Label-Free Detection of Unbound Bilirubin and Nitrophenol Explosives in Water by a Mechanosynthesized Dual Functional Zinc Complex: Recognition of Picric Acid in Various Common Organic Media.

High levels of unconjugated bilirubin (UB) in serum lead to asymptomatic and neonatal jaundice and brain dysfunctions. Herein, we have reported the detection of UB at as low as 1 µM in an aqueous alkaline medium using a Zn(II) complex. The specificity of the complex has been validated by the HPLC in the concentration window 6-90 µM, which is rare. The sensory response of the probe at physiological pH against nitro explosives developed it as an instant-acting fluorosensor for picric acid (PA) and 2,4-dinitrophenol (2,4-DNP). Spectroscopic titration provided a binding constant of 4×105  M-1 with PA. The naked eye detection was found to be 15 µM. The solid-state photoluminescent nature of the complex enabled it for PA sensing in the solid phase. Interestingly, the probe remained fluorescent in various volatile and non-volatile organic solvents. As a result, it can also detect PA and 2,4-DNP in a wide range of common organic media. NMR studies revealed the coordination of PA, 2,4-DNP, and UB to the Zn(II) center of the probe, which is responsible for the observed quenching of the probe with the analytes.

Bilirubin Molecular Species Play an Important Role in the Pathophysiology of Acute-on-Chronic Liver Failure.

Bilirubin plays a key role in early diagnosis, prognosis, and prevention of liver diseases. Unconjugated bilirubin (UCB) requires conversion to a water-soluble form through liver glucuronidation, producing monoglucuronide (BMG) or diglucuronide bilirubin (BDG) for bile excretion. This study aimed to assess the roles of bilirubin's molecular species-UCB, BMG, and BDG-in diagnosing and understanding the pathogenesis of liver cirrhosis in patients with acute-on-chronic liver failure (ACLF), compensated liver cirrhosis (LC) patients, and healthy individuals. The study included patients with ACLF and compensated LC of diverse etiologies, along with healthy controls. We collected laboratory and clinical data to determine the severity and assess mortality. We extracted bilirubin from serum samples to measure UCB, BMG, and BDG using liquid chromatography-mass spectrometry (LC-MS). The quantification of bilirubin was performed by monitoring the mass charge (m/z) ratio. Of the 74 patients assessed, 45 had ACLF, 11 had LC, and 18 were healthy individuals. Among ACLF patients, the levels of molecular species of bilirubin were UCB 19.69 µmol/L, BMG 47.71 µmol/L, and BDG 2.120 µmol/L. For compensated cirrhosis patients, the levels were UCB 11.29 µmol/L, BMG 1.49 µmol/L, and BDG 0.055 µmol/L, and in healthy individuals, the levels were UCB 6.42 µmol/L, BMG 0.52 µmol/L, and BDG 0.028 µmol/L. The study revealed marked elevations in the bilirubin species in individuals with ACLF compared to those with compensated cirrhosis and healthy controls, underscoring the progression of liver dysfunction. The correlation of BMG and BDG levels with commonly used inflammatory markers suggests a relationship between bilirubin metabolism and systemic inflammation in ACLF.

High-normal unconjugated bilirubin is associated with decreased risk of chronic kidney disease in type 2 diabetes: A real-world study.

OBJECTIVE: To investigate the association between serum unconjugated bilirubin (UCB) within normal limits and chronic kidney disease (CKD) in T2DM patients. METHOD: This cross-sectional, real-world study was performed in 8661 hospitalised T2DM patients. The subjects were stratified into quintiles based on serum UCB levels. The clinical characteristics and CKD prevalence were compared among the UCB quantile groups. The associations of serum UCB levels and quintiles with CKD were also analysed by binary logistic regression. RESULTS: After controlling for age, sex, and diabetes duration (DD), the CKD prevalence (20.4%, 12.2%, 10.6%, 8.3%, and 6.4% for the first, second, third, fourth, and fifth quintiles, respectively, p < 0.001 for trend) was significantly decreased across the serum UCB quintiles. The fully adjusted regression model showed negative associations of serum UCB levels (OR: 0.660, 95% CI: 0.585-0.744; p < 0.001 for trend) and quintiles (p < 0.001) with the presence of CKD. Compared with the subjects in the lowest UCB quintile, the risk of CKD decreased by 36.2%, 54.3%, 53.8%, and 62.1%, respectively, in those from the second to the highest UCB quintile. Additionally, C-reactive protein (CRP) levels were significantly higher in the subjects with CKD than in those without CKD (p < 0.001), and significantly decreased across the UCB quintiles (p < 0.001 for trend). CONCLUSIONS: Serum UCB levels within the normal range were significantly and negatively linked to CKD in T2DM patients. High-normal UCB may be an independent protective factor for CKD by its antioxidant and the following anti-inflammatory activities through its signalling activity, which was indicated by clearly decreased CRP levels across the UCB quintiles.

Preoperative serum bilirubin is an independent prognostic factor for curatively resected esophageal squamous cell carcinoma.

PURPOSE: This study examines prognostic value of preoperative serum bilirubin, including unconjugated bilirubin (UCB), conjugated bilirubin (CB), and total bilirubin (TB), in esophageal squamous cell carcinoma (ESCC) patients who underwent curative resection. METHODS: Between May 2010 and December 2012, a total of 351 ESCC patients were retrospectively reviewed. All the patients underwent curative resection as their primary treatment. Clinicopathological features and overall survival (OS) rate were investigated. Kaplan-Meier curves were used to calculate the OS rate, and the prognostic factors were identified by Cox regression model. Besides, the potential inhibition effect of UCB on ESCC was investigated with both in vitro and in vivo models. RESULTS: The higher-level groups of UCB, CB, and TB demonstrated longer OS than their low counterparts, with hazard ratio (HR) values of 0.567 (95% CI: 0.424-0.759), 0.698 (95% CI: 0.522-0.933), and 0.602 (95% CI: 0.449-0.807), respectively. All three forms of bilirubin were identified as independent prognostic factors for patients with ESCC, and they were found to effectively stratify the survival risk of patients at TNM stage III. In vivo and in vitro models further confirmed the inhibitory effect of unconjugated bilirubin (UCB) on the proliferation of ESCC. CONCLUSION: The findings of our study have shed new light on the prognostic value and biological functions of bilirubin in relation to ESCC. These results may contribute to a better understanding of the underlying mechanisms involved in ESCC tumorigenesis and provide potential therapeutic pathways for treating ESCC.

Expression profiling of N6-methyladenosine-modified mRNA in PC12 cells in response to unconjugated bilirubin.

BACKGROUND: Abnormal methylation of N6-methyladenosine (m6A) is reportedly associated with central nervous system disorders. However, the role of m6A mRNA methylation in unconjugated bilirubin (UCB) neurotoxicity requires further research. METHODS: Rat pheochromocytoma PC12 cells treated with UCB were used as in vitro models. After the PC12 cells were treated with UCB (0, 12, 18, and 24 µM) for 24 h, the total RNA m6A levels were measured using an m6A RNA methylation quantification kit. The expression of m6A demethylases and methyltransferases was detected through western blotting. We determined the m6A mRNA methylation profile in PC12 cells exposed to UCB (0 and 18 µM) for 24 h using methylated RNA immunoprecipitation sequencing (MeRIP-seq). RESULTS: Compared with the control group, UCB (18 and 24 µM) treatment decreased the expression of the m6A demethylase ALKBH5 and increased the expression of the methyltransferases METTL3 and METTL14, which resulted in an increase in the total m6A levels in PC12 cells. Furthermore, 1533 m6A peaks were significantly elevated and 1331 peaks were reduced in the UCB (18 µM)-treated groups compared with those in the control group. Genes with differential m6A peaks were mainly enriched in protein processing in the endoplasmic reticulum, ubiquitin-mediated proteolysis, cell cycle, and endocytosis. Through combined analysis of the MeRIP-seq and RNA sequencing data, 129 genes with differentially methylated m6A peaks and differentially expressed mRNA levels were identified. CONCLUSION: Our study suggests that the modulation of m6A methylation modifications plays a significant role in UCB neurotoxicity.

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

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

Neutrophil extracellular traps enhance procoagulant activity and thrombotic tendency in patients with obstructive jaundice.

BACKGROUND & AIMS: Patients with obstructive jaundice (OJ) are considered to be prothrombotic with increased risk of thromboembolism complications. The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) and thrombosis risk in patients with OJ is unclear. In this study, we investigated NETs formation in OJ patients and the role of elevated unconjugated bilirubin (UCB) in inducing NETs, resulting in enhanced PCA and endothelial injury. METHODS: NETs of OJ patients and healthy controls were measured. NETs PCA was assessed via coagulation time (CT), fibrin formation and purified coagulation complex production assays. Visualization of NETs and mitochondrial reactive oxygen species (MitoROS) were performed with a fluorescence microscope. We further used confocal microscopy to quantify the exposure of phosphatidylserine (PS), fibrin strands and FVa/Xa on Human umbilical vein endothelial cells (HUVECs). RESULTS: Assessment of NETs components levels revealed greater NETs production in OJ patients than in healthy controls. Importantly, OJ-NETs were responsible for enhanced PCA. UCB induced NETs formation via MitoROS accumulation and mitochondrial mobilization. HUVECs cocultured with OJ NETs lost their cell-cell junctions and consequently converted to a procoagulant phenotype. The PCA was attenuated by using DNase I alone or in combination with lactadherin. CONCLUSIONS: Our results suggest that UCB-induced NETs play a prominent role in promoting the hypercoagulable and prothrombotic state in OJ patients. The increased MitoROS accumulation in neutrophils initiated NETosis. NETs are promising targets for indicating or improving coagulation disorders in OJ patients.

Mechanism of bilirubin elimination in urine: insights and prospects for neonatal jaundice.

Despite a century of research, bilirubin metabolism and the transport mechanisms responsible for homeostasis of bilirubin in serum remain controversial. Emerging evidence on the hepatic membrane transporters and inherited disorders of bilirubin metabolism have contributed to a greater understanding of the various steps involved in bilirubin homeostasis and its associated excretory pathways. We discuss these recent research findings on hepatic membrane transporters and evaluate their significance on the newborn bilirubin metabolism and excretion. New insights gained speculate that a proportion of conjugated bilirubin is excreted via the renal system, as an alternative to the intestinal excretion, even in normal physiological jaundice with no associated pathological concerns. Finally, this paper discusses the clinical relevance of targeting the altered renal excretory pathway, as bilirubin in urine may hold diagnostic importance in screening for neonatal jaundice.

Interference of eltrombopag with bilirubin measurements on the Vitros 5600 analyzer.

BACKGROUND: Eltrombopag is a thrombopoietin-receptor agonist used to restore platelet count to hemostatic levels in chronic immune thrombocytopenia. The drug has shown to have hepatobiliary adverse effects, but also positive interference with the analytical measurement of bilirubin. Understanding the degree of interference of this drug with bilirubin testing becomes relevant in the clinical management of these patients. METHODS: Eltrombopag at concentrations ranging from 10 to 150 µg/ml was spiked into plasma samples with different baseline concentrations of bilirubin. Total bilirubin, conjugated, and unconjugated bilirubin were measured for each sample using VITROS TBILI and BuBc slides on the Vitros 5600 automated chemistry platform, and interference was assessed. RESULTS: Plasma samples spiked with eltrombopag yielded falsely elevated bilirubin measurements compared to baseline, with the degree of elevation increasing with greater concentrations of eltrombopag. Bilirubin values were increased relative to baseline across all groups, except in conjugated bilirubin measurements in samples with low baseline conjugated bilirubin. For samples with low total bilirubin at baseline, >100 µg/ml of eltrombopag resulted in an error of >+0.6 mg/dl on the measured total bilirubin. For samples with low unconjugated bilirubin at baseline, the error for the same concentrations was >+0.7 mg/dl. CONCLUSION: Our results show that, at supra-physiologically high concentrations, eltrombopag can positively interfere with bilirubin measurements on Vitros 5600 platform.

Clinical profile in schizophrenia and schizoaffective spectrum: relation with unconjugated bilirubin in a prospective and controlled study with psychopathological and psychosocial variables.

OBJECTIVE: Our objective was to assess unconjugated bilirubin (UCB) as biomarker for schizophrenia (SCZ) and schizoaffective (SAF) spectrums disorder (relapse vs. partial remission). METHODS: Eighty-eight psychotic patients completed first assessment during relapse at ward admission, half with SCZ and half with SAF disorder. Forty-four acute bipolar patients were used as controls. After 12-month follow-up, we collected longitudinal protocol (laboratory, psychopathological, and psychosocial data) from 60 patients, half with SCZ and half with SAF disorder. RESULTS: During psychotic relapse (N = 88), we found a statistically significant difference (analysis of variance [ANOVA]; p = .002), confirmed after post hoc multiple comparisons (Bonferroni) between SCZ (N = 44) and both SAF (N = 44; p = .05) and bipolar controls (N = 44; p = .05); a positive correlation (Pearson's r = .314) between UCB mean levels and Personal and Social Performance item (d) "disturbing and aggressive behaviors"; and a positive correlation (R2 = .223), with statistically significance (p = .008), between UCB mean levels and mean length of stay at the psychiatric ward in SAF patients who completed full protocol (N = 30). During partial remission (N = 60) we found: a statistically significant difference (ANOVA; p = .006), confirmed after post hoc multiple comparisons (Bonferroni) between SCZ (N = 30) and SAF (N = 30; p = .05); plus a negative correlation (Pearson's r = -.399) between UCB mean levels and Positive and Negative Syndrome Scale item G7 "psychomotor retardation." Comparing first and second assessments (paired samples t test) we found a statistically significant difference in UCB mean levels among SAF patients (p = .034). CONCLUSIONS: There is potential in the research of UCB as a biological marker for SCZ and SAF spectrums disorders during relapse and partial remission of both syndromes.

Unconjugated bilirubin and schizophrenia: a systematic review.

Schizophrenia is a complex syndrome of unknown etiology and difficult to manage. Unconjugated bilirubin has been researched as a potential biological marker of this syndrome. The objective of this review article was to gather the studies published to date on the relationship between this molecule and schizophrenia. Broad inclusion criteria have been used (PRISMA) to include as many relevant studies as possible. Fourteen studies were selected: 3 analyzed the effects of unconjugated hyperbilirubinemia in animal models; 6 demonstrated an increased incidence of schizophrenia in patients with increased unconjugated bilirubin; 2 reported an increased incidence of the disease in patients with decreased unconjugated bilirubin; and 3 linked an increased incidence of schizophrenia with an increased excretion of the oxidative product of bilirubin, the so-called biopyrrins. Because of apparently contradictory reported results, the hypothesis that the relationship between schizophrenia and unconjugated bilirubin was not linear and that there was an inflammatory dysfunction explaining this was considered. The 2 most accepted models for the pathophysiology of schizophrenia are described, and the possible role of the molecule in each is clarified. The bilirubin buffer system and its role in antioxidant defense was explored. The average levels of unconjugated bilirubin in patients with schizophrenia, schizoaffective disorder, and bipolar disorder were also compared, having been hypothesized that these diseases could be different points of a same pathological spectrum. Finally, it was concluded that unconjugated bilirubin is a promising molecule that could be used as a possible biological marker for schizophrenia, and the necessity of subsequent efforts for its research was considered.

Unconjugated bilirubin and acute psychosis: a five years retrospective observational and controlled study in patients with schizophrenia, schizoaffective and bipolar disorders.

Introduction: Unconjugated bilirubin (UCB) high levels have been correlated with schizophrenia spectrum disorders.Methods: We searched for correlation between UCB mean levels, duration of psychiatric admission, and diagnosis in 255 individuals, including 56 healthy controls and 199 acute patients (namely 44 with schizophrenia, 99 with schizoaffective disorder and 56 with bipolar disorder).Results: We found a statistically significant difference between UCB mean levels of patients with schizophrenia versus patients with schizoaffective (0.41 mg/dL vs. 0.34 mg/dL; p < .03) and bipolar disorders (0,41 mg/dL vs. 0.29mg/dL; p < .0001). We also found a statistically significant difference between UCB mean levels of patients with schizoaffective disorder and bipolar disorder (0.34 mg/dL vs. 0.28; p < .04). We also found a significant difference (p < .001) between mean admission duration of schizophrenia (29 days) versus bipolar patients (16 days). Although in a non-significant manner, the schizoaffective group got a mean admission duration value (22 days) right in between the schizophrenia and the bipolar patients.Conclusions: Our results deserve further research to access the role UCB may have in the physiopathology of acute patients with schizophrenia/schizoaffective/bipolar spectrum disorders.KeypointsUnconjugated Bilirubin (UCB) high levels are correlated with acute psychosis.UCB high levels are correlated with duration of psychiatric admission.UCB mean levels of schizophrenic patients are higher than schizoaffective patients.UCB mean levels of schizoaffective patients are higher than bipolar patients.

HIF-1α-induced xenobiotic transporters promote Th17 responses in Crohn's disease.

In Crohn's disease, pathogenic Th17-cells express low levels of CD39 ectonucleotidase and are refractory to the immunosuppressive effects of unconjugated bilirubin (UCB), an endogenous ligand for aryl-hydrocarbon-receptor (AhR). This resistance to AhR ligation might be associated with alterations in responses to hypoxia. Limited exposure to hypoxia appears beneficial in acute tissue injury. However, in protracted inflammation, hypoxemia may paradoxically result in Th17-cell activation. We report here that in vitro exposure of Th17-cells from Crohn's disease patients to hypoxia limits responsiveness to AhR stimulation by UCB, as reflected by lower CD39 levels. Blockade of hypoxia-inducible-factor-1alpha (HIF-1α) upregulates CD39 and favors Th17-cell regulatory responses. Resistance of Th17-cells to AhR signaling results, in part, from HIF-1α-dependent induction of ATP-binding cassette (ABC) transporters: multidrug-resistance-protein-1 (MDR1) and multidrug-resistance-associated-protein-4 (MRP4). Increased ABC transporters promote efflux of suppressive AhR ligands, such as UCB, from Th17-cells. Inhibition of MDR1, MRP4 and/or HIF-1α with ritonavir (RTV) reconstitutes AhR function in Th17-cells, enhancing therapeutic effects of UCB in dextran-sulfate-sodium-induced experimental colitis. Deleterious effects of hypoxia on Th17-cells in Crohn's disease can be ameliorated either by inhibiting HIF-1α or by suppressing ABC transporters to increase UCB availability as an AhR substrate. Targeting HIF-1α-ABC transporters could provide innovative therapeutic pathways for IBD.

Attenuation of neuro-inflammation improves survival and neurodegeneration in a mouse model of severe neonatal hyperbilirubinemia.

All pre-term newborns and a high proportion of term newborns develop neonatal jaundice. Neonatal jaundice is usually a benign condition and self-resolves within few days after birth. However, a combination of unfavorable complications may lead to acute hyperbilirubinemia. Excessive hyperbilirubinemia may be toxic for the developing nervous system leading to severe neurological damage and death by kernicterus. Survivors show irreversible neurological deficits such as motor, sensitive and cognitive abnormalities. Current therapies rely on the use of phototherapy and, in unresponsive cases, exchange transfusion, which is performed only in specialized centers. During bilirubin-induced neurotoxicity different molecular pathways are activated, ranging from oxidative stress to endoplasmic reticulum (ER) stress response and inflammation, but the contribution of each pathway in the development of the disease still requires further investigation. Thus, to increase our understanding of the pathophysiology of bilirubin neurotoxicity, encephalopathy and kernicterus, we pharmacologically modulated neurodegeneration and neuroinflammation in a lethal mouse model of neonatal hyperbilirubinemia. Treatment of mutant mice with minocycline, a second-generation tetracycline with anti-inflammatory and neuroprotective properties, resulted in a dose-dependent rescue of lethality, due to reduction of neurodegeneration and neuroinflammation, without affecting plasma bilirubin levels. In particular, rescued mice showed normal motor-coordination capabilities and behavior, as determined by the accelerating rotarod and open field tests, respectively. From the molecular point of view, rescued mice showed a dose-dependent reduction in apoptosis of cerebellar neurons and improvement of dendritic arborization of Purkinje cells. Moreover, we observed a decrease of bilirubin-induced M1 microglia activation at the sites of damage with a reduction in oxidative and ER stress markers in these cells. Collectively, these data indicate that neurodegeneration and neuro-inflammation are key factors of bilirubin-induced neonatal lethality and neuro-behavioral abnormalities. We propose that the application of pharmacological treatments having anti-inflammatory and neuroprotective effects, to be used in combination with the current treatments, may significantly improve the management of acute neonatal hyperbilirubinemia, protecting from bilirubin-induced neurological damage and death.

Mildly elevated unconjugated bilirubin is associated with reduced platelet activation-related thrombogenesis and inflammation in Gilbert's syndrome.

Gilbert's syndrome (GS) is associated with a mild unconjugated hyperbilirubinemia, increased circulating antioxidant capacity, and reduced cardiovascular disease (CVD) risk. The current study investigated whether mildly elevated circulating unconjugated bilirubin (UCB) is negatively associated with multiple thrombotic risk factors including platelet activity, hemostatic function, and inflammation in individuals with GS. Blood samples were collected from matched GS and control subjects (14 per group). Activation-dependent platelet surface marker expression of PAC-1 (binds to GPIIb/IIIa surface receptors on activated platelets) and CD62P (marker for P-selectin released from activated degranulated platelets) was assessed in adenosine diphosphate (ADP)-stimulated platelets using flow cytometry. Exogenous agonists, ADP, collagen, and arachidonic acid (AA), were used to stimulate platelet aggregation. A statistically significant decrease in the expression of P-selectin (P = 0.030) on activated platelets was observed in GS subjects. Collagen and AA-induced platelet aggregation were significantly (P = 0.018; P = 0.032 for respective agonists) reduced in GS versus control group. Elevated UCB (P = 0.001) and high density lipoprotein (P = 0.033) in addition to reduced low density lipoprotein (P = 0.024) and high sensitive C-reactive protein (P = 0.043) were also observed in GS when compared to the control group. Reduced P-selectin expression suggests decreased platelet activation-dependent degranulation, while reduced platelet aggregation by AA and collagen indicates a quantitative decrease in platelet aggregation consequently targeting the cyclooxygenase-1 and GP VI pathways, respectively. These findings are the first to demonstrate that the activation of platelets is mildly inhibited in individuals with GS, an effect that might contribute to protection from platelet hyperactivation-induced thrombosis and thus cardiovascular mortality in individuals with benign hyperbilirubinemia.

Dimethyl ester of bilirubin exhibits anti-inflammatory activity through inhibition of secretory phospholipase A2, lipoxygenase and cyclooxygenase.

Overproduction of arachidonic acid (AA) mediated by secretory phospholipase A2 group IIA (sPLA2IIA) is a hallmark of many inflammatory disorders. AA is subsequently converted into pro-inflammatory eicosanoids through 5-lipoxygenase (5-LOX) and cyclooxygenase-1/2 (COX-1/2) activities. Hence, inhibition of sPLA2IIA, 5-LOX and COX-1/2 activities is critical in regulating inflammation. We have previously reported unconjugated bilirubin (UCB), an endogenous antioxidant, as sPLA2IIA inhibitor. However, lipophilic UCB gets conjugated in liver with glucuronic acid into hydrophilic conjugated bilirubin (CB). Since hydrophobicity is pre-requisite for sPLA2IIA inhibition, conjugation reduces the efficacy of UCB. In this regard, UCB was chemically modified and derivatives were evaluated for sPLA2IIA, 5-LOX and COX-1/2 inhibition. Among the derivatives, BD1 (dimethyl ester of bilirubin) exhibited âˆ¼ 3 fold greater inhibitory potency towards sPLA2IIA compared to UCB. Both UCB and BD1 inhibited human 5-LOX and COX-2 activities; however only BD1 inhibited AA induced platelet aggregation. Molecular docking studies demonstrated BD1 as better inhibitor of aforesaid enzymes than UCB and other endogenous antioxidants. These data suggest that BD1 exhibits strong anti-inflammatory activity through inhibition of AA cascade enzymes which is of great therapeutic importance.

When an Analytical Interference Is a Useful Diagnostic Tool: Finding Monoclonal Gammopathies in Routine Analysis.

BACKGROUND: The daily productivity of a clinical laboratory depends on the large number of interferences that affect analytical accuracy. Obviously, they have always been considered as a very important aspect to keep accuracy under control. Nevertheless, we wondered if this aspect would be beneficial. In this article, we propose a method for finding monoclonal gammopathies that are based on the fact that the presence of paraprotein in the sample may interfere with routine laboratory assays, specifically, with the quantification of uric acid and conjugated bilirubin. METHODS: Over a 5-month period, we evaluated 18,278 sera samples of patients from primary care. None of them were suspected of having plasma cell dyscrasias (not observed hypercalcemia, renal failure, anemia, and/or lytic bone lesions). Although biochemical findings suggested paraprotein interference, we carried out serum capillary electrophoresis (CE) and quantification of immunoglobulins and serum-free light chains (SFLCs). We also confirmed the results obtained by performing the corresponding immunofixation electrophoresis (IFE). Flow cytometry analyses were conducted for immunophenotypic characterization of plasma cells from these patients. RESULTS: The proposed detection method allowed us to identify eight patients with previously undiagnosed monoclonal gammopathy. CONCLUSIONS: The results show that it is possible to use analytical interference for diagnostic purposes, and most importantly, almost all cases were identified at an early stage of the disease, when associated clinical manifestations were not yet observed.

Metastable and equilibrium phase diagrams of unconjugated bilirubin IXα as functions of pH in model bile systems: Implications for pigment gallstone formation.

Metastable and equilibrium phase diagrams for unconjugated bilirubin IXα (UCB) in bile are yet to be determined for understanding the physical chemistry of pigment gallstone formation. Also, UCB is a molecule of considerable biomedical importance because it is a potent antioxidant and an inhibitor of atherogenesis. We employed principally a titrimetric approach to obtain metastable and equilibrium UCB solubilities in model bile systems composed of taurine-conjugated bile salts, egg yolk lecithin (mixed long-chain phosphatidylcholines), and cholesterol as functions of total lipid concentration, biliary pH values, and CaCl2 plus NaCl concentrations. Metastable and equilibrium precipitation pH values were obtained, and average pKa values of the two carboxyl groups of UCB were calculated. Added lecithin and increased temperature decreased UCB solubility markedly, whereas increases in bile salt concentrations and molar levels of urea augmented solubility. A wide range of NaCl and cholesterol concentrations resulted in no specific effects, whereas added CaCl2 produced large decreases in UCB solubilities at alkaline pH values only. UV-visible absorption spectra were consistent with both hydrophobic and hydrophilic interactions between UCB and bile salts that were strongly influenced by pH. Reliable literature values for UCB compositions of native gallbladder biles revealed that biles from hemolytic mice and humans with black pigment gallstones are markedly supersaturated with UCB and exhibit more acidic pH values, whereas biles from nonstone control animals and patients with cholesterol gallstone are unsaturated with UCB.

High-normal serum bilirubin is a useful indicator to assess the risk of diabetic retinopathy in type 2 diabetes: A real-world study.

Background: To investigate the association of serum bilirubin within normal range, especially unconjugated bilirubin (UCB), with diabetic retinopathy (DR) in patients with type 2 diabetes mellitus (T2DM). Methods: In this cross-sectional, real-world study, 7617 T2DM patients were stratified into quartiles based on serum UCB levels. DR was determined by digital fundus photography and further classified into non-proliferative diabetic retinopathy (NPDR) and PDR. The associations of serum bilirubin levels and UCB quartiles with DR were investigated by logistic regression analysis. Results: After controlling for age, sex, and diabetes duration, the DR prevalence was significantly decreased across the serum UCB quartiles (40.4 %, 33.4 %, 29.7 %, 26.6 % for each quartile, respectively, p < 0.001 for trend). The subjects with DR had lower serum total bilirubin (TB) and UCB, rather than conjugated bilirubin (CB), compared with those without DR (p = 0.003 for TB, p < 0.001 for UCB, and p = 0.528 for CB, respectively), while all three types of serum bilirubin in the subjects with PDR were obviously lower than those with NPDR (p = 0.006 for TB, and p < 0.001 for UCB and CB, respectively). After adjustment for confounding factors, logistic regression demonstrated negative associations of serum TB and UCB levels, rather than CB, with the presence of DR (OR: 0.844, 95%CI: 0.774-0.920, p < 0.001 for TB; OR: 0.828, 95%CI: 0.763-0.899, p < 0.001 for UCB; and OR: 0.984, 95%CI: 0.900-1.074, p = 0.713 for CB, respectively). Additionally, a fully-adjusted analysis revealed a negative correlation between UCB quartiles and DR (p < 0.001). Conclusion: High-normal serum TB and UCB were closely associated with the decreased odds of DR, while all types of serum bilirubin were negatively correlated with the severity of DR in T2DM patients. Serum bilirubin may be used as a potential indicator to assess the risk and severity of DR in T2DM.