Ficus carica stem extract (FE) in methanol-induced hepatotoxicity in male Wistar rats. The rats were divided into two batches: 16 control rats (C) drinking tap water and 16 treated rats drinking Ficus carica stem extract for six weeks. Then, each group was divided into two subgroups, and one of them was intraperitoneally injected (i.p.) daily methanol at a dose of 2.37 g/kg body weight i.p. for 30 days, for four weeks. The results showed that FE was found to contain large amounts of polyphenols and carotenoids. The treatment with methanol exhibited a significant increase of serum hepatic biochemical parameters  and hepatic lipid peroxidation. Hepatic antioxidant enzymes, namely, SOD, CAT, and GSH-Px, were significantly decreased in methanol-treated animals. FE treatment prior to methanol intoxication has significant role in protecting animals from methanol-induced hepatic oxidative damage.The present study was aimed to investigate the antioxidant activity of  It is a highly toxic alcohol commonly found in automobile windshield washer solvent, gas line antifreeze, copy machine fluid, fuel for small stoves, and paint strippers and used as industrial solvent. Methanol, the simplest of all alcohols, is toxic to humans. In developing countries like India, methanol is a common adulterant in country liquor which increases the chance of accidental poisoning enormously. The methanol could initiate ROS formation directly via a free radical intermediate, or possibly indirectly through mechanisms like the activation and/or enhancement of ROS-producing NADPH oxidases, which has been reported for ethanol [Methanol, also known as methyl alcohol and wood alcohol, is a primary alcohol with the chemical formula CH ethanol . It is a ethanol . These factors together with the excess of formaldehyde, formed during acute intoxication, cause significant increases in lipid peroxidation. Products of lipid peroxidation are very harmful to cells ultimately causing their death and can act as second messengers of a complex chain reaction .Ficus carica (Family: Moraceae) is commonly referred to as “Fig.” Figs have been traditionally used for their medicinal benefits as metabolic, cardiovascular, respiratory, antispasmodic, and anti-inflammatory remedy [Ficus carica are traditionally used as laxative, stimulant, antitussive, emollient, and resolvent as well as against throat diseases [F. carica was chosen for its abundance of phenols, essential oils, and flavonoids, and F. carica stem has not been investigated. The aim of this study was undertaken to test the efficacy of aqueous extract prepared from F. carica stem against hepatic injury induced by methanol in rats to determine the possible use of this plant in preventing hepatic damage.Liver diseases are among the most serious health problems in the world today, and their prevention and treatment options still remain scarce despite tremendous advances in modern medicine. The pathogenesis of hepatic diseases as well as the role of oxidative stress and inflammation therein is well recognized , and cony remedy . The leadiseases , 9. A dediseases . Some rediseases . F. cari2O2), 5, 5′ dithiobis (2-nitrobenzoic acid) (DTNB), the Folin-Ciocalteu reagent, sodium carbonate (Na2CO3), and other solvents were of analytical grade and were freshly prepared in distilled water.2,2-Diphenyl-l-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), thiobarbituric acid (TBA), glutathione (GSH), oxidised glutathione (GSSG), glutathione reductase (GR), bovine serum albumin (BSA), and 2,4-dinitrophenyl hydrazine (DNPH) were purchased from Sigma Chemical, France. Trichloroacetic acid (TCA), hydrogen peroxide  for 15–20 min, and then filtered using the Whatman filter paper. The aqueous extract was given as beverage instead of tap water.The μL diluted extract solution was shaken for 5 min with 50 μL of the Folin-Ciocalteau reagent. Then 150 μL of 20% Na2CO3 was added and the mixture was shaken once again for 1 min. Finally, the solution was brought up to 790 μL by adding distilled water. After 90 min, the absorbance at 760 nm was evaluated using a spectrophotometer. Gallic acid was used as a standard for the calibration curve. The phenolic content was expressed as mg gallic acid equivalent/gram of dry extract using the linear equation based on the calibration curve.Total phenolic content was determined using the Folin-Ciocalteu method  adapted The flavonoid content in extracts was determined spectrophotometrically according to , using aF. carica stem extracts was determined using DPPH as a reagent according to the method of [μL of sample solutions in ethanol at different concentrations. The mixture was incubated for 20 min in the dark at room temperature. The scavenging capacity was determined spectrophotometrically by monitoring the decrease in absorbance at 517 nm against a blank using a spectrophotometer (Bio-Rad SmartSpec Plus). Lower absorbance of the reaction mixture indicates higher free-radical-scavenging activity. Ascorbic acid was used as positive control. The percent DPPH scavenging effect was calculated using the following equation: DPPH scavenging effect (%) = (Acontrol − Asample/Acontrol) × 100, where Acontrol is the absorbance of the control reaction containing all reagents except the tested compound. Asample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated from the graph-plotting inhibition percentage against extract concentration. Tests were carried out in triplicate.Radical-scavenging activity of ethod of  with sli3+/ascorbate/EDTA/H2O2 system in the non-site-specific assay or Fe3+/ascorbate/H2O2 in the site-specific assay. The reacting mixture of the deoxyribose assay contained in a final volume of 1 mL the following reagents: 200 μL KH2PO4–KOH (100 mM), 200 μL deoxyribose (15 mM), 200 μL FeCl3 (500 μM), 100 μL EDTA (1 mM), 100 μL ascorbic acid (1 mM), 100 μL H2O2 (10 mM), and 100 μL sample. Reaction mixtures were incubated at 37°C for 1 h. At the end of the incubation period, 1 mL of 1% (w/v) TBA was added to each mixture followed by the addition of 1 mL of 2.8% (w/v) TCA. Solutions were heated on a water bath at 80°C for 20 min to develop the pink-coloured malondialdehyde-thiobarbituric acid, MDA-TBA2 adduct, and the absorbance of the resulting solution  was measured at 532 nm. Mannitol, a classical hydroxyl radical scavenger was used as positive control. The inhibition ratio of the extract (%) was calculated using the following formula: The hydroxyl-radical-scavenging activity was determined according to the colorimetric deoxyribose oxidation by the Fenton reaction leading to malondialdehyde . The hydWistar rats weighing 180 to 200 g were obtained from the Central Pharmacy of Tunisia . The animals were quarantined and allowed to acclimatize for a week prior to experimentation. The animals were handled under standard laboratory conditions of a 12 h light/dark cycle in a temperature- and humidity-controlled room. Food and water were available ad libitum. Our Institutional Animal Care and Use Committee approved the protocols for the animal study, and the animals were cared for in accordance with the institutional ethical guidelines.Adult male albino Ficus carica stem extract (FE) for six weeks. Then, each group was divided into two subgroups, and one of them was intraperitoneally injected (i.p.) daily, for four weeks, with methanol (2.37 g/kg b.wt.) according to [−1 phosphate buffer (pH 7.4). The homogenate and supernatant were frozen at −30°C in aliquots until used for antioxidant enzymes and lipid peroxidation. The protein content of the supernatant was determined using the method of [After acclimatization, the rats were divided into two batches: 16 control rats (C) drinking tap water and 16 treated rats drinking ethod of .Serum samples were obtained by the centrifugation of blood at 4000 rpm for 15 min at 4°C and were then divided into Eppendorf tubes. Isolated sera were stored at −30°C until they were used for the analyses. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were measured using commercial kits according to the manufacturer's directions.μL of supernatants were homogenized by sonication with 50 μL of TBS, 125 μL of TCA-BHT in order to precipitate proteins and centrifuged . 200 μL of obtained supernatant were mixed with 40 μL of HCl (0.6 M) and 160 μL of TBA dissolved in Tris, and the mixture was heated at 80°C for 10 min. The absorbance of the resultant supernatant was read at 530 nm. The amount of TBARS was calculated by using an extinction coefficient of 156 × 105 mM−1 cm−1.Lipid peroxidation in the tissue homogenate was estimated by measuring thiobarbituric acid reactive substances (TBARS) and was expressed in terms of malondialdehyde (MDA) content which is the end product of lipid peroxidation, according to . In brie2O2 as substrate; glutathione peroxidase (GSH-Px) activity was measured by a modification of the colorimetric method of [2O2 as substrate and the reduced GSH.In liver tissues, SOD activity was determined according to the colorimetric method of  using thethod of  using H2P < 0.05.All values are expressed as mean ± S.E.M. The results were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons using SPSS for Windows (version 11). Differences were considered significant when  F. carica stem extract contained phenolic compounds (133 ± 3.50 mg GAE/g), in which its level was expressed as gallic acid equivalents. Total flavonoids were expressed as quercetin equivalent per g of dry plant extract. The F. carica stem extract contained flavonoid compounds (43.25 ± 2.0 mg QE/g).Total phenolics and flavonoids are shown in F. carica stem extract was determined by its capacity to scavenge the stable free radicals DPPH, which has been widely used to test free-radical-scavenging activity, and the results were compared with the scavenging ability of control samples of BHT  of linoleic acid which simultaneously attack β-carotene, resulting in bleaching of its characteristic yellow color in aqueous solution. The antioxidant activity of F. carica was evaluated using different concentrations of extracts and was compared with BHT used as reference  scavenging activity in a concentration dependant. Several polyphenols including flavonoids are found in figs [F. carica was chosen for its abundance of phenols, essential oils, and flavonoids, which are effective on bacteria through compounds produced such as resveratrol, psoralen, and bergapten. These compounds have demonstrated antibacterial activity and could be used commercially as a means of phytopathogenic bacteria control [F. carica contains alkaloids, flavonoids, coumarins, saponins, and terpenes. The extract was tested for its possible spasmolytic effect on isolated rabbit jejunum preparations, where it inhibited the contractions at the concentration, thus showing the antispasmodic effect. The relaxant effect was dose dependent with an IC50 value of 0.74 mg/mL. The observed antispasmodic effect of F. carica was reversible returning to normal spontaneous contractions within 2–4 min of washing the tissue with fresh bathing physiological solution [F. carica extract could be responsible for the radical-scavenging activity in methanol toxicity. Liver is a major site for metabolism of exogenous chemicals, resulting in the formation of metabolites which may be more or less toxic than the parent compound. The aqueous extract of F. carica stem, administered prophylactically, exhibited significant protection against methanol-induced liver injury as manifested by the reduction in toxin-mediated rise in serum transaminases, ALP, and LDH in rats. The results of [Oldenlandia umbellata administered orally to rats reversed the altered hepatic serum marker enzymes  by CCl4 to almost normal. The rise in serum levels of AST, ALT, and ALP has been attributed to the damaged structural integrity of the liver, because they are cytoplasmic in location and released into circulation after cellular damages [Coriandrum sativum at a dose of 200 mg/kg body weight to rats returned the activities of serum AST and ALT as a hepatic biomarker to normal values, which may be a consequence of the stabilization of plasma membrane as well as repair of hepatic tissue damage caused by CCl4. Lactate dehydrogenase is a well-known marker for cell toxicity. When cells are damaged due to oxidative stress or destroyed due to deficient oxygen or glucose supply, the membrane becomes permeable or may rupture which results in the leakage of this enzyme. This enzyme enters into the blood stream thus increasing its concentration in the serum [F. carica extract diminished methanol-induced oxidative stress by increasing antioxidant status. These results agree with the hypothesis that oxidative damage is neutralized when antioxidants are administered before or after the induction of oxidative stress [Phyllanthus amarus (P. amarus) leaf extract had a hepatoprotective role against ethanol-induced oxidative damage in adult male Wistar rats. P. amarus cotreatment significantly reduced the activities of plasma AST, ALT, and ALP in the ethanol-challenged rats. This investigator had reported similar observations to our study in the levels of plasma AST, ALT, and ALP activities. In the present study, methanol caused an increase in LPO level in liver tissues which is an indicator of oxidative stress. Furthermore, LPO products are measured as index of oxygen free radical. The present result of increased LPO level after methanol exposure indicates an increased oxygen free radical generation by alcohols. This result is in accordance with previous studies using methanol and other alcohols [Taraxacum officinale root, a herbal medicine, exhibited significant decrease in hepatic lipid peroxidation against liver damage mediated by alcohol in vivo. Compared with the control group, the methanol-intoxicated animals exhibited a significant decrease in SOD, CAT, and GPx levels. These changes were significantly reversed upon treatment with FE. Therefore, some polyphenols and flavonoids exert a stimulatory action on transcription and gene expression of certain enzymes. The antioxidant enzyme system plays an important role in the defence of cells against oxidative insults. Both SOD and CAT are key antioxidant enzymes that protect against oxidative stress and tissue damage [•2) into H2O2 and O2, thus participating with other antioxidant enzymes in the enzymatic defense against oxygen toxicity. CAT is a key component of the antioxidant defense system. Inhibition of this protective mechanism results in enhanced sensitivity to free-radical-induced cellular damage. The decrease of CAT may result in a lot of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxide [F. carica stem. Moreover, these findings supported the beneficial effect of F. carica in maintaining the hepatocytes integrity and function. It is conceivable that these effects may be due, at least in part, to its antioxidant activity. The antioxidant enzyme  activities were increased by FE in liver tissues when compared with those administered with methanol only. These results are in accordance with those of Srivastava and Shivanandappa [Decalepis hamiltonii, a climbing shrub that grows in the forests of peninsular India and is consumed for its health-promoting properties, prevented CCl4-induced oxidative stress in the rat liver by inhibiting lipid peroxidation and protein carbonylation, restoring the levels of antioxidant enzymes  and glutathione. The concentration of LPO decreased significantly following FE administration in the liver tissues. These results clearly show that the antioxidant property of FE reduces significantly the damage of methanol-induced liver injury and activates the biological defense system of the liver. Furthermore, FE appears to scavenge free radicals [In our study, the phenolic estimation reveals that aqueous extract of radicals . Total H in figs . On the  control , 25. The control  denoted solution –28. It csults of  are simi 420 ± 3. μg/mL sc damages  which mehe serum . In the e stress . Faremi e stress  recentlyalcohols . FE admialcohols . Recent alcohols  have proe damage . These eperoxide . GPx connandappa  which deradicals , as showF. carica have been demonstrated to possess excellent antioxidant activities by various in vitro and in vivo assays. The various in vitro antioxidant tests proved that the plant possesses components such as polyphenols and flavonoids with scavengers of free radicals. Current study also demonstrates that F. carica aqueous extract could reduce methanol-induced toxicity, particularly hepatotoxicity, by suppressing alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) activities, inhibiting lipid peroxidation and increasing antioxidant enzyme activities. The observed strong antioxidant activity of F. carica may be due to the presence of several phenolic compounds, such as flavonoids. Further investigation is needed to isolate and identify the active principle involved in antioxidant activities of this plant.In the present study, the stem extracts of


Both gastric bypass (GB) and duodenal switch with sleeve gastrectomy (DS) have been widely used as bariatric surgeries, and DS appears to be superior to GB. The aim of this study was to better understand the mechanisms leading to body weight loss by comparing these two procedures in experimental models of rats.Animals were subjected to GB, DS or laparotomy (controls), and monitored by an open-circuit indirect calorimeter composed of comprehensive laboratory animal monitoring system and adiabatic bomb calorimeter.Body weight loss was greater after DS than GB. Food intake was reduced after DS but not GB. Energy expenditure was increased after either GB or DS. Fecal energy content was increased after DS but not GB.GB induced body weight loss by increasing energy expenditure, whereas DS induced greater body weight loss by reducing food intake, increasing energy expenditure and causing malabsorption in rat models. Various bariatric surgical procedures, such as gastric banding, gastric bypass (GB) and duodenal switch with sleeve gastrectomy (DS), have been developed in order to reduce food intake and/or lead to malabsorption Various rat models of bariatric surgery have been developed in order to understand the underlying physiological mechanisms of different surgical procedures. There have been many studies in the literature reporting the surgical procedures (such as Roux-en-Y) in rats that are made as same as they are used in humans vs.DS on eating behavior and energy expenditure in rat models.GB is the most common procedure because of relatively high efficacy and safety, whereas DS seems to be even more effective, particularly in super-obese patients vs. ∼600 g induced by high-fat diet) and the experimental efforts in terms of time-consuming and financial expense  were purchased from Taconic M&B, Skensved, Denmark and housed in ventilated cages in a specific pathogen free environment with room temperature of 22°C, 40–60% relative humidity and 12 hr day/night cycle with 1 hr dusk/dawn. The rats had free access to tap water and standard rat pellet food . In our previous studies, we have reported that the male rats gained body weight mainly as a result of continuous expansion of the fat compartment after puberty (8 weeks of age with 200 g body weight), and that the male rats that were fed a high-fat diet starting at 5 weeks of age gained body weight up to ∼650 g at 40 weeks of age as a result of increased fat mass  expense . Further expense .p = 0.276). Because of markedly loss of body weight after DS, the group of DS rats, together with age-matched group of laparotomized rats , were followed up only for 8 weeks, while GB rats and the rest of laparotomized rats  were followed up for 14 weeks. In consideration of the “3Rs” for the human use of animals , rats that had been used for studies of the effects of individual surgical procedures were re-used Thirty-four rats, at 587.0±8.1 g body weight, were randomly divided into experimental (GB and DS) as well as control groups : GB (14 rats), DS (7 rats), and LAP (13 rats). The body weight was not different between the groups before surgery  . Buprenorphine (0.05 mg/kg)  was administrated as an analgesic agent subcutaneously immediately during surgery. LAP was performed through a middle-line incision with gentle manipulation of viscera. A rat model of Roux-en-Y GB procedure has been described by Stylopoulos and his colleagues 2 consumption (VO2 mL/kg/hr) and the volume of CO2 production (VCO2 mL/kg/hr) were measured by an air sample withdrawn every 5 min from each chamber through the gas dryer. The energy expenditure  was calculated according to equation: (3.815+1.232 RER) × VO2, where the respiratory exchange ratio (RER) was obtained by VCO2 divided by VO2. In order for rats to acclimate to CLAMS, they were placed in these metabolic chambers for 24 hr one week before the first CLAMS monitoring. For the measurement of eating and metabolic parameters, the rats were placed in the CLAMS for 48 hr. In order to minimalize possible effect of stress, only data from the last 24 hr in CLAMS were used for the analysis. An analysis of eating pattern in control rats over a time period from day 1 and 21 showed no significant differences in any parameters, indicating that the animals had acclimated to CLAMS  2–3 weeks after GB, DS or LAP and 14 weeks after GB, 8 weeks after DS or 8–14 weeks after LAP. The CLAMS is composed of a 4-chamber indirect calorimeter designed for the continuous monitoring of individual rats from each chamber. The eating data was generated by monitoring all feed balances every 0.5 s. In CLAMS program used in the present study, the end of an eating event  was when the balance was stable for more than 10 s and a minimum of 0.05 g was eaten. The eating parameters during daytime and nighttime (12 hr each time) for each rat included: accumulated food intake , number of meals, meal size , meal duration , intermeal interval (min), rate of eating (g/min), and satiety ratio (min/g). The intermeal interval was defined as the interval in minutes between two meals. The rate of eating was calculated by dividing meal size by meal duration. The satiety ratio, an index of non-eating time produced by each gram of food consumed, was calculated as intermeal interval divided by meal size to CLAMS . The ratFeces were collected while the rats were placed in CLAMS chambers and dried for 72 hr at 60°C. The energy density was determined by means of an adiabatic bomb calorimeter .Blood was drawn from the abdominal aorta under the anesthesia just before the animals were killed, and plasma was stored at −80°C until determination of levels of cytokines. The multiplex cytokine assay was used . It contained the following analytics: IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, granulocyte-macrophage colony stimulating factor (GM-CSF), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα).t-test or Mann Whitney U test was performed for two-group comparisons. ANOVA followed by Bonferroni test was performed for multiple comparisons. Homogeneity of regression assumption test and ANCOVA were performed for analysis of energy expenditure. SPSS version 19.0  was used. A p-value of <0.05 was considered statistically significant.The values were expressed as means ± SEM. Two-tailed independent-samples No one died after LAP alone, 6 after GB, and 2 after DS due to surgical complications, trauma and learning curve factors.LAP alone did not reduce body weight during the study period (maximum 14 weeks). GB caused approximately 20% weight loss throughout the study period (14 weeks). DS induced approximately 50% weight loss within 8 weeks .In comparison with LAP, GB increased daytime (but not nighttime) food intake  at 3 weeks, and had no effects afterwards (14 weeks postoperatively). In contrast, DS reduced nighttime (but not daytime) food intake . The food intake  at 8 weeks was not reduced because of markedly loss of the body weight after DS .GB was without effects neither on satiety ratio (min/g) nor rate of eating (g/min), whereas DS increased satiety ratio during nighttime, and decreased rate of eating during both daytime and nighttime at 2 weeks and 8 weeks postoperatively  Tables ,2.p = 0.051) (Age-matched control rats that underwent LAP only were included for comparisons because metabolic parameters are age-dependent = 0.051) .DS rats and similar regression slopes between LAP and GB or DS (p>0.05) (p>0.05)  particularly in LAP(p>0.05) . ANCOVA (p>0.05) .There was no change in the fecal energy density after GB. DS had severe diarrhea within 2 weeks postoperatively, so that it was difficult to collect the fecal samples. At 2 months, the solid feces were collected and the energy density was increased .vs. GB or DS in the plasma levels of the 11 cytokines measured . Previously, we have found that the food intake was independent on the size of stomach by comparing gastrectomy and GB in rat models vs. duodenal switch without sleeve gastrectomy in rats Unlike GB, DS does reduce the food intake. Previously, we have shown that food intake was reduced by duodenal switch alone but not by sleeve gastrectomy alone by comparing sleeve gastrectomy only Mechanisms underlying postoperative weight loss and possible regain remain unclear. A major point of controversy is whether this is due to biological or behavioral factors 2 consumption by body weight or body surface area 2 consumption and CO2 production, and expressed as kcal/hr/rat, kcal/hr/100 g body weight, and kcal/hr/cm2 body surface. Dividing the energy expenditure by body weight does not take into account differences in body composition, and therefore, the fat-free mass or lean body mass (as denominator) has often been used in both human and mouse studies. However, this could be inappropriate because brown fat can be the most metabolically active tissue in the body p values by ANOVA vs. ANCOVA (i.e. testing the body-weight independent differences) can be interpreted as that GB or DS increases the energy expenditure (possible cause) while reducing the body weight (effect), which is at odds with the positive correlation between the body weight and the energy expenditure in control animals (LAP).The most extensively used method for calculation of energy expenditure is dividing OBoth GB and DS are designed for restriction and malabsorption by creating the alimentary limb. However, DS, but not GB, caused diarrhea shortly after surgery (2 weeks) and malabsorption (measured at 2 months postoperatively) in rats, which is in line with observations in patients It should be noticed that in the present study, neither prophylactic nor postoperative antibiotics were used and none of the 11 plasmas cytokines measured was changed after surgery, indicating no or little impact of microflora and inflammation on the eating behavior and the body weight changes. Recently, a mouse study showed that specific alterations in the gut microbiota contributed to the beneficial effect of bariatric surgery on energy balance vs. sleeve with duodenal switch (one or two-staged) in the future.There are several limitations of the present study. 1) The rats used were not obese. Whether or not the postsurgical effects of these two procedures are different between normal and obese rats that are induced by high-fat diet or developed spontaneously , and which animal model of obesity best mimics the obese humans in response to the bariatric surgery could be the subjects for further research. 2) GB procedure used in rats was not exactly the same as it was applied in humans. In general, research in patients is directly clinical relevant. However, studies in animals provide much greater latitude in control and experimental manipulation of the system, and ultimately help to reveal the underlying mechanisms and to adopt the protocols and methods that are tested in animals to humans Figure S1Scatterplot of energy expenditure against body weight. LAPGB or DS: laparotomy as control for GB or DS; GS: gastric bypass; DS: Duodenal switch.(TIF)Click here for additional data file.Figure S2Adjust energy expenditure by ANCOVA. LAPGB or DS: laparotomy as control for GB or DS; GS: gastric bypass; DS: Duodenal switch. Means ± SEM.(TIF)Click here for additional data file.Table S1CLAMS measurements of normal rats. Data at day 1 and 21 one week after 24 hours training with CLAMS cage are expressed as means ± SEM. ns: not significant between day 1 vs. day 21.(DOC)Click here for additional data file.Table S2Plasma levels of cytokines. Data of rats after gastric bypass (GB) and duodenal switch (DS) compared with the age-matched laparotomy-operated groups  are expressed as means ± SEM. ns: not significant between LAPGB vs. GB or LAPDS vs. DS.(DOC)Click here for additional data file.


To determine changes in sexual behaviors and other relevant characteristics related to human immunodeficiency virus (HIV) and sexually transmitted infection (STI) risks among young Croatian adults.We surveyed adults aged 18-24 in 2005 (n = 1092) and 18-25 in 2010 (n = 1005). Both samples were probabilistic and stratified by county, settlement size, age, and gender. The samples were non-matched. Trained interviewers conducted structured face-to-face interviews in participants’ households. The part of the questionnaire assessing sensitive information was self-administered.P = 0.002), while the prevalence of condom use at most recent sexual intercourse remained stable (54% in 2005 and 54.7% in 2010). Consistent condom use also remained unchanged. About one fifth of participants (19.2% in 2005 and 20% in 2010) reported consistent condom use in the past year. At both survey points for both genders, consistent condom use was associated with age , negative attitudes toward condom use ), and condom use at first intercourse . In the observed period, HIV/AIDS knowledge was stable.A majority of participants at both survey points (85.2%-86.2%) were sexually active. Median age at sexual debut (17 years) remained unchanged. Lifetime number of sexual partners was also stable. More women than men reported only one lifetime sexual partner. The prevalence of condom use at first intercourse increased (from 62.6 to 70%, Risky sexual practices remain common among young Croatian adults. Given the recently reported STI prevalence rates in this age cohort, introduction of school-based sex education that would focus on protective behavioral and communication skills seems to be of crucial epidemiological importance. Adolescents and young adults are more vulnerable to sexually transmitted infections (STI) than other segments of the general population. Inclined to intense exploration of their sexuality , young pChlamydia trachomatis being the most frequently acquired STIs (A recent acquired immunodeficiency syndrome (AIDS) epidemic update suggested that almost half of all new human immunodeficiency virus (HIV) infections worldwide occurred among people aged 15-24 years . One stured STIs . Althougred STIs ,12. Thisred STIs ,13-17. Tred STIs .Unfortunately, there is no comprehensive sex education in Croatian public schools. The recent debates about its introduction proved highly controversial and politicized . As a reIn 2005 and 2010, two cross-sectional studies on HIV/AIDS-related attitudes, knowledge, and behaviors were carried out on national probability non-matched samples of young adults. Using data from the last census (2001), both samples were stratified according to county, settlement size, age, and gender. Consistent randomization procedures were employed in all sampling steps. In the last step, Kish’s method of the most recent birthday  was usedIn 2005, the number of participants was set equal for all 6 regions of the country to enable robust regional estimates , while i2 = 3.07, P = 0.381), the reported number of partners in the past year , condom use at most recent intercourse , and consistent condom use in the past month .The 2005 survey included 1092 women and men aged 18-24 years, while the 2010 survey included 1005 participants aged 18-25 years. This slight difference in age ranges may seem problematic for between-study comparisons, however a set of analyses suggested that this was not the case. The inclusion of 25-year-olds in 2010 did not affect comparisons of age at coital debut. All sexually active participants in the 2010 sample reported having had first sexual intercourse at the age of 24 years or younger. Furthermore, 25-year-olds did not significantly differ from 24-year-olds in the lifetime number of sexual partners  and 10.8% (2010) stated health issues, their parents’ disapproval, and other (or no) reasons. It is important to note that the structure of non-response was almost identical when refusal was attributed to the study topic  – 23.4% in 2005 and 23.8% in 2010.Participants were interviewed in their homes or, exceptionally, at some other nearby public place  where it was possible to conduct the interview without the presence of other family members. Most interviewers were young women between 25 and 35 years of age, with considerable interviewing experience. Both in 2005 and 2010, the interviewers received an additional six-hour training focused on collecting information on sensitive topics.All study procedures were approved by the Ethical Review Board of the Faculty of Humanities and Social Sciences, University of Zagreb. Prior to the interview, all participants gave verbal informed consent. Following the completion of the survey, participants were given brochures with essential HIV/AIDS information. The 2010 survey additionally included a biological part (not reported here). After participants had completed the survey, they were asked for a urine sample to be tested for chlamydia. Prior to completing the questionnaire, participants were unaware of the biological testing component.An originally developed KABP  questionnaire was used in both waves, with slight modifications. The first part of the questionnaire, administered by face-to-face interviewing, asked about socio-demographic characteristics, HIV/AIDS knowledge, attitudes toward people living with HIV/AIDS, attitudes toward gendered sexual roles, beliefs about condoms and condom use, self-esteem, and locus of control. The second part of the questionnaire, focused on sexual behaviors and other relevant experiences , was self-administered. The questionnaires consisted of 170-190 items and took on average 30 minutes to complete. The 2005 version was piloted for comprehensiveness and completion time among 100 high-school students. Slightly revised version was pre-tested in 2009 on 103 high-school and 132 university students. Instruments used for comparisons were identical in the original and the repeated study.Socio-demographic characteristics assessed were age and personal and parental education (obtained separately for mother and father), occupation, family socio-economic status, and the type of the longest place of residence . In addition, two measures of religiosity were used. Religious upbringing was measured by the following single-item indicator: “Were you brought up in the religious spirit?” A three-point scale was used to anchor answers . Personal religiosity was assessed by the frequency of attending religious services on occasions other than weddings, funerals, christenings, and the like, ranging from 1 = never to 6 = almost daily.Knowledge about HIV/AIDS was measured with the standard UNGASS indicators . Five it2005 = 0.75; α2010 = 0.83).Attitudes toward condom use were assessed by a four-item Negative Beliefs about Condom Use Scale developed by A. Štulhofer and colleagues . Items sSocio-sexual characteristics and sexual risk-taking behaviors were assessed with several single-item indicators: age at sexual debut , contraception/protection use at first and most recent sexual intercourse, condom use consistency (in the past 12 months), and number of sexual partners (lifetime and during the last 12 months). Sexual partners were defined as individuals the participant had vaginal intercourse with. Sexual orientation was assessed by asking about the gender of sexual partners (from 1 = “exclusively men” to 5 = “exclusively women”). Self-assessed HIV and STI-related risks were measured by the following questions: “How would you rate your personal risk of acquiring HIV infection?” and “How would you rate your personal risk of acquiring any other STI?” A 10-point response scale ranging from 1 (“negligible risk”) to10 (“extremely high risk”) was used for anchoring answers. Finally, participants were asked to indicate whether they had ever been diagnosed with an STI and whether they had ever been tested for HIV.2 tests were used to assess differences between genders and study waves on various indicators. t-tests were used to compare study wave and gender-specific means on several continuous measures, which were analyzed for normality of distribution. Reliability analysis and principal component analysis were carried out to assess internal consistency and dimensionality of composite indicators. Consistent condom use, as the central indicator of responsible sexual behavior among young adults with multiple partnerships, was assessed in more detail with multivariate logistic regression analysis. The analyses were carried out separately by study wave and gender. The model included the variables found relevant in previous studies , the frequency of church-going slightly but significantly decreased .Young adults sampled in 2005 and 2010 significantly differed in most socio-demographic characteristics . Althoug2 = 10.5, P < 0.001) and about 5% more knew that healthy looking partner may still be HIV positive . The scores on the overall additive index, however, remained the same in the observed period.Basic HIV/AIDS knowledge was relatively high in both study waves . With th2 = 5.22, P = 0.022). Between 2005 and 2010, the average age at sexual debut decreased significantly in statistical  but not in practical terms. When tested using non-parametric procedures, the connection between age at first intercourse and the study wave was not confirmed  . In 2010, men reported sexual debut at a mean ± standard deviation of 16.9 ± 1.86 years and women at 17.4 ± 1.59 years . In both study waves, the median age at sexual debut was 17 years (interquartile range: 16-18) for both genders. It should also be noted that a proportion of participants who reported sexual debut before the age of 17 remained stable.About 85% of participants in 2010 were sexually active . The pro= 0.095) . In 20052 = 25.0, P < 0.001) was generated primarily by a lower proportion of participants who were sexually inactive in 2010 compared to 2005, as well as a higher proportion of participants who reported 5 or more sexual partners. In both waves, more women than men reported only one sexual partner – in the past 12 months  and lifetime .The lifetime number of sexual partners did not change between the study waves. The median number was 3 . As for the number of partners in the last year, the observed difference  and in 2010 2.5 times as many men than women  reported that they had had most recent intercourse with a casual partner. Similarly, about two times more men than women reported concurrent partnerships .In 2005 and 2010, about three quarters of participants (75.9%-76.7%) had the most recent sexual intercourse with a steady partner. About one quarter of participants reported having had concurrent partnerships at least once during their lifetime (23.3%-24.1%). Differences between genders were observed for both indicators. In 2005, twice as many men , but the proportions remained fairly low .Consistent use of condoms in the past 12 months did not increase between 2005 and 2010 . About one fifth of participants reported using condoms regularly (19.2%-20%). These rates need also to be considered from the point of view of condom use failures and errors. In 2010 (the 2005 wave did not assess problems with condom use), as many as 34.1% of participants reported delayed condom application at least once in the past year and 18.1% condom breakage. Men were more likely to report condom breakage  and 2010 study .Negative beliefs about condom use remained low. Compared to women, men scored higher on the scale, showing less positive attitudes and beliefs toward condoms in both the 2005 . In 2005, more women than men judged the risk of becoming infected with STIs as negligible . In 2010, the same tendency was observed with HIV risk self-assessment, with 80.3% of women and 71.5% of men judging their risk of HIV infection as negligible   .women2005 = 0.74, P = 0.004; ORwomen2010 = 0.72, P < 0.001; ORmen2005 = 0.73, P < 0.001; ORmen2010 = 0.80, P = 0.006) and negative attitudes toward condom use  were associated with lower odds of consistent condom use. Condom use at first sexual intercourse was by far the strongest predictor. Among women, it increased the odds of consistent condom use 3.84-4.64 times (P < 0.001). Among men, an association of similar magnitude was found .Consistent condom use was associated with age, condom use at first sexual intercourse, and attitudes toward condom use . In bothP = 0.006). In 2010, consistent condom use in women was associated with mother’s education – women who reported that their mothers had only elementary education were 85% less likely to consistently use condoms than women with college-educated mothers . Among men, lower odds of consistent condom use in 2010 were associated with having more lifetime sexual partners   .The primary aim of this study was to examine the change in HIV and STI-related sexual risk taking in the population of young Croatian adults during the 2005-2010 period. Our results corroborate the findings from earlier studies among Croatian adolescents and late adolescents ,14,16-18Contrary to the often repeated fear of hyper-sexualization of youth in Croatia, the median age at sexual debut remained stable since the late 1980s ,26. A reFrom epidemiological perspective, however, the fact that a majority of young adults have multiple sexual partners remains of primary importance – regardless of whether the number is increasing or not. In the absence of consistent condom use, such behavior carries substantial STI risks ,12. AccoIn the context of contemporary sexuality, characterized by ubiquitous pre-marital sexual activity and sequential sexual relationships, a regular use of condoms remains one of the central protective behaviors. In multivariate analyses, three indicators were found to be robust (stable across time) and gender non-specific predictors of consistent condom use: younger age, positive attitudes toward condom use, and condom use at sexual debut.Corroborating earlier findings ,15,17, cHIV incidence and prevalence remain comparably low in Croatia, with less than 0.1% infections and, on average, 10-15 new HIV infections per million a year in the last 10 years . This isSeveral study limitations need to be addressed. Despite adherence to standard procedures that maximize anonymity and confidentiality, validity of the findings reported in this article is limited by self-reporting. Even though proportions of individuals who refused to participate in the study because of its subject were almost identical in the 2005 and 2010 waves, a larger total non-response in 2010 suggests selection biases that may have affected generalizability and comparability of the findings. However, some recent analyses indicate that non-response does not necessarily increase biases , while sFully aware that the five-year timeframe may not be long enough to detect possible shifts in attitudinal and behavioral patterns, we firmly believe that continuous monitoring of the dynamics of HIV relevant knowledge, attitudes, and behaviors is a public health imperative. Systematic study of sexual risk taking, particularly among young people who are the most vulnerable segment of the general population, is crucial not only for tracing epidemiological trends but also for informing and guiding future prevention and intervention programs in the country. The present study is hopefully one of the first steps in this direction.


Salmonella enterica with these resources may result in the contamination of produce and disease outbreaks. Therefore, surface water was examined for the distribution of S. enterica at multiple time points from 4 sites on the upper Suwannee River. Isolates were confirmed by detection of the invA gene, and 96% of all samples were positive for the bacterium. Most probable number enumeration ranged from <18 to 5400 MPN/100 mL. Genetic diversity of these isolates (n=110) was compared to other environmental (n=47) or clinical (n=28) strains and to an online library (n=314) using DiversiLab rep-PCR. All strains showed >60% similarity and distributed into 16 rep-PCR genogroups. Most (74%) of the Suwannee River isolates were clustered into two genogroups that were comprised almost exclusively (97%) of just these isolates. Conversely, 85% of the clinical reference strains clustered into other genogroups. However, some Suwannee River isolates (12%) were clustered with these primarily clinically-associated genogroups, supporting the hypothesis that river water can serve as a disease reservoir and that pathogenic strains may persist or possibly originate from environmental sources.The Suwannee River spans the Florida/Georgia border to the Gulf of Mexico, and contributes to regional irrigation and recreational activities. Association of  Salmonella enterica serotypes [Salmonella were primarily attributed to contaminated poultry meat and eggs, but other sources include soil, factory surfaces, animal feces, and raw meats [Nontyphoidal salmonellosis is the leading cause of bacterial foodborne illness in the US and contributed to approximately 33% of all foodborne-related deaths in 2009 . The disaw meats . More reaw meats –8 and otaw meats  have beeaw meats .Salmonella [Salmonella from irrigation sources has been shown to adhere to plants and survive for long periods of time [Irrigation water may play an important role in contaminating soil and produce with lmonella , 15. Stolmonella , 17, butlmonella . Laboratlmonella  and flowlmonella , and Sal of time .S. enterica [S. enterica in 79.2% of samples [S. enterica densities directly correlated with water temperature  and precipitation levels  and also increased 62% in summer compared to spring months. This survey also identified serovars with environmental sources that were associated with disease in this region. However, the genetic relationship of strains from clinical and environmental sources was not examined.The Suwannee River is a central feature of the Suwannee River watershed and is the largest source of free-flowing fresh water to the Gulf of Mexico . The regenterica . This reenterica , 23, and samples . S. enteS. enterica isolates from aquatic environments show a wider diversity of serotypes than those recovered from animal sources [S. enterica from environmental sources is still relatively unknown. Therefore, the present study examined the genetic profiles of S. enterica recovered from the Suwannee River to those of strains from clinical and other environmental sources. The DiversiLab repetitive extragenic palindromic PCR (rep-PCR) analysis was used to evaluate genetic similarity of Salmonella strains because it shows enhanced discrimination compared to other methods such as pulsed field gel electrophoresis [S. enterica from Suwannee River surface water samples comprised a diverse population that was genetically distinct from strains from other environmental sources and from most strains of clinical origin. This research establishes an S. enterica database that should serve as reference for routine monitoring and source tracking in future outbreaks.  sources –26, but phoresis –29. ResuS. enterica isolates were recovered from surface water samples of the Suwannee River (n = 110), as described below. Reference strains included a total of 186 S. enterica isolates from both clinical (n = 31) and environmental (n = 45) sources and strains that were kindly provided by ABC Research, Inc., Dr. Mickie Parish and Margaret Richards, as described in Supplemental Table 1 (available online at doi:10.1155/2011/461321). All strains were stored at −80°C in Luria Bertani NaCl (1%) broth (LBN) and 50% glycerol and subcultured on LBN agar (LA) for genetic characterization.Suwannee River surface water samples were collected monthly from January to June 2003. Sampling locations included public access sites at the Big Shoals State Park, FL (BP), Stephen Foster State Folk Culture Center in White Springs, FL (WS), Spirit of Suwannee Campground boat ramp (SP), and the Florida Sheriffs Boys Ranch boat ramp (BR). Samples were collected in sterile, glass containers by skimming the surface water and transported in coolers on ice to the laboratory, stored for no more than 24 hours at 4°C, and processed as described below. All media were from Difco Scientific, Inc. unless otherwise specified.Salmonella water samples  were used to determine most probable number (MPN) by addition to equal volumes of sterile 1% buffered peptone water (BPW) at 2x or 1x (for 1 mL samples) concentration in triplicate [Salmonella-positive samples were confirmed by species-specific PCR or DNA probe identification of presumptive positive colonies in XLD and LA, as described below. All confirmed isolates were frozen as mentioned above and stored at –80°C. The MPN/100 mL of each sample from the Suwannee River was determined by the number of replicate enrichment cultures for each dilution that was confirmed by species-specific identification of presumptive positive colonies, as described below.iplicate . Broth cSalmonella on XLD agar plates were confirmed by DNA probe colony hybridization and by PCR. For DNA probe assay, colonies were grown overnight on LA at 37°C and transferred by overlay to filter paper (Whatman no. 541), as previously described [μg/mL) in SSC and washed in SSC. Filters were hybridized at 56°C in buffer with an alkaline-phosphatase-labeled  DNA probe (5′>CTGGTTGATTTCCTGATCGC>3′) derived from the S. enterica invA gene. Filters were washed with 1x SSC at 56°C, followed by washes at room temperature to remove unbound probe, and developed in the presence of NBT/BCIP substrate .All samples with typical colony morphology for escribed . Brieflyμl of PBS and incubated for 7 min at 100°C. Samples were centrifuged at 13,000 rpm for 3 min, and the supernatant was transferred to a new microcentrifuge tube. Extracted DNA in the supernatant (2 μl) was combined with 23 μl of master mix (Eppendorf), forward primer INVE (5′-TGCCTACAAGCATGAAATGG-3′), and reverse primer INVA (5′-AAACTGGACCACGGTGACAA-3′) for PCR amplification on a Mastercycler Gradient Thermal Cycler (Eppendorf). The following conditions were used: initial denaturation for 3 min. at 94°C, followed by 30 cycles of 1 min. at 94°C, 1 min. at 56°C, and 1 min. at 72°C, with the final extension of 15 min. at 72°C. Samples were combined with 2 μl of 6x loading dye (Promega) and run on 1% agarose gels for visualization of PCR products by ethidium bromide staining. Positive Salmonella bands had a length of 457 base pairs. For PCR confirmation, broth cultures (1 mL) were extracted by boiling cells from a suspension of one colony in 400 S. enterica isolates was performed according to manufacturer's specifications for the Salmonella (BioMerieux). Briefly, genomic DNA from isolates grown on LA plates was extracted using UltraClean Microbial DNA Isolation Kit . DNA concentration was determined by spectrometry  and stored at −20°C. PCR amplification used S. enterica rep-PCR DNA Fingerprinting Kit (BioMerieux) with AmpliTaq DNA polymerase and GeneAmp 10x PCR Buffer I with Mg Cl2 . Each PCR reaction (25 μL) included 2 μL of the S. enterica primer mix, 0.5 μL of the AmpliTaq DNA polymerase, 2.5 μL of the Buffer I, 18 μL of the Master mix, and approximately 2 μL (50 ng/μL) of template DNA. Amplification was performed in a Mastercycler Gradient Thermal Cycler (Eppendorf) according to the conditions indicated in the S. enterica rep-PCR Fingerprinting kit. All rep-PCR amplicons were screened by electrophoresis in a 1.5% agarose gel (Fisher Scientific) in Tris-acetate-EDTA buffer containing ethidium bromide. All amplification products were stored at −20°C. Sample analysis was performed using amplicons (1 μL) loaded onto the DNA microfluidics Labchip and for capillary electrophoresis by the Agilent 2100. Electrophoretograms were analyzed by DiversiLab system software (BioMeriuex) for strain comparison of the DNA similarity to the DiversiLab rep-PCR online library for S. enterica (n = 352).DiversiLab Rep-PCR analysis of The Chi-square test was used to compare segregation of rep-PCR genogroups with strain origin  and serovar .Salmonella was isolated from the surface water at all sites that were sampled on the Suwannee River .Although statistics were not possible because MPN was not performed from replicate samples from each site, the results did show that N/100 mL . InteresS. enterica recovered from the Suwannee River was evaluated by the DiversiLab rep-PCR system. These isolates were compared to other S. enterica strains from both clinical and environmental sources and to the DiversiLab S. enterica library. This method divides S. enterica strains into two main clusters that segregate subspecies III from subspecies I strains (n = 10) of the same strains (n = 6) generally showed at least 95% similarity (data not shown). Therefore, isolates with >95% similarity were considered to be clonal by this assay. A total of 499 strains were examined by rep-PCR, and all strains were >60% similar by this assay. Strains segregated into 16 genogroups using the criteria of >85% DNA similarity for more than two strains, while 14 strains were ungrouped .The genetic relatedness among isolates of  strains . ReplicaS. enterica strains recovered from the Suwannee River were quite diverse and were distributed among 10 genogroups, while 6 isolates did not cluster with other strains . None of the Suwannee isolates clustered with subspecies 3 (Arizonae). Significant (P < 0.001) genetic relatedness was observed among the Suwannee River isolates by rep-PCR, and these strains were generally distinct from strains derived from other sources. Most (74%) of the Suwannee River strains segregated into only two genogroups, namely, 10 and 15. Each of these groups contained only one isolate from non-Suwannee sources, including one from a clinical case and another from a tomato. Conversely, 69% of strains from the DiversiLab Library, consisting of isolates from clinical sources, were distributed into genogroups 5, 6, 11, and 13, while only 12% of Suwannee River isolates were included in any of these groups. Interestingly, Suwannee River isolates were also distinct from strains derived from other environmental sources including those from central Florida, such as frogs, oranges, or lakes  were found at all sites . SimilarS. enterica isolates (n = 30) recovered from the Suwannee River in a prior study in 1998-1999 [n = 9), Muenchen (n = 3), Rubislaw (n = 6), Braenderup (n = 2), and one strain each of Montevideo, Newport, Johannesburg, and Cubana. Some strains were untypeable strains (n = 3) or rough (n = 3). Specific serotypes and source of strains are shown in S. enterica derived from the Suwannee River and elsewhere, and strains (70%) with the same serovar were frequently clonal (>95% similar) with one or more strains from independent sources. However, rep-PCR also revealed genetic distinctions among strains of the same serovar, as serotypes frequently segregated into multiple genogroups (S. enterica by serotyping but demonstrated 96% DNA similarity to other Suwannee River isolates that were serotyped as Braenderup (UF-8 and UF-9) in genogroup 9 ). Similarly, a rough S. enterica strain (UF-29) demonstrated >95% DNA similarity with two Suwannee River isolates serotyped as Inverness (UF-28 and UF-30) within genogroup 10. A high level of DNA similarity (>90%) was also noted among several Suwannee River isolates with serotype Rubislaw and another un-typeable strain (UF-3) within genogroup15. However, two un-typeable  strains did not cluster with any other isolates at the 85% level of DNA similarity. A single clinical S. enterica Inverness serotype clustered with 39 Suwannee River isolates in genogroup 10, including 8 Suwannee River isolates that were also Inverness serotype. The remaining Suwannee River isolate that serotyped as Inverness was found in genogroup 5 that also included Suwannee River isolates which serotyped as Muenchen and Johannesburg. Several of the Suwannee River strains indicated very high levels (≥90%) of DNA relatedness to important clinical serovars such as Typhimurium, Montevideo, and Muenchen.998-1999  were sernogroups . FurtherS. enterica was widely distributed in the upper Suwannee River and was recovered from multiple locations at different points of time. Serotyping and genetic analyses illustrated the diversity of these strains but also showed that some genotypes may be more prevalent in the Suwannee River than others, as >70% of strains clustered into only two genogroups. Furthermore, these genogroups were distinct as most strains derived from clinical and other environmental sources were excluded. Interestingly, strains recovered from central Florida lakes in a preliminary study using similar methodology were much more likely to cluster with strains from clinical sources. Although the number of strains in this study is too small to draw conclusions about these populations, results suggest that genotypes of S. enterica may be differentially distributed in Florida aquifers.Salmonella strain were highly reproducible and always clustered with >95% similarity. Wise et al. [The DiversiLab rep-PCR assay showed some agreement with serotyping, as serotypes from different sources frequently clustered together. However, genetic typing also indicated that some stains with the same serotype were genetically diverse. For example, strains from the Suwannee River with serotype Inverness were mostly (8 of 9) in genogroup 10, which also included a clinical Inverness strain, but one of the Suwannee River Inverness isolates diverged to genogroup 5 and was more genetically related to strains with serotype Muenchen. Prior descriptions of rep-PCR typing have also shown mixed results. Weigel et al.  noted gre et al.  recentlye et al.  found itSalmonella serovars in the Florida aquifers is still unknown, the present study identified 8 serovars  and 10 genogroups from this region. A prior investigation in 1989 reported 14 Salmonella serovars derived from the Suwannee River that included Allandale, Aqua, Braenderup, Daytona, Gaminara, Hartford, Inverness, Montevideo, Muenchen, Paratyphi B, Saintpaul, subspecies IV, Tallahassee, and Typhimurium [The top 10 disease-associated serotypes in 2009 included Newport, Typhimurium, Javiana, Muenchen, Heidelberg, Montevideo, Oranienburg, and St. Paul. While the true diversity of himurium . Anotherhimurium . Thus, MSalmonella from the Little River in southern Georgia portion of the upper Suwannee River watershed [S. enterica subsp. Arizonae, which was not recovered from the Suwannee River either by serotype or rep-PCR genotype. They concluded that recovery of Muenchen in the watershed was epidemiologically significant, as its incidence is increasing in human cases in Georgia (34% increase over 10 years). Furthermore, the Muenchen strains from the Suwannee River were associated with a predominantly clinical (genogroup 5) genotype. Recent Salmonella outbreaks in Florida have identified Braenderup [Salmonella strains in the Suwannee River that were genetically associated with strains from clinical sources.The present study conducted in north Florida showed some similarity to a recent survey of atershed . Both reaenderup , Javianaaenderup , St. Pauaenderup . No JaviSalmonella in possible irrigation sources and suggest the presence of potential pathogens. Franz and Van Bruggen [Salmonella in Florida, there are striking differences in the human population densities associated with upstream versus downstream sites. The upstream sites are in closer proximity to the source of this river and greater distance from agriculture and other human impact. For example, the most upstream site at Big Shoals is in a state park and is essentially unpopulated with no agriculture in close proximity to the river, while the other sites are in direct contact with human populations. These differences may explain the 10- to 100-fold increase in the MPN mL−2 at downstream sites relative to upstream. However, there were no striking differences observed in terms of diversity of Salmonella from the different sites on Suwannee River. Future studies are needed to examine the complex environmental parameters, especially in relationship to nutrient availability, agricultural input, wildlife distribution, flow rates, rainfall and other factors, that may impact the microbial diversity and survival of Salmonella.Although these investigations are not a systematic or exhaustive survey of Suwannee River watershed, they demonstrate the diversity of  Bruggen  reportedSalmonella enterica strains were based on DiversiLab rep-PCR and included Suwannee River isolates (n=110) from this study, which were compared to other environmental (n=47) or clinical (n=28) strains and to an online DiversiLab library (n=314). Analysis of 499 environmental and clinical Salmonella Isolates was assembled into a dendrogram based on rep-PCR fingerprinting profiles at ≥85% DNA similarity level, using DiversiLab software. Genogroups (n=16) were comprised of clusters with >2 strains each. Salmonella isolates from the Suwannee River clustered into 10 genogroups, but the majority of strains were found Genogroups 10 and 15.Genogroups for S. enterica genogroups were derived from DiversiLab rep-PCR as described in the Materials and Methods. Strains included Suwannee River isolates from this study (highlighted), which were compared to other environmental or clinical strains. Dendrogram was assembled from rep-PCR fingerprinting profiles at ≥85% DNA similarity level, using DiversiLab software. The strains comprised a total of 16 genogroups with >2 strains each. Gel-like images, serovar identity, isolation source, and identification number for each Salmonella isolate are provided. Abbreviations are used for descriptions of sampling locations on the Suwannee River and include: BS , WS (White Springs), SP (Spirit of Suwannee), and BR (Boy's Ranch). Additional strains from our collection were from ATCC or provided by Dr. Parish, and strain source is indicated when known. DiversiLab strains are designated by SE number.”Legend for Figure 1S should be changed to read “Click here for additional data file.


Background: Menstrually related migraine (MRM) affects more than                    half of female migraineurs. Because such migraines are often predictable, they                    provide a suitable target for treatment in the mild pain phase. The present                    study was designed to provide prospective data on the efficacy of almotriptan                    for treatment of MRM.Methods: Premenopausal women with MRM were randomized to                    almotriptan (N = 74) or placebo                        (N = 73), taken at onset of the                    first perimenstrual migraine. Patients crossed over to the other treatment for                    the first perimenstrual migraine of their second cycle, followed by a two-month                    open-label almotriptan treatment period.Results: Significantly more patients were pain-free at two hours                    , pain-free from                    2–24 hours with no rescue medication , and pain-free from                    2–24 hours with no rescue medication or adverse events                     with almotriptan versus                    placebo. Nausea (p = .0007) and                    photophobia (p = .0083) at                    two hours were significantly less frequent with almotriptan. Almotriptan                    efficacy was consistent between three attacks, with 56.2% of patients                    pain-free at two hours at least twice. Adverse events were similar with                    almotriptan and placebo.Conclusion: Almotriptan was significantly more effective than                    placebo in women with MRM attacks, with consistent efficacy in longer-term                    follow-up. Menstrually related migraine (MRM) affects >50% of female                migraineurs, with <10% having migraines exclusively in the                perimenstrual period (PMP) ,3. NeuroWomen with MRM may benefit from short-term prevention ,6, but tIn a retrospective analysis of data from women with MRM from a randomized,                comparative study of almotriptan versus zolmitriptan for acute treatment of                migraine, almotriptan was effective and well tolerated , while aThe primary objective was to demonstrate the superiority of almotriptan over                    placebo in the percentage of patients free of pain two hours after drug intake                    during an MRM attack. Secondary objectives were to investigate the efficacy and                    tolerability of almotriptan versus placebo in women with MRM, and the                    consistency of treatment with almotriptan during open-label follow-up.The study (EudraCT: 2005-000244-90) was a two-month, multicenter, randomized,                    double-blind, placebo-controlled crossover trial followed by a two-month active                    treatment open-label follow-up evaluation to assess consistency. The study was                    performed in the neurology, headache or gynecology departments at seven                    hospitals in Italy, in accordance with Good Clinical Practice. The protocol was                    approved by the ethics committees of all participating centers and written                    informed consent was obtained from each patient.Patients enrolled in the study attended the clinic for five study visits,                    including the screening assessment. Each visit other than the screening visit                    took place two to six days after each migraine attack recorded in the study. At                    each visit patients recorded migraine pain intensity, adverse events and                    concomitant medications in a diary. Patients were randomized 1:1 to receive double-blind oral almotriptan malate or                    identical, matching placebo. Patients were assigned a randomization number in                    ascending order by the Statistics Group within Almirall SA., according to the                    relevant standard operating procedure. The randomization list was                    computer-generated, and randomization data were kept strictly confidential,                    accessible only by authorized persons. Data were unblinded only when the trial                    was completed and the data verified and locked. The success of blinding was not                    formally evaluated during the study.Study medication was self-administered at the onset of the first migraine attack                    occurring during the PMP . Participants took one 12.5 mg tablet, preferably in                    the first hour after pain onset or, if possible, when the pain was still of mild                    intensity, and not more than two hours after pain onset. Patients then crossed                    over to the other double-blind treatment  for the first                    migraine in the PMP of their second menstrual cycle. Two boxes  per                    patient were provided, each containing two tablets of study medication or                    placebo in sealed blisters. One tablet was to be used for the treatment of the                    attack, with the other to be used only in case of recurrence. In the open-label                    phase, all patients received almotriptan for the first PMP migraine in their                    third and fourth menstrual cycles. Patients were almotriptan-naive women aged 18–50 years, from any ethnic                    group, who experienced MRM, as defined according to the criteria of the                    International Headache Society . PatientAt baseline, patients were also required to provide a verifiable diary of their                    migraine history covering at least their preceding two menstrual cycles, their                    medical history in general and their latest medication history.Headache pain intensity was rated by patients on a 4-point scale:                    . Migraine-associated symptoms                    , migraine duration and                    use of rescue medication were recorded throughout the study. Rescue medications                    included nonsteroidal anti-inflammatory drugs and paracetamol, and other agents                    that investigators considered to be appropriate for each patient at their                    center. Triptans and ergotamine-containing drugs were not permitted as rescue                    medication. Tolerability was assessed in terms of adverse events, physical                    examinations and vital signs. According to sample size calculations, 130 evaluable patients were required to                    demonstrate superiority of almotriptan over placebo, and thus it was planned to                    screen and randomize 160 patients to account for an estimated 20% of                    withdrawals after randomization. A sample size of 130 patients (pairs of                    observations) achieves 90% power to detect an odds ratio (OR) of 2.00                    using a two-sided McNemar test with a significance level of 0.05 and the                    proportion of patients free of pain at two hours as primary endpoint. The OR is                    equivalent to a difference between two paired proportions of 0.250, which occurs                    when the proportion of patients responding to almotriptan and not to placebo is                    0.500 and the proportion of patients responding to placebo and not to                    almotriptan is 0.250. The proportion of discordant pairs is 0.750.The primary endpoint was the percentage of patients free of pain at two hours                    after drug intake. Secondary endpoints included percentage of patients pain-free                    at time points from 0.25 to 24 hours after drug intake; percentage of patients                    being sustained pain-free ; percentage of patients being SPF with no adverse events                    (SNAE); rate of recurrence (defined as onset of a new attack within 24 hours of                    successful drug treatment of the first migraine attack); percentage of patients                    with rescue medication intake; evolution of migraine-associated symptoms                    . The evaluation of primary endpoint and all the secondary endpoints, with the                    exception of “duration of migraine attack”, was performed by                    means of a generalized linear model implemented with binomial distribution,                    log-link function and generalized estimating equations. The model effects were                    treatment sequence ,                    treatment  and the period (first and second). As this                    was a crossover study, a compound symmetry variance–covariance matrix                    was employed to account for clustered data (repeated measures). p values. The primary efficacy population                    was the modified intent-to-treat (mITT) population, defined as all patients who                    received at least one dose of study medication and for whom data on the primary                    endpoint were available (i.e. double-blind phase completers). A per-protocol                    analysis, from which patients with major protocol violations were excluded, was                    also performed. The safety population included all patients who received at                    least one dose of study medication. A post-hoc analysis was conducted to assess                    the efficacy of almotriptan in comparison with placebo in the subgroups of                    patients with pain of moderate/severe (intensity                    score = 2−3) or mild (intensity                    score = 1) intensity at headache onset.Results were reported as RRs with the associated 95% confidence interval                    (CI) and two-tailed N = 74) or                    placebo–almotriptan (PLA/ALM)                    (N = 73)  and were included in the safety                    analysis. Baseline characteristics were similar in the two treatment arms;                    overall, the mean (± standard deviation [SD]) age of patients was                    34.9 ± 8.0 years, and all patients were Caucasian. The                    double-blind phase was completed by 63/74 (85.1%) and 59/73                    (80.8%) patients in the ALM/PLA and PLA/ALM arms, respectively, and                    these patients formed the mITT population                    (N = 122). The most common reasons for                    withdrawal were loss to follow-up                    (N = 8) and personal request                        (N = 5)  and 176 in the moderate/severe                    subgroup . The study was conducted between May 2005 and October 2008. In total, 194 patients                    were screened, of whom 147 were randomized to almotriptan–placebo                    (ALM/PLA) ((N = 73) . Of the  (N = 5) . The perOf the 122 patients who entered the open-label phase, 105 (86.1%)                    completed the study. The most common reason for withdrawal in the open-label                    phase was loss to follow-up.N = 59/122] vs. 26.2%                        [N = 32/122]; RR, 1.81 ; p = .0008)                        . In                    the post-hoc analysis, almotriptan was associated with a significantly higher                    percentage of patients free of pain at two hours in the mild subgroup                     but not in the                    moderate/severe subgroup  (Almotriptan was associated with a significantly higher percentage of patients                    free of pain at two hours after drug intake compared with placebo (48.4%                        [         . The res= .1100) . Figurep = .0022); the result was also                    statistically significant in the moderate/severe subgroup  but failed to                    achieve significance in the mild subgroup  (p = .0061) (p = .0304) but not in                    the mild subgroup .Overall, 36.1% of patients were classed as SPF while receiving                    almotriptan, compared with 17.2% while receiving placebo                        (= .0766) . Similar= .0061) . Again, N = 48/122) while receiving                    almotriptan and 59.8%                    (N = 73/122) while receiving placebo                    . In the mild and                    moderate/severe subgroups, the percentages were 33.3%                        (N = 12/36) vs. 59.4%                        (N = 19/32)  and                    41.9% (N = 36/86) vs.                    60.0% (N = 54/90) , respectively. The                    duration of migraine attack was significantly shorter in patients receiving                    almotriptan compared with placebo , but was not                    significantly different between the two treatment arms in either the mild or                    moderate/severe subgroups   . Table N = 17/59)                    during almotriptan treatment and 31.3%                    (N = 10/32) during placebo treatment                        (p = .9349).Migraine recurrence within 24 hours of successful treatment occurred in 28.8                    % of patients . Similarly, the incidence of photophobia at two hours                    post-medication  was significantly lower during almotriptan treatment                    compared with placebo. No between-group differences in vomiting, phonophobia or                    osmophobia at two hours after drug intake were reported. The incidence of nausea at two hours after drug intake was significantly lower                    while patients were receiving almotriptan compared with placebo . The percentage ranged from 0.9–1.0%                    at 15 minutes post-intake to 64.8–71.8% at 24 hours. The                    percentages of patients classed as SPF and SNAE were 40.0–41.8%                    and 36.2–39.1%, respectively . Rescue                    medication was used by 17.1–17.3% of patients in the mITT                    population, 12.5–13.9% in the mild subgroup and                    18.9–20.0% in the moderate/severe subgroup. The efficacy of                    almotriptan in terms of percentage of patients pain-free at two hours, SPF, and                    SNAE was highly consistent between the three attacks recorded in the study.                    Overall, 59 of 105 patients (56.2%) responded to treatment in at least                    two of three migraine attacks (i.e. were pain-free at two hours post-intake at                    least twice out of three attacks), while 42 (40.0%) and 39                    (37.1%) patients were SPF and SNAE, respectively, at least twice out of                    three cycles.The recurrence rate in the open-label phase was 29.5–33.9%. The                    incidence of migraine-associated symptoms was generally numerically lower in the                    open-label phase than in the double-blind phase. At two hours after intake, the                    incidences were nausea, 14.4–14.5%; vomiting,                    1.0–2.7%; osmophobia, 1.8–3.8%; photophobia,                    21.2–24.5%; and phonophobia, 18.2–20.2%.N = 122), there was no                    significant difference in the incidence of TEAEs between almotriptan and placebo                    . In the open-label                    phase, TEAEs were reported in 9/132 patients (6.8%). Overall,                    42% of TEAEs were considered by the investigator to be definitely,                    possibly, or probably related to the study drug. All TEAEs were graded as mild                    or moderate, and none led to study discontinuation during either study phase. No                    individual TEAE was reported in >5% of patients, and no serious                    adverse events or deaths were reported during the study. No clinically relevant                    changes in vital signs occurred within or between treatment arms.During the double-blind phase, treatment-emergent adverse events (TEAEs) occurred                    in 8/132 patients (6.1%) in the safety population during almotriptan                    treatment and 8/132 patients (6.1%) during placebo treatment. In the                    mITT population , the percentage of pain-free patients at two hours with                almotriptan was maintained or even increased. Results for secondary                endpoints—including percentage of pain-free patients over time, sustained                freedom from pain with or without adverse events, use of rescue medication and                occurrence of migraine symptoms—confirmed the efficacy of almotriptan in the                overall patient population. Results in the post-hoc analyses of the mild and                moderate/severe subgroups also showed a benefit for almotriptan, although some                results did not achieve statistical significance. It should be noted, however, that                the study was not powered to detect differences between almotriptan and placebo in                the post-hoc analyses.The results of the present study are highly consistent with those from a previous                retrospective analysis of almotriptan in women with MRM, in which 44.9% of                patients receiving almotriptan were pain-free at two hours after medication intake                (48.4% in the present study), with a recurrence rate of 32.8%                (28.8% in the present study)  migraine                excluded. One possible limitation of the study is the analysis of treatment                consistency, for which a more individual intrapatient assessment may have been                preferred. The present results do show, however, that 56% of patients can be                expected to respond to almotriptan for at least two of three migraine attacks.In conclusion, the results of this double-blind, placebo-controlled, crossover study                demonstrated the efficacy of almotriptan in ameliorating the symptoms of migraine                and confirmed its superiority over placebo in women with MRM attacks, which are                usually found to be of longer duration and less responsive to acute treatment than                nonmenstrual attacks . Data fr


Objective. To examine the prevalence and correlates of vitamin D deficiency and inadequacy among US women of childbearing age. Methods. Data from 1,814 female participants (20–44 y) in the 2003–2006 NHANES were analyzed to estimate the age-adjusted prevalence and prevalence ratios with 95% confidence intervals (CIs) for vitamin D deficiency (defined as serum 25-hydroxyvitamin D [25(OH)D] <12.0 ng/mL) and inadequacy (defined as 25(OH)D: 12.0–<20.0 ng/mL). Results. The age-adjusted prevalence was 11.1% (95% CI: 8.8–14.0%) for vitamin D deficiency and 25.7% (95% CI: 22.3–29.5%) for vitamin D inadequacy. Race/ethnicity other than non-Hispanic white and obesity were associated with increased risks, whereas dietary supplement use, milk consumption of ≥1 time/day, and potential sunlight exposure during May-October were associated with decreased risks for both vitamin D deficiency and inadequacy (P < 0.05). Current smoking and having histories of diabetes and cardiovascular disease were also associated with an increased risk for vitamin D deficiency (P < 0.05). Conclusions. Among women of childbearing age, periconceptional intervention programs may focus on multiple risk factors for vitamin D deficiency and inadequacy to ultimately improve their vitamin D nutrition. Vitamin D, a lipid-soluble vitamin or steroid prehormone, is known to play an important role in bone metabolism through regulation of calcium and phosphate homeostasis. During the past few years, the influence of vitamin D on nonskeletal biologic processes has been expanded to a wide array of health outcomes including cardiovascular disease, insulin resistance and diabetes, cancer, respiratory infection, innate and adaptive immunity, and both cardiovascular and all-cause mortality –9.For women of childbearing age, maternal vitamin D malnutrition imposes multiple health impacts on both women and their offspring –12. AdeqLimited evidence has shown that pregnant women and neonates residing in the northern USA are at high risk for vitamin D insufficiency regardless the use of vitamin supplementation , 29. A rData for this analysis came from the 2003–2006 National Health and Nutrition Examination Survey (NHANES), which uses a multistage stratified sampling design to collect data from the noninstitutionalized civilian US population on a 2-year cycle basis. Survey participants were initially interviewed at home and then invited to a mobile examination center (MEC), where they received various examinations and provided blood samples for laboratory tests. In the NHANES 2003–2006, all procedures involving human subjects were approved by the Research Ethics Review Board of the National Center for Health Statistics and written informed consent was obtained from all participants. Details about the NHANES survey design and methods are reported elsewhere .We examined interview and laboratory data from female participants aged 20–44 years. The pregnancy status of a woman was determined as (1) current pregnancy which was confirmed either by a positive laboratory pregnancy test or by a self-reported pregnancy at the time of MEC exam, (2) nonpregnant with a history of pregnancy, and (3) never pregnant. Participants whose pregnancy status could not be ascertained were excluded from our analyses.125I RIA kit, DiaSorin Corporation, Stillwater, MN), which has been described in detail in the laboratory procedure manuals of the NHANES website [Participants' serum specimens were frozen and stored at <–70°C until analysis. Serum concentrations of 25(OH)D were measured using the DiaSorin radioimmunoassay procedure (DiaSorin Corporation 25(OH)D  website . The 25(2). Smoking status was categorized as current smokers (participants who had smoked at least 100 cigarettes during their lifetime and were still smoking) and nonsmokers (participants who had smoked less than 100 cigarettes during their lifetime or those who had smoked at least 100 cigarettes during their lifetime but had stopped). Physical activity was calculated as an average daily metabolic equivalent task (MET)-hour index that summed transportation, household, and leisure-time physical activity, and participants were dichotomized as having physical activity (MET-hour index >0) and physically inactive (MET-hour index = 0). Alcohol consumption was categorized as any alcohol users (participants who had an average daily drinks of >0 during the past 12 months) and abstainers . Healthcare access was assessed by asking participants: (1) whether they were covered by health insurance or some other kind of health care plan including those obtained through employment or purchased directly as well as government programs like Medicare and Medicaid that provide medical care or help pay medical bills , and (2) whether, during the past 12 months, they had seen a doctor or other health care professional about their health at a doctor's office, a clinic, hospital emergency room, at home or some other place .The demographic covariates in our analyses were participants' age , race/ethnicity , and education level . Major food sources of vitamin D intake were assessed by asking participants how often in the previous 30 days they had consumed milk , or they had eaten fish . Potential sunlight exposure was assessed using the 6-month time period during which they were examined (categorized as November 1 through April 30 and May 1 through October 31). Dietary supplement use was assessed by asking participants whether they had taken any dietary supplements during the previous month . Body mass index (BMI) for currently pregnant women was calculated from self-reported, prepregnancy weight and height, and the BMI for nonpregnant women was calculated from measured weight and height following standardized protocol and instruments , and current asthma. The mean systolic and diastolic blood pressure (SBP and DBP) were established by using the average of the last two readings of SBP or DBP for participants whose blood pressure was measured three or four times, or using the last reading for participants whose blood pressure was measured twice, or using the only reading for participants whose blood pressure was measured only once. Participants who reported taking antihypertension medications or who had SBP ≥140 mmHg or DBP ≥90 mmHg were defined as having hypertension . PhysiciP value for multiple comparisons and used SUDAAN  to account for the complex sampling design.Of 2,443 female NHANES participants aged 20–44 years, we excluded 349 women who responded “do not know/not sure,” refused to answer, or had missing responses to the question regarding their pregnancy status. After further excluding those who had missing values for their 25(OH)D concentrations or other study covariates, 1,814 women remained in our analyses. The estimates for the mean concentration of serum 25(OH)D and the prevalence of vitamin D deficiency and inadequacy were weighted and age-adjusted to the 2000 US female population aged 20–44 years. We used log-linear regression models with robust variance estimator to estimate unadjusted and multivariate-adjusted prevalence ratios with 95% confidence intervals (CIs). We used a Bonferroni corrected Of all study participants, 435 were currently pregnant and 1,034 reported having a history of pregnancy. Their mean age was 32.6 years (95% CI: 32.2–33.1). Approximately 66.8% of women were non-Hispanic white, 12.1% non-Hispanic black, 10.0% Mexican American, and 11.0% in other racial/ethnic group. About 14.6% of women had less than a high school education, 21.2% were high school graduates or equivalents, and 64.2% attained an educational level of greater than a high school diploma.P < 0.008, P < 0.001 for linear trends, Figures P < 0.017, P < 0.017).The mean 25(OH)D concentration was 24.1 ng/mL (95% CI: 23.0–25.1 ng/mL), the prevalence of vitamin D deficiency was 11.1% (95% CI: 8.8–14.0%), and the prevalence of vitamin D inadequacy was 25.7% (95% CI: 22.3–29.5%). These measures did not differ significantly by age . By raceP < 0.001 for all, P < 0.001 for all, 2) as among women with a normal BMI (P < 0.001 for both). Additionally, the adjusted prevalence ratio for vitamin D deficiency was 1.5 times as high among women with diabetes, 2.3 times as high among women with cardiovascular disease, and 1.4 times as high among women who were current smokers as among their respective counterparts.By race/ethnicity, the multivariate-adjusted prevalence ratio for vitamin D deficiency was 8.3 times as high among non-Hispanic black women, 4.1 times as high among Mexican American women, and 5.4 times as high among women in other racial/ethnic groups as among non-Hispanic white women . Although education of > high school diploma, physical activity, any alcohol use, insurance coverage, and having had a health care visit were all associated with decreased likelihoods of vitamin D deficiency and inadequacy in unadjusted models, they were not significant contributors to the full models when controlling for all study covariates. In contrast, milk consumption ≥1 time/day, dietary supplement use, and potential sunlight exposure during May-October were significantly and independently associated with lower prevalence ratios of vitamin D deficiency and inadequacy regnancy . A longiregnancy , a findiregnancy , 19. Howregnancy , 30, 36.Our study showed that multiple factors were associated with suboptimal vitamin D status in women of childbearing age. The significant racial/ethnic disparities that we found in the prevalence of vitamin D deficiency/inadequacy were in agreement with those reported previously both in the general population as well as in pregnant women, showing non-Hispanic whites had the highest adjusted mean 25(OH)D concentration, followed by Mexican Americans, and non-Hispanic blacks had the lowest , 36–38. In the human body, about 50–90% of vitamin D comes from the biosynthesis of vitamin D3  in the skin from 7-dehydrocholesterol that requires sunlight (ultraviolet radiation), and the remainder comes from a limited number of foods  and from dietary supplements. A previous study based on data from the third NHANES (1988–1994) reported that seasonality, milk consumption, and dietary supplement use were significant and independent determinants of hypovitaminosis D in women of childbearing age , which iLow vitamin D levels are linked to increased risk for high blood pressure, insulin resistance and diabetes, and cardiovascular disease in the general population , 6–9. Si2 was an independent predictor of vitamin D deficiency and inadequacy among women of childbearing age. Smoking has been associated with a low bone mass and an increased risk for osteoporotic fracture [2D and parathyroid hormone [Multiple health-related behavioral factors have been shown to affect pregnancy outcomes. For example, physical inactivity in mothers is associated with increased risks for obesity and obesity-related chronic conditions such as diabetes mellitus and hypertension, and pregnant women who are obese are at increased risk for miscarriage, pregnancy-induced hypertension and preeclampsia, gestational diabetes, and thromboembolism, and at an increased risk of having children with macrosomia, spontaneous intrauterine demises, or delivered by Cesarean section which carries an increased risk for wound infection –44. Matefracture . Brot et hormone . Our resNutritional assessment, education, and counseling are important components of periconception care. In the present study, although the prevalence of vitamin D deficiency and inadequacy decreased linearly across educational levels, education was not a significant contributor to the models after controlling for potential confounders. Also, we found that increasing use of health care services was not a significant determinant of vitamin D status either. However, these findings should be viewed with caution because the effectiveness of nutritional education and counseling was not assessed in this study. This study has several limitations. First, the causal relationships between factors and vitamin D deficiency/inadequacy cannot be established from our cross-sectional study design. Second, the drift in serum 25(OH)D measurements over the time has been reported. However, we did not find significant differences in the prevalence of vitamin D deficiency and inadequacy between the NHANES 2003-2004 and 2005-2006 survey periods, suggesting that our results may not have been affected in a meaningful way by the variability of serum 25(OH)D assay . In conclusion, our results demonstrate that achieving an optimal vitamin D level remains a distant target for women of childbearing age in the United States. Given that adequate vitamin D concentrations in pregnant women are associated with healthy reproductive outcomes, periconceptional intervention programs may focus on raising vitamin D levels in these women and efforts to prevent vitamin D deficiency-linked health outcomes should begin during routine gynecologic care prior to conception and continue through the postpartum period. The multiple risk factors for vitamin D deficiency and inadequacy that we identified in this study should be useful in identifying at-risk groups of women who may be targeted by these programs.