Unconjugated Bilirubin Concentrations Research Articles

CST3 alleviates bilirubin-induced neurocytes' damage by promoting autophagy.

High concentrations of unconjugated bilirubin (UCB) have toxic effects. The aim of our study was to find a way to elevate UCB tolerance or inhibit its toxicity in neurocytes. It has been reported that cystatin C (CST3) concentrations have a significant positive correlation with total bilirubin (TB) levels and a negative correlation with albumin levels. In addition, CST3 can directly bind UCB, decrease human umbilical vein endothelial cells' permeability, improve blood-brain barrier integrity after ischemic brain injury in mice, and induce autophagy. We hypothesized that CST3 could increase the solubility of UCB, decrease permeability of neurocytes, induce autophagy of neurocytes, and alleviate bilirubin-induced damage. To verify our hypothesis, we measured TB and conjugated bilirubin levels, and the permeability and autophagy of neurocytes treated with UCB and CST3. Our findings suggest that CST3 can protect against UCB-induced damage in neurocytes and that autophagy played an important role in this process.

The pyroptosis mechanism of ototoxicity caused by unconjugated bilirubin in neonatal hyperbilirubinemia

When activated by unconjugated bilirubin (UCB), inflammatory mediators such as IL − 18 and TNF contribute to the neurotoxicity and ototoxicity observed in severe neonatal hyperbilirubinemia. However, in cell and molecular level, the regulation and mechanism of UCB-induced ototoxicity are remained unclear. In this study, 7-day-old mammary rats were exposed to various concentrations of UCB to imitate the infant auditory damage. The auditory brainstem response result (ABR) indicated severe hearing loss, which occurred with increasing concentration. Morphological analysis of organotypic cochlear cultures treated with different concentrations of UCB indicated that auditory nerve fibers (ANF) were demyelinated and the density of spiral ganglion neurons (SGN) were decreased. In addition, HEI-OC1 cells treated with different concentrations of UCB showed severe necrosis by Flow Cytometry. The morphologic feature of pyroptosis has been observed by scanning electronic microscope. Cleaved Caspase-1, GSDMD and NLRP3 expression were significantly increased in cochlear explants with UCB-induced. To further clarify the molecular mechanism of UCB-induced inner ear cell pyroptosis, specific inhibitors of pyroptosis were applied, the protein associated with pyrotosis such as Cleaved Caspase-1, GSDMD, ASC, IL-18 and NLRP3 were significantly lower than the group with UCB alone. All the data above indicated that ERK /NLRP3/GSDMD signaling pathway involved in UCB-induced ototoxicity.

Blood unconjugated bilirubin and tacrolimus are negative predictors of specific cellular immunity in kidney transplant recipients after SAR-CoV-2 inactivated vaccination

The immunogenicity of SARS-CoV-2 vaccines is poor in kidney transplant recipients (KTRs). The factors related to poor immunogenicity to vaccination in KTRs are not well defined. Here, observational study demonstrated no severe adverse effects were observed in KTRs and healthy participants (HPs) after first or second dose of SARS-CoV-2 inactivated vaccine. Different from HPs with excellent immunity against SARS-CoV-2, IgG antibodies against S1 subunit of spike protein, receptor-binding domain, and nucleocapsid protein were not effectively induced in a majority of KTRs after the second dose of inactivated vaccine. Specific T cell immunity response was detectable in 40% KTRs after the second dose of inactivated vaccine. KTRs who developed specific T cell immunity were more likely to be female, and have lower levels of total bilirubin, unconjugated bilirubin, and blood tacrolimus concentrations. Multivariate logistic regression analysis found that blood unconjugated bilirubin and tacrolimus concentration were significantly negatively associated with SARS-CoV-2 specific T cell immunity response in KTRs. Altogether, these data suggest compared to humoral immunity, SARS-CoV-2 specific T cell immunity response are more likely to be induced in KTRs after administration of inactivated vaccine. Reduction of unconjugated bilirubin and tacrolimus concentration might benefit specific cellular immunity response in KTRs following vaccination.

A Case of Acute Intravascular Hemolysis During Dialysis: A Quiz

A Case of Acute Intravascular Hemolysis During Dialysis: A Quiz

Quantitative effects of bilirubin structural photoisomers on the measurement of direct bilirubin via the vanadate oxidation method.

Exposing blood serum samples to ambient white light-emitting diode (WLED) light may accelerate bilirubin photoisomer production. We previously demonstrated the quantitative effect of bilirubin configurational isomers (BCI) on direct bilirubin (DB) value using the vanadate oxidation method. However, the effects of bilirubin structural photoisomers (BSI) remain unclear. In Study 1, the relationship between WLED irradiation time and BSI production was examined. Serum samples from five neonates were irradiated with WLED light for 0, 10, 30, 60 and 180 min. Bilirubin isomer concentration and BSI production rates were calculated. In Study 2, we performed quantitative investigation of BSI effect on DB values: Differences in DB, BCI and BSI values before and after irradiation were calculated as ⊿DB, ⊿BCI and ⊿BSI, respectively. Assuming the coefficient of BCI affecting DB values was 'a', relational expression was ⊿DB = a*⊿BSI + 0.19*⊿BCI. Serum samples from 15 neonates were irradiated with green LED light for 10 and 30s. The respective bilirubin isomer levels were measured, and the coefficient was derived. In Study 1, the median BSI production rate was 0.022 mg/dL per min in specimens with an unconjugated bilirubin concentration of 10.88 mg/dL. In Study 2, assuming that ⊿DB-0.19*⊿BCI was Y and ⊿BSI was X, the relational expression was Y = 0.34X-0.03 (R2 = 0.87; p < .01) and a = 0.34. Under ambient WLED light, serum sample generated 1.3 mg/dL BSIs in 1 h. Approximately 34% (0.44mg/dL) of BSI concentrations was measured as DB when using the vanadate oxidation method according to the above equation.

Method for Measuring Bioactive Molecules in Blood by a Smartphone Using Bioluminescent Ratiometric Indicators.

Bioluminescent indicators facilitate determination of bioactive molecules in blood samples with high sensitivity. Using a bright luciferase, its bioluminescence (BL) can be easily detected by conventional light sensing devices. In this chapter, we describe a protocol to measure bioactive molecules in blood by taking the BL images with a smartphone camera. We exemplify the measurement of unconjugated bilirubin (UCBR) concentration in the blood of mice using a ratiometric bioluminescent UCBR indicator, BABI (bilirubin assessment with a bioluminescent indicator), and a smartphone camera. We show the UCBR concentration is easily determined through measuring the variance in the BL color with a smartphone camera. This method provides a practical method to lead to future point-of-care diagnosis with quick and simple procedures.

Surface-Enhanced Raman Scattering-Active Gold-Decorated Silicon Nanowire Substrates for Label-Free Detection of Bilirubin.

Bilirubin (BR) is a product of hemoglobin breakdown, and its increasing levels in the blood may indicate liver disorders and lead to jaundice. Kernicterus is most dangerous in newborns when the unconjugated BR concentration can quickly rise to toxic levels, causing neurological damage and even death. The development of an accurate, fast, and sensitive sensor for BR detection will help reduce diagnostic time and ensure successful treatment. In this study, we propose a new method for creating a surface-enhanced Raman scattering (SERS)-active substrate based on gold-decorated silicon nanowires (Au@SiNWs) for sensitive label-free BR detection. Gold-assisted chemical etching of crystalline silicon wafers was used to synthesize SiNWs, the tops of which were then additionally decorated with gold nanoparticles. The low detection limit of model analyte 4-mercaptopyridine down to the concentration of 10-8 M demonstrated the excellent sensitivity of the obtained substrates for SERS application. The theoretical full-wave electromagnetic simulations of Raman scattering in the Au@SiNW substrates showed that the major contribution to the total SERS signal comes from the analyte molecules located on the SiNW surface near the gold nanoparticles. Therefore, for efficient BR adsorption and SERS detection, the surface of the SiNWs was modified with amino groups. Label-free detection of BR using amino modified Au@SiNWs with high point-to-point, scan-to-scan, and batch-to-batch reproducibility with a detection limit of 10-6 M has been demonstrated. Artificial urine, mimicking human urine samples, was used as the matrix to get insights into the influence of different parameters such as matrix complexity on the overall BR SERS signal. The signal stability was demonstrated for 7 days after adsorption of BR with a concentration of 5 × 10-5 M, which is the required sensitivity for clinical applications.

Low concentrations of bilirubin protect from dopaminergic neuron loss in an ex vivo model of Parkinson's disease

Disease-modifying therapy for Parkinson's disease (PD) is still an unmet medical need. Hence, we investigated the protective effects of unconjugated bilirubin (UCB) in an ex vivo PD model and identified the protective UCB concentrations threshold.

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

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

A survey of the reactivity of in vitro diagnostic bilirubin reagents developed in Japan using artificially prepared bilirubin materials: A comparison of synthetic delta, unconjugated, and taurine-conjugated bilirubin.

In vitro diagnostic bilirubin reagents based on oxidation with bilirubin oxidase or vanadic acid for total and direct-reacting bilirubin are widely used in Japan; however, their reactivity to unconjugated and conjugated bilirubin and delta bilirubin has not been completely disclosed by manufacturers. We used artificially prepared bilirubin materials to investigate the reactivity with four in vitro diagnostic bilirubin reagents. Porcine unconjugated bilirubin solution, chemically synthesized ditaurobilirubin solution, and chemically synthesized delta bilirubin solution were used as surrogates of naturally occurring unconjugated bilirubin, conjugated bilirubin, and delta bilirubin, respectively. The total bilirubin and direct-reacting bilirubin concentrations were measured by three bilirubin oxidase methods and one vanadic acid method, and the observed concentrations were compared with those obtained by the diazo-based reference measurement procedure. The unconjugated bilirubin and delta bilirubin concentrations were similar when any of the four in vitro diagnostic bilirubin reagents were used during total bilirubin measurement. This was consistent with reference measurement procedure and exhibited a converged inter-method variation. Compared with reference measurement procedure, significantly low ditaurobilirubin concentrations were observed by the in vitro diagnostic bilirubin reagents despite the converged inter-method variation. In delta bilirubin measurement, some reagents reacted doubtfully with unconjugated bilirubin, while showed lower ditaurobilirubin concentrations than its corresponding total bilirubin concentration. Reactivity with delta bilirubin was different for each method including reference measurement procedure. Some reagents were developed to react less with delta bilirubin and others to strongly react with delta bilirubin. We revealed the reactivity of IVD-TB and IVD-DB reagents to artificially prepared bilirubin materials, and their consistency with reference measurement procedure. The delta bilirubin data results vary depending on the reagents used.

The influence of recombinant interferon on morphological and biochemical parameters of calves’ blood in early postnatal period

The main mortality of young cattle due to infectious diseases mostly occurs in the first two months of the life, which is resulted from the calves' immune system immaturity. In order to correct the immunological status, various immunomodulators are used, interferons in particular. The aim of the study was to assess the effect of recombinant bovine interferon-α2d on the biochemical and morphological parameters of the calves' blood in the early postnatal period. Two groups of young animals were formed. Calves from the 1st group (G1,n=10) was twice injected interferon intramuscularly with the interval between injections - 7 days (500 th. MU). The 2nd group was a control (G2, n=10), the injection of the immunostimulant was replaced with saline. Before the beginning (on the 7th day of life) and at the end of the experiment (on the 21st day of life), the blood was taken for the biochemical and morphological analysis. In G1 and G2, the concentration of unconjugated bilirubin at the beginning of the experiment was 2.2 ± 0.2 and 2.1 ± 0.3 mmol/L respectively, in the G1 interferon concentration decreased to 1.1 ± 0.1 mmol/L, which was 2 times less than in the G2 (P ≤ 0.01). The activity of alkaline phosphatase at the beginning of the study was 613.8 ± 54.2 in the G2 and 514.6 ± 22.4 units/L in the G1. By the 21st day of life, the level of it decreased by 16.2% (431, 1 ± 38.0 units/L) in the G1, and it was less by 31.3% (P ≤ 0.05) than the G2 (627.3 ± 77.6 units/ L). The ALT activity at the beginning of the experiment was 4.0 ± 0.8 unit/ L in the G2 and 6.1 ± 1.2 units/L in the G1. After two weeks in the G2 level of ALT increased to 6.0 ± 2.0 U / L, while in calves from the G1, on the contrary, it was decreased in 2.4 times (P≤0.05) - 2.5 ± 0.1 U / L. The AST activity in the intact group by the 21st day was 16.0 ± 8.0 units/ L, which was 8.4 times higher (P≤0.05) than in the G1 (1.9 ± 0.4). Under the influence of interferon, there was the increase in the average concentration of leukocytes (by 13.2%), as well as the decrease in the relative number of neutrophils by 36.8% (P≤0.05). The absolute number of agranulocytes increased by 37.7% (P≤0.05) compared to the control one (5.3 ± 0.5 * 109/ L) and it was 7.3 ± 0.7 * 109/ L. Thus, parenteral injection of recombinant bovine interferon-α2d to calves in the early postnatal period causes the decrease in transaminase activity and bilirubin concentration, as well as the increase in the number of cells responsible for the humoral immune response.

Ratiometric Bioluminescent Indicator for Simple and Rapid Diagnosis of Bilirubin.

Bilirubin in human blood is highly important as a general index of one's physical condition because its concentration changes under the influence of several diseases. In particular, in newborns, jaundice is one of the most common diseases involving unconjugated bilirubin (UCBR), causing serious symptoms such as nuclear jaundice and deafness. Therefore, a frequent measurement of the UCBR levels in the blood is important. Here, we report a ratiometric bioluminescent indicator, BABI (bilirubin assessment with a bioluminescent indicator), that changes the emission color from blue to green depending on the UCBR concentration in a sample. Owing to the use of a bioluminescence signal that has a higher signal-to-noise ratio than the absorption and fluorescence signal, BABI enables highly sensitive and quantitative detection of UCBR for small blood samples using a smartphone camera. The establishment of a UCBR measurement assay using BABI provides the possibility of a simple and rapid method for blood-based diagnosis using bioluminescent indicators and a versatile mobile device.

The Extent of Intracellular Accumulation of Bilirubin Determines Its Anti- or Pro-Oxidant Effect.

Background: Severe hyperbilirubinemia can cause permanent neurological damage in particular in neonates, whereas mildly elevated serum bilirubin protects from various oxidative stress-mediated diseases. The present work aimed to establish the intracellular unconjugated bilirubin concentrations (iUCB) thresholds differentiating between anti- and pro-oxidant effects. Methods: Hepatic (HepG2), heart endothelial (H5V), kidney tubular (HK2) and neuronal (SH-SY5Y) cell lines were exposed to increasing concentration of bilirubin. iUCB, cytotoxicity, intracellular reactive oxygen species (ROS) concentrations, and antioxidant capacity (50% efficacy concentration (EC50)) were determined. Results: Exposure of SH-SY5Y to UCB concentration > 3.6 µM (iUCB of 25 ng/mg) and >15 µM in H5V and HK2 cells (iUCB of 40 ng/mg) increased intracellular ROS production (p < 0.05). EC50 of the antioxidant activity was 21 µM (iUCB between 5.4 and 21 ng/mg) in HepG2 cells, 0.68 µM (iUCB between 3.3 and 7.5 ng/mg) in SH-SY5Y cells, 2.4 µM (iUCB between 3 and 6.7 ng/mg) in HK2 cells, and 4 µM (iUCB between 4.7 and 7.5 ng/mg) in H5V cells. Conclusions: In all the cell lines studied, iUCB of around 7 ng/mg protein had antioxidant activities, while iUCB > 25 ng/mg protein resulted in a prooxidant and cytotoxic effects. UCB metabolism was found to be cell-specific resulting in different iUCB.

The Association between Serum Bilirubin Levels and Colorectal Cancer Risk: Results from the Prospective Cooperative Health Research in the Region of Augsburg (KORA) Study in Germany.

Emerging studies have suggested that bilirubin, particularly unconjugated bilirubin (UCB), has substantial anti-inflammatory and antioxidant properties that protect against oxidative stress-associated diseases such as cancer. Few observational studies have investigated the etiological role of bilirubin in colorectal cancer (CRC) development. In this case-control study, nested in the population-based prospective cohort of the Cooperative Health Research in the Region of Augsburg (KORA) study in south Germany, pre-diagnostic circulating UCB concentrations were measured by high-performance liquid chromatography in 77 CRC cases and their individually matched controls. Multivariable unconditional logistic regression was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for associations between log-transformed UCB levels (log-UCB), standardized per one-standard-deviation (one-SD) increment, and CRC risk. The models were a priori stratified by sex based on previous evidence. In the fully adjusted models, each one-SD increment in log-UCB was indicative of a positive association with CRC risk (OR, 1.20; 95% CI, 0.52–2.79) among men, and of an inverse association (OR, 0.76; 95% CI, 0.34–1.84) among women (Pheterogeneity = 0.4 for differences between men and women). We found little evidence for sex-specific associations of circulating bilirubin with CRC risk, and further studies are needed to confirm or refute the potential associations.

Gilbert syndrome with systemic lupus erythematosus presenting with persistent unconjugated hyperbilirubinemia: A case report.

Gilbert syndrome (GS) is a hereditary unconjugated hyperbilirubinemia that results from mutations in the bilirubin uridine diphosphate-glucuronosyltransferase (UGT1A1) gene. To the best of our knowledge, there are currently no reports that focus on patients with systemic lupus erythematosus (SLE) coexisting with GS. The present study aimed to evaluate the clinical characteristics and genotype of UGT1A1 in a Chinese patient with SLE and GS. Complete medical records and laboratory data were reviewed for a patient with SLE referred to Ruijin Hospital (Shanghai, China) for treatment between March 2016 and January 2020. Genetic analysis of the UGT1A1 gene was performed by PCR amplification and Sanger sequencing. The serum total bilirubin and unconjugated bilirubin concentrations on admission were 96.2 and 86.8 µmol/l, respectively. The homozygous mutation c.1456T>G (p.Y486D) in exon 5 was detected in this patient. The patient had a good response to phenobarbital orally at a dose of 30 mg/day and a decrease in serum bilirubin was observed. Elevated unconjugated hyperbilirubinemia in SLE needs to be differentiated from other diseases, such as GS, which can be diagnosed by UGT1A1 genetic sequencing.

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

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

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

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

Microglia Susceptibility to Free Bilirubin Is Age-Dependent.

Increased concentrations of unconjugated bilirubin (UCB), namely its free fraction (Bf), in neonatal life may cause transient or definitive injury to neurons and glial cells. We demonstrated that UCB damages neurons and glial cells by compromising oligodendrocyte maturation and myelination, and by activating astrocytes and microglia. Immature neurons and astrocytes showed to be especially vulnerable. However, whether microglia susceptibility to UCB is also age-related was never investigated. We developed a microglia culture model in which cells at 2 days in vitro (2DIV) revealed to behave as the neonatal microglia (amoeboid/reactive cells), in contrast with those at 16DIV microglia that performed as aged cells (irresponsive/dormant cells). Here, we aimed to unveil whether UCB-induced toxicity diverged from the young to the long-cultured microglia. Cells were isolated from the cortical brain of 1- to 2-day-old CD1 mice and incubated for 24 h with 50/100 nM Bf levels, which were associated to moderate and severe neonatal hyperbilirubinemia, respectively. These concentrations of Bf induced early apoptosis and amoeboid shape in 2DIV microglia, while caused late apoptosis in 16DIV cells, without altering their morphology. CD11b staining increased in both, but more markedly in 2DIV cells. Likewise, the gene expression of HMGB1, a well-known alarmin, as well as HMGB1 and GLT-1–positive cells, were enhanced as compared to long-maturated microglia. The CX3CR1 reduction in 2DIV microglia was opposed to the 16DIV cells and suggests a preferential Bf-induced sickness response in younger cells. In conformity, increased mitochondrial mass and NO were enhanced in 2DIV cells, but unchanged or reduced, respectively, in the 16DIV microglia. However, 100 nM Bf caused iNOS gene overexpression in 2DIV and 16DIV cells. While only arginase 1/IL-1β gene expression levels increased upon 50/100 nM Bf treatment in long-maturated microglia, MHCII/arginase 1/TNF-α/IL-1β/IL-6 (>10-fold) were upregulated in the 2DIV microglia. Remarkably, enhanced inflammatory-associated microRNAs (miR-155/miR-125b/miR-21/miR-146a) and reduced anti-inflammatory miR-124 were found in young microglia by both Bf concentrations, while remained unchanged (miR/21/miR-125b) or decreased (miR-155/miR-146a/miR-124) in aged cells. Altogether, these findings support the neurodevelopmental susceptibilities to UCB-induced neurotoxicity, the most severe disabilities in premature babies, and the involvement of immune-inflammation neonatal microglia processes in poorer outcomes.

Dubin-Johnson Syndrome in Pregnancy: Case Report and Literature Review

Dubin-Johnson Syndrome in Pregnancy: Case Report and Literature Review

A novel accurate LC-MS/MS method for quantitative determination of Z-lumirubin

Although phototherapy (PT) is a standard treatment for neonatal jaundice, no validated clinical methods for determination of bilirubin phototherapy products are available. Thus, the aim of our study was to establish a such method for clinical use. To achieve this aim, a LC-MS/MS assay for simultaneous determination of Z-lumirubin (LR) and unconjugated bilirubin (UCB) was conducted. LR was purified after irradiation of UCB at 460 nm. The assay was tested on human sera from PT-treated neonates. Samples were separated on a HPLC system with a triple quadrupole mass spectrometer detector. The instrument response was linear up to 5.8 and 23.4 mg/dL for LR and UCB, respectively, with submicromolar limits of detection and validity parameters relevant for use in clinical medicine. Exposure of newborns to PT raised serum LR concentrations three-fold (p < 0.01), but the absolute concentrations were low (0.37 ± 0.16 mg/dL), despite a dramatic decrease of serum UCB concentrations (13.6 ± 2.2 vs. 10.3 ± 3.3 mg/dL, p < 0.01). A LC-MS/MS method for the simultaneous determination of LR and UCB in human serum was established and validated for clinical use. This method should help to monitor neonates on PT, as well as to improve our understanding of both the kinetics and biology of bilirubin phototherapy products.