Unconjugated Bilirubin Levels Research Articles

Dental management of bilirubin encephalopathy patient: A case report and review

Patients with special health care needs often present with compromised oral health for which they need dental treatment. Such patients and often their guardians find it difficult to maintain oral health due to various reasons such as decreased manual dexterity and preference for soft diet. Bilirubin encephalopathy is a condition occurring as a result of neonatal jaundice/hyperbilirubinemia. Elevated levels of unconjugated bilirubin at birth often leads to brain damage manifesting as auditory and motor defects such as athetoid cerebral palsy. This paper reports one such case of dental management of a known case of bilirubin encephalopathy.

Monitoring the Response of Hyperbilirubinemia in the Mouse Brain by In Vivo Bioluminescence Imaging.

Increased levels of unconjugated bilirubin are neurotoxic, but the mechanism leading to neurological damage has not been completely elucidated. Innovative strategies of investigation are needed to more precisely define this pathological process. By longitudinal in vivo bioluminescence imaging, we noninvasively visualized the brain response to hyperbilirubinemia in the MITO-Luc mouse, in which light emission is restricted to the regions of active cell proliferation. We assessed that acute hyperbilirubinemia promotes bioluminescence in the brain region, indicating an increment in the cell proliferation rate. Immunohistochemical detection in brain sections of cells positive for both luciferase and the microglial marker allograft inflammatory factor 1 suggests proliferation of microglial cells. In addition, we demonstrated that brain induction of bioluminescence was altered by pharmacological displacement of bilirubin from its albumin binding sites and by modulation of the blood–brain barrier permeability, all pivotal factors in the development of bilirubin-induced neurologic dysfunction. We also determined that treatment with minocycline, an antibiotic with anti-inflammatory and neuroprotective properties, or administration of bevacizumab, an anti-vascular endothelial growth factor antibody, blunts bilirubin-induced bioluminescence. Overall the study supports the use of the MITO-Luc mouse as a valuable tool for the rapid response monitoring of drugs aiming at preventing acute bilirubin-induced neurological dysfunction.

Modulation of bilirubin neurotoxicity by the Abcb1 transporter in the Ugt1-/- lethal mouse model of neonatal hyperbilirubinemia.

Moderate neonatal jaundice is the most common clinical condition during newborn life. However, a combination of factors may result in acute hyperbilirubinemia, placing infants at risk of developing bilirubin encephalopathy and death by kernicterus. While most risk factors are known, the mechanisms acting to reduce susceptibility to bilirubin neurotoxicity remain unclear. The presence of modifier genes modulating the risk of developing bilirubin-induced brain damage is increasingly being recognised. The Abcb1 and Abcc1 members of the ABC family of transporters have been suggested to have an active role in exporting unconjugated bilirubin from the central nervous system into plasma. However, their role in reducing the risk of developing neurological damage and death during neonatal development is still unknown.To this end, we mated Abcb1a/b-/- and Abcc1-/- strains with Ugt1-/- mice, which develop severe neonatal hyperbilirubinemia. While about 60% of Ugt1-/- mice survived after temporary phototherapy, all Abcb1a/b-/-/Ugt1-/- mice died before postnatal day 21, showing higher cerebellar levels of unconjugated bilirubin. Interestingly, Abcc1 role appeared to be less important.In the cerebellum of Ugt1-/- mice, hyperbilirubinemia induced the expression of Car and Pxr nuclear receptors, known regulators of genes involved in the genotoxic response.We demonstrated a critical role of Abcb1 in protecting the cerebellum from bilirubin toxicity during neonatal development, the most clinically relevant phase for human babies, providing further understanding of the mechanisms regulating bilirubin neurotoxicity in vivo. Pharmacological treatments aimed to increase Abcb1 and Abcc1 expression, could represent a therapeutic option to reduce the risk of bilirubin neurotoxicity.

Modulation of Hemolytic and Hemoglobin/Heme Scavenging Profiles in Sickle Cell Anemia, Hereditary Spherocytosis and Paroxysmal Nocturnal Hemoglobinuria

Sickle Cell Anemia (SCA), Hereditary Spherocytosis (HS) and Paroxysmal Nocturnal Hemoglobinuria (PNH) can share some clinical complications, such as leg ulcers and priapism, due to hemolytic processes. Intravascular hemolysis (IVH) results in the release of cell-free hemoglobin (Hb) and heme into the circulation, potentially causing depletion of nitric oxide, oxidative stress and inflammation. We compared the hemolytic profile and heme/Hb scavenging profiles of patients with SCA on hydroxyurea therapy (SCAHU; 15-30 mg/kg/day for ≥ 2 months) or not (SCA), hereditary spherocytosis (HS), splenectomized HS (SHS) and paroxysmal nocturnal hemoglobinuria not on eculizumab therapy (PNH). Blood samples were collected from patients and from healthy volunteers (CON). Plasma levels of heme, haptoglobin (Hp) and hemopexin (Hpx) were quantified by colorimetric assay or ELISA. Fetal hemoglobin (HbF) was determined in the SCA groups by HPLC. Reticulocytes (RET) and cell counts were determined using an automated flow cytometry hematology system and serum levels of lactate dehydrogenase (LDH) and unconjugated bilirubin (UCB) were quantified by a modular colorimetric automation system. As expected, plasma heme was significantly elevated in the SCA group, compared to the CON group and with intermediate values in the SCAHU group (57.4±9.3, 24.5±2.9, 43.2±4.5µM, respectively, p<0.05, N≥10), correlating significantly and negatively with HbF levels in the SCA patients (r=-0.94, p<0.01). Accordingly, RET and UCB were significantly increased in SCA, compared to CON, with SCAHU presenting intermediate values (RET: 16.2±1.4; 1.9±0.1; 11.5±1.2%; UCB: 2.1±0.7; 0.2±0.0; 1.44±0.2 mg/dL; respectively, p<0.01, N≥10), indicating increased red cell turnover and heme catabolism in SCA. No significant elevations in plasma heme were observed in the HS and SHS groups, compared to CON (35.3±3.8 and 34.5±10.0µM; P>0.05, N≥10), although RET and UCB levels were significantly elevated in HS (8.27±1.07 % and 1.72±0.57 mg/dL, P<0.01, N≥13), but not SHS (2.80±0.17 % and 0.99±0.66 mg/dL, N≥11), perhaps indicating a redistribution of extravascular hemolysis (EHV) to low-level IVH in SHS. Surprisingly, plasma heme was not significantly elevated in PNH individuals studied (36.5±4.0 µM, N≥10), even though concentrations of plasma LDH were the highest in this patient group (1196±305 U/L; compared to 182±8, 352±73, 407±38. 211±19 and 182±28 U/L for CON, SCA, SCAHU, HS, SHS, respectively, P<0.01; N≥10) and LDH correlated significantly with plasma heme in this group (r=0.905, P<0.01 for PNH). RET and UCB presented and intermediate values in PNH (5.35±1.21% and 0.84±0.18 mg/dL, P<0.05, N≥10, compared to CON). To investigate whether hemolytic profiles may be modulated by the Hb and heme scavengers, Hp and Hpx, we measured these proteins in the circulations of patients. Hp was significantly depleted in SCA, SCAHU, HS and PNH (1.0±0.4, 1.0±0.6, 37.8±0.16, 12.3±10.2, respectively, compared to 142.9±20.6 mg/dL in CON, p<0.05; N≥10), while SHS presented similar levels to those of CON (189.9±38.8 mg/dL, N=5). In contrast, somewhat surprisingly, Hpx levels varied considerably in each patient group, with a significant depletion only observed in the SHS group (12.2±0.3, 15.9±5.4, 15.0±3.8, 6.0±1.5, 1.5±0.4, 17.8±5.0mg/dL for CON, SCA, SCAHU, HS, SHS, PNH, respectively; P<0.05, SHS compared to CON, N≥10). As such, plasma heme was slightly lower in PNH than in SCA, in association with very high plasma LDH, suggesting that either IVH is slightly less severe or that Hb/heme scavenging is more efficient in the PNH population studied than in SCA. While EHV may be abolished by splenectomy in HS, intermediate heme levels may suggest that some IVH, or reduced heme catabolism, may still occur in some of these patients. Interestingly, depleted Hp stores were coupled with maintained (or even elevated) Hpx levels in SCA and PNH, indicating that hemolytic processes may stimulate Hpx production, potentially via a heme-oxygenase-1 (HO-1)-dependent pathway. Furthermore, significantly diminished levels of Hpx in SHS suggest a role for the spleen in Hpx maintenance, possibly due to the importance of this organ for HO-1 activity. In conclusion, while parameters suggestive of hemolysis can be observed in SCA, HS and PNH, specific profiles of hemolytic markers and heme catabolism indicate that hemolytic events may be modulated differently in these diseases. DisclosuresConran:Bayer AG: Research Funding. Almeida:Jassen & Cilag: Other: Currently employed with.

Gilbert’s syndrome: protective effect on endothelial dysfunction

Abstract Objective Gilbert’s syndrome (GS), is a benign condition characterized by unconjugated hyperbilirubinemia related to a decreased hepatic glucuronidating activity without symptoms and signs of liver disease or overt hemolysis. In the present study, we aimed to assess circulating levels of asymmetric dimethylarginine (ADMA), pentraxin-3 (PTX-3) and high sensitivity C-reactive protein (hs-CRP) between patients with GS and controls and determine the correlation of unconjugated bilirubin (UCB) levels with these molecules as prognostic factors for vascular risk stratification and endothelial dysfunction. Methods Forty two patients with GS and 37 age and sex matched control subjects were enrolled in this study. The diagnosis of GS was made by unconjugated hyperbilirubinemia (1 mg/dL or &gt;17.1 μmol/L) on at least two occasions with normal values of other liver function tests, normal hepatic imaging, and absence of hemolysis. Results Serum ADMA, PTX-3 and hs-CRP levels were significantly lower in GS than the healthy controls (p=0.037, p=0.025 and p=0.040, respectively). In correlation analysis, UCB was negatively correlated with ADMA, PTX-3 and hs-CRP (r=−0.239, p=0.034; r=−0.280, p=0.012 and r=−0.224, p=0.047, respectively). Discussion and conclusion The present study showed for the first time that decreased levels of ADMA, PTX-3 and hs-CRP may prove the protective effects of hyperbilirubinemia on the endothelial dysfunction.

Relation between unconjugated bilirubin and RDW, neutrophil to lymphocyte ratio, platelet to lymphocyte ratio in Gilbert's syndrome.

BackgroundUnconjugated bilirubin (UCB) plays a protective role in coronary artery disease. Red cell distribution width (RDW), neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) are inflammatory biomarkers and higher levels are related to atherosclerosis and adverse cardiovascular events.AimWe aimed to investigate the relation between UCB levels and RDW, NLR, PLR in people with Gilbert’s syndrome (GS). Materials and methodsWe selected 2166 subjects (1082 with GS and 1084 healthy controls) from a database having 33,695 people. RDW, NLR and PLR were investigated in the subjects with GS and compared with the healthy controls. Linear regression analysis was used to evaluate the relation between variables.ResultsNLR and PLR were higher in the subjects with GS compared to the controls (p < 0.001). RDW was similar in both groups (p = 0.318). UCB was negatively correlated with lymphocyte counts (p = 0.040), and positively correlated with RDW (p < 0.001) and PLR (p = 0.037) in the subjects with GS. There was no significant correlation between UCB and NLR (p = 0.078). RDW (p < 0.001) and lymphocyte counts (p = 0.030) were significantly associated with UCB levels in the regression analysis conducted in the subjects with GS.ConclusionThere is a negative association between UCB and NLR, PLR due to low amounts of lymphocyte counts, which causes increased risk of CVD. These results suggest that the cardio-protective effect of UCB is due to both anti-oxidative and anti-inflammatory ways indirectly.

Fluorescent protein-based detection of unconjugated bilirubin in newborn serum.

Increased serum levels of unconjugated bilirubin are associated with the development of brain damage in newborns. In current clinical settings, there are no methods for directly determining serum levels of unconjugated bilirubin. UnaG, a fluorescent protein from Japanese eel muscle that specifically binds to unconjugated bilirubin was used in this study. Linear regression analysis was carried out to compare unconjugated bilirubin levels measured by UnaG and conventional bilirubin oxidase methods. Unconjugated bilirubin levels in the serum of newborns who were untreated or treated with phototherapy were compared. Effects of interfering factors in the serum (conjugated bilirubin, hemoglobin, and lipid) on unconjugated bilirubin concentration measured by the UnaG method were also evaluated. Unconjugated bilirubin levels measured by the UnaG method were highly correlated with those determined by the bilirubin oxidase assay. Unconjugated bilirubin levels determined by bilirubin oxidase and UnaG assays were similar in serum samples containing conjugated bilirubin. The performance of the UnaG assay was unaffected by phototherapy and the presence of serum hemoglobin and lipid emulsion. These results demonstrate the clinical applicability of the UnaG method for direct measurement of unconjugated bilirubin levels in newborn serum.

Hemato-Biochemical Alterations in Water Buffaloes Clinically Infected with Bovine Theileriosis before and after tTreatment by Buparvaquone

Many studies have been done about theileriosis in cows and derelict its effect in water buffaloes. So this study was carried out to ascertain the changes in hematology and serum constituents in buffaloes affected with bovine theileriosis in Egypt. A total of 19 buffaloes of age 2-5 years were included in the study. Other animals of same age were kept as healthy controls. The affected animals showed signs and symptoms of bovine theileriosis included high fever, enlarged prescapular lymph nodes, corneal opacity, lacrimation, etc. Blood smears confirm the presence of T. annulata piroplasms in red blood cells. Hematological studies revealed decrease in erythrocytic count, packed cell volume and hemoglobin before treatment of theileria infected buffaloes, normocytic normochromic in theileria infected buffaloes and macrocytic hypochromic at one week post buparvaquone administration. Leucogram revealed leucocytosis. theileria infected buffaloes revealed increase in the serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and serum levels of total bilirubin, unconjugated bilirubin (UB), conjugated bilirubin (CB), creatinine and blood urea nitrogen level. In contrary, total serum proteins and albumin showed significant decrease. Antioxidants results revealed a significant increase in Malondialdehyde (MDA) activity and a significant decease in the activity of catalase (CAT) in T. annulata clinically infected buffaloes. These alterations were improved by treatment the diseased animals by buparvaquone 2 weeks post administration.

Unconjugated bilirubin elevation impairs the function and expression of breast cancer resistance protein (BCRP) at the blood-brain barrier in bile duct-ligated rats.

Liver failure is associated with dyshomeostasis of efflux transporters at the blood-brain barrier (BBB), which contributes to hepatic encephalopathy. In this study we examined whether breast cancer resistance protein (BCRP), a major efflux transporter at the BBB, was altered during liver failure in rats. Rats underwent bile duct ligation (BDL) surgery, and then were sacrificed after intravenous injection of prazosin on d3, d7 and d14. The brains and blood samples were collected. BCRP function at the BBB was assessed by the brain-to-plasma prazosin concentration ratio; Evans Blue extravasation in the brain tissues was used as an indicator of BBB integrity. The protein levels of BCRP in the brain tissues were detected. Human cerebral microvessel endothelial cells (HCMEC/D3) and Madin-Darby canine kidney cells expressing human BCRP (MDCK-BCRP) were tested in vitro. In addition, hyperbilirubinemia (HB) was induced in rats by intravenous injection of unconjugated bilirubin (UCB). BDL rats exhibited progressive decline of liver function and HB from d3 to d14. In the brain tissues of BDL rats, both the function and protein levels of BCRP were progressively decreased, whereas the BBB integrity was intact. Furthermore, BDL rat serum significantly decreased BCRP function and protein levels in HCMEC/D3 cells. Among the abnormally altered components in BDL rat serum tested, UCB (10, 25 μmol/L) dose-dependently inhibit BCRP function and protein levels in HCMEC/D3 cells, whereas 3 bile acids (CDCA, UDCA and DCA) had no effect. Similar results were obtained in MDCK-BCRP cells and in the brains of HB rats. Correlation analysis revealed that UCB levels were negatively correlated with BCRP expression in the brain tissues of BDL rats and HB rats as well as in two types of cells tested in vitro. UCB elevation in BDL rats impairs the function and expression of BCRP at the BBB, thus contributing to hepatic encephalopathy.

Cardiovascular changes in patients with non-severe Plasmodium vivax malaria

BackgroundCardiovascular system involvement in patients with Plasmodium vivax malaria has been poorly addressed. The aim of this study was to evaluate cardiac structures and function, and serum markers of cardiovascular injury in patients with the non-severe form of vivax malaria in Manaus, Amazonas State, Brazil. Methods and resultsWe prospectively evaluated 26 patients with vivax malaria in an outpatient referral hospital and compared results with a control group of 25 gender- and age-matched healthy individuals. Patients underwent clinical evaluation, laboratory tests, and transthoracic echocardiography at first evaluation (day zero, D0) and seven days (D7) after malaria diagnosis. At D0 echocardiography showed higher left ventricular (LV) systolic diameter (28.8±2.82 vs 30.9±4.03mm; p=0.037) and LV diastolic volume (82.4±12.3 vs 93.8±25.9ml; p=0.05), and lower LV ejection fraction (Teicholz method: 73.2±6.59 vs 68.4±4.87%; p=0.004) in patients compared to controls. Right ventricle (RV) fractional area change (54.7±5.11 vs 50.5±6.71%; p=0.014) was lower, and RV myocardial performance index (0.21±0.07 vs 0.33±0.19; p=0.007), and pulmonary vascular resistance (1.13±0.25 vs 1.32±0.26 Woods unit; p=0.012) were higher in patients than controls. Patients presented higher serum levels of unconjugated bilirubin (0.24±0.15 vs 1.30±0.89mg/dL; p<0.001), soluble vascular cell adhesion molecule–1 (sVCAM-1; 453±143 vs 1983±880ng/mL; p<0.001), N-terminal prohormone brain natriuretic peptide (0.59±0.86 vs 1.08±0.81pg/mL; p=0.045), and troponin T (861±338 vs 1037±264pg/mL; p=0.045), and lower levels of plasma nitrite (13.42±8.15 vs 8.98±3.97μM; p=0.016) than controls. Most alterations had reversed by D7. ConclusionPatients with non-severe Plasmodium vivax malaria present subclinical reversible cardiovascular changes.

Relationship between plasma bilirubin level and oxidative stress markers in HIV-infected patients on atazanavir- vs. efavirenz-based antiretroviral therapy.

Chronic oxidative stress (OS) may play a role in cardiovascular disease in HIV-infected patients, and increased bilirubin levels may have a beneficial role in counteracting OS. Atazanavir (ATV) inhibits UDP-glucuronosyl-transferase 1A1 (UGT1A1), thus increasing unconjugated bilirubin levels. We aimed to compare changes in OS markers in patients on ATV/ritonavir (ATV/r)- vs. efavirenz (EFV)-based first-line antiretroviral therapy (ART). A multicentre, prospective cohort study of HIV-infected patients who started first-line ART with either ATV/r or EFV was conducted. Lipoprotein-associated phospholipase A2 (Lp-PLA2), myeloperoxidase (MPO) and oxidized low-density lipoprotein (oxLDL) were measured for 145 patients in samples obtained at baseline and after at least 9 months of ART during which the initial regimen was maintained and the patient was virologically suppressed. The change in OS markers was modelled using multiple linear regressions adjusting for baseline values and confounders. After adjustment for baseline variables, patients on ATV/r had a significantly greater decrease in Lp-PLA2 [estimated difference -16.3; 95% confidence interval (CI) -31.4, -1.25; P = 0.03] and a significantly smaller increase in OxLDL (estimated difference -21.8; 95% CI -38.0, -5.6; P < 0.01) relative to those on EFV, whereas changes in MPO were not significantly different (estimated difference 1.2; 95% CI -14.3, 16.7; P = 0.88). Adjusted changes in bilirubin were significantly greater for the ATV/r group than for the EFV group (estimated difference 1.33 mg/dL; 95% CI 1.03, 1.52 mg/dL; P < 0.01). Changes in bilirubin and changes in OS markers were significantly correlated. When compared with EFV, ATV/r-based therapy was associated with lower levels of oxidative stress biomarkers, which was in part attributable to increased bilirubin levels.

Statin Intolerance in a Patient with Gilberts Syndrome and Hypercholesterolemia

Abstract Background: Statin intolerance especially myalgias can be a serious problem. Whilst it is well know that drugs that compete for Cytochrome 450 system can result in myalgias there is sparse data on the role of glucuronidation of statins contributing to statin intolerance We report on a 60 year old male with Hypercholesterolemia (HC) who was referred for management of his HC since he had statin intolerance manifesting as myalgias and was shown to have Gilbert’s Syndrome. Case Report: Investigation of this patient revealed he had Familial Combined Hyperlipidemia with a LDL-cholesterol of 189 mg/dl. He was also diagnosed with Gilbert’s Syndrome since he had elevated unconjugated bilirubin with no evidence of liver disease or hemolysis. The combination of Niacin, Cholestyramine and ezetimibe resulted in a successful decrease in his LDL-cholesterol to 114 mg/dl. Discussion: We believe that his Gilberts Syndrome resulted in an impairment in glucuronidation of statin drugs resulting in an increase in free drug levels and myalgias. We caution that clinicians should consider this possibility when confronted with a patient with both isolated elevations of unconjugated bilirubin and increase LDL-cholesterol levels before commencing statin therapy.

Longer telomeres in chronic, moderate, unconjugated hyperbilirubinaemia: insights from a human study on Gilbert's Syndrome.

Bilirubin (BR) is a natural endogenous compound with a potent bioactivity. Gilbert’s Syndrome (GS) is a benign hereditary condition of increased unconjugated bilirubin (UCB) in serum and serves as a convenient model for studying the effects of BR in humans. In absence of liver disease, increased UCB levels are inversely associated to all-cause mortality risk, especially from cardiovascular diseases (CVDs). On the other hand, telomere malfunction is linked to a higher risk of CVDs. To our knowledge, there is no data on whether UCB is linked to telomere length in healthy or diseased individuals In the present study we have observed a relationship between mildly increased serum UCB and telomere length. We used an in vivo approach, assessing telomere length in PBMCs from individuals with GS (n = 60) and matched healthy controls (n = 60). An occurrence of longer telomeres was observed in male individuals chronically exposed to increased UCB, as well as in Gunn rats, an animal model of unconjugated hyperbilirubinaemia. Previously identified differences in immunomodulation and redox parameters in individuals with GS, such as IL-6, IL-1β and ferric reducing ability of plasma, were confirmed and proposed as possible contributors to the occurrence of longer telomeres in GS.

Unconjugated Bilirubin Is a Novel Prognostic Biomarker for Nasopharyngeal Carcinoma and Inhibits Its Metastasis via Antioxidation Activity.

Distant metastasis is the most common cause of treatment failure and mortality in nasopharyngeal carcinoma (NPC) patients. Thus, it is important to understand the mechanism of NPC metastasis and identify reliable prognostic factors. In this study, we investigated the prognostic value of unconjugated bilirubin (UCB), which was previously considered a byproduct of heme catabolism, in NPC patients and examined the effects of UCB on NPC metastasis. The receiver operating characteristic analysis-generated UCB cutoff point for DMFS was 9.7 μmol/L. We found that higher UCB levels were significantly associated with favorable distant metastasis-free survival (DMFS, 93.3% vs. 84.2%, P < 0.001) in NPC patients and was an independent predictor for DMFS (HR, 0.416; 95% confidence interval, 0.280-0.618; P < 0.001). We next found that UCB treatment impaired the invasion capability of NPC cells and potently inhibited lung metastasis of NPC cells in nude mice. Further investigation showed that UCB inhibited reactive oxygen species production, which is involved in the repression of ERK1/2 activation and matrix metalloproteinase-2 (MMP-2) expression. Moreover, lower levels of ERK1/2 phosphorylation and MMP-2 expression were observed in the NPC lung metastases of nude mice administered UCB. Taken together, our results indicate that UCB is a significantly favorable factor for DMFS in NPC patients and may play an important role in NPC chemoprevention.

Analysis of uridine diphosphate glucuronosyl transferase 1A1 gene mutations in neonates with unconjugated hyperbilirubinemia.

This study was carried out to analyze uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) gene mutations in neonates with unconjugated hyperbilirubinemia, from two different ethnic groups. Polymerase chain reaction and gene sequencing were used to analyze the differences in genotypes and allele frequencies of different gene mutations among the ethnic groups; this was followed by checking their correlation with the serum bilirubin level and the occurrence of unconjugated hyperbilirubinemia in neonates. Our results reveal that the UGT1A1 mutant genotype, 211G>A, is distributed differently in the case vs control groups, as well as in the Zhuang vs Han ethnic groups. Moreover, this difference is statistically significant (P < 0.05); the total serum bilirubin (TSB) and unconjugated bilirubin (UCB) levels in patients carrying the single homozygous mutation, 211G>A, were markedly higher than that in patients without the mutation (P < 0.05). Furthermore, the TSB and UCB levels were significantly different between patients carrying single or compound 211G>A heterozygous mutation, (TA)6/7, and 1941C>G/2042C>G heterozygous mutation, and patients without mutation (P > 0.05). Our findings suggest that the 211G>A mutation in the first exon may be a risk factor for unconjugated hyperbilirubinemia in Zhuang and Han neonates. The serum bilirubin levels seem to be affected by the homozygosity or heterozygosity of the UGT1A1 gene mutation; 211G>A homozygous mutation is an important factor that causes a rise in bilirubin in neonates with unconjugated hyperbilirubinemia.

Protective Effect of Aged Garlic Extract Against the Oxidative Stress Induced by Acute Ionizing Irradiation on Hepatic Antioxidant Enzymes in Rats

Ionizing radiations damage cells, tissues and organs among which the liver through a cascade of molecular events that are triggered by reactive oxygen species (ROS), lipid peroxidation and nitrogen species (NS). Aged Garlic Extract (AGE) has been demonstrated to possess free radical scavenging capacity and antioxidant activity. Therefore, the present study has been focused in analyzing the properties of AGE against the lipid peroxidation and oxidative damages of rat liver tissues induced by acute radiation. Eight groups, five healthy male rats each were used (20 irradiated and 20 Sham Irradiated), among which some were receiving via gavages distilled water, the others AGE at different doses (25 mg/kg and 50 mg/kg) and the rest vitamin E + Alpha Lipoic Acid. Then, biochemical analyses, lipid peroxidation, total Protein and antioxidants assessment were made from blood samples and liver tissue homogenates. Exposure of rats to gamma radiation caused a significant increase in the level of Malondialdehyde, Nitrite, transaminase enzymes, alkaline phosphatase, and Bilirubin (Total Serum Bilirubin, Direct Bilirubin and Unconjugated Bilirubin) level while a significant decrease was recorded in serum total proteins, glutathione content, superoxide dismutase, catalase activities and total protein level. AGE treated rats revealed a significant improvement in all previous parameters. From these results, it can be concluded that AGE may have significant anti-radiation properties in rat’s liver after radiation exposure.

GBT440, a Potent Anti-Sickling Hemoglobin Modifier Reduces Hemolysis, Improves Anemia and Nearly Eliminates Sickle Cells in Peripheral Blood of Patients with Sickle Cell Disease

▪Background: Sickle cell disease (SCD) is caused by polymerization of Hemoglobin S, resulting in red blood cell (RBC) sickling, RBC destruction, vaso-occlusive episodes and end-organ damage. No direct anti-polymerization, mechanism-based, preventive therapy is available. GBT440 is a novel small molecule hemoglobin modifier which increases hemoglobin oxygen affinity. In vitro and in vivo studies have shown it to be a potent and direct anti-sickling agent with high specificity for hemoglobin, and that 10-30% hemoglobin modification could be both safe (not compromising oxygen delivery) and effective (preventing HbS polymerization). We hypothesized that a potent antisickling hemoglobin modifier should rapidly interrupt RBC hemolysis, improve anemia and potentially become a safe and effective long-term disease-modifying therapy. We therefore explored safety, pharmacokinetics and pharmacodynamics and potential efficacy in healthy volunteers and SCD patients.Methods: This prospective, randomized, placebo-controlled, double blind, parallel group phase I/II study enrolled healthy volunteers (HV) ages 18-55, and homozygous HbSS SCD patients ages 18-60 with baseline Hb levels ³6 g/dL and ²10 g/dL and without vaso-occlusive crisis or transfusion within 30 days of screening. The study was conducted in two parts: part A tested single ascending doses and part B multiple ascending doses of study drug with 6:2 randomization (GBT440:placebo). Doses administered were: part A, HV cohorts 100mg to 2800 mg, SCD cohort 1000 mg; part B, HV cohorts 300 mg to 900 mg once daily for 15 days, SCD cohort 700 mg once daily for 28 days. The primary endpoint was safety. Secondary endpoints included pharmacokinetics (PK) and pharmacodynamics (PD). Clinical indices of hemolysis were prespecified as endpoints and are described for the SCD multiple dose cohort (700 mg QD for 28 days). Descriptive statistics were used to analyze data.Results: As of July 24 2015, 64 healthy volunteers (HVs) and 16 SCD patients had been enrolled. 54 healthy volunteers had completed the study, 2 were discontinued due to mild-moderate nonserious adverse events (headache, rash) and 8 were in follow-up; 8 SCD patients had completed Part A of the study and 8 were in Part B follow-up. No SCD patients were discontinued; one part B subject had a dose reduction from 700 mg to 400 mg (due to abdominal discomfort). GBT440 was generally well-tolerated, most adverse events were mild, there were no deaths, and there was one serious adverse event (AE) of acute painful crisis in a placebo subject. There were no AEs related to tissue hypoxia. GBT440 showed dose proportional PK, a terminal half-life of 1.5-3 days, high partitioning into RBCs (RBC:Plasma 60-90:1), and a dose dependent increase in hemoglobin oxygen affinity in both HV and SCD patients. In the multiple dose SCD cohort, all patients were evaluable at 28 days (age range 21-52 years; 2 were on hydroxyurea; 3 males/5 females). GBT440-treated patients showed increased hemoglobin, hematocrit and erythrocyte counts with corresponding decreases in LDH, unconjugated bilirubin, reticulocytes and erythropoietin levels with trends evident as early as day 8 compared to no changes in the placebo group (Figure 1 and Table 1). Analysis of peripheral blood smears revealed a marked reduction in sickle cells with GBT440 treatment.Conclusions: Single, oral, daily dosing with GBT440 was well tolerated across a wide dose range and demonstrated dose proportional and predictable PK and PD. GBT440 demonstrated proof of mechanism with a dose-dependent increase in hemoglobin oxygen affinity without causing tissue hypoxia. GBT440 demonstrated proof of concept in SCD patients with rapid reduction in RBC hemolysis and improved oxygen delivery to tissues as evidenced by reduced erythropoietin level, and a marked reduction in circulating sickle cells. These results support further clinical investigation of GBT440 as a potential disease-modifying therapy for SCD.Table 1Mean change to Day 28 (SEM)GBT440PLAHemoglogin (g/dL)0.8 (0.2)-0.5 (0.4)% Sickle cells in peripheral blood (%)-83 (9)19 (4)Erythrocyte count (1012/L)0.5 (0.1)-0.1 (0.2)Reticulocyte count (%)-2.2 (1)0.8 (1.6)LDH (% change from baseline)-12 (7)-9 (1)Unconjugated bilirubin (% change from baseline)-25 (10)-9.6 (16)Erythropoietin (U/L)-29 (16)21 (33)GBT = GBT440 treated subject; PLA = placebo subject; SEM = standard error of the mean [Display omitted] DisclosuresLehrer-Graiwer:Global Blood Therapeutics: Employment, Equity Ownership. Howard:Pfizer: Consultancy; Novartis: Consultancy, Other: Travel Grant; Aes-Rx: Consultancy. Layton:Agios: Consultancy; Novartis: Consultancy; Glaxo Smith Kline: Consultancy. Mant:Quintiles: Employment, Equity Ownership. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Koller:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Patel:Global Blood Therapeutics: Employment, Equity Ownership. Ramos:Global Blood Therapeutics: Employment, Equity Ownership.

A Case of Doxycycline and Cefuroxime-Induced Liver Injury

Introduction: Drug induced liver injury (DILI) is a relevant topic in clinical practice. Doxycycline and Cefuroxime are commonly used and well tolerated. We describe a case of DILI from Doxycycline and Cefuroxime. Case presentation: A 56 year old male presented with fever. Past history was significant for a tick bite treated for possible Lyme disease with Doxycycline which he did not tolerate after 7 days followed by Cefuroxime for 2 weeks. He had another 2 week course of Cefuroxime after another tick bite. He had no relevant family history. He drank alcohol once a week. Physical examination was unremarkable. Laboratory tests revealed ALT: 704 U/L, AST: 612 U/L, total bilirubin: 1.9 mg/dL, direct bilirubin: 0.6 mg/dL, indirect bilirubin: 1.3 mg/dL, ALP: 589 U/L, GGT: 848 U/L, INR: 0.9 and albumin: 3.7 g/dL. EBV IGM antibody was equivocal at 36.8 U/ml. Blood cultures and serologies for viral hepatitis, Lyme disease, CMV, Babesia microti and Anaplasma phagocytophilum were negative. Abdominal ultrasound was negative for biliary dilation. A liver biopsy revealed portal and lobular chronic inflammation with mild cholestasis compatible with DILI. EBV in situ hybridization was negative. His liver function tests started to improve within days after stopping Cefuroxime. Discussion: Doxycycline is among the class of tetracyclines that can increase the level of unconjugated bilirubin. It has been shown to be safer and less hepatotoxic than Tetracycline. Doxycycline is reported to cause acute liver failure, hepatocellular necrosis, and cholestasis. Cefuroxime is a second generation cephalosporin that has also been reported to cause cholestasis, ischemic hepatitis, and liver failure. We believe that our patient suffered from Doxycycline and Cefuroxime DILI. The 3 courses of antibiotics could have been the culprit for the portal and lobular chronic inflammation on biopsy. There are few case reports about the hepatotoxic effects of Doxycycline and Cefuroxime. Physicians should be mindful of this adverse drug event since these are commonly used medications.

Neuroprotective Core Measure 6: Protecting Skin - Neuroprotective Care in the Newborn: Does Skin Protect the Immature Brain From Hyperbilirubinemia?

Hyperbilirubinemia continues to pose a significant and common problem in the newborn period. Exposure of the brain to high levels of unconjugated bilirubin leads to acute bilirubin encephalopathy and kernicterus, especially in preterm infants. Given the shared embryological origin of the skin (epidermis) and brain, we hypothesized that cutaneous binding of unconjugated bilirubin to skin (i.e., jaundice) might protect the immature brain. Support for this hypothesis requires direct quantification of binding of unconjugated bilirubin to cutaneous structures. Bilirubin binding was tested using a series of in vitro experiments wherein newborn skin and vernix caseosa were exposed to physiologically-relevant solutions of bilirubin. Tissue binding was assessed spectrophotometrically and via bilirubin autofluorescence. Study findings indicate the following: (1) unconjugated bilirubin binds quickly and avidly to thin films of vernix caseosa; (2) bilirubin binds to human epidermis in vitro via a mechanism involving dermal diffusion; (3) unconjugated bilirubin localizes to the dermis and epidermis as shown by autofluorescence; and (4) topical application of vernix caseosa to the epidermis augments bilirubin binding; i.e., increases jaundice. These findings are consistent with a physiological neuroprotective role for the skin in shielding the immature brain from high levels of unconjugated bilirubin. New therapies based on these results are envisioned with the goal of increasing cutaneous bilirubin binding (jaundice) thereby protecting the developing brain and facilitating bilirubin excretion with phototherapy.

Exome-Wide Association Study Identifies New Low-Frequency and Rare UGT1A1 Coding Variants and UGT1A6 Coding Variants Influencing Serum Bilirubin in Elderly Subjects: A Strobe Compliant Article.

Genome-wide association studies (GWASs) have identified loci contributing to total serum bilirubin level. However, no exome-wide approaches have been performed to address this question. Using exome-wide approach, we assessed the influence of protein-coding variants on unconjugated, conjugated, and total serum bilirubin levels in a well-characterized cohort of 773 ambulatory elderly subjects from Italy. Coding variants were replicated in 227 elderly subjects from the same area. We identified 4 missense rare (minor allele frequency, MAF < 0.5%) and low-frequency (MAF, 0.5%-5%) coding variants located in the first exon of the UGT1A1 gene, which encodes for the substrate-binding domain (rs4148323 [MAF = 0.06%; p.Gly71Arg], rs144398951 [MAF = 0.06%; p.Ile215Val], rs35003977 [MAF = 0.78%; p.Val225Gly], and rs57307513 [MAF = 0.06%; p.Ser250Pro]). These variants were in strong linkage disequilibrium with 3 intronic UGT1A1 variants (rs887829, rs4148325, rs6742078), which were significantly associated with total bilirubin level (P = 2.34 × 10(-34), P = 7.02 × 10(-34), and P = 8.27 × 10(-34)), as well as unconjugated, and conjugated bilirubin levels. We also identified UGT1A6 variants in association with total (rs6759892, p.Ser7Ala, P = 1.98 × 10(-26); rs2070959, p.Thr181Ala, P = 2.87 × 10(-27); and rs1105879, p.Arg184Ser, P = 3.27 × 10(-29)), unconjugated, and conjugated bilirubin levels. All UGT1A1 intronic variants (rs887829, rs6742078, and rs4148325) and UGT1A6 coding variants (rs6759892, rs2070959, and rs1105879) were significantly associated with gallstone-related cholecystectomy risk. The UGT1A6 variant rs2070959 (p.Thr181Ala) was associated with the highest risk of gallstone-related cholecystectomy (OR, 4.58; 95% CI, 1.58-13.28; P = 3.21 × 10(-3)). Using an exome-wide approach we identified coding variants on UGT1A1 and UGT1A6 genes in association with serum bilirubin level and hyperbilirubinemia risk in elderly subjects. UGT1A1 intronic single-nucleotide polymorphisms (SNPs) (rs6742078, rs887829, rs4148324) serve as proxy markers for the low-frequency and rare UGT1A1 variants, thereby providing mechanistic explanation to the relationship between UGT1A1 intronic SNPs and the UGT1A1 enzyme activity. UGT1A1 and UGT1A6 variants might be potentially associated with gallstone-related cholecystectomy risk.