Maternal Diet High in Linoleic Acid Alters Offspring Lipids and Hepatic Regulators of Lipid Metabolism in an Adolescent Rat Model.

Linoleic acid (LA), an n-6 polyunsaturated fatty acid (PUFA), is essential for fetal growth and development. A maternal high LA (HLA) diet alters cardiovascular development in adolescent rats and hepatic function in adult rats in a sex-specific manner. We investigated the effects of an HLA diet on adolescent offspring hepatic lipids and hepatic lipid metabolism gene expression, and the ability of the postnatal diet to alter these effects. Female Wistar Kyoto rats were fed low LA (LLA; 1.44% energy from LA) or high LA (HLA; 6.21% energy from LA) diets during pregnancy and gestation/lactation. Offspring, weaned at postnatal day (PN) 25, were fed LLA or HLA and euthanised at PN40 (n = 6-8). Maternal HLA increased circulating uric acid, decreased hepatic cholesterol and increased hepatic Pparg in males, whereas only hepatic Srebf1 and Hmgcr increased in females. Postnatal (post-weaning) HLA decreased liver weight (% body weight) and increased hepatic Hmgcr in males, and decreased hepatic triglycerides in females. Maternal and postnatal HLA had an interaction effect on Lpl, Cpt1a and Pparg in females. These findings suggest that an HLA diet both during and after pregnancy should be avoided to improve offspring disease risk.

Spiky Silver-Iron Oxide Nanohybrid for Effective Dual-Imaging and Synergistic Thermo-Chemotherapy.

Nanophotothermal therapy based on nanoparticles (NPs) that convert near-infrared (NIR) light to generate heat to selectively kill cancer cells has attracted immense interest due to its high efficacy and being free of ionizing radiation damage. Here, for the first time, we have designed a novel nanohybrid, silver-iron oxide NP (AgIONP), which was successfully tuned for strong absorbance at NIR wavelengths to be effective in photothermal treatment and dual-imaging strategy using MRI and photoacoustic imaging (PAI) in a cancer model in vivo and in vitro, respectively. We strategically combine the inherent anticancer activity of silver and photothermal therapy to render excellent therapeutic capability of AgIONPs. In vitro phantoms and in vivo imaging studies displayed preferential uptake of folate-targeted NPs in a cancer mice model, indicating the selective targeting efficiency of NPs. Importantly, a single intravenous injection of NPs in a cancer mice model resulted in significant tumor reduction, and photothermal laser resulted in a further substantial synergistic decrease in tumor size. Additionally, biosafety and biochemical assessment performed in mice displayed no significant difference between NP treatment and control groups. Overall, our folic acid AgIONPs displayed excellent potential in the simultaneous application for safe and successful targeted synergistic photothermal treatment and imaging of a cancer model.

A Bioactive Disintegrable Polymer Nanoparticle for Synergistic Vascular Anticalcification.

Although poly(aspartic acid) (PASP), a strong calcium chelating agent, may be potentially effective in inhibition of vascular calcification, its direct administration may lead to side effects. In this study, we employed polysuccinimide, a precursor of PASP, to prepare targeted polysuccinimide-based nanoparticles (PSI NPs) that not only acted as a prodrug but also functioned as a carrier of additional therapeutics to provide powerful synergistic vascular anticalcification effect. This paper shows that chemically modified PSI-NPs can serve as effective nanocarriers for loading of hydrophobic drugs, in addition to anticalcification and antireactive oxygen species (anti-ROS) activities. Curcumin (Cur), with high loading efficiency, was encapsulated into the NPs. The NPs were stable for 16 h in physiological conditions and then slowly dissolved/hydrolyzed to release the therapeutic PASP and the encapsulated drug. The drug release profile was found to be in good agreement with the NP dissolution profile such that complete release occurred after 48 h at physiological conditions. However, under acidic conditions, the NPs were stable, and Cur cumulative release reached only 30% after 1 week. Though highly effective in the prevention of calcium deposition, PSI NPs could not prevent the osteogenic trans-differentiation of vascular smooth muscle cells (VSMCs). The presence of Cur addressed this problem. It not only further reduced ROS level in macrophages but also prevented osteogenic differentiation of VSMCs in vitro. The NPs were examined in vivo in a rat model of vascular calcification induced by kidney failure through an adenine diet. The inclusion of Cur and PSI NPs combined the therapeutic effects of both. Cur-loaded NPs significantly reduced calcium deposition in the aorta without adversely affecting bone integrity or noticeable side effects/toxicity as examined by organ histological and serum biochemistry analyses.

Changes in Essential Fatty Acids and Ileal Genes Associated with Metabolizing Enzymes and Fatty Acid Transporters in Rodent Models of Cystic Fibrosis.

Cystic fibrosis (CF), the result of mutations in the CF transmembrane conductance regulator (CFTR), causes essential fatty acid deficiency. The aim of this study was to characterize fatty acid handling in two rodent models of CF; one strain which harbors the loss of phenylalanine at position 508 (Phe508del) in CFTR and the other lacks functional CFTR (510X). Fatty acid concentrations were determined using gas chromatography in serum from Phe508del and 510X rats. The relative expression of genes responsible for fatty acid transport and metabolism were quantified using real-time PCR. Ileal tissue morphology was assessed histologically. There was an age-dependent decrease in eicosapentaenoic acid and the linoleic acid:α-linolenic acid ratio, a genotype-dependent decrease in docosapentaenoic acid (n-3) and an increase in the arachidonic acid:docosahexaenoic acid ratio in Phe508del rat serum, which was not observed in 510X rats. In the ileum, Cftr mRNA was increased in Phe508del rats but decreased in 510X rats. Further, Elvol2, Slc27a1, Slc27a2 and Got2 mRNA were increased in Phe508del rats only. As assessed by Sirius Red staining, collagen was increased in Phe508del and 510X ileum. Thus, CF rat models exhibit alterations in the concentration of circulating fatty acids, which may be due to altered transport and metabolism, in addition to fibrosis and microscopic structural changes in the ileum.

Sexual dimorphism: increased sterol excretion leads to hypocholesterolaemia in female hyperbilirubinaemic Gunn rats.

Circulating bilirubin is associated with reduced serum cholesterol concentrations in humans and in hyperbilirubinaemic Gunn rats. However, mechanisms contributing to hypocholesterolaemia remain unknown. Therefore, this study aimed to investigate cholesterol synthesis, transport and excretion in mutant Gunn rats. Adult Gunn and control rats were assessed for daily faecal sterol excretion using metabolic cages, and water was supplemented with [1-13 C]-acetate to determine cholesterol synthesis. Bile was collected to measure biliary lipid secretion. Serum and liver were collected for biochemical analysis and for gene/protein expression using RT-qPCR and western blot, respectively. Additionally, serum was collected and analysed from juvenile rats. A significant interaction of sex, age and phenotype on circulating lipids was found with adult female Gunn rats reporting significantly lower cholesterol and phospholipids. Female Gunn rats also demonstrated elevated cholesterol synthesis, greater biliary lipid secretion and increased total faecal cholesterol and bile acid excretion. Furthermore, they possessed increased hepatic low-density lipoprotein (LDL) receptor and SREBP2 expression. In contrast, there were no changes to sterol metabolism in adult male Gunn rats. This is the first study to demonstrate elevated faecal sterol excretion in female hyperbilirubinaemic Gunn rats. Increased sterol excretion creates a negative intestinal sterol balance that is compensated for by increased cholesterol synthesis and LDL receptor expression. Therefore, reduced circulating cholesterol is potentially caused by increased hepatic uptake via the LDL receptor. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome). KEY POINTS: Female adult hyperbilirubinaemic (Gunn) rats demonstrated lower circulating cholesterol, corroborating human studies that report a negative association between bilirubin and cholesterol concentrations. Furthermore, female Gunn rats had elevated sterol excretion creating a negative intestinal sterol balance that was compensated for by elevated cholesterol synthesis and increased hepatic low-density lipoprotein (LDL) receptor expression. Therefore, elevated LDL receptor expression potentially leads to reduced circulating cholesterol levels in female Gunn rats providing an explanation for the hypocholesterolaemia observed in humans with elevated bilirubin levels. This study also reports a novel interaction of sex with the hyperbilirubinaemic phenotype on sterol metabolism because changes were only reported in females and not in male Gunn rats. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome).

Compression Socks Reduce Running-Induced Intestinal Damage.

ABSTRACT: Zadow, EK, Edwards, KH, Kitic, CM, Fell, JW, Adams, MJ, Singh, I, Kundur, A, Johnstone, ANB, Crilly, J, Bulmer, AC, Halson, SL, and, and Wu, SSX. Compression socks reduce running-induced intestinal damage. J Strength Cond Res 36(9): 2461-2464, 2022-Exercise is associated with a reduction in splanchnic blood flow that leads to the disruption of intestinal epithelium integrity, contributing to exercise-induced gastrointestinal syndrome. Strategies that promote intestinal blood flow during exercise may reduce intestinal damage, which may be advantageous for subsequent recovery and performance. This study aimed to explore if exercise-associated intestinal damage was influenced by wearing compression garments, which may improve central blood flow. Subjects were randomly allocated to wear compression socks ( n = 23) or no compression socks (control, n = 23) during a marathon race. Blood samples were collected 24 hours before and immediately after marathon and analyzed for intestinal fatty acid-binding protein (I-FABP) concentration as a marker of intestinal damage. The magnitude of increase in postmarathon plasma I-FABP concentration was significantly greater in control group (107%; 95% confidence interval [CI], 72-428%) when compared with runners wearing compression socks (38%; 95% CI, 20-120%; p = 0.046; d = 0.59). Wearing compression socks during a marathon run reduced exercise-associated intestinal damage. Compression socks may prove an effective strategy to minimize the intestinal damage component of exercise-induced gastrointestinal syndrome.

Diagnostic Sensitivity of Plasma Endothelin-1 for Cerebral Vasospasm After Subarachnoid Hemorrhage, a Multicentre Double-Blind Study.

The purpose of this study is to establish the diagnostic sensitivity of Endothelin-1 for risk stratification and screening of clinical vasospasm after subarachnoid hemorrhage.This is a multicentre, observational study, correlating daily blood Endothelin-1 with clinical variables. Binary logistic regression used to examine if Endothelin-1 levels could be used to predict clinical vasospasm. Bivariate modelling used to explore associations between patient characteristics and vasospasm. A Receiver Operating Curve used to explore cut-off values for Endothelin-1. Sensitivity and specificity was used to validate the cut-point found in the pilot study. A total of 96 patients were enrolled over two years. Median Endothelin-1 was higher for patients who experienced clinical vasospasm except for day-5, where median endothelin for patients without vasospasm was higher (3.6 IQR = 5.3), compared to patients with vasospasm (3.3 IQR = 8.5) although differences were not significant. The Receiver Operating Curve analysis confirmed that day-5 Endothelin-1 was not a good indicator of vasospasm, with an area under the curve of 0.506 (95% CI: 0.350-0.663, p = 0.938). The levels of Endothelin-1 in blood do not discriminate patients who may develop symptomatic vasospasm. The high variability in Endothelin-1 levels, aligns with the pathophysiological variability of most biomarkers, decreasing their ability to predict a clinical event.

Gilbert's Syndrome and the Gut Microbiota - Insights From the Case-Control BILIHEALTH Study.

The heme catabolite bilirubin has anti-inflammatory, anti-oxidative and anti-mutagenic effects and its relation to colorectal cancer (CRC) risk is currently under evaluation. Although the main metabolic steps of bilirubin metabolism, including the formation of stercobilin and urobilin, take place in the human gastrointestinal tract, potential interactions with the human gut microbiota are unexplored. This study investigated, whether gut microbiota composition is altered in Gilbert's Syndrome (GS), a mild form of chronically elevated serum unconjugated bilirubin (UCB) compared to matched controls. Potential differences in the incidence of CRC-associated bacterial species in GS were also assessed. To this end, a secondary investigation of the BILIHEALTH study was performed, assessing 45 adults with elevated UCB levels (GS) against 45 age- and sex-matched controls (C). Fecal microbiota analysis was performed using 16S rRNA gene sequencing. No association between mildly increased UCB and the composition of the gut microbiota in this healthy cohort was found. The alpha and beta diversity did not differ between C and GS and both groups showed a typical representation of the known dominant phyla. Furthermore, no difference in abundance of Firmicutes and Proteobacteria, which have been associated with the mucosa of CRC patients were observed between the groups. A sequence related to the Christensenella minuta strain YIT 12065 was identified with a weak association value of 0.521 as an indicator species in the GS group. This strain has been previously associated with a lower body mass index, which is typical for the GS phenotype. Overall, sex was the only driver for an identifiable difference in the study groups, as demonstrated by a greater bacterial diversity in women. After adjusting for confounding factors and multiple testing, we can conclude that the GS phenotype does not affect the composition of the human gut microbiota in this generally healthy study group.

Serum metabolomics analysis reveals increased lipid catabolism in mildly hyperbilirubinemic Gilbert's syndrome individuals.

BACKGROUND: The protective role of mildly elevated bilirubin against CVD and diabetes mellitus type 2 (DMT2) is associated with a favorable lipid phenotype. As the mechanistic understanding of this protection in humans remains elusive, we aimed to assess the metabolomics profile of mildly hyperbilirubinemic (Gilbert's syndrome; GS) individuals especially targeting lipid catabolism. METHODS AND RESULTS: Using NMR serum metabolomics of 56 GS individuals and 56 age and gender-matched healthy controls, GS individuals demonstrated significantly greater concentrations of acetylcarnitine (+20%, p < 0.001) and the ketone bodies, 3-hydroxybutyric acid (+132%, p < 0.001), acetoacetic acid (+95%, p < 0.001) and acetone (+46%, p < 0.001). Metabolites associated with an increased mitochondrial lipid metabolism such as citrate (+15%, p < 0.001), anaplerotic amino acid intermediates and creatinine were significantly greater and creatine significantly reduced in GS individuals. Stimulators of lipid catabolism including AMPK (+59%, p < 0.001), pPPARα (+24%, p < 0.001) and T3 (+9%, p = 0.009) supported the metabolomics data while concomitantly blood glucose and insulin (-33%, p = 0.002) levels were significantly reduced. We further showed that the increased lipid catabolism partially mediates the favorable lipid phenotype (lower triglycerides) of GS individuals. Increased trimethylamine (+35%, p < 0.001) indicated changes in trimethylamine metabolism, an emerging predictor of metabolic health. CONCLUSION: We showed an enhanced lipid catabolism in mildly hyperbilirubinemic individuals, novel evidence as to why these individuals are leaner and protected against chronic metabolic diseases emphasizing bilirubin to be a promising future target in obese and dyslipidemia patients.

Oxidative Stress and Related Biomarkers in Gilbert's Syndrome: A Secondary Analysis of Two Case-Control Studies.

Bilirubin is an important antioxidant and a modulator of biological functions. However, most of the protection against oxidative stress was shown in vitro or ex vivo. The aim of this case-control study was to investigate whether subjects with Gilbert's syndrome (GS) experience different levels of lipid and protein oxidation (as well as differences in oxidative stress related markers) compared to healthy controls. GS subjects (n =119) demonstrated higher serum levels of unconjugated bilirubin (p < 0.001), a lower BMI (p < 0.001), 37% higher antioxidant potential assessed as ferric reducing ability potential (p < 0.001), higher advanced oxidation protein products (p < 0.01) andlower apolipoprotein B (p < 0.05), hs-C-reactive protein (p < 0.05), interleukin 6 (p < 0.001) and interleukin 1 beta (p < 0.05) values compared to healthy controls (n =119). Furthermore, the resting heart rate was significantly lower in the GS group (p < 0.05). Stronger protective effects for GS subjects were demonstrated in the older subgroup (n =104, average age 50 years) compared to those of the younger group (n =134, average age 27 years). Although not all markers related to oxidative stress were different between the groups (e.g., malondialdehyde, homocysteine, oxLDL, and myeloperoxidase; p >0.05), the observed differences contribute to the explanation of why GS serves as an important protector in the pathogenesis of metabolic, oxidative stress related diseases.

Effect of Silymarin Treatment on Circulating Bilirubin and Cardiovascular Disease Risk Factors in Healthy Men: A Single-Blind, Randomized Crossover Trial.

This clinical trial (ACTRN12619001296123) investigated the impact of silymarin (Legalon®) on circulating bilirubin concentration, lipid status, systemic inflammation, and antioxidant status. The study design was a randomized, placebo-controlled, single-blind crossover trial of healthy men (18-65 years), conducted at Griffith University, Gold Coast, Australia. Participants were recruited from Griffith University and were randomized to silymarin (140 mg silymarin capsules thrice daily) or placebo (3 capsules containing mannitol taken daily) for 14 days followed by a ≥4-week washout and crossover to the other arm. The main outcomes were whether silymarin treatment would increase serum bilirubin concentration by >0.29 mg/dL, change serum lipid status (cholesterol and triglycerides), inflammation (c-reactive protein), and antioxidant capacity (ferric reducing ability of plasma) compared with baseline. Silymarin consumption (n = 17) did not affect serum concentrations of unconjugated bilirubin (0.73 versus 0.67 mg/dL, P = .79), cholesterol (185 versus 189 mg/dL, P = .19), triglycerides (94.2 versus 92.3 mg/dL, P = .79), c-reactive protein (0.17 versus 0.09 mg/dL, P = .23), or antioxidant status (6.61 versus 6.67 mg Fe2+ /dL, P = .40). These findings challenge previous reports and manufacturer claims of hyperbilirubinemia following silymarin treatment and are critical to guiding researchers toward an effective means to mildly elevate bilirubin, which evidence suggests could protect from cardiovascular disease.

Peripherally Inserted Central catheter iNnovation to reduce Infections and Clots (the PICNIC trial): a randomised controlled trial protocol.

INTRODUCTION: Peripherally inserted central catheters (PICCs) are vital for the delivery of medical therapies, but up to 30% of PICCs are associated with complications such as deep vein thrombosis or infection. The integration of antimicrobial and hydrophobic catheter materials, and pressure-activated valves, into polyurethane PICCs are innovations designed to prevent infective and/or thrombotic complications. METHODS AND ANALYSIS: A multicentre, parallel group, superiority randomised controlled trial with two experimental arms ((1) hydrophobic PICC (with pressure-activated valve); (2) chlorhexidine gluconate-impregnated PICC (with external clamp)) and one control group ((3) conventional polyurethane PICC (with external clamp)). Recruitment of 1098 adult and paediatric patients will take place over 2 years at three tertiary-referral hospitals in Queensland, Australia. Patients are eligible for inclusion if their PICC is to be inserted for medical treatment, with a vascular size sufficient to support a 4-Fr PICC or larger, and with informed consent. The primary outcome is PICC failure, a composite of thrombotic (venous thrombosis, breakage and occlusion) and infective complications (PICC-associated bloodstream infection and local infection). Secondary outcomes include: all-cause PICC complication; thrombotic complications; infective complications; adverse events (local or systemic reaction); PICC dwell time; patient/parent satisfaction; and healthcare costs. Differences between both intervention groups and the control group will be compared using Cox proportional hazards regression. Effect estimates will be presented as HRs with corresponding 95% CI. ETHICS AND DISSEMINATION: Ethical approval from Queensland Health (HREC/QCHQ/48682) and Griffith University (Ref. No. 2019/094). Results will be published. TRIAL REGISTRATION NUMBER: ACTRN12619000022167.

Mitochondrial Function, Fatty Acid Metabolism, and Body Composition in the Hyperbilirubinemic Gunn Rat.

Background: Circulating bilirubin is associated with reduced adiposity in human and animal studies. A possible explanation is provided by in vitro data that demonstrates that bilirubin inhibits mitochondrial function and decreases efficient energy production. However, it remains unclear whether hyperbilirubinemic animals have similar perturbed mitochondrial function and whether this is important for regulation of energy homeostasis. Aim: To investigate the impact of unconjugated hyperbilirubinemia on body composition, and mitochondrial function in hepatic tissue and skeletal muscle. Materials and Methods: 1) Food intake and bodyweight gain of 14-week old hyperbilirubinemic Gunn (n = 19) and normobilirubinemic littermate (control; n = 19) rats were measured over a 17-day period. 2) Body composition was determined using dual-energy X-ray absorptiometry and by measuring organ and skeletal muscle masses. 3) Mitochondrial function was assessed using high-resolution respirometry of homogenized liver and intact permeabilized extensor digitorum longus and soleus fibers. 4) Liver tissue was flash frozen for later gene (qPCR), protein (Western Blot and citrate synthase activity) and lipid analysis. Results: Female hyperbilirubinemic rats had significantly reduced fat mass (Gunn: 9.94 ± 5.35 vs. Control: 16.6 ± 6.90 g, p < 0.05) and hepatic triglyceride concentration (Gunn: 2.39 ± 0.92 vs. Control: 4.65 ± 1.67 mg g-1, p < 0.01) compared to normobilirubinemic controls. Furthermore, hyperbilirubinemic rats consumed fewer calories daily (p < 0.01) and were less energetically efficient (Gunn: 8.09 ± 5.75 vs. Control: 14.9 ± 5.10 g bodyweight kcal-1, p < 0.05). Hepatic mitochondria of hyperbilirubinemic rats demonstrated increased flux control ratio (FCR) via complex I and II (CI+II) (Gunn: 0.78 ± 0.16 vs. Control: 0.62 ± 0.09, p < 0.05). Similarly, exogenous addition of 31.3 or 62.5 µM unconjugated bilirubin to control liver homogenates significantly increased CI+II FCR (p < 0.05). Hepatic PGC-1α gene expression was significantly increased in hyperbilirubinemic females while FGF21 and ACOX1 was significantly greater in male hyperbilirubinemic rats (p < 0.05). Finally, hepatic mitochondrial complex IV subunit 1 protein expression was significantly increased in female hyperbilirubinemic rats (p < 0.01). Conclusions: This is the first study to comprehensively assess body composition, fat metabolism, and mitochondrial function in hyperbilirubinemic rats. Our findings show that hyperbilirubinemia is associated with reduced fat mass, and increased hepatic mitochondrial biogenesis, specifically in female animals, suggesting a dual role of elevated bilirubin and reduced UGT1A1 function on adiposity and body composition.

Effect of warming and infusion of red blood cell concentrates on markers of haemolysis: An ex vivo simulation study.

BACKGROUND: Transfusion of red blood cell (RBC) concentrates is a common procedure to restore blood volume and tissue oxygen delivery in patients with trauma. Although RBC warmers may prevent hypothermia, some warming or infusion equipment may lead to haemolysis and patient injury. OBJECTIVES: The aim of this study was to test the effect of (i) RBC warming and (ii) administration via manual vs. pump infusion on haemolysis. METHODS: This experimental ex vivo study studied haemolysis markers of RBC injury. The sample consisted of 90 RBC infusions in two simulations, randomly, 45 warmed RBC infusions and 45 nonwarmed RBC infusions, in two or three stages: before the intervention (baseline-warming, N= 45; nonwarming, N= 45), after water bath warming at 42 °C (warmed, N= 45), and then after the warmed or nonwarmed RBCs were infused by manual or pump infusion at a rate of 100 mL/h (infusion-warming, N= 45; nonwarming, N= 45). RESULTS: Warmed RBCs showed significantly lower total haemoglobin (Hb) and haematocrit levels and increase in free Hb levels, haemolysis levels, and lactate dehydrogenase (LDH) activity (all p<0.05) than baseline RBCs. Pump infusion RBCs were associated with reduced total Hb and increased free Hb, haemolysis, and potassium (K) levels (all p<0.05) compared with warmed RBCs. In contrast, manual infusion of warmed RBCs resulted in significantly reduced total Hb levels and increased LDH activity (both <0.05). After infusion, total Hb, free Hb, haematocrit, haemolysis, and LDH values were significantly different for warmed vs. nonwarmed RBCs (p<0.05). CONCLUSIONS: Haemolysis biomarkers increase with RBC warming and infusion, especially when using infusion pumps. Critically ill patients should be carefully monitored for possible complications during and after RBC infusion.

Pharmacokinetics of bilirubin-10-sulfonate and biliverdin in the rat.

BACKGROUND AND PURPOSE: Biliverdin (BV) administration induces antioxidant and anti-inflammatory effects, with previous reports also identifying anti-anaphylactic potential. Interestingly however, intra-duodenal administration of BV in rats leads to the formation of bilirubin-10-sulfonate (BRS), which might be responsible for BV's purported effects. EXPERIMENTAL APPROACH: This study aimed to assess the intravenous, intraperitoneal and intraduodenal pharmacokinetics of BRS and BV in order to assess their therapeutic potential in future studies. Bile and venous blood were intermittently collected before and after administration, which was subsequently analysed using liquid chromatography-mass spectrometry for quantification of bile pigment concentrations. KEY RESULTS: Interestingly, i.p. BRS administration led to a greater circulating concentration and had a reduced excretion rate, which resulted in a substantially elevated AUC180 when compared to BV administration. Furthermore, BRS was excreted intact in the bile, in contrast to BV which was excreted after chemical reduction and conjugation. Intraperitoneal and intraduodenal administration substantially increased blood BRS concentrations (p<0.05), however, the bioavailability of BV was higher than BRS following i.p. administration (i.p. BV 28.4%, BRS 15.5%) but lower following i.d. administration (i.d. BV 0.04%, BRS 0.07%), over 180 minutes. When BRS was administered i.v., BRS had a significantly (p<0.05) longer distribution (191.9 vs 54.1 minutes) half-life compared to BV, and significantly reduced (p<0.05) volume of distribution (0.026 vs 0.145 L kg-1). As a consequence, intraperitoneal and intraduodenal administration resulted in significantly greater blood concentrations of BRS (p<0.05) over 180 minutes. Therefore, BRS may be more likely to induce antioxidant or molecular effects, when compared to BV, due to greater concentrations and a longer half-life. CONCLUSIONS AND IMPLICATIONS: Cumulatively, these data demonstrate that BRS has a superior pharmacokinetic profile when compared to BV, which is a result of its resistance to hepatic metabolism and excretion. These data therefore provide a basis to explore the capacity of BRS to protect from inflammatory pathology.

Circulating bilirubin levels and risk of colorectal cancer: serological and Mendelian randomization analyses.

BACKGROUND: Bilirubin, a byproduct of hemoglobin breakdown and purported anti-oxidant, is thought to be cancer preventive. We conducted complementary serological and Mendelian randomization (MR) analyses to investigate whether alterations in circulating levels of bilirubin are associated with risk of colorectal cancer (CRC). We decided a priori to perform analyses separately in men and women based on suggestive evidence that associations may differ by sex. METHODS: In a case-control study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC), pre-diagnostic unconjugated bilirubin (UCB, the main component of total bilirubin) concentrations were measured by high-performance liquid chromatography in plasma samples of 1386 CRC cases and their individually matched controls. Additionally, 115 single-nucleotide polymorphisms (SNPs) robustly associated (P < 5 × 10-8) with circulating total bilirubin were instrumented in a 2-sample MR to test for a potential causal effect of bilirubin on CRC risk in 52,775 CRC cases and 45,940 matched controls in the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), the Colon Cancer Family Registry (CCFR), and the Colorectal Transdisciplinary (CORECT) study. RESULTS: The associations between circulating UCB levels and CRC risk differed by sex (Pheterogeneity = 0.008). Among men, higher levels of UCB were positively associated with CRC risk (odds ratio [OR] = 1.19, 95% confidence interval [CI] = 1.04-1.36; per 1-SD increment of log-UCB). In women, an inverse association was observed (OR = 0.86 (0.76-0.97)). In the MR analysis of the main UGT1A1 SNP (rs6431625), genetically predicted higher levels of total bilirubin were associated with a 7% increase in CRC risk in men (OR = 1.07 (1.02-1.12); P = 0.006; per 1-SD increment of total bilirubin), while there was no association in women (OR = 1.01 (0.96-1.06); P = 0.73). Raised bilirubin levels, predicted by instrumental variables excluding rs6431625, were suggestive of an inverse association with CRC in men, but not in women. These differences by sex did not reach formal statistical significance (Pheterogeneity ≥ 0.2). CONCLUSIONS: Additional insight into the relationship between circulating bilirubin and CRC is needed in order to conclude on a potential causal role of bilirubin in CRC development.

Biliverdin and bilirubin sulfonate inhibit monosodium urate induced sterile inflammation in the rat.

BACKGROUND: Biliverdin, a by-product of haem catabolism, possesses potent endogenous antioxidant and anti-inflammatory properties. Bilirubin-C10-sulfonate (BRS), an active metabolite formed after enteral administration of BV in the rat, also possess antioxidant properties. Therefore, we investigated the anti-inflammatory and antioxidant activity of BV and BRS in an in vivo model of monosodium urate induced sterile inflammation. METHODS: Subcutaneous air pouches were created on the dorsal flanks of Wistar rats (10-12 weeks of age). Prior to stimulation of the 6-day old pouch with monosodium urate (25 mg), groups were pre-treated with intraperitoneal BRS (27 mg/kg) and BV (27 mg/kg). Total and differential leukocyte counts were determined in pouch fluid aspirate at 1, 6, 12, 24 and 48 h after monosodium urate stimulation. Biliverdin (BV), BRS and unconjugated bilirubin (UCB) concentrations in the serum and pouch fluid were quantified using liquid chromatography-mass spectrometry. Pouch fluid cytokine concentrations (IL-1ß, IL-1α, TNF-α, IL-17A, IL-12, GM-CSF, IL-33, IFN-γ, IL-18, IL-10, MCP-1, CXCL-1 and IL-6) were assessed after 6 h. In addition, 24 h protein carbonyl and chloramine concentrations were assessed in pouch fluid using ELISA and spectrophotometry, respectively. RESULTS: BRS and BV significantly (p < 0.05) inhibited leukocyte (total, neutrophil and macrophage) infiltration into the pouch fluid from 6 to 48 h. For example, after 6 h neutrophil counts decreased following BRS (0.32 ± 0.11 × 106 cells mL-1) and BV (0.17 ± 0.03 × 106 cells mL-1) compared to MSU only (3.51 ± 1.07 × 106 cells mL-1). Both BV and BRS significantly (p < 0.05) reduced pouch GM-CSF (BV: 5.8 ± 1.2 pg mL-1, BRS: 6.9 ± 1.5 pg mL-1 vs MSU only: 13.0 ± 1.9 pg mL-1) and MCP-1 concentrations at 6 h (BV: 1804 ± 269 pg mL-1, BRS: 7927 ± 2668 pg mL-1 vs MSU only: 17,290 ± 4503 pg ml-1), whilst BV additionally inhibited IL-6 (4354 ± 977 pg mL-1 vs MSU only: 25,070 ± 5178 pg mL-1) and IL-18 (17.6 ± 2.0 pg mL-1 vs MSU only: 81.5 ± 19.9 pg mL-1) concentrations at 6 h (p < 0.05). Despite these differences, no change in pouch chloramine or protein carbonyl concentrations occurred at 24 h (p > 0.05). Serum BV concentrations rapidly diminished over 6 h, however, BRS was readily detected in the serum over 48 h, and in pouch fluid over 12 h. CONCLUSIONS: This study is the first to elucidate anti-inflammatory activity of BRS and the efficacy of BV administration in a model of gouty inflammation. Reduced leukocyte infiltration and cytokine production in response to sterile inflammation further support the importance of these molecules in physiology and their therapeutic potential in sterile inflammation.

Targeted cryoprecipitate transfusion in severe traumatic haemorrhage.

BACKGROUND: Severe traumatic haemorrhage is the leading cause of death in young adults. Trauma Induced Coagulopathy is a complex and multifactorial phenomenon associated with severe traumatic haemorrhage. Fibrinogen is one of the first coagulation factors to become depleted in TIC and evidence suggests that severely injured trauma patients with hypofibrinogenaemia have poor outcomes. It is postulated that early fibrinogen replacement can improve clinical outcomes. This study investigated cryoprecipitate transfusion in hyopfibrinogeneamic trauma patients. METHODS: This retrospective, single center, observational study investigated the use of cryoprecipitate in severely injured trauma patients admitted to an Australian Level I Trauma Centre. The primary outcome was time to administration of cryoprecipitate after identification of hypofibrinogenaemia using ROTEM (FIBTEM A5). Data collected included demographics, ISS, laboratory values of coagulation and blood product usage. RESULTS: 71 patients received cryoprecipitate with a median time of 61 minutes [IQR 37-93] from FIBTEM A5 result to initial cryoprecipitate administration. At 24 hours following admission to ED, Clauss Fibrinogen levels increased by 1.30g/L [IQR 0.45-1.85] and FIBTEM A5 assay increased by 8mm [IQR 3.0-11.3]. Changes in both variables were highly significant (p<0.001) and Clauss Fibrinogen versus FIBTEM A5 values showed moderate to strong correlation (R=0.75-0.80). CONCLUSION: This study demonstrated that early administration of cryoprecipitate was both feasible and efficacious in fibrinogen replacement in severe traumatic haemorrhage. High-level evidence supporting cryoprecipitate or fibrinogen concentrate replacement with regards to efficacy and feasibility is required to guide future clinical practice. This study provided baseline data to inform the design of further clinical trials investigating fibrinogen replacement in traumatic haemorrhage.

The impact of bilirubin ditaurate on platelet quality during storage.

Bilirubin ditaurate (BRT), a conjugated bilirubin analogue, has demonstrated anti-platelet characteristics following acute ex vivo exposure. Scavenging of mitochondrial superoxide and attenuation of granule exocytosis suggested a potential benefit for including BRT for storage. With no reports of cytotoxicity following acute exposure, the impact of 35µM BRT on platelet function was investigated, in clinically suppled units, for up to seven days. Exposure to 35µM BRT significantly reduced mitochondrial membrane potential and increased glucose consumption until exhaustion after 72 hours. Platelet aggregation and activation was significantly impaired by BRT. Mitochondrial superoxide production and phosphatidylserine expression were significantly elevated following glucose exhaustion, with decreased viability observed from day five onwards. Lactate accumulation and loss of bicarbonate, support a metabolic disturbance, leading to a decline of quality following BRT inclusion. Although acute ex vivo BRT exposure reported potentially beneficial effects, translation from acute to chronic exposure failed to combat declining platelet function during storage. BRT exposure resulted in perturbations of platelet quality, with the utility of BRT during storage therefore limited. However, these are the first data of prolonged platelet exposure to analogues of conjugated bilirubin and may improve our understanding of platelet function in the context of conjugated hyperbilirubinemia.

Development of an intravaginal ring for the topical delivery of Aurora kinase A inhibitor, MLN8237.

Human papilloma virus (HPV) is the main culprit in cervical cancers. Although the HPV vaccine is now available, the slow and gradual process for HPV cancers to form means little will change, even for vaccinated individuals. This warrants the development of new therapeutic strategies in both the newly diagnosed and recurrent patients. We have previously shown that Alisertib (MLN8237), an Aurora A kinase inhibitor, potently and selectively kills HPV-positive cervical cancer cells. However, Alisertib is known for its unfavorable side effects when administered systemically. A targeted delivery approach is therefore warranted. The topical delivery of drugs to the cervix for the treatment of cervical cancer is an underexplored area of research that has the potential to significantly improve therapeutic outcome. Here, we design a novel topical drug delivery system for localized delivery in the vaginal tract using intravaginal silicone rings loaded with Alisertib. We assessed the suitability of the drug for the application and delivery method and develop a high-performance liquid chromatography method, then show that the vaginal rings were effective at releasing Alisertib over an extended period of time. Furthermore, we showed that Alisertib-loaded vaginal rings did not induce overt inflammation in the mouse vaginal tract. Our work has major translational implications for the future development of vaginal ring devices for the topical treatment of cervical cancer.