Plasma Unconjugated Bilirubin Research Articles

Effects of Hydroxyurea Treatment on Haemolysis in Patients with Sickle Cell Disease at Muhimbili National Hospital, Tanzania

Tanzania is one of the countries with a high burden of sickle cell disease (SCD). Haemolytic anaemia is a clinical feature of SCD, and has been linked to major complications leading to morbidity and mortality. Treatment with hydroxyurea (HU) has shown to induce foetal haemoglobin (HbF) which in turn decreases haemolysis in patients. This study aimed to investigate the effects of HU on haemolysis in SCD patients attending Muhimbili National Hospital, Tanzania by comparing their haemolytic parameters before and after therapy. Patients meeting the criteria were initiated on HU therapy for 3 months. Two haemolytic biomarkers: unconjugated plasma bilirubin levels and absolute reticulocyte counts were measured from patients’ blood samples at baseline and after 3 months of HU therapy and compared. Both absolute reticulocyte counts and indirect plasma bilirubin levels significantly declined after HU therapy. Median (IQR) plasma unconjugated bilirubin levels dropped significantly from 20.3 (12.7–34.4) μmol/L to 14.5 (9.6–24.1) μmol/L (p < 0.001) and mean (SD) absolute reticulocyte counts dropped significantly from 0.29 (0.1) x 109/L to 0.17 (0.1) x 109/L (p < 0.001) after therapy, thus, a decline in both haemolytic biomarkers after treatment was observed. This study found a potential for use of HU therapy in managing SCD patients in our settings evidenced by improvements in their haemolytic parameters. Clinical trials with a lager sample size conducted for a longer time period would be beneficial in guiding towards the inclusion of HU in treatment protocols for the Tanzanian population.
 Keywords: Sickle cell disease; hydroxyurea; haemolysis; foetal haemoglobin

Oral Acute & Sub-Chronic Toxicity Assessment of Ethanol Leaf Extract of Simarouba glauca

Traditional herbal medicine and their preparations have been widely used for thousands of years and are still in use in developing and developed countries owing to their medicinal values and their presumed relative safety. This belief that medicinal plants are not toxic or are with less side effect due to their natural origin is debatable; hence this study was conducted to evaluate the safety and (or) toxicity of Ethanol leaf extract of Simarouba glauca (EESG) on liver, kidney and heart functions of Wistar rats. The oral acute toxicity of EESG was evaluated in line with Lorke’s method. The sub-chronic toxicity of EESG was carried out according to the OECD guidelines with modification and using a total of twenty-four (24) male Wistar rats; divided into four groups of six rats each, following a two-week acclimatization. Test rats were orally administered EESG at doses of 500, 1000 and 2000 mg/kg body weight respectively daily for thirty (30) days, while the control was given only feed and water ad libitum. At the end of the experiment, the rats were fasted overnight and sacrificed under chloroform anesthesia; relevant biochemical and histopathology analyses were carried out. The data obtained from the oral acute test indicate that the LD50 was above 5000 mg/kg and there was no death recorded. There were significant increases (P ˂ 0.05) in percentage (%) body weight of rats administered respective doses of EESG. There were significant reductions (P˂0.05) in mean liver: body weight ratio of rats administered EESG 500 and 2000 mg/kg respectively, significant reductions (P˂0.05) in mean kidney: body weight ratios of rats given EESG 1000 and 2000 mg/kg respectively; significant reductions (P˂0.05) in mean heart: body weight ratios of test rats administered EESG 2000 mg/kg; whereas others were not significantly different (P˃0.05) relative to their respective control. Plasma ALT and GGT activities of rats administered respective dose of EESG were significantly reduced (P˂0.05); plasma ALP activities were significantly elevated (P˂0.05) relative to the control after 30 days. There were no significant differences (P˃0.05) in plasma total proteins and albumin levels. Plasma total and unconjugated bilirubin of rats administered respective dose of EESG were not significantly different (P˃0.05); whereas, rats given EESG recorded significant reduction in plasma conjugated bilirubin. Plasma urea was significantly elevated (P˂0.05) in rats administered EESG 1000 and 2000 mg/kg respectively. Test rats given EESG 500 and 1000 mg/kg respectively recorded significant elevations in plasma creatinine and rats given EESG 2000 mg/kg recorded significant decrease in plasma creatinine levels; others were not significantly different relative to the control. Plasma chloride and potassium ion levels of rats administered respective doses of EESG were not significantly different (P˃0.05); significant reduction (P˂0.05) in plasma sodium ions concentration in all group compared to the control. Plasma calcium ion levels in all group were not significantly different (P˃0.05); whereas there were significant reductions (P˂0.05) in plasma bicarbonate ion levels relative to their respective controls. Although plasma ALP activity were significantly elevated, there were no elevations in specific liver function enzymes and no visible hepatocellular damage. Furthermore, the conspicuous elevations observed in plasma urea and creatinine levels do not exclusively indicate EESG-induced organ injury. Therefore, it is suggestive that EESG was not significantly toxic to the to the liver, kidney and heart respectively and may be administered at lower doses in further studies.

Prebiotics for the prevention of hyperbilirubinaemia in neonates.

Hyperbilirubinaemia occurs in approximately two-thirds of all newborns during the first days of life and is frequently treated with phototherapy. Although generally seen as safe, there is rising concern regarding phototherapy and its potentially damaging effects on DNA and increased side effects particularly for preterm infants. Other methods, such as enteral feeding supplementation with prebiotics, may have an effective use in the management of hyperbilirubinaemia in neonates. To determine whether administration of prebiotics reduces the incidence of hyperbilirubinaemia among term and preterm infants compared with enteral supplementation of milk with distilled water/placebo or no supplementation. We used the standard search strategy of Cochrane Neonatal to search the Cochrane Central Register of Controlled Trials (CENTRAL 2018, Issue 5), MEDLINE via PubMed (1966 to 14 June 2018), Embase (1980 to 14 June 2018), and CINAHL (1982 to 14 June 2018). We also searched clinical trials databases, conference proceedings, and the reference lists of retrieved articles for randomised controlled trials (RCTs) and quasi-randomised trials. We considered all RCTs that studied neonates comparing enteral feeding supplementation with prebiotics versus distilled water/placebo or no supplementation. Two reviewers screened papers and extracted data from selected papers. We used a fixed-effect method in combining the effects of studies that were sufficiently similar. We then used the GRADE approach to assess the quality of the evidence. Three small studies evaluating 154 infants were included in this review. One study reported a significant reduction in the risk of hyperbilirubinaemia and rate of treatment with phototherapy associated with enteral supplementation with prebiotics (risk ratio (RR) 0.75, 95% confidence interval (95% CI) 0.58 to 0.97; one study, 50 infants; low-quality evidence). Meta-analyses of two studies showed no significant difference in maximum plasma unconjugated bilirubin levels in infants with prebiotic supplementation (mean difference (MD) 0.14 mg/dL, 95% CI -0.91 to 1.20, I² = 81%, P = 0.79; two studies, 78 infants; low-quality evidence). There was no evidence of a significant difference in duration of phototherapy between the prebiotic and control groups, which was only reported by one study (MD 0.10 days, 95% CI -2.00 to 2.20; one study, 50 infants; low-quality evidence). The meta-analyses of two studies demonstrated a significant reduction in the length of hospital stay (MD -10.57 days, 95% CI -17.81 to -3.33; 2 studies, 78 infants; I² = 0%, P = 0.004; low-quality evidence). Meta-analysis of the three studies showed a significant increase in stool frequency in the prebiotic groups (MD 1.18, 95% CI 0.90 to 1.46, I² = 90%; 3 studies, 154 infants; high-quality evidence). No significant difference in mortality during hospital stay after enteral supplementation with prebiotics was reported (typical RR 0.94, 95% CI 0.14 to 6.19; I² = 6%, P = 0.95; 2 studies; 78 infants; low-quality evidence). There were no reports of the need for exchange transfusion and incidence of acute bilirubin encephalopathy, chronic bilirubin encephalopathy, and major neurodevelopmental disability in the included studies. None of the included studies reported any side effects. Current studies are unable to provide reliable evidence about the effectiveness of prebiotics on hyperbilirubinaemia. Additional large, well-designed RCTs should be undertaken in neonates that compare effects of enteral supplementation with prebiotics on neonatal hyperbilirubinaemia with supplementation of milk with any other placebo (particularly distilled water) or no supplementation.

Circadian Clock Gene Bmal1 Regulates Bilirubin Detoxification: A Potential Mechanism of Feedback Control of Hyperbilirubinemia.

Controlling bilirubin to a low level is necessary in physiology because of its severe neurotoxicity. Therefore, it is of great interest to understand the regulatory mechanisms for bilirubin homeostasis. In this study, we uncover a critical role for circadian clock in regulation of bilirubin detoxification and homeostasis.Methods: The mRNA and protein levels of Bmal1 (a core clock gene), metabolic enzymes and transporters were measured by qPCR and Western blotting, respectively. Luciferase reporter, mobility shift and chromatin immunoprecipitation were used to investigate transcriptional gene regulation. Experimental hyperbilirubinemia was induced by injection of bilirubin or phenylhydrazine. Unconjugated bilirubin (UCB) and conjugated bilirubin were assessed by ELISA.Results: We first demonstrated diurnal variations in plasma UCB levels and in main bilirubin-detoxifying genes Ugt1a1 and Mrp2. Of note, the circadian UCB levels were antiphase to the circadian expressions of Ugt1a1 and Mrp2. Bmal1 ablation abrogated the circadian rhythms of UCB and bilirubin-induced hepatotoxicity in mice. Bmal1 ablation also decreased mRNA and protein expressions of both Ugt1a1 and Mrp2 in mouse livers, and blunted their circadian rhythms. A combination of luciferase reporter, mobility shift, and chromatin immunoprecipitation assays revealed that Bmal1 trans-activated Ugt1a1 and Mrp2 through specific binding to the E-boxes in the promoter region. Further, Bmal1 ablation caused a loss of circadian time-dependency in bilirubin clearance and sensitized mice to chemical induced-hyperbilirubinemia. Moreover, bilirubin stimulated Bmal1 expression through antagonism of Rev-erbα, constituting a feedback mechanism in bilirubin detoxification.Conclusion: These data supported a dual role for circadian clock in regulation of bilirubin detoxification, generating circadian variations in bilirubin level via direct transactivation of detoxifying genes Ugt1a1 and Mrp2, and defending the body against hyperbilirubinemia via Rev-erbα antagonism. Thereby, our study provided a potential mechanism for management of bilirubin related diseases.

Antioxidant bilirubin works in multiple ways to reduce risk for obesity and its health complications

Gilbert syndrome (GS) is a common genetic variant in which plasma unconjugated bilirubin levels are elevated throughout life, in the absence of hepatic pathology.1 This typically reflects decreased hepatic capacity...

Bilirubin, a novel endocrine hormone with fat burning properties

Plasma bilirubin levels have been shown to be inversely related to obesity, diabetes, and cardiovascular events. Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of the bile pigment. In the present study, we found that bilirubin has a new function as an endocrine hormone that can signal to the fat burning nuclear receptor, peroxisome proliferator‐activated receptor α (PPARα). The bilirubin‐PPARα mediated effects cause a significant decrease in lipid accumulation in adipose and liver of mice. Bilirubin is conjugated in the liver by UDP‐glucuronosyltransferase 1‐1 (UGT1A1) protein and then excreted into bile. Furthermore, we found that blockade of the UGT1A1 by either anti‐sense morpholinos or by genetic mutation caused significantly raised plasma unconjugated bilirubin levels which also heightened PPARα transcriptional activity in liver and adipose. Ultimately, increasing plasma unconjugated bilirubin decrease adiposity and lower blood glucose levels. These data indicate a new function for bilirubin as an endocrine hormone that can be regulated in the liver to control adiposity and insulin resistance in peripheral tissues. Targeting of UGT1A1 in the liver may offer future implications in the treatment of obesity and type II diabetes.Support or Funding InformationThis research was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL125445 (T.D.H.) and PO1HL‐51971 (D.E.S) and National Institute of General Medical Sciences P20GM‐104357 (D.E.S.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Repeated AAV-mediated gene transfer by serotype switching enables long-lasting therapeutic levels of hUgt1a1 enzyme in a mouse model of Crigler-Najjar Syndrome Type I.

Adeno-associated virus (AAV) -mediated gene therapy is a promising strategy to treat liver-based monogenic diseases. However, two major obstacles limit its success: first, vector dilution in actively dividing cells, such as hepatocytes in neonates/children, due to the non-integrating nature of the vector; second, development of an immune response against the transgene and/or viral vector. Crigler-Najjar Syndrome Type I is a rare monogenic disease with neonatal onset, caused by mutations in the liver-specific UGT1 gene, with toxic accumulation of unconjugated bilirubin in plasma, tissues and brain. To establish an effective and long lasting cure, we applied AAV-mediated liver gene therapy to a relevant mouse model of the disease. Repeated gene transfer to adults by AAV-serotype switching, upon neonatal administration, resulted in lifelong correction of total bilirubin (TB) levels in both genders. In contrast, vector loss over time was observed after a single neonatal administration. Adult administration resulted in lifelong TB levels correction in male, but not female Ugt1-/- mice. Our findings demonstrate that neonatal AAV-mediated gene transfer to the liver supports a second transfer of the therapeutic vector, by preventing the induction of an immune response and supporting the possibility to improve AAV-therapeutic efficacy by repeated administration.

Prebiotics for the prevention of hyperbilirubinaemia in neonates

Background Hyperbilirubinaemia occurs in approximately two-thirds of all newborns during the first days of life and is frequently treated with phototherapy. Although generally seen as safe, there is rising concern regarding phototherapy and its potentially damaging effects on DNA and increased side effects particularly for preterm infants. Other methods, such as enteral feeding supplementation with prebiotics, may have an effective use in the management of hyperbilirubinaemia in neonates. Objectives To determine whether administration of prebiotics reduces the incidence of hyperbilirubinaemia among term and preterm infants compared with enteral supplementation of milk with distilled water/placebo or no supplementation. Search methods We used the standard search strategy of Cochrane Neonatal to search the Cochrane Central Register of Controlled Trials (CENTRAL 2018, Issue 5), MEDLINE via PubMed (1966 to 14 June 2018), Embase (1980 to 14 June 2018), and CINAHL (1982 to 14 June 2018). We also searched clinical trials databases, conference proceedings, and the reference lists of retrieved articles for randomised controlled trials (RCTs) and quasi-randomised trials. Selection criteria We considered all RCTs that studied neonates comparing enteral feeding supplementation with prebiotics versus distilled water/placebo or no supplementation. Data collection and analysis Two reviewers screened papers and extracted data from selected papers. We used a fixed-effect method in combining the effects of studies that were sufficiently similar. We then used the GRADE approach to assess the quality of the evidence. Main results Three small studies evaluating 154 infants were included in this review. One study reported a significant reduction in the risk of hyperbilirubinaemia and rate of treatment with phototherapy associated with enteral supplementation with prebiotics (risk ratio (RR) 0.75, 95% confidence interval (95% CI) 0.58 to 0.97; one study, 50 infants; low-quality evidence). Meta-analyses of two studies showed no significant difference in maximum plasma unconjugated bilirubin levels in infants with prebiotic supplementation (mean difference (MD) 0.14 mg/dL, 95% CI -0.91 to 1.20, I² = 81%, P = 0.79; two studies, 78 infants; low-quality evidence). There was no evidence of a significant difference in duration of phototherapy between the prebiotic and control groups, which was only reported by one study (MD 0.10 days, 95% CI -2.00 to 2.20; one study, 50 infants; low-quality evidence). The meta-analyses of two studies demonstrated a significant reduction in the length of hospital stay (MD -10.57 days, 95% CI -17.81 to -3.33; 2 studies, 78 infants; I² = 0%, P = 0.004; low-quality evidence). Meta-analysis of the three studies showed a significant increase in stool frequency in the prebiotic groups (MD 1.18, 95% CI 0.90 to 1.46, I² = 90%; 3 studies, 154 infants; high-quality evidence). No significant difference in mortality during hospital stay after enteral supplementation with prebiotics was reported (typical RR 0.94, 95% CI 0.14 to 6.19; I² = 6%, P = 0.95; 2 studies; 78 infants; low-quality evidence). There were no reports of the need for exchange transfusion and incidence of acute bilirubin encephalopathy, chronic bilirubin encephalopathy, and major neurodevelopmental disability in the included studies. None of the included studies reported any side effects. Authors' conclusions Current studies are unable to provide reliable evidence about the effectiveness of prebiotics on hyperbilirubinaemia. Additional large, well-designed RCTs should be undertaken in neonates that compare effects of enteral supplementation with prebiotics on neonatal hyperbilirubinaemia with supplementation of milk with any other placebo (particularly distilled water) or no supplementation.

NADPH Oxidase Activity in Retinal Pigmented Epithelium as a Target for Prevention of Age-related Macular Degeneration

Considerable evidence points to macular oxidative stress as a key driver of age-related macular degeneration (AMD). In particular, retinal pigmented epithelial (RPE) cells subjected to oxidative stress are prone to apoptosis or senescence; lose their tight junctions; decrease their production of factors which provide trophic support for retinal photoreceptors, suppress choroidal neovascularization, and control complement activation; and boost their production of vascular endothelial growth factor (VEGF), a major stimulant to neovascularization – all effects which are consistent with the pathogenesis of AMD. There is good reason to suspect that NADPH oxidase activity in RPE cells is the key source of the oxidative stress that compromises RPE function in AMD. Studies with other cell lines indicate that a number of risk factors for AMD – including cigarette smoke, homocysteine, hyperglycemia, cadmium, and up-regulated activation of the alternative complement cascade – have the ability to stimulate NADPH oxidase activity, as does all-trans-retinal, a suspected pathogenic factor in AMD. Hence, measures which can down-regulate NADPH oxidase activity may have potential for preventing or controlling AMD. These may include high-dose statins – found to reverse features of early AMD in about half of patients in a recent pilot study – and spirulina, a rich source of the chromophore phycocyanobilin (PhyCB ). PhyCB is a biliverdin derivative that can mimic the intracellular physiological role of bilirubin as an NADPH oxidase inhibitor. Patients with Gilbert syndrome or relatively high plasma unconjugated bilirubin may be at lower risk for AMD owing to bilirubin’s inhibitory impact on NADPH oxidase; this prediction could be readily tested in epidemiological studies.

Is the unbound concentration of atazanavir of interest in therapeutic drug monitoring?

To date, therapeutic drug monitoring (TDM) is carried out with antiretrovirals and is usually based on total concentrations (Ct ). However, for some patients, TDM does not reflect efficacy or the avoidance of toxicity as is the case for atazanavir (ATV), a HIV protease inhibitor. As the unbound concentration (Cu ) is the pharmacological active form, the aim of the study was to evaluate the value of Cu and the unbound fraction (fu , fu = Cu /Ct ) for the TDM of ATV. The variability of Cu and the corresponding fu of ATV was explored in 43 patients treated with ATV for an average of 13.5 months. Cu was determined by coupling ultrafiltration and liquid chromatography. As ATV is highly bound to alpha-1 acid glycoprotein (AAG), the correlation between fu and AAG was also evaluated. The viral load was monitored to evaluate the patients' virologic response, while total plasma bilirubin and unconjugated plasma bilirubin were used as biomarkers of ATV toxicity. Median trough Cu and Ct were 37.9 μg/L (Interquartile range (IQR) 20.6-94.9 μg/L) and 628.6 μg/L (IQR 362.7-1078.1 μg/L), respectively. fu , Cu and Ct showed high variability, but the fu variability was not correlated with the AAG level. The unbound concentration and fraction were unrelated to the virologic response (P = 0.21 and P = 0.65 for Cu and fu , respectively) nor to the unconjugated bilirubin (Pearson correlation coefficient (ρ), ρ = 0.22; P = 0.17 for Cu ). Neither total nor unbound concentrations of ATV fully explained hyperbilirubinaemia or virologic failure. From this study, we conclude that unbound ATV did not appear to be more relevant than Ct .

Optimizing Exchange Transfusion for Severe Unconjugated Hyperbilirubinemia: Studies in the Gunn Rat

BackgroundSevere unconjugated hyperbilirubinemia carries the risk of neurotoxicity. Phototherapy (PT) and exchange transfusion (ET) are cornerstones in the treatment of unconjugated hyperbilirubinemia. Studies to improve ET efficacy have been hampered by the low application of ET in humans and by the lack of an in vivo model. The absence of an appropriate animal model has also prevented to determine the efficacy of adjunct or alternative treatment options such as albumin (Alb) administration.AimTo establish an in vivo model for ET and to determine the most effective treatment (combination) of ET, PT and Alb administration.MethodsGunn rats received either PT, PT+Alb, ET, ET+PT, ET+PT+Alb or sham operation (each n = 7). ET was performed via the right jugular vein in ∼20 min. PT (18 µW/cm2/nm) was started after ET or at T0. Albumin i.p. injections (2.5 g/kg) were given after ET or before starting PT. Plasma unconjugated bilirubin (UCB), plasma free bilirubin (Bf), and brain bilirubin concentrations were determined.ResultsWe performed ET in 21 Gunn rats with 100% survival. At T1, ET was profoundly more effective in decreasing both UCB −44%, p<0.01) and Bf −81%, p<0.05) than either PT or PT+Alb. After 48 h, the combination of ET+PT+Alb showed the strongest hypobilirubinemic effect (−54% compared to ET).ConclusionsWe optimized ET for severe unconjugated hyperbilirubinemia in the Gunn rat model. Our data indicate that ET is the most effective treatment option, in the acute as well as the follow-up situation.

Antihypertensive Actions of Moderate Hyperbilirubinemia: Role of Superoxide Inhibition

Moderate (approximately 2-fold) increases in plasma unconjugated bilirubin levels are able to attenuate the development of angiotensin II (Ang II)-dependent hypertension. To determine the specific role of decreases in superoxide production to the blood pressure-lowering effects of moderate hyperbilirubinemia (MHyB), we performed this study, in which the Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin was given to Ang II-infused mice in the presence and absence of moderate hyperbilirubinemia. Apocynin (14mM) was administered in the drinking water prior to treatment with UDP-glucuronosyltransferase 1A1 antisense morpholino (16 μg/kg), which was administered by intravenous injection every third day. Treatments were started before the implantation of Ang II-containing minipumps (1μg/kg/min) and continued throughout the protocol. Ang II infusion increased blood pressure to 145±2mm Hg. Apocynin treatment alone reduced blood pressure to 135±5mm Hg, whereas MHyB alone decreased blood pressure to 118±5mm Hg in Ang II-infused mice. Prior inhibition of NADPH oxidase with apocynin did not result in a further decrease in blood pressure in MHyB mice, which averaged 117±3mm Hg (n = 6 mice per group). In aortic preparations, apocynin treatment decreased Ang II-mediated superoxide production from 2433±120 relative light units (RLU)/min/mg to 1851±126 RLU/min/mg (n = 4 mice per group), which was similar to levels observed in MHyB mice alone (1473±132 RLU/min/mg) or in combination with apocynin (1503±115 RLU/min/mg). Our results indicate that MHyB lowers blood pressure by a mechanism that is partially dependent on the inhibition of superoxide production.

Antihypertensive Actions of Moderate Hyperbilirubinemia: Role of Superoxide Inhibition

Our lab has previously demonstrated that moderate (~ 2 fold) increases in plasma unconjugated bilirubin levels are able to attenuate the development of Angiotensin II (Ang II) dependent hypertension. In order to determine the specific role of decreases in superoxide production to the blood pressure lowering affects of moderate hyperbilirubinemia (MHyB), we performed the current study in which the NADPH oxidase inhibitor, apocynin, was given to Ang II infused mice in the presence and absence of moderate hyperbilirubinemia. Apocynin (14 mM) was administered in the drinking water prior to treatment with UGT1A1 antisense morpholino (ASM; 16 μg/kg) which was administered via intravenous injection every third day. Treatments were started before the implantation of Ang II containing minipumps (1μg/kg/min) and continued throughout the 7 day protocol. Ang II infusion increased blood pressure to 145 ± 2 mmHg. Apocynin treatment alone reduced blood pressure to 135 ± 5 while MHyB alone decreased blood pressure to 118 ± 5 in Ang II infused mice. Prior inhibition of NADPH oxidase with apocynin did not result in a further decrease in blood pressure in MHyB mice which averaged 117 ± 3 (n=6/group). In aortic preparations, apocynin treatment decreased Ang II mediated superoxide production from 2433 ± 120 to 1851 ± 126 RLU/min/mg (n=4/group) which was similar to levels observed in MHyB mice alone 1473 ± 132 or in combination with apocynin 1503 ± 115. Our results indicate that MHyB lowers blood pressure via a mechanism that is independent of the inhibition of superoxide production; however, we cannot exclude the possibility that other antioxidant properties of bilirubin may be important to the antihypertensive actions in Ang II dependent hypertension. This work was supported by grants from the National Heart, Lung and Blood Institute, HL088421 and PO1HL‐51971.

Combined Treatment Strategies for Unconjugated Hyperbilirubinemia in Gunn Rats

We recently demonstrated that acceleration of the gastrointestinal transit by polyethylene glycol (PEG) treats unconjugated hyperbilirubinemia in jaundiced Gunn rats. It is unclear whether acceleration of gastrointestinal transit also (partly) underlies the therapeutic effects of established hypobilirubinemic treatments or whether PEG cotreatment might enhance these effects. We treated Gunn rats with phototherapy (17 μW/cm2/nm), orlistat (200 mg/kg chow), ursodeoxycholate (5 g/kg chow), or calcium phosphate (CaP) (20 g/kg chow) either as single treatment or in combination with PEG. Three weeks of phototherapy, orlistat, ursodeoxycholic acid, or CaP treatment decreased plasma unconjugated bilirubin (UCB) levels by 47, 27, 28, and 45%, respectively (each p < 0.001), without a significant impact on gastrointestinal transit time. PEG cotreatment accelerated the gastrointestinal transit in all treatment groups, which resulted in an additive hypobilirubinemic effect of -20% and -26% (final plasma UCB -67 and -53%, respectively) in phototherapy- and orlistat-treated animals. PEG cotreatment did not enhance the hypobilirubinemic effect of ursodeoxycholic acid or CaP. We conclude that phototherapy, orlistat, ursodoxycholic acid, and CaP do not exert their hypobilirubinemic effect via acceleration of the gastrointestinal transit. PEG cotreatment enhanced the hypobilirubinemic effects of phototherapy and of orlistat treatment. Current results support a clinical trial to evaluate PEG cotreatment during phototherapy.

Pilot study of omega-3 fatty acid supplements in sickle cell disease

In a previous retrospective study, it was observed that the greater the amounts of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the blood, the lesser the number of complications of sickle cell disease (SCD) and the higher the steady state haemoglobin level. SCD causes ischaemia-reperfusion injury and inflammation; which can be ameliorated by a metabolite of DHA that down-regulates expression of pro-inflammatory genes. The objectives of this prospective pilot study were to evaluate the effects of DHA and EPA supplements in SCD, and test the hypothesis that these effects are mediated partly by reducing inflammation. Oral DHA and EPA supplements were given to 16 SCD patients for 6 months. We then compared pre- and post-supplementation values of number of crisis, steady state Hb, plasma unconjugated bilirubin and three indices of inflammation: plasma interleukin-6, blood neutrophil and platelet counts. There was a significant reduction in the plasma level of unconjugated bilirubin, and the number of sickle cell crisis; but not in the markers of inflammation. The pilot data suggest that DHA and EPA supplements reduce the number of crisis and steady state haemolysis in SCD; but provide no evidence that these effects are mediated by reducing inflammation.

Neonatal Hyperbilirubinemia and Organic Anion Transporting Polypeptide-2 Gene Mutations

The aim of this study was to investigate the genotypic distribution of organic anion transporting polypeptide 2 (OATP-2) gene mutations and the relationship with hyperbilirubinemia of unknown etiology. Polymerase chain reaction, restriction fragment length polymorphism, and agarose gel electrophoresis techniques were used for detection of OATP-2 gene mutations in 155 newborn infants: 37 with unexplained hyperbilirubinemia, 65 with explained hyperbilirubinemia, and 53 without hyperbilirubinemia. In the OATP-2 gene, we identified A→G transitions at nucleotide positions 388 and 411 and observed six polymorphic forms. The 388/411-411 mutation was the most common form (43%) in subjects with hyperbilirubinemia of unknown etiology. Male sex [odds ratio (OR): 3.08] and two polymorphic forms of the OATP-2 gene [the 388/411-411 A→G mutation (OR: 3.6) and the 388-411 mutation (OR: 2.4)] increased the risk of neonatal hyperbilirubinemia. In male infants with the 388 A→G mutation of the OATP-2 gene, the levels of unconjugated bilirubin in plasma were significantly increased compared with those observed in females. The polymorphic forms of 388 nucleotide of the OATP-2 gene were identified as risk factors for hyperbilirubinemia of unknown etiology.

Omega-3 Fatty Acids Reduce the Number of Crisis and Steady State Hemolysis In Sickle Cell Disease

AbstractAbstract 1624 Background and Study Objectives.Previous studies showed that: (1) increased blood levels of the omega-3 (n3) fatty acids (FA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are associated with increased steady-state Hb levels and reduced prevalence of the complications of sickle cell disease (SCD); (2) EPA and DHA reduced the number of crisis in a pilot group of 5 HbSS patients. The primary aim of this study was to assess the effects of EPA and DHA in a larger number of patients. SCD causes recurrent vaso-occlusion, ischemia-reperfusion injury and inflammation (Osarogiagbon et al. Blood. 2000, 96: 314–320). A metabolite of DHA (10,17S-docosatriene) protects against ischemia-reperfusion injury by inhibiting leukocyte infiltration and expression of pro-inflammatory genes (Marcheselli et al, J Bio Chem. 2003; 278: 43807–43817. Our hypothesis was that anti-inflammatory properties contribute to the mechanism(s) of action of DHA/EPA in SCD. The secondary objective was to test this hypothesis. Study Design and Methods.Following Institutional Review Board approval and informed consent, 20 HbSS patients (10 Males, 10 Females) were enlisted to receive oral EPA 15mg /kg/day and DHA 10 mg/kg/day with standard treatment of SCD, and reviewed at 2-monthly intervals for 6 months. Standard treatment included folic acid 5 mg daily, pyrimethamine-sulfadoxine (25mg/500mg) 2–3 tablets monthly for malaria prophylaxis, and appropriate management of acute clinical events. To assess the placebo effect of regular clinical monitoring, another group of 8 HbSS patients (2 Males, 6 Females) were enlisted to be reviewed 2-monthly for 6 months, but observed on standard treatment only. Inclusion criteria for both groups were 3 or more crisis/yr and age>5yrs. Exclusion criteria: pregnancy, hydroxyurea or regular transfusion therapy, any other disease e.g diabetes. To reduce the confounding effect of individual variability in severity of SCD, the study was so designed that each participant was his/her own control. Using the Mann-Whitney test, we compared pre- and post-treatment numbers of crisis over 6 months, steady state levels of Hb and plasma unconjugated bilirubin. To test the hypothesis that anti-inflammatory effects contribute to the mode of action DHA/EPA, we compared 3 indices of inflammation pre-and post-treatment: plasma level of interleukin-6 measured by ELISA, peripheral blood neutrophil and platelet counts determined with an automated cell counter. Results.In the omega-3 FA group 4 participants dropped out or were withdrawn from the study: 1 developed recurrent transient ischemic attacks and started regular blood transfusion to prevent stroke; 1 had recurrent severe crisis and began hydroxyurea therapy; 2 were not compliant with follow-up and omega-3 FA therapy. For the 16 evaluable participants (7M and 9F aged 9–33 yrs) the median number of crisis over 6 months reduced from 3 to 0 (p<0.0001) and steady state unconjugated bilirubin from 14.95 umol/l to 8.6 umol/l (p = 0.03). There were no significant differences between pre and post-treatment median neutrophil count (4.94 × 109/l, 4.36 × 109/l, p = 0.32) platelet count (318 × 109/l, 255 × 109/l, p= 0.057) Hb level (7.7 g/dl, 7.5 g/dl, p=1.0) and IL-6 (10.5 pg/ml, 12 pg/ml, p = 0.59).In the standard treatment group, 3 participants (2M, 1F) were not compliant with follow-up. The evaluable 5 female HbSS patients, age 17–26 yrs, had no significant change between months 0 and 6 median number of crisis (1.5, 1, p = 0.12) steady state Hb (7.6 g/dl, 6.6 g/dl, p = 0.54) unconjugated bilirubin 24 umol/l, 30 umol/l, p = 0.84), neutrophil count (5.45 × 109/, 4.66 × 109/, p = 0.15) and platelet count 359 × 109/, 411 × 109/, p = 0.84). Months 0 and 6 plasma IL-6 concentrations could be measured in only 3 participants in the standard treatment group. Since this number is too small for statistical analysis, the respective 0 and 6 month values for the 3 individuals are provided: 8 pg/ml and 7 pg/ml, 9 pg/ml and 9.6 pg/ml, 18 pg/ml and 22 pg/ml. Conclusions.The data suggest that EPA and DHA reduce the number of crisis and steady state hemolysis in SCD, and do not support the hypothesis that these effects are mediated by inhibiting inflammation. Disclosures:No relevant conflicts of interest to declare.

Acceleration of the gastrointestinal transit by polyethylene glycol effectively treats unconjugated hyperbilirubinaemia in Gunn rats

Background and aimsSeveral conditions that delay gastrointestinal transit are associated with unconjugated hyperbilirubinaemia. We hypothesised that the gastrointestinal transit time is directly related to plasma unconjugated bilirubin (UCB) concentrations, and...

Inhibition of bilirubin metabolism induces moderate hyperbilirubinemia and attenuates ANG II-dependent hypertension in mice

Population studies indicate that moderate hyperbilirubinemia is associated with reduced incidence of cardiovascular diseases, including hypertension. Despite this correlative evidence, no studies have directly tested the hypothesis that moderate increases in plasma bilirubin levels can attenuate the development of hypertension. This hypothesis was tested by treating mice with Indinavir, a drug that competes with bilirubin for metabolism by UDP-glucuronosyltransferase 1A1 (UGT1A1). Treatment of mice with Indinavir (500 mg x kg(-1) x day(-1), gavage) resulted in a twofold increase in plasma unconjugated bilirubin levels. Next, we determined the effect of Indinavir-induced changes in plasma bilirubin on the development of ANG II-dependent hypertension. Moderate hyperbilirubinemia was induced 3 days before the implantation of an osmotic minipump that delivered ANG II at a rate of 1 microg x kg(-1) x min(-1). ANG II infusion increased mean arterial pressure (MAP) by 20 mmHg in control mice but by only 6 mmHg in mice treated with Indinavir (n = 6). Similar to Indinavir treatment, direct infusion of bilirubin (37.2 mg x kg(-1) x day(-1) i.v.) resulted in a twofold increase in plasma bilirubin levels and also attenuated the development of ANG II-dependent hypertension. Moderate hyperbilirubinemia resulted in an increase in plasma nitrate/nitrite levels, which averaged 36 +/- 2 vs. 50 +/- 7 microM in ANG II vehicle vs. Indinavir-treated mice (n = 5). Moderate hyperbilirubinemia resulted in attenuation of vascular oxidative stress as determined by dihydroethidium staining of aortic segments. These results indicate that moderate hyperbilirubinemia prevents ANG II-dependent hypertension by a mechanism that may involve decreases in vascular oxidative stress.

Effective Treatment of Unconjugated Hyperbilirubinemia With Oral Bile Salts in Gunn Rats

We tested the hypothesis that oral administration of bile salts, which are known to increase the biliary excretion of unconjugated bilirubin (UCB), decreases unconjugated hyperbilirubinemia in the Gunn rat model. Adult Gunn rats were fed a standard diet or the same diet supplemented with 0.5 weight % ursodeoxycholic acid (UDCA) or cholic acid (CA) for 1 or 6 weeks. UCB and urobilinoids, a family of intestinal UCB breakdown products, were determined in plasma, feces, or both. After 6 weeks of treatment, tracer 3H-UCB was administered intravenously to determine steady-state UCB kinetics over the next 60 hours. One-week treatment with UDCA or CA decreased plasma UCB concentrations by 21% and 30%, respectively (each P < .01). During the first 4 days of treatment, both UDCA and CA increased the combined fecal excretion of UCB and urobilinoids (+52% and +32%, respectively; each P < .01). Prolongation of treatment to 6 weeks caused a persistent decrease in plasma UCB concentrations to approximately 40% below baseline (each bile salt P < .001). (3)H-UCB kinetic studies showed that UDCA and CA administration decreased UCB pool size (-33% and -32%, respectively; each P < .05) and increased UCB fractional turnover (+33% and +25%, respectively; each P < .05). Dietary bile salt administration induces a large, persistent decrease in plasma UCB concentrations in Gunn rats. Both UDCA and CA enhance UCB turnover by increasing its fecal disposal. These results support the application of oral bile salt treatment in patients with unconjugated hyperbilirubinemia.