daily for 5 days. A high fat breakfast or dinner was served 30 min before dosing. One capsule was administered with 240 mL of water. Blood samples were drawn before and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 h after taking the ninth dose using vacutainers containing heparin and sodium fluoride. Within 30 min of collection, the samples were centrifuged at 2,600 g for 10 minutes at 4°C and the plasma transferred to labeled polypropylene tubes containing 50 μL of 17% phosphoric acid and stored at −80°C until analysis.In the single dose study, the volunteers (n = 14) were randomly allocated to receive a single dose of either one 5 mg nifedipine capsule or one 5 mg nifedipine capsule plus one capsule of LipoCol Forte; after a washout of 7 days, the subjects received a single dose of the other drug. The subjects were fasted at least 10 h before dosing. The investigational products were administered with 240 mL of water. Blood samples were collected prior to the drug administration and at 0.167, 0.333, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 14, 16, and 24 h after dosing using photophobic vacutainers containing heparin. Within 30 minutes of collection, samples were centrifuged at 2,600 g for 10 minutes at 4°C and the plasma transferred to labeled photophobic polypropylene tubes and stored at −80°C.Healthy subjects (n = 13) took one capsule of LipoCol Forte orally with 240 mL water at 8 a.m.; blood samples were collected before and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 h after administration of LipoCol Forte. After a washout of 4 days, the subjects took gemfibrozil (two 300 mg Lopid capsules) with 240 mL water at 7 AM and 7 PM for 2 days, then, on the third day, took two gemfibrozil capsules at 7 AM and one LipoCol Forte capsule with 240 mL water at 8 AM and blood samples were collected as above. The volunteers were fasted overnight before dosing and a high fat breakfast or dinner was served 0.5 h after administration of gemfibrozil. In addition, a high fat breakfast was served 0.5 h before, and a meal was served 4 and 10 h after administration of LipoCol Forte. Blood samples were prepared as described in the previous section .The volunteers (μm) with a mobile phase composed of acetonitrile (A) and 0.2% formic acid (B) using a gradient of 40%–75% (v/v) of solvent B at a flow rate of 1.0 mL/min with a postcolumn split volume ratio of 2/10 to the mass detector. To measure the metabolic activity of each CYP isoenzyme, the following marker substrates were quantified by running ESI+ MRM or ESI− MRM analyses: the peak areas of m/z 180 → 110 for phenacetin, m/z 152 → 110 for acetaminophen, m/z 240 → 184 for bupropion, m/z 256 → 238 for hydroxybupropion, m/z 269 → 170 for tolbutamide, m/z 285 → 186 for hydroxytolbutamide, m/z 217 → 188 for (S)-mephenytoin, m/z 233 → 190 for (S)-4-hydroxymephenytoin, m/z 272 → 171 for dextromethorphan, m/z 258 → 157 for dextrorphan, m/z 289 → 97 for testosterone, and m/z 305 → 269 for 6β-hydroxytestosterone.For the analysis of CYP450 enzyme markers, chromatographic separation carried out on a Biosil ODS column  injected onto the LC/MS/MS system and separation was performed on a Biosil ODS column  with a mobile phase of acetonitrile/0.2% acetic acid (81/19) at a flow rate of 1.0 mL/min. All quantitative analyses were performed in ESI+ MRM mode: the peak areas of m/z 406 → 286 for lovastatin, m/z 423 → 303 for lovastatin acid, and m/z 296 → 215 for diclofenac.For the analysis of lovastatin and lovastatin acid in plasma, plasma samples (0.5 mL) were acidified with 0.2 M potassium dihydrogen phosphate and extracted with 4 mL of diethylether containing an internal standard . After centrifugation, the diethylether layer was transferred to a clean tube and evaporated to dryness at ambient temperature under nitrogen and the residue reconstituted with 200 μL) was injected onto the LC/MS/MS system and separation was performed on a Biosil ODS column  with a mobile phase of acetonitrile/1% formic acid (70/30) at a flow rate of 1.0 mL/min. All quantitative analyses were performed in ESI+ MRM mode: the peak areas of m/z 347 → 315 for nifedipine and m/z 559 → 440 for atorvastatin.For the analysis of nifedipine in plasma, plasma samples (0.2 mL) were extracted with 0.4 mL of acetonitrile containing an internal standard . After centrifugation, the supernatant (50 Cmax), time to reach the peak concentration (Tmax), area under the plasma concentration versus time curve from time zero to the time of the last quantifiable concentration (AUCt0−), AUC from time zero to the time extrapolated to infinity (AUC0−inf), half-life (T1/2), mean residence time (MRT), apparent clearance (CL/F), and apparent volume of distribution (Vd/F). The plasma concentration-time data were used to calculate the pharmacokinetic parameters of lovastatin, lovastatin acid, and nifedipine by the noncompartment method using WinNonlin . The pharmacokinetics were characterized, when appropriate, by the peak concentration in the plasma . Statistical differences were evaluated by analysis of variance, with a level of significance of 0.05. Pairwise comparisons between groups were made using Fisher's least-significant difference test.50 values of about 9–16 μM, but was less effective (IC50 > 50 μM) on CYP1A2 and CYP2D6. In contrast, the three extracts from LipoCol Forte, Xuezhikang, and Cholestin exhibited higher inhibitory potencies for all six CYP450 enzymes, with IC50 values of about 1–8 μM. In particular, the three extracts had comparable inhibitory potencies to typical inhibitors of CYP1A2 and CYP2C19, but were less effective than other CYP inhibitors.The amounts (mg/capsule) of lovastatin in the RYR products LipoCol Forte, Xuezhikang, and Cholestin were measured . Methano3H-digoxin as substrate. PSC833, a P-gp inhibitor, at the concentration of 25 μM resulted in the highest inhibition of digoxin uptake by MDCK-MDR1 cells, but the extracts of LipoCol Forte, Xuezhikang, and Cholestin (adjusted to 25 μM lovastatin based on the content shown in μM)  of lovastatin, showing higher absorption of lovastatin in the presence of the RYR product extracts.The inhibitory effects of the RYR product extracts on P-gp was first investigated in MDCK-MDR1 cells using n in μM) . The inhvastatin . The resCmax for lovastatin and lovastatin acid increased proportionally with the dose of LipoCol Forte  and a multiple dose study (1 LipoCol Forte capsule twice daily for 5 days). In the single dose study, the AUC and ol Forte , showingol Forte , showingBoth lovastatin and the dihydropyridine calcium antagonist nifedipine are primarily metabolized by CYP3A4 . Both drCmax for lovastatin acid were significantly increased.A combination of a fibrate and a statin is effective in patients with mixed lipid disorders. Since an interaction between the fibrate gemfibrozil and lovastatin has been reported , possiblDespite the fact that RYR products contain lovastatin and, in some countries, can be classified as drugs, in Asia, they are still considered as a food or dietary supplement and are widely available to the public. A number of studies have demonstrated the cholesterol lowering efficacy of RYR products –7, whichLovastatin is metabolized by CYP3A4  and is aI1/IC50 or I2/IC50, in which I1 and I2 are the maximal plasma concentration at steady-state at the highest clinical dose and the oral molar concentration  at the luminal side of gastrointestinal, respectively [I1/IC50 values for RYR product to inhibit CYP3A4 and P-gp are about 0.001 and 0.0001 for CYP3A4 and p-gp, respectively. Both are below the suggested cut-off value of 0.1 [I2/IC50 values for RYR product to inhibit CYP3A4 and P-gp are 18 and 2.1, respectively. According to FDA guidance, this suggests the likelihood of in vivo interaction between RYR and CYP3A4 substrates, based on the I2/IC50 value of larger than 10 [I2/IC50 ratio at this dose. Alternatively, solubility problem may lead to false-positive estimation utilizing the proposed I2/IC50 criteria [It is suggested that the potential drug interactions can be evaluated in vitro by calculating the values of ectively –34. The ectively , 11. Dese of 0.1 , 34, sug than 10 , 34. Pre than 10 . Howevercriteria , 37. NonGiven that occurrence of RYR-associated adverse reactions has usually been reported in patients with concomitant comedications –21, possA number of drugs, including fibrates, azole anti-fungals, macrolide antibiotics, anti-arrhythmics, and protease inhibitors, may increase the plasma levels of statins, through the inhibition of CYP3A4, P-gp, or OATP1B1 , 31. GivPharmacokinetics of lovastatin and its active metabolite, lovastatin acid, are linear in the dose range of 1 to 4 LipoCol Forte capsules taken as a single dose and no significant accumulation was observed after multiple dosing. Extracts of RYR products  are more effective than pure lovastatin in inhibiting the activities of CYP450 enzymes and P-gp in vitro, whereas the use of one LipoCol Forte capsule does not change the pharmacokinetics of nifedipine in vivo. Nevertheless, the interaction between RYR products and substrates of CYP1A2 and CYP2C19 is worth an attention. On the other hand, the concomitant use of gemfibrozil with LipoCol Forte increases the plasma concentrations of lovastatin acid. It should be cautious when RYR products are used with drugs that can interact with lovastatin.


Feed samples from the Finnish national feed control programme and suspected carry-over samples from a feed manufacturer were analysed in parallel with an existing liquid chromatography method coupled with ultraviolet detection. All feed control samples were negative in LC-UV, but with the developed MS method, monensin, salinomycin, and narasin were detected at concentrations of <0.025–0.73 mg/kg, <0.025–0.027 mg/kg, and <0.025–1.6 mg/kg, respectively. In suspected carry-over samples after an output of 2.0 tonnes of unmedicated feed in the pelletizer line, the concentrations of monensin, salinomycin, and narasin varied from undetected to 16 mg/kg. In the mixer line, after 3.2 tonnes of unmedicated feed output, the concentrations of monensin, salinomycin, and narasin varied from undetected to 2.4 mg/kg.A liquid chromatography-mass spectrometric method was developed and validated to determine six polyether ionophores  in feed samples. The method developed was very straightforward, involving extraction with 84% acetonitrile of the coccidiostats from the feed samples and filtration of the raw extract prior to chromatographic analysis. Method validation included the determination of selectivity, linearity, specificity, repeatability, the limit of detection, limit of quantification, decision limit (CC Eimeria. They are added to feeds as their sodium salts at levels of 75–125 mg/kg , 60–125 mg/kg (monensin), 20–70 mg/kg , 60–70 mg/kg (narasin), 5 mg/kg (maduramicin), and 20–25 mg/kg (semduramicin). Broiler chickens receive coccidiostats via feed during their entire lifespan, whereas for egg-laying birds the use of coccidiostats is forbidden. Anticoccidial drugs can also be administered, for example, to cattle to improve feed efficacy, that is, to increase their body mass. Due to the extensive use of coccidiostats, the safety of these compounds has been tested in many animal experiments . Although monensin, salinomycin, narasin, and maduramicin are forbidden for laying hens, these compounds were not found to have significant effects, for instance, on egg production or shell thickness when used at the permitted levels  Lasalocid, monensin, and salinomycin are used for laying birds up to the age of 12–16 weeks, depending on the additive. None of the coccidiostats are licensed for the use in egg-laying birds and therefore eggs should be free from coccidiostats contamination. Nonetheless, residues of polyether ionophores have been detected in European eggs at levels from 0.3 to <40 μg/kg . In addig [μg/kg , 16. Howμg/kg), most of the methods have been validated at the mg/kg level. In addition, apart from the study of Delahaut et al. [Several methods used to determine polyether ionophores in feed are based on high performance liquid chromatography (HPLC) with fluorescence detection , 18 or Ut et al. , the valt et al.  analysedt et al. , 14. ForThe European Commission has established official maximum contents of polyether ionophores in feed  . Based oA cereal mixture  was used in the validation study. The validated method was used in analysis of poultry feed from the Finnish national feed control programme (2008: 8 samples) and suspected carry-over samples from a European feed manufacturer . The carry-over of monensin, salinomycin, and narasin from medicated feed to unmedicated feed was investigated. Three batches of unmedicated feed were collected after one batch of medicated feed. Unmedicated feed samples from the mixer line were collected after 1 min (0.8 tonnes output), 2.5 min (2.0 tonnes), and 4 min (3.2 tonnes). In proportion, unmedicated feed samples from the pelletizer line were collected after 3 min (0.6 tonnes output), 5 min (1.0 tonnes), and 10 min (2.0 tonnes). μg/mL & 10 μg/mL; lasalocid, semduramicin; 8 μg/mL & 80 μg/mL) were prepared in methanol. Standard stock solutions for HPLC (0.5 mg/mL) were prepared in methanol. Working standard solutions for HPLC were prepared in methanol : water . Methanol, acetonitrile, acetic acid , ammonium acetate, and sulphuric acid were purchased from J. T. Baker . Vanillin (99%) was purchased from Fluka Chemie . A vanillin postcolumn reagent was freshly prepared daily by dissolving 30 g vanillin in 970 mL methanol/sulphuric acid . The water used was purified with a Millipore Milli-Q Plus System . Standards of monensin , salinomycin (~96%), and narasin (~97%) were purchased from Sigma , lasalocid  from Fluka Chemie , maduramicin  from Alpharma , and semduramicin from BVL . Standard stock solutions (1 mg/mL) and working standard solutions for LC-MS/MS analysis  into an autosampler vial. Samples were ground with a laboratory mill . Then, 25 g of ground sample was extracted with 100 mL of 84% acetonitrile in water for two hours using a VKS-75 horizontal shaker  at room temperature. Samples in the validation study were spiked with different concentrations of polyether ionophores standard solutions just before extraction. As the matrix of carry-over samples was different than matrix used in the validation, the samples were also spiked before extraction to ensure suitability of the method for these samples. The extracted samples were filtered through S&S 602 H 1/2 filter paper , and the filtered extracts were stored at +4°C until analysis. One millilitre of extract was evaporated to dryness under nitrogen at 40°C. The sample was dissolved in 1 mL of mobile phase (acetonitrile: 2 mM ammonium acetate containing 2% acetic acid (95 : 5 v/v)) and filtered using a 0.2-18(2) (5 μm), 3.0 × 150 mm . The flow rate of the mobile phase was 0.5 mL/min and the injection volume was 10 μL. A positive ionisation mode was used with an ESI probe. The parameters of the MS were optimized using a standard solution. The best response was recorded with the following parameters: capillary voltage 3.75 kV, source temperature 130°C and desolvation temperature 250°C. The optimized cone voltages and collision gas energies for each of the analysed coccidiostats are presented in Polyether ionophores were analysed with a Waters Alliance 2695 liquid chromatograph  connected to a MicroMass Quattro Micro triple-quadrupole mass spectrometer . The separation of polyether ionophores was conducted as described by Jestoi et al. . The anaμm syringe filter  into an autosampler vial.The LC-UV method for polyether ionophores was modified from the method AM-AA-CR-J424-AC-791 . Brieflyμm). The flow rate was 0.8 mL/min for the mobile phase  and 0.7 mL/min for the vanillin reagent. The injection volume was 50 μL. In this method, the limit of detection (LOD) for monensin, salinomycin, and narasin was 2 mg/kg, 2 mg/kg, and 3 mg/kg, respectively, and the respective limits of quantification (LOQ) for these analytes were 4 mg/kg, 4 mg/kg, and 5 mg/kg.Monensin, salinomycin, and narasin were analysed using an Agilent 1100 liquid chromatograph  and postcolumn reagent pump . Coccidiostats were detected at 520 nm. The analytical column was a Shandon 250 × 4.6 mm Hypersil ODS (5 α), detection capability (CCβ), limit of detection (LOD), and limit of quantification (LOQ). Six replicates of samples spiked at three concentrations  and calibration curves with and without the matrix were analysed on three separate days. The calibration curves were constructed with external standards by injecting standards prepared in the mobile phase and matrix at five different concentrations . The blank matrix was spiked with corresponding amounts of standards and prepared as regular samples.Method validation included the determination of selectivity, linearity, specificity, reproducibility, recovery, the decision limit  the slopes of the five-point calibration curves obtained with and without the matrix . Due to the presence of significant matrix effects (P < 0.05), the calibration curves for all compounds were prepared in the matrix. The matrix effect, calculated as the ratio of signal suppression to enhancement (SSE%) as described in Sulyok et al. [The selectivity of the developed method was tested by comparing . LODs (blank sample mean response + 3 × standard deviation) for lasalocid, monensin, salinomycin, narasin, maduramicin, and semduramicin were 0.0029 mg/kg, 0.0042 mg/kg, 0.0047 mg/kg, 0.0026 mg/kg, 0.0011 mg/kg, and 0.0022 mg/kg, respectively. The corresponding LOQs (blank sample mean response + 10 × standard deviation) for the compounds analysed were 0.0075 mg/kg, 0.011 mg/kg, 0.012 mg/kg, 0.0071 mg/kg, 0.0029 mg/kg, and 0.0060 mg/kg, respectively. Although lower concentrations of the analytes could be quantified with the method, for practical reasons the LOQs used for the analytes were the lowest spiking level of 0.025 mg/kg for monensin, salinomycin, narasin, and maduramicin, and 0.080 mg/kg for lasalocid and semduramicin. CCα and CCβ values were calculated using data on within-laboratory reproducibility from this study (data not shown). CCα and CCβ values for monensin, salinomycin, narasin, and maduramicin were 0.025–0.027 mg/kg and 0.029–0.033 mg/kg, respectively. CCα values for lasalocid and semduramicin were 0.37 mg/kg and 0.24 mg/kg, respectively, and the corresponding CCβ values, in turn, were 0.50 mg/kg and 0.29 mg/kg , which was also tested to be linear over the range. For that reason, carry-over samples could be analysed with any dilution. The carry-over samples were also spiked and the result was corrected by recovery, if the recovery % was over the range recoveries obtained during the validation. Lasalocid, maduramicin, and semduramicin were not detected in any samples analysed with LC-MS/MS. In samples from the feed control programme the concentrations of monensin and narasin were <0.025–0.73 mg/kg and <0.025–1.6 mg/kg, respectively . SalinomIn the suspected carry-over samples from the pelletizer line, after an output of 2.0 tonnes of unmedicated feed, the level of monensin was 8 mg/kg and 16 mg/kg in two turkey feeds. In two broiler feeds, the concentration of narasin after 2.0 tonnes of unmedicated feed output was 16 mg/kg and 17 mg/kg. Carry-over was also detected in the mixer line, but it was minor compared to the pelletizer line. After 3.2 tonnes of unmedicated feed output, the concentration of monensin in feed for turkey and broiler was 0.58–2.4 mg/kg and <0.025–0.27 mg/kg, respectively. The concentration of narasin was 0.16–0.28 mg/kg in turkey feed and 0.094–1.5 mg/kg in broiler feed. Salinomycin was detected in nine samples (<0.025–0.027 mg/kg) from both the pelletizer and mixer lines. All the results for these suspected carry-over samples are presented in The samples mentioned in α, and CCβ values demonstrated that the method is capable of quantifying polyether ionophores in feed below the maximum concentrations stated in Commission Directive 2009/8/EC [The developed LC-MS/MS method proved to be sensitive and repeatable in the analysis of six polyether ionophores, as shown in the validation results. The obtained LOD, LOQ, CC009/8/EC . The European Union has not set any recommendation concerning the performance of analytical methods or the interpretation of results for contaminants in feed. Accordingly, the developed LC-MS/MS method was validated according to Commission Decision 2002/657/EC , which h+ and K+. Complex formation occurs by intramolecular hydrogen bonding between a carboxylic group at one end of the molecule and a terminal alcohol group at the other [As with other ionophoric compounds, coccidiostats can also form electrically neutral pseudo-macrocyclic complexes with several metal cations, such as Nahe other . Polyethhe other –40. Sodihe other , 27. In The most widely used method to compensate for matrix effects in LC-MS methods is the use of an internal standard, since this allows the response of an analyte of interest to be normalized, for example, compensating for possible variations during sample preparation, injection, chromatography, and matrix effects. The best choice for an internal standard would be the isotope-labelled analogues of coccidiostats, which would have identical chemical and structural properties to those of the analyte . We obseAs the operator control samples from the feed mill demonstrated, cross-contamination is possible during manufacture. Unfortunately, the levels of monensin and narasin in medicated feed were not available. According to the legislation, monensin can be added at concentrations of between 100 and 125 mg/kg to feed for broilers and between 60 and 100 mg/kg to feed for turkeys. Narasin can be added at concentrations of between 60 and 70 mg/kg to feed for broilers. Based on these addition levels, 10–20% of the added monensin was still left after three unmedicated feed batches (2.0 tonnes) in the pelletizer line . In addiThe results determined with the developed LC-MS/MS method and the existing LC-UV method were compared. In those carry-over samples in which the concentrations of coccidiostats were more than 10 mg/kg, the results determined with both methods were similar. As expected, significant differences between the results of two analytical methods were observed at trace levels of coccidiostats, since the LOQ values of the LC-MS/MS method (0.025–0.080 mg/kg) were much lower than those of the LC-UV method (4-5 mg/kg). Samples from the national feed control programme were collected from feed batches that should not contain any polyether ionophores. With the LC-UV method, samples were previously found to be negative for monensin, salinomycin, and narasin. However, with the LC-MS/MS method, both monensin and narasin were detected at concentrations ranging from <0.025–0.73 mg/kg and <0.025–1.6 mg/kg, respectively. It is notable that narasin was found in one sample at the concentration of 1.6 mg/kg, which exceeded the permissible content (0.7 mg/kg). Moreover, in those carry-over samples that were found to be negative for monensin and narasin with the LC-UV method, both compounds were detected with the LC-MS/MS method. Thus, the developed LC-MS/MS method proved to be suitable for detecting trace amounts of polyether ionophores in feed.In conclusion, a new LC-MS/MS method was developed for the determination of six polyether ionophores at trace levels. Sample preparation was minimized, because the raw extracts were only filtered before the LC run. The use of mass spectrometry enabled polyether ionophores to be analysed without precolumn or postcolumn derivatization. The validation data indicate that the developed method is reliable in the determination of coccidiostats in feed at the existing contamination levels. The LC-MS/MS method demonstrated its efficiency in the analysis of coccidiostats at trace levels against the conventional LC method. In feed samples that tested negative with the LC-UV method, concentrations of coccidiostats of up to 2.10 mg/kg were detected with LC-MS/MS. The suspected carry-over of coccidiostats during the manufacture of feed was tested by analysing unmedicated feed batches produced after medicated feed. Coccidiostats could be still detected after 3.2 tonnes of unmedicated feed output.