Inflammatory response and bile secretion pathways were further analyzed. Colon content and hepatic BAs were quantified by LC-MS (n = 8–10 samples/group). Gut organoids were treated with CBD  with or without TNFα and lipopolysaccharide (LPS) followed by mRNA extraction and qPCR to assess CBD-induced changes to inflammatory markers. The expression of 78 out of 114 inflammatory response pathway genes were reduced in CBD-treated OVX mice relative to vehicle (VEH)-treated OVX mice. In contrast, 63 of 111 inflammatory response pathway genes were increased in CBD-treated sham surgery (SS) mice compared to VEH-treated SS group and 71 of 121 genes were increased due to ovariectomy. CBD did not alter BA profiles in colon content or liver. CBD repressed Tnf and Nos2 expression in intestinal organoids in a dose-dependent manner. In conclusion, CBD suppressed colonic inflammatory gene expression in E2-deficient mice but was pro-inflammatory in E2-sufficient mice suggesting CBD activity in the intestine is E2-dependent.Cannabidiol (CBD)  treatment was shown to improve metabolic outcomes in ovariectomized (OVX) mice deficient in 17β-estradiol (E2). Herein, CBD effects on intestinal and hepatic bile acids (BAs) and inflammation were investigated. Following RNA sequencing of colon tissues from vehicle (VEH)- or CBD-treated sham surgery (SS) or OVX mice ( In the United States, women typically enter perimenopause in their mid-to-late forties and reach menopause at the average age of 51 ,4,5,6,7.Cannabis sativa L.). In preclinical studies, CBD was shown to reduce inflammation [®) is a FDA-approved for treatment of epilepsy-related disorders in both children and adults [Lactobacillus species [Lactobacilli bacteria improve gut barrier integrity leading to the lower levels of inflammatory cytokines in the gut, circulation, and bone [Cannabidiol (CBD) is a non-psychotropic phytocannabinoid derived from the industrial hemp plant . Oasis Prime HLB 1cc Cartridges (30 mg) were purchased from Waters  for filtration of phospholipids from liver samples. Corning Costar Spin-X centrifuge tubes with 0.22 μm nylon membrane  were used to filter BA extracts prior to injecting. CBD isolate was purchased from Bluebird Botanicals . Twelve unconjugated, 9 taurine conjugated, and 6 glycine conjugated BAs were ordered from Sigma-Aldrich Inc. , Cayman Chemical , or Steraloids Inc. . Deuterated internal standards of BAs were purchased from Cayman Chemical. BA and CBD information are detailed in n = 10 mice/group) for 18 weeks (5 days per week excluding weekends). A calculated volume of VEH (sesame oil) or CBD  was mixed with 100 mg of powdered peanut butter to prepare individual peroral doses, which were consumed within 1 min of offering. Mice were euthanized by CO2 asphyxiation and liver, and intestinal tissues were collected as previously described [Animal study protocols were approved by Rutgers institutional animal care and use committee under protocol# PROTO201900041. The details of the mouse study were previously described . Brieflyescribed . Individn = 4 mice/group) was extracted using RNeasy Plus Universal Mini Kit . The concentration, quality, and integrity of total RNA was determined using a Nano Drop 2000 spectrophotometer  and RNA samples were provided to Azenta  for Standard RNA-Seq processing. Paired-end sequencing was performed using Illumina HiSeq 2 × 150 bp paired-end configuration yielding 124,739 Mbases and 415,802,210 reads.Total ribonucleic acid (RNA) from colon tissue   with thePCA plots to compare similarity/difference among RNA-Seq datasets for biological replicates within group and between groups were generated using Metaboanalyst 5.0 . The fol®  for 4 min. Homogenized tissue was transferred to microcentrifuge tubes with 300 μL of pre-dried deuterated internal standards . Protein was precipitated with 99.9% acetonitrile and 1% formic acid (800 μL) and vortexed for 30 s and then placed on an orbital shaker for 1 h at 4 °C. Samples were centrifuged at 13,000× g for 10 min at 4 °C and supernatant was transferred to glass scintillation vials. Pellets remaining after centrifugation was resuspended in 1 mL 80% methanol and sonicated for 1 min with a Qsonica sonicator Q700 with chiller fitted with cuphorn and 8-tube holder ; 55% AMP, 30 s on, 59 s off, then a final 30 s on). Sonicated samples were centrifuged at 16,000× g for 20 min at 4 °C and supernatants were pooled and dried under speed vacuum at room temperature overnight. Samples were resuspended in 300 μL 50% methanol, placed on orbital shaker for 30 min, vortexed for 2 min, and filtered through Corning® Costar® Spin-X® microcentrifuge tube filters  for 5 min at 16,000× g. Samples were transferred into sampler vials  with 300 μL inserts  for HPLC analysis. Concentrations (µg/mg tissue) were determined by dividing final concentrations by tissue weights used for extraction. For each liver sample, the limit of detection (LOD), the limit of quantification (LOQ), and the coefficient of variance (CV) are presented in BAs were extracted from individual liver samples (n = 9–10/group). Frozen liver tissue was sectioned on dry ice and 50–60 mg of median lobe was transferred into 2 mL bead beating tubes with 4 stainless steel beads  and 300 μL of water. Samples were homogenized using a 1600 MiniGn = 8–9/group) were collected into microfuge tubes by flushing the lumen of the colon with 1× PBS (pH 7.4). The colon content was freeze-dried in a FreeZone 1.0 L Benchtop lyophilizer  overnight to evaporate PBS and dry weight (mg) of colon content was recorded. To subtract weight contributed by salts in PBS, 3 tubes containing 1 mL of 1× PBS were freeze-dried and their mean weight was subtracted from dried colon content weights. Deuterated internal standards  were resuspended in 50% methanol and 300 µL was added to pre-weighed microfuge tubes and dried in speed vacuum , after which colon content (15–30 mg) was added. Then 600 μL of 90% acetonitrile/9.9% water/0.1% formic acid (v/v/v) was added to internal standards and dry colon content, vortexed for 1 min, and left on a benchtop shaker at 4 °C for 1 h. After extraction, samples were centrifuged at 15,000× g for 10 min and the supernatant was collected into a clean microfuge tube. For the second round of extraction, 700 μL of 50% methanol/50% water (v/v) was added to the pellet, vortexed for 2 min, and extract was placed in QSonica sonicator Q700  at 65% amplitude for 2 min. Samples were placed on shaker at 4 °C for 45 min and then centrifuged at 12,000× g for 10 min. Supernatants were transferred to microfuge tubes and extraction was repeated with 600 μL of 90% acetonitrile and 0.1% formic acid. Samples were vortexed for 30 sec and placed in QSonica sonicator at 65% amplitude for 1 min. Samples were placed on shaker at 4 °C for 45 min and then centrifuged at 12,000× g for 10 min. Supernatants from first and second extractions were pooled and solvent was evaporated to dryness using a speed vacuum  and resuspended in 300 μL of 50% methanol. Samples were sonicated at 65% amplitude for 1 min and then filtered using 0.2 μm filters . Filtrates were centrifuged at 12,000× g for 10 min and transferred to HPLC vials  fitted with 300 μL inserts .Individual colon content samples , the limit of quantification (LOQ), and the coefficient of variance (CV) are presented in Data was generated using an Alliance e2695 HPLC system coupled to a 2998 Photodiode array detector and an Acquity QDa detector mass spectrometer equipped with an electrospray interphase , an autosampler, and a Vacuubrand pump . For each sample, technical duplicates (10 μL) were injected. The instrument and processing methods have been previously described [2 asphyxiation and ileal tissue was collected for crypt isolation according to established methods [g for 3 min, PBS was aspirated, Cultrex was added to obtain a density of 150 crypts per 25 µL volume, and 48-well plates were seeded with 25 µL Cultrex per well. The plate was incubated in a 37 °C, 5% CO2 incubator  for 30 min to allow the polymerization of Cultrex then 250 µL of 1× complete growth medium (CGM) [n = 1) and treatments were performed in triplicate. Organoids from passages 10–11 were used, and the experiment was performed twice.Eight-month-old WT C57BL/6J female mice were euthanized by CO methods . Crypts um (CGM)  was adde2O stock; 100 ng/mL in CGM; Cat#78069, STEMCELL). TNFα and LPS alone served as a positive control for inflammation. Samples were dried in speed vacuum  and resuspended in CGM media the day of treatment. Organoids were treated for 24 h, CGM was removed, and 500 µL Cultrex organoid harvesting solution  was added per well. Organoids (6 wells/treatment) were collected into 15 mL conical tubes precoated with 1× PBS and left to incubate on ice for 1 h to dissolve Cultrex. Samples were centrifuged at 500× g for 5 min at 4 °C, washed with 2 mL of 1× PBS, supernatant was removed, 800 µL of Qiazol was added, and samples were transferred to 1.7 mL microfuge tubes with two 2.8 mm stainless steel beads and frozen at −80 °C until RNA extraction. Samples were thawed on ice followed by vortexing for 30 s, then RNA was extracted using RNeasy plus universal mini kit .CBD (1 mg/mL) was dissolved in 100% methanol and then calculated volumes of this stock were used to obtain 100, 250, or 500 µM CBD concentrations (in 250 µL/well) as well as these same CBD concentrations in combination with lipopolysaccharide  and TNFα (100 µg/mL sterile ddH2) for 2 h. After media and MTT solutions were removed, viable organoids appeared purple/black. Then 50 µL of 2% sodium dodecyl sulfate (SDS) was added to each well and the plate was returned to the incubator  for 1 h. After incubation, 150 μL of pure dimethyl sulfoxide (DMSO) was mixed into each well and incubated for 4 h or overnight to solubilize the formazan crystals. Once solubilization was complete, 200 μL from each well was transferred to a microplate and absorbance was measured in a multimode plate reader  at 562 nm.MTT, 3--2,5-diphenyltetrazolium bromide reagent  was diluted 5 mg/mL in sterile 1× PBS per manufacturer’s instructions. Concurrently, treatments were performed on an additional 48-well plate of organoids for MTT assay to assess viability. Three wells were used per treatment, including 100% DMSO as a positive control for toxicity. At 24 h post-treatment, 27.5 µL of MTT solution was added to the 250 µL of 1× CGM in each well, then placed in an incubator  as previously described . RNA extNos2 (Mm00440502_m1), Tnfα (Mm00443258_m1), Il6 (Mm00446190_m1), and Il1b (Mm004342228_m1). Hmbs (Mm01143545_m1) was used as the house keeping gene.TaqMan™ assay primers  used were: p < 0.05.Data were analyzed using GraphPad Prism 8 software . The ROUT test was used to detect and remove any outliers. Normality and variance were tested before choosing parametric or non-parametric tests. To detect differences in liver qPCR and BA analysis, two-way ANOVA was used followed by Benjamini–Hochberg post hoc test with FDR adjustment, q < 0.05 was considered significant. Non-parametric BA data were analyzed by Kruskal–Wallis test followed Benjamini–Hochberg post hoc test with FDR adjustment. For organoid experiments, one-way ANOVA was performed followed by Tukey post hoc test, and the significance level was n = 4/group) was performed to investigate differential gene expression due to OVX surgery or CBD treatment. The PCA plot showed that samples within surgery and treatment groups clustered together  of which 1334 genes were upregulated and 1255 were downregulated . A compa+VEH vs. SS+CBD, and SS+VEH vs. OVX+VEH using the gene ontology biological process (GOBP) database. For each of these comparisons, 1000 significantly altered GOBP pathways were identified after FDR correction and the top 20 pathways are shown in Il1b, Il6, Tnf) and ileum  in SS and/or OVX mice [+VEH vs. OVX+CBD (pathway ranked 323), SS+VEH vs. SS+CBD (pathway ranked 219), and SS+VEH vs. OVX+VEH (pathway ranked 729). Inflammatory pathways were not detected when DEGs were mapped using KEGG.Gene ontology (GO) enrichment analysis was performed with ShinyGO for OVX+VEH vs. OVX+CBD, SSOVX mice . To findEpha2) were changed by CBD in the same direction indicating that the changes were independent of E2 status  in common, indicating that these changes to the bile secretion pathway were CBD-induced and unrelated to surgery , primary BAs (PBAs), secondary BAs (SBAs), and conjugated BAs were similar between groups regardless of surgery or CBD treatment . CBD did, or Il6 . CompareIl6 (Il6 .a and LPS (TL) treatment of ileal organoids induced the gene expression of inflammatory markers Nos2 and Tnf  in both CBD-treated OVX and SS groups relative to VEH-treated controls [Ocln and Tjp1, while the latter was increased in CBD-treated OVX mice, which suggested CBD compromised gut barrier integrity in the E2-sufficient state but was beneficial in E2-deficiency [The anti-inflammatory effects of CBD isolate or CBD-rich extracts have been reported , but thecontrols . Notablyficiency . It remaThe loss of E2-producing ovary cells with increasing age in perimenopause through postmenopause leads to elevated oxidative stress, which induces inflammation . In younPtges, which encodes PGE2 synthase, was downregulated in the colon tissue of CBD-treated OVX mice compared to VEH-treated OVX mice (CBD is a potent antioxidant . BesidesOVX mice .Hif1α was upregulated in VEH-treated OVX mice compared to VEH-treated SS mice (Hif1α was downregulated in CBD-treated OVX mice compared to the VEH-treated mice (The presence of ROS species is sensed and monitored by the hypoxia inducible factor (HIF) pathway . In hypo SS mice  and exhited mice  and had Importantly, a physiologically beneficial level of ROS is required for pathogen resistance and cell signaling . The excIl1ß, Cxcl1, and Cxcl2) and a decreased expression of Muc2, suggesting an induction of intestinal inflammation [While CBD isolate was used in this study, hemp-derived (defined as having <0.3% tetrahydrocannibinol) extract preparations contain other phytocannabinoids as well as terpenes and flavonoids ,64. Compammation . Male miThe endocannabinoid system functions to maintain the homeostasis of central and peripheral tissues and displays cross-talk with estrogen signaling . EndogenCnr2, which encodes CB2, compared to VEH-treated OVX mice and was associated with decreased bone loss [Cnr2 was upregulated in VEH-treated OVX mice compared to the SS group and CBD treatment lead to a downregulation in the OVX mice (CBD is a partial agonist for CB2, which is mainly expressed in immune cells and peripheral tissues . CB2 is one loss . In the OVX mice . CBD actCorynebacterium parvum-primed and unprimed male mice were treated with CB2 agonist WIN 55212-2 or with CB2 antagonist SR141716A, the levels of pro-inflammatory cytokines in serum was suppressed [Cnr2 expression in bone [Cnr2 expression in the colon (When ppressed , suggest in bone  but decrhe colon B and in . The CBD-induced increase in bile secretion genes is likely due to it being a lipophilic compound that requires bile-mediated micelle formation for intestinal absorption [BAs are synthesized in the liver, stored in the gallbladder, and secreted into the duodenum for the digestion of lipophilic compounds . 95% of sorption . An incrsorption . There wIn conclusion, the RNA-Seq analysis of colon tissues allowed a comprehensive investigation of CBD- and OVX-induced transcriptome changes. CBD had a potent anti-inflammatory effect in colon tissues of E2-deficient OVX mice but may contribute to inflammation in intact, E2-sufficient females. To better delineate the CBD mechanisms of action in E2-deficient and -sufficient states, additional experiments are needed to follow up on the extensive gene expression changes. The gut organoid data suggest that CBD may have a direct anti-inflammatory effect on the intestinal epithelium. Future gut organoid studies that test CBD in the absence and presence of E2 treatment would contribute to understanding CBD actions in E2-deficient and -sufficient states. In both the SS and OVX groups, CBD induced similar changes to genes related to bile secretion, indicating changes that were independent of E2 status. OVX or CBD treatment did not alter BA levels in liver or colon content, suggesting that previously observed CBD-induced changes in ileal and serum BAs  are more


Four 20% oil-in-water emulsions with overlapping particle size distributions contained either partially crystalline solid  or liquid  lipid droplets at 37°C and either the colloidally acid-stable Tween 80 (2.2%) or acid-unstable Span 60 (2.5%) emulsifier. Experimental meals were fed to the TIM-1, and jejunal and ileal dialysates were analyzed over 6 h to measure free FA concentration. Cumulative FA bioaccessibility was significantly higher for the liquid stable emulsion compared to all others (p < 0.05), which did not differ (p > 0.05). Emulsified TAG physical state was associated with differences in overall bioaccessibility (higher for liquid state TAG) in the colloidally stable emulsions, but this difference was blunted in droplets susceptible to acidic flocculation. In contrast, human postprandial TAG concentrations did not differ significantly between the emulsions. The discrepancy may relate to differences in in vivo gastric emptying (GE) as evidenced by ultrasonography. When the in vivo differences in GE were accounted for in follow-up TIM-1 experiments, the findings aligned more closely. Cumulative FA bioaccessibility for the liquid stable emulsion no longer differed significantly from the other emulsions, and SU’s bioaccessibility was the lowest, consistent with the in vivo observations. This work highlights the potential for TAG physical state and colloidal stability to interactively alter behavior in the gastrointestinal tract with implications for FA absorption, and the importance of establishing and improving in vitro–in vivo correlations in food-nutrition research.It is poorly understood how the physical state of emulsified triacylglycerol (TAG) alters colloidal behavior in the gastrointestinal tract to modulate lipid digestion and absorption. We, therefore, aimed to investigate the individual and combined effects on fatty acid (FA) bioaccessibility using the dynamic TIM-1  Excess energy intake, including dietary fat, is a leading cause of obesity  and a riIn vitro digestion methods have yielded interesting insights in this area. Crystalline TAGs induce partial coalescence and different gastric structures that have been associated with attenuated in vitro lipolysis (in vitro digestibility was higher for liquid canola-oil-based emulsions stabilized with non-ionic emulsifiers (Tween 20 and Poloxamer 188) than for high SFC emulsions with fully hydrogenated canola stearin   , 18 and and SDS) . Aside fvia mechanical peristaltic forces and the addition of surface-active molecules that changes the interfacial area available for lipolysis  or palm olein (liquid at 37°C), and either Tween 80 (acid-stable emulsifier) or Span 60 (acid-unstable emulsifier). These emulsions were previously consumed by participants in a randomized crossover study in which different gastric microstructures were observed directly by ultrasonography and associated with different rates of GE  tract occurs ipolysis . As an eipolysis , 23. Theipolysis . Human-lipolysis –27. MRI ipolysis . Steingoipolysis , but wites of GE .In vitro digestion models allow gaining mechanistic insights into how food structures change in the gastrointestinal tract to differentially affect postprandial metabolism. The dynamic TIM-1 multicompartment system simulates the luminal conditions of the upper GI tract from the stomach, duodenum, and jejunum to the ileum, integrating physiological factors including meal transit time, peristaltic contractions, enzyme concentration, and pH changes , porcine bile extract [95% ], bovine pancreas trypsin powder , anhydrous ethanol, HCl (37%), NaHCO3 (≥99%), hydroxypropylmethyl cellulose powder (HPMC), NaCl (≥99%), KCl (≥99%), and CaCl2 (≥96%). Fresh porcine bile was collected from Conestoga Meat Packers  at the time of slaughter, pooled and filtered (cheesecloth) into allocated single-use portions and stored at −30°C.Palm stearin , palm oil olein—organic , sorbitan monostearate , polyoxyethylene sorbitan monooleate , and deionized water were used to prepare the emulsions. We used the following reagents from Sigma-Aldrich  for the TIM-1 experiments: lipase from porcine pancreas , pepsin from porcine gastric mucosa , α-amylase from ®  at 12,000 min–1 for 2 min and then passed through a microfluidizer  three times at 125 MPa with the machine’s piping immersed in a water reservoir at ∼95°C, using portable immersion heaters. The resulting fine emulsion with palm olein (LS and LU) was held at room temperature (21 ± 1°C), and those with palm stearin (SS and SU) were placed in a pre-cooled jar, submerged in ice water for 20 min, and then refrigerated (5°C) for at least 18 h to induce crystallization of the lipid droplets. Before all analyses and experiments, the emulsions were warmed to 37°C for 1 h. All emulsions were stored and used within 7 days after confirming that PSDs and melting behavior remained consistent over this period (data not shown).The study emulsions consisted of LS (liquid lipid-acid stable emulsifier), LU (liquid lipid-acid unstable emulsifier), SS (solid lipid-acid stable emulsifier), and SU (solid lipid-acid unstable emulsifier). To determine the effects of droplet TAG crystallinity, they were formulated with palm stearin  or palm olein (liquid at 37°C). They were also formulated with either Tween 80 or Span 60 to be either colloidally stable or unstable, respectively, in the presence of gastric acidity. The emulsifier concentrations were selected to achieve overlapping particle size distributions (PSDs) based on preliminary experiments. Span 60 is a relatively low HLB value (4.7) emulsifier selected based on previous observations that monomodal PSDs could be obtained for undercooled and maximally crystallized palm stearin emulsions at concentrations permissible for consumption in the corresponding human studies . The tot4,3, and surface-weighted, D3,2, and mean diameters using refractive indices of 1.45 and 1.33 for the lipid and water phases, respectively. The emulsion droplet surface charge  was measured by a particle electrophoresis instrument . Emulsion SFC was measured at 37°C according to AOCS official method Cd 16b-93 using a Bruker Minispec PC/20 series pulsed nuclear-magnetic-resonance spectrophotometer . A differential scanning calorimeter  determined the melting behavior of each emulsion, as previously described (The FA composition of the bulk lipids and emulsifiers was determined by gas chromatography (Agilent 7890A GC) with flame ionization detection   after exescribed , by heatin vitro digestion experiments confirmed and compared the colloidal behavior of each emulsion during exposure to simulated gastric digestion fluid (SGF) as described previously  dynamically simulated digestive conditions and was used to investigate the role of droplet SFC and colloidal stability on lipid bioaccessibility, the precursor to lipid absorption. According to the fed-state protocol (g at 4°C), and the supernatant was collected. Gastric electrolyte solution (GES 1×) was prepared with 53 ± 0.5 g GES 10× and 450 ± 5 g water, and gastric enzyme solution by mixing (by magnetic stirring for 10 min at room temperature) 249 ± 3 g GES 1×, 2.5 ± 0.1 ml 1 M sodium acetate buffer (pH = 5), 167 mg lipase, 480 mg pepsin, and 13.5 mg amylase and stored at 0–5°C. The small intestine electrolyte solution was prepared from 88 ± 1 g SIES 25× and 1920 ± 10 g water. Bile was warmed to 37°C in a 60°C water bath before use. Jejunal secretions were prepared by mixing 88 ± 1 g SIES 25× and 1,720 ± 10 g water followed by 206 ± 2 g of bile, and ileal secretions were 1,606 g of SIES 1×. Gastric start solution [5 ± 0.1 g gastric enzyme solution (0.40% HPMC and 0.04% bile powder)], duodenal start solution , jejunal start residue solution , and ileal start residue solution (140 ± 1 g SIES 1×) were added directly into the compartments. The “feed meal” consisted of 100 g emulsion, 90 g GES 1×, 50 g water, and 11 mg amylase and was prepared in a beaker, maintained at 37 ± 1°C. The meal and the 50 g rinse water were added to the stomach compartment already conditioned with 10 g GES. Semi-permeable, 0.05-μm pore size capillary membranes  attached at the jejunal and ileal compartments collected the dialysates  at 30, 60, 90, 120, 180, 240, 300, and 360 min. These were weighed and sampled into 1.5-ml Eppendorf tubes and stored at −20°C until FFA analysis.The TNO protocol , TIM-1 sAll dialysate samples were thawed at room temperature, and lipids were extracted by adding 50 μL of sample to an Eppendorf tube with 50 μL 1 M HCL and 500 μL hexane and centrifuged at 14,000 RPM for 30 min . The double-layered supernatant was added to 500 μL of hexane, and the tube was rinsed with another 500 μL of hexane. Non-esterified fatty acids (NEFA) were quantified in 96-well plates in duplicate for each TIM-1 run (performed in triplicate) according to the manufacturer instructions  using an enzymatic colorimetric method assay. The cumulative FFA bioaccessibility (%) was the total accumulated lipid bioaccessibility at each time point .T1/2 using Eq. 1 . Thus, the LS emulsion was subsequently digested in the TIM-1 using an adjusted GE T1/2 of 124 min.where  in vivo  GE T1/2 All experiments were performed in at least triplicate, with analytical duplicates, using samples prepared within 7 days. Microsoft Excel  was used to plot the FFA standard curve and to calculate cumulative % bioaccessibility for the TIM-1. GraphPad Prism 8  was used for all calculations and statistical analysis. Area under the curve (AUC) values were calculated using the trapezoid rule. A shifted logistic equation fits the cumulative FA bioaccessibility using GraphPad Prism 8  to model the FFA generation after lipolysis over time as follows :Casymp is the asymptotic plateau representing the maximum bioaccessible FFAs, k is the rate constant of FFA release per unit time , and tc is the induction time   . Initialk), and induction time (tc) values were analyzed by one-way ANOVA. Data were normally distributed (passed the Shapiro–Wilk test), there were no missing values, and statistical significance was set at p < 0.05. The hourly TIM-1 FFA and AUC values were correlated with the hourly and AUC postprandial TAG response of 15 healthy male adult participants who consumed the same test emulsions across four study visits following an overnight fast, separated by least 6 days  with time  and treatment  as the two factors followed by Bonferroni’s multiple comparison testing when treatment and/or time × treatment interactions were significant. AUC, 6 h % bioaccessibility, rate constant (t 6 days  using Pep > 0.05). The melting behavior of SS and SU (p < 0.05) peak melting temperatures of 52.2 ± 0.1 and 52.0 ± 0.1°C were observed for SS and SU, respectively. DSC also confirmed the absence of crystallinity in LS and LU as no melting events were observed.Compared to palm olein, palm stearin contained significantly more palmitic acid (C16:0) (58.7 ± 0.0 versus 36.4 ± 0.1%) and less oleic acid (C18:1c9) (26.8 ± 0.1 versus 45.2 ± 0.0%) , leadingS and SU  confirms4,3) and surface area-weighted  mean diameters (p > 0.05), but not within each emulsifier. The Span 60 emulsion (∼−43 mV) charge was close to ∼−56 mV as reported previously for 10% palm stearin emulsions with 0.4% Span 60  and LU (p = 0.037) before digestion , SS (p = 0.023), and SU (p = 0.0003). LS also had a significantly higher overall bioaccessibility than LU (p < 0.0001), SS (p < 0.0001), and SU (p < 0.0001). Differences were specifically observed for LS compared to all other treatments at 180, 240, and 300 min, based on FFA concentration (p < 0.05), based on cumulative FA bioaccessibility  for each emulsion was collected over the 6-h TIM-1 digestion . Accordintration , and at sibility .p = 0.07), although LS was at least 52% higher than all other emulsions. AUC values of the cumulative bioaccessibility results were significantly different (p = 0.03), but there were no pairwise differences based on post-hoc testing (p > 0.05). There were neither differences in terms of bioaccessible fraction nor in the rate constant values obtained from the shifted logistic model (p < 0.05). At 6 h, LS had at least 24 and 39% higher bioaccessible fraction and rate constants (k), compared to all other emulsions. Similar induction times were observed (p > 0.05) due to the TIM-1 pre-set GE rate. Regardless of the analysis method, there were no significant differences in FA concentration, AUC, or time point differences among LU, SS, and SU (p > 0.05). Postprandial TAG values did not return to baseline by 6 h for two of the emulsions (LS and LU), and lipolysis was incomplete after 6-h TIM-1 for all of the emulsions   , but the = 0.74) .T1/2 was set to 124 min for LS and was kept at the default 80 min for SU. This difference was intentionally proportional to the observed longer GE in vivo for LS versus SU  compared to LS. The lower bioaccessibility for SS compared to LS, specifically, provides the strongest indication that SFC was predominantly responsible for the differences since these emulsions were both stable during gastric digestion. Similar observations have been reported showing that acid-stable emulsions formulated with canola oil had higher TAG hydrolysis compared with those containing crystalline TAG    . For exa ∼12.4%) . In that ∼12.4%) . Similarola oil) . In the igestion . Accordiigestion . HoweverIn vitro,  and acid instability (LS > LU) have the potential to attenuate lipolysis independently. Nonetheless, the combination of crystalline TAGs and acid-unstable emulsifier (SU) resulted in the greatest gastric instability  and the trend toward the lowest bioaccessibility compared to all other emulsions.In vivo, no significant differences were observed in TAG iAUC , but TIM-1 bioaccessibility was higher for LS. The trends in TIM-1 AUC values based on FFA concentration also do not agree with those based on the human postprandial TAG AUC. Therefore, the human study results do not concur with the observed higher TIM-1 bioaccessibility for LS. However, both in vitro and in vivo models suggest that acidic flocculation in the presence of TAG crystallinity has an attenuating influence. The TIM-1 FFA AUC value for SU was 50, 26, and 20% lower than LS, SS, and LU, respectively, but only LS was statistically different from the other treatments. As previously reported, human PPL AUC TAG data for SU trended to be 29, 32, and 22% lower compared to LS, SS, and LU, respectively, with no appreciable differences between LS and SS or SU   and the degree of associated structural changes in the stomach. In this case, when GE differences were accounted for, TIM-1 bioaccessibility more closely reflected the outcomes observed in vivo. Effectively, this refinement in in vitro transit corrected for an apparent major augmentation of lipid bioaccessibility for LS. In vivo, the lipid phase of the LS (and SS) emulsions emptied the stomach earlier than the separated lipids from SU and LU, triggering the ileal brake which led to a decrease in gastric motility (p > 0.05), substantial differences were observed in satiety hormones and participant ratings.According to ultrasound imaging, the acid-stable emulsions (LS and SS) remained dispersed in the stomach, as intended, and had significantly faster GE than SU and LU, which extensively flocculated and phase-separated in the stomach . In vivo and SU) . As abov and SU)  and lackresponse , 36, 51,motility , alterinin vitro–in vivo correlation with adjusted GE half times highlights the utility of non-invasive ultrasonography to obtain physiologically relevant GE rates to improve the predictability of in vitro digestion models. Other reasons in vitro results may not mirror human findings, which arise due to various pre- and post-absorptive processes  led to the highest TIM-1 digestibility, although there were no differences in the shifted logistic model parameters. The trends observed based on AUC values from the in vitro and in vivo methods did not concur. Specifically, TIM-1 did not indicate the same treatment differences observed in vivo. It was hypothesized that this was because of in vivo GE differences between the emulsions. Indeed, when the TIM-1 GE rate was adjusted to reflect the differences in emptying observed in vivo, the in vitro TIM-1 FFA response more closely resembled the human lipemic response, with the combination of solid lipid and acid-unstable emulsifier leading to the lowest digestibility both in vivo and in vitro. This provides insight into the importance of considering gastric meal structures and corresponding differences in GE in future in vitro digestion studies. Overall, emulsion TAG properties can be tailored for different gastrointestinal behaviors and in vitro and in vivo methods should be integrated for a comprehensive understanding of how food properties influence metabolic response.This study investigated the individual and combined effects of TAG physical state and colloidal acid stability of palm-based oil-in-water emulsions on The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the University of Guelph Human Research Ethics Board. The participants provided their written informed consent to participate in this study.SH and AW designed the research and analyzed the data. SH, RC, YL, NR, ZZ, and PN conducted the research. SH, MR, and AW interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Aspergillus sections and SARS-CoV-2, as well as mycotoxin analysis, screening of azole resistance, and cytotoxicity measurement were also conducted. Total bacteria contamination was 80 CFU·m−2 in settled dust samples, reached 849 CFU·m−2 in EDC and 20,000 CFU·m−2 in swabs, and ranged from 5000 to 10,000 CFU·m−2 in filters. Gram-negative bacteria (VRBA) were only observed in in settled dust samples (2.00 × 105 CFU·m−2). Regarding Aspergillus sp., the highest counts were obtained in DG18 (18.38%) and it was not observed in azole-supplemented SDA media. SARS-CoV-2 and the targeted Aspergillus sections were not detected. Mycophenolic acid was detected in one settled dust sample. Cytotoxic effects were observed for 94.4% filters and 5.6% EDC in A549 lung epithelial cells, and for 50.0% filters and 5.6% EDC in HepG2 cells. Future studies are needed in this occupational setting to implement more focused risk management measures. Cemeteries are potential environmental reservoirs of pathogenic microorganisms from organic matter decomposition. This study aimed to characterize the microbial contamination in three cemeteries, and more specifically in grave diggers’ facilities. One active sampling method (impingement method) and several passive sampling methods  were employed. The molecular detection of  In Portugal, cemeteries are perceived as historical and religious monuments where people usually go to remember their lost loved ones . The burAmong all the European countries, employers are required to prevent and assess exposure to occupational risks . HoweverWith the SARS-CoV-2 pandemic, and the great number of COVID-19 cases and deaths during the first waves of the pandemic, several countries faced an inordinate stress on crematoriums and cemeteries for disposal of the dead. Consequently, an increased concern was raised regarding occupational exposure of gravediggers to virus and other microbial agents. The pandemic crisis has also highlighted other specific occupational sectors of frontline workers, beyond healthcare workers, such as firefighters and waste collection workers. These occupational settings were recently characterized in Portugal regarding the occupational exposure to microbial contaminants, including SARS-CoV-2 . The pandemic situation has prompted countries around the globe to adopt nationwide confinements to contain the spread of the SARS-CoV-2 virus. While some countries are now starting to reopen, precautions are being taken, and vaccination rates are increasing, the presence and transmission of the virus in environments where people gather is still an issue, and the risk of viral infection remains. Escherichia coli isolates in water samples [The identification of occupational settings at higher risk of exposure to multi-drug resistant microbiota will enable the adoption of adequate exposure prevention measures . Regardi samples  and othe samples  collecte samples . HoweverAspergillus fumigatus originating from the environment have been reported [The emergence of fungal resistance to medical antifungal agents (mostly azoles) and the increasing incidence of azole-resistant disease due to resistant reported ,13,14,15reported ,16,17. Areported , creatinAspergillus sections and SARS-CoV-2, as well as mycotoxin analysis, screening of azole resistance and cytotoxicity measurement were also conducted to better estimate the health risks of exposure and to identify possible relations between the risk factors.Since the lack of data regarding cemeteries’ environment limits the implementation of suitable preventive measures for cemetery workers, in this study, we characterize the microbial contamination in three cemeteries, and more specifically in grave diggers’ facilities, in the Lisbon urban area, by air and passive sampling. The molecular detection of −2 in EDC, from 5.0 × 103 to 1.0 × 104 CFU·m−2 in filters, from 0 to 2.0 × 104 CFU·m−2 in swabs, and in settled dust samples the count was 80 CFU·m−2. Gram-negative bacteria load (VRBA) varied from 0 CFU·m−2 in EDC samples, filter samples, and swab samples, to a count of 2.00 × 105 CFU·m−2 in settled dust samples. The greatest median value for bacterial contamination was found in surface swab samples (1.05 × 101 CFU·m−2) whereas Gram-negative counts (2.00 × 101 CFU·m−2) were on settled dust samples.Total bacteria contamination ranged from 0 to 849 CFU·m5 CFU·m−2 on MEA and 1.15 × 105 CFU·m−2 on DG18 in filter samples; 4.40 × 105 CFU·m−2 on MEA and 1.30 × 105 CFU·m−2 on DG18 in floor surface swabs and 1.90 × 104 CFU·m−2 in MEA and 2.70 × 105 CFU·m−2 in DG18 in EDC samples. Settled dust had the highest fungal contamination, ranging from 1.15 × 101 CFU·m−2 on MEA and 1.00 × 101 CFU·m−2 on DG18 , followed by EDC samples , settled dust samples (45.00% DG18) and surface swab samples (18.18% MEA). Penicillium was the prevalent genera in swab samples , followed by EDC samples  settled dust  and filter samples  (Trichoderma sp. was the most common in settled dust samples (34.78% MEA) , followed by EDC samples  and surface swab samples . The lowest fungal diversity was observed in settled dust samples . 5% DG18) . Trichod78% MEA) .Aspergillus sp., the highest value of the genera was obtained in DG18 (18.38%) comparatively with MEA (4.11%). In DG18, the most contaminated matrixes with Aspergillus sp. were filter samples (27.71%). The genus was also present in surface swab samples (23.08%), EDC (11.25%), and settled dust (25.00%). On MEA, the matrices with the highest values of the genera were surface swab samples (4.55%), followed by EDC (2.79%) and filters (2.50%). The genus was not identified in settled dust samples , Nidulantes (7.9%), and Aspergilli (0.2%), while on MEA 3 sections were reported, as follows: Fumigati (3.7%). Nidulantes (0.4%) and Nigri (0.1%). As for sections identification on EDC, 2 Aspergillus sections were detected on MEA (0.6% Fumigati and 2.2% Nigri) and 2 sections on DG18 (7.6% Circumdati and 3.7% Nidulantes). On filters, 2 Aspergillus sections were identified on MEA (0.5% Fumigati and 2% Nidulantes), while on DG18, 3 sections were identified . In swabs samples, 2 sections were identified, namely section Fumigati on MEA (4.6%) and section Nidulantes on DG18 (23.1%). In settled dust the section Circumdati was dominant (25%). A greater number of distinct sections was observed in samples from settled dust filters in both MEA (0.5% Fumigati and 2% Nidulantes) and DG18  (On DG18 3 ergilli) .Cladosporium in VCZ (1.4 × 105 CFU·m−2·day−1 in EDC), SDA (1.1 × 105 CFU·m−2 in swabs), and ITZ (7.0 × 104 CFU·m−2·day−1 in EDC), followed by C. sitophila in SDA (1.0 × 105 CFU·m−2 in swabs) and ITZ (6.0 × 104 CFU·m−2·day−1 in EDC). Regarding the Aspergillus genus, it was only observed in SDA media, with the observed sections being Nidulantes (1.1 × 102 CFU·m−2·day−1 in EDC), and Nigri   .Aspergillus sections investigated. Considering all the environmental samples collected, SARS-CoV-2 was not detected as well as the four From the total of 64 samples analyzed only one settled dust sample showed contamination by a single mycotoxin. The mycophenolic acid was the mycotoxin detected with a valued below the limit of quantification (20 μg/kg).2/mL; N = 18) and EDC  were assessed by the MTT test for cellular metabolic activity. The obtained results are depicted in Sample dilutions from filters , ITZ  and VCZ  media .From the comparison of the fungal contamination between sampling methods, statistically significant differences were detected on MEA, and it was verified that the filters and swabs were the ones with the highest counts .Regarding the diversity of species found considering MEA, it was found that the settled dust  was the sampling method in which greater diversity was detected, as it was the one with the highest values of Shannon and Simpson indices. Of note, when the species richness (number of species) exceeds 10, Simpson’s index values are mainly influenced by the uniformity between the detected amounts of each species .Aspergillus sections were more frequently observed in DG18, since this medium restricts the growth of other fungi with higher growth rates, such as Mucorales order [Fumigati section was only observed in MEA corroborating also the results found in previous developed studies [To our knowledge this is the first attempt to assess cemeteries’ occupational environment focusing on microbial contamination and mycotoxins exposure. In addition, it was the first time that a comprehensive sampling campaign, as well as the assays employed, were used in this specific setting. The sampling approach was similar to our previous studies in different occupational environments in Portugal. The use of passive sampling methods to assess exposure, in what concerns the microbial contamination, allows to overcome the expected fluctuation due to a wide range of factors such as humidity levels, ventilation, human occupancy and their activities, environmental characteristics, water infiltrations, and outdoor air ,20. Addies order ,22. Fumi studies ,24.Aspergillus sections distribution observed, was different in the different sampling methods as expected, since besides the media and the indoor environment assessed, also the sampling method influence the sections distribution [Aspergillus sections reported  have toxigenic potential [Fumigati, Nigri, and Aspergilli) [Aspergillus sections are considered indicators of harmful fungal contamination [Fungal contamination, and more specifically ribution . The Aspotential , and somergilli) ,27. Thesmination , and a rAspergillus sp. in either tested azole concentration . Interestingly, the presence of fungi in azole media was only confirmed by EDC sampling. The species recovered (Cladosporium sp. and C. sitophila) have no clinical relevance, and no conclusions on their antifungal resistance profile can be drawn, as the used azole breakpoint values are not defined for the species.The screening of azole resistance revealed the absence of 4 CFU·m−2·day−1) and swabs (2.90 × 105 CFU·m−2·day−1), compared to settled dust (5.40 × 101 CFU·g−1·day−1).The correlations between sampling devices  in different media revealed that fungal counts recovered from EDC and swabs were better correlated regarding DG18 and SDA, whereas from settled dust, MEA was best correlated with SDA. This might be related with fungal total contamination, which was higher in the azole screening for EDC  and the raw materials being used and handled [As reported only one settled dust sample showed contamination by mycophenolic acid, produced mainly by  handled ,31. Howe handled .4 CFU·m−2) compared to EDC (8.49 × 102 CFU·m−2). Gram-negative bacteria contamination and fungal contamination (MEA and DG18) were similar among filters and EDC, thus, probably not responsible for the observed differences in cytotoxicity. Fungal diversity, however, might explain differences in cytotoxicity. For example, Aspergillus sp. is highly cytotoxic [Aspergillus sections [Aspergillus sp. distribution in filters (compared to EDC) consisted of sections Nidulantes , Circumdati  and Aspergilli . Of these, Nidulantes is among the most pathogenic to humans Aspergillus species, being mycotoxigenic  [Aspergillus species, aflatoxins and ochratoxins, can act towards target cells, cellular structures, and their internal processes [In this study, lung epithelial cells were used as a model for exposure by inhalation, and HepG2 cells as a model for hepatotoxicity . Cells wytotoxic , with disections ,36,37. Ilotoxin) . Most furocesses .2/mL in A549 cells, and from 0.125 to 1 cm2/mL in HepG2 cells. Previous studies reported a cytotoxicity of Aspergillus metabolites in A549 cells as ranging from 44 to 61 µM [The IC50 levels were 0.016 g/mL in one EDC only, both in A549 cells and HepG2 cells. Regarding filters, IC50 levels ranged from 0.25 to 1 cmto 61 µM . Moreoveto 61 µM .Overall, this study provided a useful contribute to unveil the microbial contamination in cemeteries and estimate workers exposure to the microbial contamination characterized, since the sampling locations were defined based on the tasks developed by the workers and places where they spent more time. This is a very specific context with no available data published on microbiological contamination and exposure of the workers involved in the activities developed. Additionally, the data produce allowed to characterize exposure and identify some measures to prevent exposure. It was also possible to reinforce the positive features of the sampling approaches followed (combining active with the passive sampling methods), as well as the assays applied (use of different culture media). Future studies are needed in this occupational setting regarding mycotoxins and cytotoxicity measurement to identify potential fungal producers and or triggers for mycotoxin production allowing to implement more focused risk management measures.This study sampling campaign was conducted between May and June 2021 in three cemeteries located in Lisbon . There wA multi-sampling approach protocol was performed by using active and passive sampling methods and through normal working days . An impiSettled dust was collected through a vacuum cleaner  with 1 × 4 collector filter attached  and a composite sample of the settled dust filters was obtained by vacuuming all the identified sampling sites ,42. Floo2) of each filter used from vacuuming. A composite sample with the settled dust was washed in a ratio of 1 g per 9.1 mL of 0.1% Tween 80 saline (0.9% NaCl) for 30 min at 250 rpm [Swabs obtained from swabbing the floor were washed with 1 mL of 0.1% Tween 80 saline (0.9% NaCl) for 30 min on the orbital shaker . The same procedure was applied on a piece  as previously published [After incubation at 27 °C for 5–7 days for fungi (MEA and DG18) and at 30 °C (TSA) and 35 °C (VRBA) for 7 days for mesophilic bacteria and coliforms (Gram negative bacteria), respectively, microbial quantification was achieved  was used, and a pan-azole-resistant A. fumigatus  was used as positive control. After three of incubation, fungal colonies were counted and prepared for microscopically identification [The extracts from the passive sampling methods from cemeteries were screened for azole resistance using an adaptation of the EUCAST method and breakpoints for umigatus ,46. For fication . Concerning SARS-CoV-2, composite surface samples were obtained by swabbing defined areas from each sampling site in each cemetery , Male and female locker room, drivers’ and gravediggers’ locker room) , using sAir samples of 600 L were obtained in all sampling locations employing the impinger Coriolis μ air sampler  with a flow rate of 300 L/min collected into a conical vial containing 5 mL Buffer NVL  component). https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html, accessed on 23 September 2020), (Samples were kept refrigerated (until 4 °C) for a maximum of 24 h before RNA was extracted from the obtained sample (1.5 mL in surface samples and 5 mL in air samples) with the NZY Viral RNA Isolation kit, from Nzytech, following to manufacturer’s instructions. One step-RT qPCR was performed using NZYSpeedy One-step RT-qPCR probe Master Mix and specific procedures were followed  with prir 2020), . qPCR waAspergillus sections  following the steps as previous published [Samples extracts (8.8 mL) from passive sampling (excluding surface swabs) were used for molecular detection of ublished   Trypsin 0.53 mM EDTA was used for cell detachment. After cell counting , 100 µL cell suspension (densities of 2.5 × 105 cells/mL) was transferred to a 96-well plate and exposed to diluted samples for 48 h at 5% CO2, 37 °C, and humid atmosphere. The cytotoxicity was measured , and the lowest sample concentration dropping absorption to <50% of cell metabolic activity (IC50) was considered the threshold toxicity level.The cytotoxic effect of filter and EDC samples was evaluated on samples’ extracts using the 3--2,5-diphenyltetrazolium bromide (MTT) assay at 510 nm, as previously described . The celip is the proportion (ni/n) of individuals of one particular species found (ni) divided by the total number of individuals found (n).Data obtained were analyzed using SPSS statistical software, version 26.0 for Windows. The results were considered significant at the 5% significance level. To test the normality of the data, the Shapiro–Wilk test was used. To study the relationship between fungal counts (MEA and DG18) and fungal counts on azole resistance screening  in each sampling method, Spearman correlation coefficient was applied, since the assumption of normality was not observed. To compare the fungal contamination and azole resistance screening between the sampling methods, the Kruskal–Wallis test was applied, since the normality assumption was not observed. To assess species diversity, Simpson and Shannon indices, given by


Vancomycin is a glycopeptide antibiotic used for prophylaxis and treatment of infections caused by methicillin-resistant Staphylococcus aureus. Although major organ sizes and functions mature during infancy, pharmacokinetic studies, especially those focused on infants, are limited. Changes in extracorporeal membrane oxygenation-related drug disposition largely contribute to changes in pharmacokinetics. Here, pharmacokinetic profiles of vancomycin in an infant receiving extracorporeal membrane oxygenation therapy are presented. A two-month-old Japanese infant with moderately decreased renal function was started on 12.0 mg/kg vancomycin every 8 h from day X for prophylaxis of pneumonia during extracorporeal membrane oxygenation therapy. As the trough concentration of vancomycin observed on day X+3 was 27.1 μg/mL, vancomycin was then discontinued. The trough concentration decreased to 18.6 μg/mL 24 h after discontinuation, and 9.0 mg/kg vancomycin every 12 h was restarted from day X+5. On day X+6, the trough concentration increased to 36.1 μg/mL, and vancomycin therapy was again discontinued. On day X+7, the trough concentration decreased to 22.4 μg/mL. The pharmacokinetic profiles of vancomycin based on first-order conditional estimation in this infant were as follows: plasma clearance = 0.053 L/kg/hour, distribution volume = 2.19 L/kg, and half-life = 29.5 h. This research reported the prolonged half-life of vancomycin during extracorporeal membrane oxygenation in infants with moderately decreased renal function. Staphylococcus aureus [Vd) is generally affected by body weight. The Food and Drug Administration has proposed classifying the pediatric population as neonates (birth to 1 month of life), infants (1–24 months), children (2–11 years), and adolescents (12–18 years) based on the complex changes and the anatomical, biochemical, and physiological differences related to age [Vd of VCM at each growth process in the pediatric population [Vancomycin (VCM) is a glycopeptide antibiotic used in the treatment of resistant Gram-positive infections, especially those caused by methicillin-resistant s aureus . The tots aureus . Moreoved to age . Pediatrfants 1–2 months, Extracorporeal membrane oxygenation (ECMO) is a cardiopulmonary support system used in patients with potentially reversible respiratory or cardiac failure and works by increasing the circulating blood volume and transiently altering renal function . ECMO inTo date, several studies have investigated the effects of ECMO on the PK of VCM in the pediatric population ,9. Howev3/μL, normal range: 3.3–8.6 × 103/μL; CRP: 6.2 mg/dL, normal range: <0.1 mg/dL). For a prophylaxis of pneumonia, VCM  was administered as an intravenous (IV) infusion over 60 min immediately after collecting one set of suction phlegm cultures. The initial VCM dose was 55 mg (12.5 mg/kg) administered every 8 h . The serum trough concentrations were measured using the PETINIA method . A trough sample was obtained 30 min before VCM administration. Since serum trough concentrations of VCM were 27.1 μg/mL at 13:00 on day X+3, VCM administration at 21:00 on day X+3, 5:00 and 13:00 on day X+4 was discontinued. At 24 h after the first trough measurement, the serum trough concentration of VCM decreased to 18.6 μg/mL at 13:00 on day X+4, and the VCM dose was restarted from day X+5 at 40 mg (9.0 mg/kg) every 12 h. After conducting a clamp test for ECMO weaning on days X+5 and X+6, an elevated WBC count (16.9 × 103/µL) and an increased CRP level (22.7 mg/dL) were noted. Despite reducing the VCM dose from day X+4, the serum concentration of VCM increased to 36.1 μg/mL on day X+6, and VCM was again discontinued. The next day (day X+7), the serum trough concentration decreased to 22.4 μg/mL. An increase in serum creatinine (SCr) and blood urea nitrogen (BUN), and a decrease in renal function was observed from day X to day X+7 . GFR was calculated as follows: GFR (mL/min/1.73 m2) = 0.45 × height (cm)/SCr (mg/dL) [A two-month (65 days)-old Japanese male infant  had a history of circulatory insufficiency of ventricular septal defect and interruption of the aortic arch. After Norwood and right ventricle-to-pulmonary artery conduit surgery (day X−4), lactic acidosis  was observed. In addition, a decline in blood pressure 101/30 mmHg) and saturation of percutaneous oxygen level (40–50%) were observed. Since the patient was in a complicated state with poor ventilation and severe neonatal circulatory failure, intensive care including Veno-Arterial (VA) ECMO therapy  were started on day X−4. Since sepsis, disseminated intravascular coagulation, and convulsions were observed, administration of 60 mg meropenem  every 8 h (h) were started on day X−4 as empiric therapy. On day X, increased white blood cell (WBC) count and C-reactive protein (CRP) levels were observed (WBC: 9.5 × 101/30 mmHgas 55 mg .5 mg/kg t1/2 and k based on observed serum VCM concentrations of 27.1 μg/mL (C0) and 18.6 μg/mL (C) were calculated using the equation of C = C0 × ekt−. The t1/2 and k were 29.5 h and 0.024 h−1, respectively. The Vd was calculated to be 2.19 L/kg using the equation of S is the salt index (1.0), F is bioavailability of VCM (1.0), and τ is the duration of VCM administration (8 h). The CL was calculated to be 0.053 L/kg/h using the equation of CL = k × Vd. C is the subsequent VCM concentration, C0 is the initial VCM concentration, e is the base of the natural logarithm, k is the elimination rate constant, t is the time interval between the initial and subsequent VCM concentrations.The PK parameters based on observed serum VCM concentrations on day X+3 (27.1 μg/mL) and day X+4 (18.6 μg/mL) were calculated using first-order conditional estimation because VCM administrations were skipped during these periods. The −kτ×e−kτ , where SVd. These findings indicate the importance of extended dosing intervals and close monitoring of VCM concentrations in infants treated with ECMO. Regarding PK study of neonates receiving VCM during ECMO therapy, Amaker et al. reported the PK profiles of VCM in 12 neonates undergoing ECMO , and the results were compared with previously published data. They showed increased Vd and t1/2 and decreased plasma CL levels [Vd and lower plasma CL and a significantly prolonged t1/2 in neonates receiving ECMO therapy [To date, one case report and two PK studies investigating the optimal dosage of VCM in pediatric patients treated with ECMO have been published ,14,15; hL levels . In addi therapy . Althoug therapy , these tVd of VCM [Vd of VCM in neonates and infants who have a smaller circulating blood volume compared with older children and adults because of a larger priming/blood volume ratio [Vd. Exposure to the ECMO circuit results in an inflammatory response, which often results in increased Vd by capillary leak and edema. In addition, the renin-angiotensin system in the kidney can be upregulated, possibly related to non-pulsatile blood flow seen in VA EMCO. Upregulation of the renin-angiotensin system alters handling of fluids and can change the ratio of fluids in the body fluid compartments [Body composition and organ maturation in the pediatric population develop during the first two years of age ,18. Totad of VCM . The volme ratio . In thisme ratio . Combinime ratio ,22. Regaartments .t1/2 of VCM in neonates receiving ECMO therapy, indicating that a dose of 20 mg/kg once daily is recommended for VCM [Vd and decreased total CL [Vd with decreased total CL [Vd or total CL [Administering VCM 10–20 mg/kg every 8–48 h depending on postmenstrual age, weight, and SCr has commonly been recommended for infants to achieve an area under the blood concentration-time curve (AUC) of 400 mg × h/L ,25. Howe for VCM . Therefototal CL . Two othtotal CL ,28. Anottotal CL . AlthougNephrotoxicity is a major adverse event associated with VCM. The incidence of nephrotoxicity further increases in pediatric patients who achieve a VCM trough concentration of >15 mg/L  and are This manuscript is a case report on the PK profile of VCM in infants and provides manuscript limitations to be addressed in the future. First, this is a case report of only one infant patient with very few available concentrations. Moreover, there is no PK model of VCM for only infant patients. Therefore, further study is needed to rigorously estimate the PK parameters of infant patients with ECMO therapy. Second, the AUC as well as trough concentrations are considered for any dosage recommendation to minimize treatment failure in the neonate population . Howevert1/2, and further studies to determine the adequate dosage of VCM in infants receiving ECMO therapy are needed. Moreover, early and frequent monitoring of VCM concentrations is important in infants receiving both nephrotoxic agents and ECMO therapy. This report is expected to facilitate the further development of VCM therapy for infants receiving ECMO therapy.In conclusion, this manuscript reported the PK profile of VCM during ECMO in an infant with moderately impaired renal function. Our case report indicates that a once-daily dose of VCM might be appropriate due to prolonged


Background: Anthracyclines such as doxorubicin remain a primary treatment for hematological malignancies and breast cancers. However, cardiotoxicity induced by anthracyclines, possibly leading to heart failure, severely limits their application. The pathological mechanisms of anthracycline-induced cardiac injury are believed to involve iron-overload-mediated formation of reactive oxygen species (ROS), mitochondrial dysfunction, and inflammation. The dietary thione, ergothioneine (ET), is avidly absorbed and accumulated in tissues, including the heart. Amongst other cytoprotective properties, ET was shown to scavenge ROS, decrease proinflammatory mediators, and chelate metal cations, including Fe2+, preventing them from partaking in redox activities, and may protect against mitochondrial damage and dysfunction. Plasma ET levels are also strongly correlated to a decreased risk of cardiovascular events in humans, suggesting a cardioprotective role. This evidence highlights ET’s potential to counteract anthracycline cardiotoxicity. Methods and Findings: We investigated whether ET supplementation can protect against cardiac dysfunction in mice models of doxorubicin-induced cardiotoxicity and revealed that it had significant protective effects. Moreover, ET administration in a mouse breast cancer model did not exacerbate the growth of the tumor or interfere with the chemotherapeutic efficacy of doxorubicin. Conclusion: These results suggest that ET could be a viable co-therapy to alleviate the cardiotoxic effects of anthracyclines in the treatment of cancers. Despite their use as a standard for effective curative chemotherapy for decades, their clinical application can be severely limited due to dose-specific cardiotoxic side effects, which range from asymptomatic echocardiographic changes to severe cardiomyopathy and heart failure . These c failure .While the exact pathological mechanisms of anthracycline cardiotoxicity are not fully understood, it is known to involve multiple pathways, including excessive oxidative stress , activatThe maintenance of redox homeostasis is critical to the preservation of normal cardiomyocyte function , and the2+), preventing their redox activities [The effectiveness and widespread application of anthracyclines in cancer therapy mean their removal is not a viable solution; hence, the absence of an effective prophylactic cardioprotectant against anthracyclines highlights an important therapeutic gap. Prior studies have shown that a low-molecular-weight dietary thione, ergothioneine (ET), may act as a physiological cytoprotectant , hercynine, and hercynine-d9, were provided by Tetrahedron . Doxorubicin and hydrochloride salt (>99%) was purchased from LC Laboratories . All other chemicals were purchased from Sigma-Aldrich , unless otherwise stated.L-ergothioneine (>98%), L-ergothioneine-dMice  were randomly assigned to cages in groups of 4–5 animals and provided with water and standard mice chow  ad libitum. The animals were kept in a barrier animal facility with a 12 h/12 h light–dark cycle. Animals were cared for in accordance with the protocols and guidelines stipulated in the Institutional Animal Care and Use Committee (IACUC), National University of Singapore. All the procedures were performed with prior approval by the IACUC, NUS, protocol No. R17-1242.w/v NaCl) intraperitoneally once weekly for 3 weeks (cumulative dose of 18 mg/kg). Animals were monitored during the administration and for a further 5 weeks with transthoracic echocardiographic measurements  taken at baseline and weeks 1, 2, 3, 5, and 8. For ET-treated animals, 70 mg/kg ET  were administered with 6 mg/kg doxorubicin  into the female mouse mammary fat pad. All mice were given the breast tumor injections and randomly assigned to groups (n = 6/group), before being given either saline (vehicle control) alone, doxorubicin alone, or doxorubicin with ET administration  suspension  and analyzed with Vevo® 2100 software, version 1.7.0, as established previously [v/v isoflurane, Baxter, Singapore), B-mode recordings were obtained from a long-axis view of the LV to analyze cardiac left ventricular volumes and left ventricular ejection fraction (LVEF). To assess global cardiac dysfunction (both systolic and diastolic) in doxorubicin-induced cardiomyopathy, myocardial performance index (MPI), a parameter defined as the sum of the isovolumic contraction time and relaxation time divided by the ejection time, was determined by Doppler echocardiography of left ventricular inflow and outflow [Animal cardiac function was assessed using a high-frequency ultrasound system Vevoeviously ,50. In b outflow ,52. EchoRNA was isolated from heart samples using the NucleoSpin TriPrep kit with on-column DNase digestion . Extracted RNAs were quantified and checked by 260/280 nm measurement using the Synergy H1 spectrophotometer with Take 3 microvolume plate . Reverse-transcription PCR (RT-PCR) was performed using QuantiTect RT kit , and quantitative PCR (qPCR) was performed using BlitzAmp Hotstart qPCR mix  with an Applied Biosystem 7500 Real-Time PCR system . Primers sequences are listed in 9 and hercynine-d9 internal standards and ultrapure water  to 100 µL final volume. For plasma ET, samples were precipitated with 50 µL of methanol at −20 °C overnight. For heart, tissue was homogenized with a motorized pellet pestle in 150 µL of ultrapure water containing internal standards. After homogenization, 750 µL of ice-cold methanol was added and samples were vortexed for 30 s before incubating at −20 °C overnight. Precipitated plasma and heart tissue samples were then centrifuged  and supernatants were evaporated under a stream of N2 gas before reconstituting in 100 µL ultrapure water. Any debris was removed by centrifugation before transferring into silanized glass inserts with vials  for analysis by liquid chromatography–mass spectrometry (LC-MS/MS). ET or hercynine levels were normalized against the accurate mass of tissue and expressed in mass per wet weight of tissue ± SEM.We mixed 10 µL of plasma or ~10 mg of heart tissue (accurately weighed) with ET-d2) in a volume of 100 µL for 1 h. Isolated DNA was hydrolyzed at 37 °C with 1 U of benzonase , 3 mU phosphodiesterase, and 4 U alkaline phosphatase in 200µL of buffer for 6 h. Internal standards were added before hydrolysis. The reaction was quenched with 5 volumes of ice-cold methanol, vortexed, and stored at −20 °C overnight. All samples were centrifuged  and supernatants were evaporated under a stream of N2 gas before reconstituting in 60 µL of ultrapure water. Any precipitates were removed by centrifugation before transferring into silanized glass inserts with vials for LC-MS/MS analysis.Levels of 8-hydroxyguanosine (8OHG) and 8-hydroxydeoxyguanosine (8OHdG) were measured in the heart tissue as biomarkers of oxidative damage to RNA and DNA, respectively. Approximately 20 mg of heart tissue was used to extract RNA and DNA using TRIzol reagent  as per the manufacturer’s protocol. All procedures were performed on ice to reduce artefactual oxidation. Isolated RNA was dissolved in RNase-free water, while DNA was dissolved in 160 µL of 10 mM Tris pH 8.0 buffer at 4 °C overnight. We enzymatically hydrolyzed 50 µg of RNA at 37 °C using 20 µg RNase A, 1 mU phosphodiesterase , and 2 U alkaline phosphatase  in buffer  and incubated with 100 µL 1:200 primary antibody (Oxyblot kit) for 2 h with shaking. Plate was then washed and incubated with 100 µL 1:400 secondary antibody (Oxyblot kit) for 1 h. Plate was washed and 100 µL of TMB  was added. The reaction was stopped with 100 µL of 2 M H2SO4 and absorbance was determined at 450 nm using the Synergy H1 spectrophotometer . Carbonylation levels were quantified against a standard curve.Diluted plasma (25x in PBS) and TRIzol-extracted heart proteins were derivatized with 4 mM DNPH (in 2 M HCl) as per manufacturer’s instructions using Oxyblot Protein Oxidation Detection Kit . We left 2 µg of proteins in 200 µL 100 mM pH 9.6 bicarbonate buffer to bind to the ELISA plate overnight at 4 °C. The plate was washed 5 times with PBS and blocked with 100 µL Pierce Protein-Free T20 buffer for 1 h. Plate was washed with PBS-T . Samples were kept at 10 °C in the autosampler during analysis. For ET and hercynine analysis, 2 µL of the processed samples were injected into a Cogent Diamond-Hydride column  maintained at 40 °C. Solvent A was acetonitrile in 0.1% v/v formic acid in ultrapure water. Chromatography was carried out at a flow rate of 0.5 mL/min using the following gradient elution: 1.5 min of 98% solvent B, followed by a 4 min gradual decrease in solvent B to 95%. The column was washed with 5% solvent B for 2.5 min and re-equilibrated with 98% solvent B for 4 min. The total run time was 12 min. The retention times for guanosine, 8OHG, dG, and 8OHdG were 3.7, 4.3, 4.8, and 6.1 min, respectively.For the 8OHG and 8OHdG analysis, 10 µL of the processed samples were injected into an Accucore PFP column  maintained at 30 °C. Solvent A was acetonitrile; Solvent B was 0.1% For all targets, MS was carried out under positive ion ESI and a multiple reaction monitoring mode. Capillary voltage was set at 3200 V, and gas temperature was kept at 350 °C. Nitrogen sheath gas pressure for nebulizing sample was at 50 psi, and gas flow was set at 12 L/min. Ultra-high purity nitrogen was used as collision gas. The optimized precursor to product ion transitions and their respective fragmentor voltages and collision energies are listed in p < 0.05 considered statistically significant.Data were tabulated using Microsoft Excel . Statistical analyses  with Tukey’s multiple comparison test or 2-way ANOVA, with multiple column comparison) were performed, and graphs were generated using GraphPad Prism version 9.2.0 . Unless otherwise stated, all data are expressed as mean ± standard error, with Administration of 6 mg/kg doxorubicin weekly for 3 weeks  in C57BL-6J mice led to significant decreases in animal weights  comparedLeft ventricular systolic function, as assessed by an echocardiographic assessment of the change in LVEF, is a primary measure of cardiac function ,49. As eA trend towards an increase in the mRNA expression of OCTN1 was observed in the heart following administration of doxorubicin compared with saline controls; however, this was not significant A. No chaIt should be noted that as with prior studies , detectThe levels of ET in the plasma also followed a similar trend to the heart tissues, with a significant drop in ET levels following doxorubicin administration A. HoweveThe levels of 8OHdG, 8OHG, and protein carbonyls were measured in the heart tissues as established biomarkers of oxidative damage to DNA, RNA, and protein, respectively ,53. A slIn the BALB-C breast cancer model, the body weights of animals between the saline and ET control groups (without doxorubicin) were not different. However, doxorubicin administration caused a significant loss of body weight and premature death in these animals, regardless of ET supplementation A. As witFollowing euthanasia, the excised breast tumors were measured with a caliper to estimate the volume and were subsequently weighed  mRNA in human heart [We established that an optimized cumulative dose of 18 mg/kg doxorubicin over 3 weeks was able to induce cardiotoxicity and significantly decrease LVEF in the C57BL-6J mice A. Althouan heart , suggestan heart ,61, whican heart ,63. Convan heart ,48. Suppan heart . As seenDoxorubicin administration also significantly increased MPI scores, an assessment of global cardiac dysfunction that combines both systolic and diastolic performance  relativeA significant decrease in the basal heart levels of ET  was observed with doxorubicin administration alone, despite the observed trend toward an increase in OCTN1 expression A. This m2+ or the prevention of mitochondrial dysfunction in cardiomyocytes. Iron plays a key role in doxorubicin cardiotoxicity through the formation of doxorubicin–Fe complexes [2+and Cu2+ [A trend of increasing oxidative damage to DNA and RNA A,B was oomplexes  and ironomplexes ,79. Convomplexes . ET can and Cu2+ , formingand Cu2+ . Indeed,and Cu2+ . Similarand Cu2+  and cardand Cu2+ ,83. Howeand Cu2+ ,85, indiand Cu2+ ,86, therIn our breast cancer model, supplementation with ET did not have any significant effect on tumor size or weight, both alone or with doxorubicin, indicating that ET does not exacerbate tumor growth, nor does it interfere with the chemotherapeutic efficacy of doxorubicin, unlike other antioxidants ,20,21. TWhile further studies are needed to elucidate the mechanisms of protection by ET and to compare its protective actions with that of dexrazoxane ,23, thisAnthracyclines such as doxorubicin remain a primary chemotherapeutic against hematological malignancies and tumors, such as breast cancer and sarcomas, due to their effectiveness. However, counteracting their cardiotoxic effects remains a major challenge. We demonstrated that the unique cytoprotective thione, ET, which can accumulate in heart tissues, decreases the cardiotoxic effects of doxorubicin in our mouse model. Moreover, using a murine breast cancer model, we established that ET did not interfere with the chemotherapeutic efficacy of doxorubicin nor exacerbate the growth of the breast tumor. Although further work is still needed to elucidate the protective mechanisms against doxorubicin, this study highlights the potential for ET to be used as an agent for alleviating anthracycline cardiotoxicity in cancer patients, which certainly warrants further attention.


Hospital readmissions after creation of an ileostomy are common and come with a high clinical and financial burden. The aim of this review with pooled analysis was to determine the incidence of dehydration-related and all-cause readmissions after formation of an ileostomy, and the associated costs.A systematic literature search was conducted for studies reporting on dehydration-related and overall readmission rates after formation of a loop or end ileostomy between January 1990 and April 2021. Analyses were performed using R Statistical Software Version 3.6.1.n = 82,451 patients). The pooled incidence of readmissions due to dehydration was 6% (95% CI 0.04–0.09) within 30 days, with an all-cause readmission rate of 20% (CI 95% 0.18–0.23). Duration of readmissions for dehydration ranged from 2.5 to 9 days. Average costs of dehydration-related readmission were between $2750 and $5924 per patient. Other indications for readmission within 30 days were specified in 15 studies, with a pooled incidence of 5% (95% CI 0.02–0.14) for dehydration, 4% (95% CI 0.02–0.08) for stoma outlet problems, and 4% (95% CI 0.02–0.09) for infections.The search yielded 71 studies (One in five patients are readmitted with a stoma-related complication within 30 days of creation of an ileostomy. Dehydration is the leading cause for these readmissions, occurring in 6% of all patients within 30 days. This comes with high health care cost for a potentially avoidable cause. Better monitoring, patient awareness and preventive measures are required.The online version contains supplementary material available at 10.1007/s10151-022-02580-6. Hospital readmissions after creation of an ileostomy are common and impede patient convalescence . ReasonsDehydration is often cited as a leading cause for stoma-related readmissions, due to fluid and electrolyte losses . DehydraThe reported incidence of readmission particularly in relation to dehydration varies –8, probaThis review was conducted in line with the Cochrane Handbook for systematic reviews of In Reporting following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines . The stuStudies were considered for inclusion if they met the following criteria: (1) patients with a newly created loop or end ileostomy for any indication; (2) assessment of readmissions related to dehydration, or overall number of readmissions, or other reasons for readmission after creation of an ileostomy; (3) studies were cohort, case-matched studies, or randomized clinical trials. The exclusion criteria were: (1) reviews, letters, expert opinions, commentaries, case reports, or case series with less than 10 cases; (2) language other than English; (3) lack of the sufficient data or outcomes of interest; (4) visits just to the emergency department; (5) studies reporting only on complications of revised ileostomies (with exception of readmissions for a revision of a newly created ileostomy); (6) second stage ileostomies in a three-stage ileo-anal pouch procedure; (7) colostomies, jejunostomies, non-intestinal stomas, and ghost ileostomies; (8) duplicate studies.Two reviewers (IV and MS) independently reviewed titles and abstracts, followed by full-text revision. Disagreements were resolved by consensus discussion between the two reviewers (IV and MS).Data were extracted independently by two authors (IV and MS) and included the following variables: year of publication, country, study design, number of patients, characteristics of included patients, indication for the ileostomy, type of surgery, number of elective procedures, number of open procedures, type of stoma (loop/end), overall number of readmissions, number of readmissions related to dehydration, other reasons for readmissions, duration, and cost of readmissions related to dehydration.The indications for an ileostomy were recorded and were classified as colorectal disease if they included bowel cancer, inflammatory bowel disease, diverticulitis, or familiar adenomatous polyposis.Readmissions were defined as an unplanned return to the hospital with an overnight stay for any reason. This did not include elective or planned readmissions.The following were accepted as readmission related to dehydration: a clinician-reported diagnosis of dehydration, or high output stoma . It did not include anastomotic leaks, which were reported separately.Whilst the primary outcome was readmission within 30 days related to dehydration after creation of an ileostomy readmission for other timeframes was also summarised. Secondary outcomes included number of all-cause readmissions, other common indications for readmission, duration, and cost associated with readmission.All included studies were assessed for methodological quality and risk of bias. For cohort studies, the Newcastle Ottawa quality assessment scale was used to assess risk of bias . For ranI2 and τ2 statistics, and the data were considered significant if the p value (τ2) was < 0.1 with low, moderate, and high for I2 values of 25%, 50%, and 75%.Quantitative analysis was performed using RStudio  with a random-effects model. For the outcome measures, pooled weighted proportions with corresponding 95% CIs were calculated using inversed variance weighting. Heterogeneity was assessed using the In total, 3508 articles were screened on title and abstract, with 3143 articles not meeting our inclusion criteria. A further 294 studies were excluded after full-text review leaving 71 studies  for analysis, with 62 studies able to be included in a quantitative meta-analysis , Fig. I2 = 96%, τ2 = 0.16 p < 0.01), Fig. I2 = 97%, τ2 = 1.38 p < 0.01) of patients  [A total of 46 studies reported on readmission within 30 days of ileostomy creation , 60–66. gure S1) , 64, 66.I2 = 98%, τ2 = 3.76 p < 0.01). Other indications for admission included stoma outlet issues in 4%  and infection (excluding anastomotic leaks) in 4%  .Other indications for readmission within 30 days were reported in 15 studies Table  1, 2, 1, 64, 66 I2 = 39%, τ2 = 0.02 p = 0.14), with the pooled proportion of all-cause readmission being 27%  . Dehydration was the indication for readmission in 40% of all patients admitted during this timeframe , Figure S5.Readmission within 60 days of ileostomy creation was reported in 6 studies , 49, 67.I2 = 92%, τ2 = 0.83 p < 0.01) and stoma outlet issues in 3% , Figure S7.Of the five papers reporting on other indications for readmission, Figure S6 , 39, 67,I2 = 65%, τ2 = 0.46 p < 0.01), Figure S8 [I2 = 92%, τ2 = 1.25 p < 0.01), Figure S9 [I2 = 0%, τ2 = 0 p = 0.67), Figure S10 [Eight studies reported on readmission related to dehydration between the time frame of ileostomy creation and closure (range 2–9 months) , 68–70. igure S8 , 68–70. igure S9 , 57, 70.gure S10 , 57, 70.I2 = 53%, τ2 = 0.49 p = 0.12) were admitted for dehydration, 2%  for stoma outlet problems, and 1%  for infection .Of the 3 papers reporting specific indications for readmission during this time frame , 57, 70,Ten studies reported on duration of readmission, as summarised in Table Two studies reported readmission due to dehydration within 30 days of stoma creation, with a cost ranging between $2750 and $5924 per patient , 8. If tShaffer et al. reported a total cost of $4,520 per patient for readmission within 30 days for any indication. After implementation of an intervention programme to improve monitoring, these costs were reduced to $508 per patient [Tyler et al. reported a mean associated charge for readmission of $33,363  for readmissions within 30 days after a colorectal resection. In patients with an ileostomy, acute renal failure and fluid and electrolyte disorders were the second most common cause of readmission 17.4%) after surgical complications directly related to the procedure (19.3%) [% after sIn the present systematic review and meta-analysis, the readmission rate within 30 days after stoma creation is 20%, with dehydration as the leading cause, occurring in around 6% of patients , 58, 61.This high readmission rate following the creation of an ileostomy is consistent with previous published data. However, data examining the factors associated with readmission are still limited to small cohorts, single institutions, or are from reports often of poor quality , 2, 11. p < 0.0001) [Dehydration is most common in the early post-operative period, with the highest incidence of reduced kidney function within the first 3–6 months after surgery , 68, 69. 0.0001) . DehydraRisk factors for dehydration include: stoma output more than 1 L at discharge , the preReadmissions are costly and may be avoidable to some extent. This is particularly the case for dehydration, since better monitoring and timely intervention might prevent extensive fluid loss. Improved inpatient coaching and outpatient follow-up care have been shown to reduce readmission , 30, 64.There are some limitations to this review. In most studies, readmission rates were not the primary outcome of the study. This might have led to under-reporting. There was significant heterogeneity between the different studies, making the results prone to information bias. This heterogeneity can partly be attributed to the variety of ileostomy indications in different patient populations, and the time span of 30 years in this systematic review which might include changes in indication and management of an ileostomy. In addition, the definition of dehydration and the method of diagnosis varied; for example in some studies, coded diagnoses were used to identify patients with dehydration. In this review, the majority of the ileostomies were created in an elective setting –48, 59. One out of five patients is readmitted after creation of an ileostomy. Dehydration is the leading cause for these readmissions, occurring in one-third of patients within 30 days. This comes with high health care costs. Better monitoring, patient awareness, and preventive measures are required.Supplementary file1 (DOCX 13212 KB) Supplementary Figure 1: Proportion of readmission for dehydration of overall readmissions within 30 days. Supplementary Figure 2: Most common causes of readmission within 30 days A. dehydration B Stoma outlet problems C. Infection. Supplementary Figure 3: Readmissions for dehydration within 60 days. Supplementary Figure 4: Overall readmissions within 60 days. Supplementary Figure 5: Proportion of readmission related to dehydration of overall readmissions. Supplementary Figure 6: All causes readmission dehydration within 60 days. Supplementary Figure 7: All causes readmission dehydration within 60 days. Supplementary Figure 8: Readmissions related to dehydration between stoma creation and closure. Supplementary Figure 9: Overall readmissions between stoma creation and closure. Supplementary Figure 10: Proportion of readmission for dehydration of overall readmissions. Supplementary Figure 11: Most common causes of readmission between stoma creation and closure A. dehydration B. Stoma outlet problems C. Stoma infection.Below is the link to the electronic supplementary material.