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.

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.

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.

A Role for Folate in Microbiome-Linked Control of Autoimmunity.

The microbiome exerts considerable control over immune homeostasis and influences susceptibility to autoimmune and autoinflammatory disease (AD/AID) such as inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes (T1D), psoriasis, and uveitis. In part, this is due to direct effects of the microbiome on gastrointestinal (GI) physiology and nutrient transport, but also to indirect effects on immunoregulatory controls, including induction and stabilization of T regulatory cells (T reg). Secreted bacterial metabolites such as short-chain fatty acids (SCFA) are under intense investigation as mediators of these effects. In contrast, folate (vitamin B9), an essential micronutrient, has attracted less attention, possibly because it exerts global physiological effects which are difficult to differentiate from specific effects on the immune system. Here, we review the role of folate in AD/AID with some emphasis on sight-threatening autoimmune uveitis. Since folate is required for the generation and maintenance of T reg , we propose that one mechanism for microbiome-based control of AD/AID is via folate-dependent induction of GI tract T reg , particularly colonic T reg, via anergic T cells (T an). Hence, folate supplementation has potential prophylactic and/or therapeutic benefit in AID/AD.

Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis.

We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised ∼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised ∼1.2% of total cells. Further Treg-enrichment (∼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.

Immune Privilege: The Microbiome and Uveitis.

Immune privilege (IP), a term introduced to explain the unpredicted acceptance of allogeneic grafts by the eye and the brain, is considered a unique property of these tissues. However, immune responses are modified by the tissue in which they occur, most of which possess IP to some degree. The eye therefore displays a spectrum of IP because it comprises several tissues. IP as originally conceived can only apply to the retina as it contains few tissue-resident bone-marrow derived myeloid cells and is immunologically shielded by a sophisticated barrier - an inner vascular and an outer epithelial barrier at the retinal pigment epithelium. The vascular barrier comprises the vascular endothelium and the glia limitans. Immune cells do not cross the blood-retinal barrier (BRB) despite two-way transport of interstitial fluid, governed by tissue oncotic pressure. The BRB, and the blood-brain barrier (BBB) mature in the neonatal period under signals from the expanding microbiome and by 18 months are fully established. However, the adult eye is susceptible to intraocular inflammation (uveitis; frequency ~200/100,000 population). Uveitis involving the retinal parenchyma (posterior uveitis, PU) breaches IP, while IP is essentially irrelevant in inflammation involving the ocular chambers, uveal tract and ocular coats (anterior/intermediate uveitis/sclerouveitis, AU). Infections cause ~50% cases of AU and PU but infection may also underlie the pathogenesis of immune-mediated "non-infectious" uveitis. Dysbiosis accompanies the commonest form, HLA-B27-associated AU, while latent infections underlie BRB breakdown in PU. This review considers the pathogenesis of uveitis in the context of IP, infection, environment, and the microbiome.

Activation of dendritic cells by crosslinked collagen hydrogels (artificial corneas) varies with their composition.

Activated T cells are known to promote fibrosis, a major complication limiting the range of polymeric hydrogels as artificial corneal implants. As T cells are activated by dendritic cells (DC), minimally activating hydrogels would be optimal. In this study, we evaluated the ability of a series of engineered (manufactured/fabricated) and natural collagen matrices to either activate DC or conversely induce DC apoptosis in vitro. Bone marrow DC were cultured on a series of singly and doubly crosslinked hydrogels (made from recombinant human collagen III [RHCIII] or collagen mimetic peptide [CMP]) or on natural collagen-containing matrices, MatrigelTM and de-cellularised mouse corneal stroma. DC surface expression of major histocompatibility complex Class II and CD86 as well as apoptosis markers were examined. Natural matrices induced low levels of DC activation and maintained a "tolerogenic" phenotype. The same applied to singly crosslinked CMP-PEG gels. RHCIII gels singly crosslinked using either N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide with the coinitiator N-hydroxy succinimide (EDC-NHS) or N-cyclohexyl-N-(2-morpholinoethyl)carbodiimide metho-p-toulenesulfonate with NHS (CMC-NHS) induced varying levels of DC activation. In contrast, however, RHCIII hydrogels incorporating an additional polymeric network of 2-methacryloyloxyethyl phosphorylcholine did not activate DC but instead induced DC apoptosis, a phenomenon observed in natural matrices. This correlated with increased DC expression of leukocyte-associated immunoglobulin-like receptor-1. Despite low immunogenic potential, viable tolerogenic DC migrated into and through both natural and manufactured RHCIII gels. These data show that the immunogenic potential of RHCIII gels varies with the nature and composition of the gel. Preclinical evaluation of hydrogel immunogenic/fibrogenic potential is recommended.

TGF-ß1-activated type 2 dendritic cells promote wound healing and induce fibroblasts to express tenascin c following corneal full-thickness hydrogel transplantation.

We showed previously that 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide hydrochloride (EDC) cross-linked recombinant human collagen III hydrogels promoted stable regeneration of the human cornea (continued nerve and stromal cell repopulation) for over 4 years. However, as EDC cross linking kinetics were difficult to control, we additionally tested a sterically bulky carbodiimide. Here, we compared the effects of two carbodiimide cross linkers-bulky, aromatic N-cyclohexyl-N0-(2-morpholinoethyl)-carbodiimide (CMC), and nonbulky EDC-in a mouse corneal graft model. Murine corneas undergoing full-thickness implantation with these gels became opaque due to dense retro-corneal membranes (RCM). Corneal epithelial cytokeratin 12 and alpha smooth muscle actin indicative of functional tissue regeneration and wound contraction were observed in RCM surrounding both hydrogel types. However, quantitatively different levels of infiltrating CD11c+ dendritic cells (DC) were found, suggesting a hydrogel-specific innate immune response. More DC infiltrated the stroma surrounding EDC-N-hydroxysuccinimide (NHS) hydrogels concurrently with higher fibrosis-associated tenascin c expression. The opposite was true for CMC-NHS gels that had previously been shown to be more tolerising to DC. In vitro studies showed that DC cultured with transforming growth factor ß1 (TGF-ß1) induced fibroblasts to secrete more tenascin c than those cultured with lipopolysaccharide and this effect was blocked by TGF-ß1 neutralisation. Furthermore, tenascin c staining was found in 40- to 50µm long membrane nanotubes formed in fibroblast/DC cocultures. We suggest that TGF-ß1 alternatively activated (tolerising) DC regulate fibroblast-mediated tenascin c secretion, possibly via local production of TGF-ß1 in early wound contraction, and that this is indirectly modulated by different hydrogel chemistries.

Transmission Electron Microscopy Data on drusen-like deposits in the retinal degeneration sTg-IRBP: HEL mouse model.

Histology (H&E) and transmission electron microscopy (TEM) data are provided showing age-related changes in the retinal structure of sTg-IRBP:HEL mice. These include substantial photoreceptor loss, atrophy of the retinal pigment epithelium, Bruch׳s membrane disruption and thickening, along with the presence of drusenoid deposits and changes in basal laminar infoldings. These features resemble some of those key characteristics found in the course of human dry (atrophic) age-related macular degeneration (AMD), particularly with regard to drusen. Hence, we believe the sTg-IRBP:HEL mouse model represents a useful and promising archetype for future study of the mechanism of drusen formation in AMD.

Mild hyperbilirubinaemia as an endogenous mitigator of overweight and obesity: Implications for improved metabolic health.

BACKGROUND AND AIMS: Mild endogenous elevation of unconjugated bilirubin (UCB) as seen in Gilbert's syndrome (GS), might mitigate cardiovascular disease (CVD) risk factors including overweight/obesity. This study aimed to determine whether hyperbilirubinaemia is linked to improved anthropometric data and lipid profile. METHODS: Our study considered GS and age-/gender-matched healthy controls (n = 248). Additionally, obese female type 2 diabetic patients (DM2) (n = 26) were included as a "disease control group". RESULTS: BMI, hip circumference (HC), and lipid profile were significantly lower in GS. UCB was inversely correlated with BMI (p <0 .001), HC as well as with fat mass (FM) and lipid variables (p < 0.05). Moreover, DM2 patients had significantly lower UCB compared to GS and healthy controls. Older GS subjects (≥35 years) had significantly reduced anthropometric data and improved lipid profile. CONCLUSIONS: Our results propose that the health promoting potential of mild hyperbilirubinaemia may extend to protection from age-related weight gain and dyslipidaemia.

Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

BACKGROUND: Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. METHODS AND RESULTS: Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 µmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. CONCLUSIONS: Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease.

Characteristics of the heme catabolic pathway in mild unconjugated hyperbilirubinemia and their associations with inflammation and disease prevention.

Heme catabolism exerts physiological functions that impact health through depressing inflammation. Upon reactive pathway progression, as in Gilbert's Syndrome (GS; UGT1A1*28 polymorphism), aggravated health effects have been determined. Based on lower inflammation and improved metabolic health reported for GS, inter-group differences in heme catabolism were explored. Therefore, a case-control study including 120 fasted, healthy, age- and gender matched subjects with/without GS, was conducted. Genetic expressions of HMOX-1 and BLVRA were measured. Additionally participants were genotyped for those polymorphisms that are known (UGT1A1*28) or likely (HMOX-1 microsatellites) to impact bilirubinemia. Intracellular interleukins (IL-6, IL-1ß, TNFα), circulatory C-reactive protein (CRP), serum amyloid A (SAA) and haptoglobin (Hpt) were analysed as inflammatory markers. To assess intracellular heme oxygenase 1 (HO-1) isolated PBMCs were used. In GS vs. C, inflammation markers were significantly decreased. This was supported by an altered heme catabolism, indirectly reflecting in elevated unconjugated bilirubin (UCB; main phenotypic feature of GS) and iron, decreased hemopexin (Hpx) and Hpt and in up-regulated biliverdin reductase (BLVRA) gene expressions. Moreover, HMOX (GT)n short alleles were non-significantly more prominent in female GS individuals. Herewith, we propose a concept to elucidate why GS individuals encounter lower inflammation, and are thus less prone to oxidative-stress mediated diseases.

Features of an altered AMPK metabolic pathway in Gilbert's Syndrome, and its role in metabolic health.

Energy metabolism, involving the ATP-dependent AMPK-PgC-Ppar pathway impacts metabolic health immensely, in that its impairment can lead to obesity, giving rise to disease. Based on observations that individuals with Gilbert's syndrome (GS; UGT1A1(*)28 promoter mutation) are generally lighter, leaner and healthier than controls, specific inter-group differences in the AMPK pathway regulation were explored. Therefore, a case-control study involving 120 fasted, healthy, age- and gender matched subjects with/without GS, was conducted. By utilising intra-cellular flow cytometry (next to assessing AMPKα1 gene expression), levels of functioning proteins (phospho-AMPK α1/α2, PgC 1 α, Ppar α and γ) were measured in PBMCs (peripheral blood mononucleated cells). In GS individuals, rates of phospho-AMPK α1/α2, -Ppar α/γ and of PgC 1α were significantly higher, attesting to a boosted fasting response in this condition. In line with this finding, AMPKα1 gene expression was equal between the groups, possibly stressing the post-translational importance of boosted fasting effects in GS. In reflection of an apparently improved health status, GS individuals had significantly lower BMI, glucose, insulin, C-peptide and triglyceride levels. Herewith, we propose a new theory to explain why individuals having GS are leaner and healthier, and are therefore less likely to contract metabolic diseases or die prematurely thereof.

Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases.

Bilirubin, the principal tetrapyrrole, bile pigment and catabolite of haem, is an emerging biomarker of disease resistance, which may be related to several recently documented biological functions. Initially believed to be toxic in infants, the perception of bilirubin has undergone a transformation: it is now considered to be a molecule that may promote health in adults. Data from the last decade demonstrate that mildly elevated serum bilirubin levels are strongly associated with reduced prevalence of chronic diseases, particularly cardiovascular diseases (CVDs), as well as CVD-related mortality and risk factors. Recent data also link bilirubin to other chronic diseases, including cancer and Type 2 diabetes mellitus, and to all-cause mortality. Therefore, there is evidence to suggest that bilirubin is a biomarker for reduced chronic disease prevalence and a predictor of all-cause mortality, which is of important clinical significance. In the present review, detailed information on the association between bilirubin and all-cause mortality, as well as the pathological conditions of CVD, cancer, diabetes and neurodegenerative diseases, is provided. The mechanistic background concerning how bilirubin and its metabolism may influence disease prevention and its clinical relevance is also discussed. Given that the search for novel biomarkers of these diseases, as well as for novel therapeutic modalities, is a key research objective for the near future, bilirubin represents a promising candidate, meeting the criteria of a biomarker, and should be considered more carefully in clinical practice as a molecule that might provide insights into disease resistance. Clearly, however, greater molecular insight is warranted to support and strengthen the conclusion that bilirubin can prevent disease, with future research directions also proposed.

Bilirubin and related tetrapyrroles inhibit food-borne mutagenesis: a mechanism for antigenotoxic action against a model epoxide.

Bilirubin exhibits antioxidant and antimutagenic effects in vitro. Additional tetrapyrroles that are naturally abundant were tested for antigenotoxicity in Salmonella. Un-/conjugated bilirubin (1 and 2), biliverdin (4), bilirubin and biliverdin dimethyl esters (3 and 5), stercobilin (6), urobilin (7), and protoporphyrin (8) were evaluated at physiological concentrations (0.01-2 µmol/plate; 3.5-714 µM) against the metabolically activated food-borne mutagens aflatoxin B1 (9) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (10). Compound 8 most effectively inhibited the mutagenic effects of 9 in strain TA102 and 10 in TA98. Compound 7 inhibited 9-induced mutagenesis in strain TA98 most effectively, while 1 and 4 were promutagenic in this strain. This is likely due to their competition with mutagens for phase-II detoxification. Mechanistic investigations into antimutagenesis demonstrate that tetrapyrroles react efficiently with a model epoxide of 9, styrene epoxide (11), to form covalent adducts. This reaction is significantly faster than that of 11 with guanine. Hence, the evaluated tetrapyrroles inhibited genotoxicity induced by poly-/heterocyclic amines found in foods, and novel evidence obtained in the present investigation suggests this may occur via chemical scavenging of genotoxic metabolites of the mutagens investigated. This may have important ramifications for maintaining health, especially with regard to cancer prevention.

Anti-genotoxic potential of bilirubin in vivo: damage to DNA in hyperbilirubinemic human and animal models.

The bile pigment bilirubin is a known antioxidant and is associated with protection from cancer and cardiovascular disease (CVD) when present in too strong concentrations. Unconjugated bilirubin (UCB) might also possess anti-genotoxic potential by preventing oxidative damage to DNA. Moderately elevated bilirubin levels are found in individuals with Gilbert syndrome and more severe in the hyperbilirubinemic Gunn rat model. This study was therefore aimed to assess the levels of oxidative damage to DNA in Gilbert syndrome subjects and Gunn rats compared to matched controls. Seventy-six individuals (age- and sex-matched) were allocated into Gilbert syndrome (UCB ≥17.1 µmol/L; n = 38) or control groups (UCB < 17.1 µmol/L; n = 38). In addition, 40 Gunn rats were used to support the results of the human trial. Single-cell gel electrophoresis (SCGE) assay measuring standard conditions (strand breaks, apurinic/apyrimidinic sites) and formamidopyrimidine glycosylase (FPG)-sensitive sites was conducted in human peripheral blood mononuclear cells (PBMC) and rat PBMCs, colon, and hepatocytes. Furthermore, urinary 8-oxo-2'-deoxyguanosine (8oxodGuo, DNA oxidation) and 8-oxo-guanosine (8oxoGuo, RNA oxidation) were measured in humans. The Gilbert syndrome and Gunn rat groups had significantly higher UCB levels (P < 0.001) than the corresponding controls. No further differences in damage to DNA or RNA were detected between the two groups, except higher strand breaks (PBMCs) in Gunn rats when compared with controls. However, when demographic effects were analyzed, lower 8oxodGuo concentrations were detected in the human group with a BMI ≥25 kg/m(2) (1.70 ± 0.67 vs. 1.38 ± 0.43 nmol/mmol creatinine, P < 0.05), although this group showed lower UCB levels than normal weight subjects. This study suggests that the disease preventative effect of UCB is unrelated to DNA oxidation/strand breaks in human and animal models of hyperbilirubinaemia.

Haem catabolism: a novel modulator of inflammation in Gilbert's syndrome.

BACKGROUND: Moderately elevated unconjugated bilirubin concentrations protect against inflammatory diseases and are present in individuals with Gilbert's syndrome. This study examined the relationship between circulating haem oxygenase catabolites, unconjugated bilirubin, carboxy haemoglobin, iron and inflammatory parameters. MATERIALS AND METHODS: Seventy-six matched individuals were allocated to Gilbert's syndrome (GS) or control group (unconjugated bilirubin ≥ or < 17.1 µM). Iron, carboxy haemoglobin and high-sensitivity C-reactive protein were analysed using routine diagnostic tests. Unconjugated bilirubin and haem were analysed using high-performance liquid chromatography. The cytokines IL-1ß, TNF-α and IL-6 were assessed using high-sensitivity enzyme-linked immunosorbent assays. RESULTS: Gilbert's syndrome subjects had significantly greater levels of unconjugated bilirubin (P < 0.05), carboxy haemoglobin (P < 0.05), iron (P < 0.05), IL-1ß (P < 0.05), a significantly lower body mass index (P < 0.05) and IL-6 concentrations (P < 0.05) vs. controls. Regression analysis revealed that unconjugated bilirubin mainly explained IL-1ß results (16%), and body mass index+IL-6 predicted 26% of the variance in C-reactive protein concentrations. CONCLUSIONS: A positive relationship between unconjugated bilirubin and free plasma haem, iron and carboxy haemoglobin indicated a positive feedback loop of haem oxygenase induction possibly mediated by unconjugated bilirubin. Furthermore, reduced body mass index in Gilbert's syndrome individuals was linked to reduced inflammation status, which could be influenced by circulating haem oxygenase catabolites and contribute to reduced risk of noncommunicable diseases in this population.

In vitro DNA-damaging effects of intestinal and related tetrapyrroles in human cancer cells.

Epidemiological studies report a negative association between circulating bilirubin concentrations and the risk for cancer and cardiovascular disease. Structurally related tetrapyrroles also possess in vitro anti-genotoxic activity and may prevent mutation prior to malignancy. Furthermore, few data suggest that tetrapyrroles exert anti-carcinogenic effects via induction of cell cycle arrest and apoptosis. To further investigate whether tetrapyrroles provoke DNA-damage in human cancer cells, they were tested in the single cell gel electrophoresis assay (SCGE). Eight tetrapyrroles (unconjugated bilirubin, bilirubin ditaurate, biliverdin, biliverdin-/bilirubin dimethyl ester, urobilin, stercobilin and protoporphyrin) were added to cultured Caco2 and HepG2 cells and their effects on comet formation (% tail DNA) were assessed. Flow cytometric assessment (apoptosis/necrosis, cell cycle, intracellular radical species generation) assisted in revealing underlying mechanisms of intracellular action. Cells were incubated with tetrapyrroles at concentrations of 0.5, 5 and 17µM for 24h. Addition of 300µM tertiary-butyl hydroperoxide to cells served as a positive control. Tetrapyrrole incubation mostly resulted in increased DNA-damage (comet formation) in Caco2 and HepG2 cells. Tetrapyrroles that are concentrated within the intestine, including protoporphyrin, urobilin and stercobilin, led to significant comet formation in both cell lines, implicating the compounds in inducing DNA-damage and apoptosis in cancer cells found within organs of the digestive system.

In vitro antioxidant capacity and antigenotoxic properties of protoporphyrin and structurally related tetrapyrroles.

The antioxidant properties of protoporphyrin IX and related tetrapyrroles are poorly characterized. Therefore, eight tetrapyrroles, five of which are produced in vivo, were tested to assess their antioxidant capacities in the Salmonella reverse mutation, TEAC, FRAP and ORAC assays. Tertiary-butyl hydroperoxide (tert-BOOH) in the presence or absence of metabolic activation (±S9) was added to Salmonella strain TA102 together with the test compounds. In the absence of metabolic activation, the order of effectiveness was protoporphyrin > biliverdin > bilirubin ditaurate > bilirubin > biliverdin dimethyl ester > stercobilin > bilirubin dimethyl ester > urobilin. In the presence of S9, the effectiveness was reversed, with urobilin > biliverdin dimethyl ester > bilirubin dimethyl ester > stercobilin > biliverdin > bilirubin > bilirubin ditaurate > protoporphyrin. In the antioxidant capacity assays FRAP, TEAC and ORAC, mainly bilirubin, bilirubin ditaurate, biliverdin and protoporphyrin showed antioxidant activity. This study reports that previously untested tetrapyrroles of related structure prevent oxidatively induced genotoxicity, and for some, novel underlying mechanisms of antioxidant action were revealed. These results support the physiological importance and biological relevance of tetrapyrroles including protoporphyrin that might act as antioxidants, protecting from oxidatively induced DNA damage, particularly in the tissues/organs where they accumulate.

Determination of the in vivo prebiotic potential of a maize-based whole grain breakfast cereal: a human feeding study.

Epidemiological studies have shown an inverse relationship between risk of CVD and intake of whole grain (WG)-rich food. Regular consumption of breakfast cereals can provide not only an increase in dietary WG but also improvements to cardiovascular health. Various mechanisms have been proposed, including prebiotic modulation of the colonic microbiota. In the present study, the prebiotic activity of a maize-derived WG cereal (WGM) was evaluated in a double-blind, placebo-controlled human feeding study (n 32). For a period of 21 d, healthy men and women, mean age 32 (sd 8) years and BMI 23·3 (sd 0·58) kg/m2, consumed either 48 g/d WG cereal (WGM) or 48 g placebo cereal (non-whole grain (NWG)) in a crossover fashion. Faecal samples were collected at five points during the study on days 0, 21, 42, 63 and 84 (representing at baseline, after both treatments and both wash-out periods). Faecal bacteriology was assessed using fluorescence in situ hybridisation with 16S rRNA oligonucleotide probes specific for Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum/perfringens subgroup, Lactobacillus-Enterococcus subgroup and total bacteria. After 21 d consumption of WGM, mean group levels of faecal bifidobacteria increased significantly compared with the control cereal (P = 0·001). After a 3-week wash-out period, bifidobacterial levels returned to pre-intervention levels. No statistically significant changes were observed in serum lipids, glucose or measures of faecal output. In conclusion, this WG maize-enriched breakfast cereal mediated a bifidogenic modulation of the gut microbiota, indicating a possible prebiotic mode of action.