Bilirubin Species Research Articles

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.

A simplified HPLC-based method for measuring unconjugated bilirubin, bilirubin-monoglucuronide, bilirubin-diglucuronide, and delta-bilirubin in plasma with increased conjugated bilirubin

Prolonged icterus is an important and common problem in neonatology and accurate determination of the different bilirubin species, including differentiation between delta-bilirubin and mono- and di-conjugated bilirubin, is useful for diagnostic purposes. However, most bilirubin measurements routinely performed in the clinical laboratory are hampered by the lack of separation of the four bilirubin fractions (unconjugated bilirubin, mono-conjugated bilirubin, di-conjugated bilirubin, and delta-bilirubin). Herein, we propose a high-performance liquid chromatography-based method, independent of commercially available standards or reliable molar absorption coefficients, for the determination of bilirubin fractions in blood samples from icteric patients. The method is a robust and reliable candidate for a semi-automatized setup for measuring the various bilirubin fractions in a specialized laboratory handling samples from clinics with expertise in biliary disease.

Serum Unbound Bilirubin Versus Transcutaneous Bilirubin in Jaundiced Term Neonates

Transcutaneous bilirubin (TcB) consists of the skin-deposited bilirubin. Free bilirubin speaks to the protein-unbound bilirubin (UB) that can go into the tissues. We expected to portray the connection between UB-TcB and study the section of UB into the skin. We tentatively enlisted 40 neonates and we estimated TcB, UB and all out serum bilirubin. Different locales TcB estimation was performed. TcB has a comparative relationship with UB and TSB.There is asignifigant relationship with TSB, DSB(direct serum bilirubin) between embittered term neonates when contrasted with solid term neonates. There is a critical relationship between free bilirubin in embittered term neonates when contrasted with solid ter.mTcB test doesn't appear to straightforwardly supplant UB estimation. Be that as it may, TcB and UB are connected ,UB adds to the skin bilirubin statement yet it isn't the main bilirubin species estimated by transcutaneous bilirubinometry.

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The ‘omics’ approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics.

Relationship between transcutaneous bilirubin and circulating unbound bilirubin in jaundiced neonates

BackgroundTranscutaneous bilirubin (TcB) consists of the skin-deposited bilirubin. Free bilirubin represents the protein-unbound bilirubin (UB) that is able to pass into the tissues. We aimed to describe the relationship UB-TcB and study the passage of UB into the skin. MethodsWe prospectively enrolled 194 neonates and we measured TcB, UB, serum bilirubin and albumin. Multiple sites TcB measurement was performed, bilirubin-albumin equilibrium constant and plasma bilirubin avidity (PBA) were calculated. ResultTcB has a similar correlation with UB and TSB. There is a quadratic relationship between UB and TcB (R2=0.48; p<0.001), remaining significant (β for UB2=−0.8; p<0.001. β for UB=1.1; p<0.001) after adjustment for gestational age, birth weight, postnatal age and albumin (Adj-R2=0.72). UB contributes to the skin bilirubin deposition, as there are significant correlations between albumin and TcB (r=−0.202; p=0.01) and between PBA and ΔTcB (r=0.323; p=0.017). ConclusionTcB assay does not seem to directly replace UB measurement. However, TcB and UB are linked by a quadratic relationship: UB contributes to the skin bilirubin deposition but it is not the only bilirubin species measured by transcutaneous bilirubinometry.

Computational chemical analysis of unconjugated bilirubin anions and insights into pKa values clarification

The pKa, the negative logarithm of the acid dissociation equilibrium constant, of the carboxylic acid groups of unconjugated bilirubin in water is a discussed issue because there are quite different experimental values reported. Using quantum mechanical calculations we have studied the conformational behavior of unconjugated bilirubin species (in gas phase and in solution modeled implicitly and explicitly) to provide evidence that may clarify pKa values because of its pathophysiological relevance. Our results show that rotation of carboxylate group, which is not restricted, settles it in a suitable place to establish stronger interactions that stabilizes the monoanion and the dianion to be properly solvated, demonstrating that the rationalization used to justify the high pKa values of unconjugated bilirubin is inappropriate. Furthermore, low unconjugated bilirubin (UCB) pKa values were estimated from a linear regression analysis.

Controversies in bilirubin biochemistry and their clinical relevance

Despite a century of research, several clinically relevant areas of bilirubin biochemistry remain controversial, poorly understood, or unrecognized. These include: (i) The structure and molecularity of bilirubin under physiological environments such as membranes, brain tissue and when bound to proteins. Related to this is the large number of structurally different bilirubin species that may occur in blood under pathological conditions and their potential effects on measurements of bilirubin and free bilirubin. (ii) The mechanism of phototherapy, the neurotoxicity of the photoisomers produced and their influence on measurements of bilirubin and free bilirubin. (iii) The role of membrane transporters in the passage of unconjugated bilirubin across the placenta, intestine, vascular epithelium, blood-brain barrier, and into the liver. (iv) Biochemical mechanisms of bilirubin toxicity, pharmacologic prevention of kernicterus, the contribution of bilirubin to antioxidant defenses, and the practical value of free bilirubin measurements for identifying infants at most risk of kernicterus.

Revalidation and rationale for high pKa values of unconjugated bilirubin

BackgroundOur prior solvent partition analysis, published in 1992, yielded pKa values for unconjugated bilirubin of about 8.1 and 8.4, but these results have been challenged and studies by other methods have suggested pKa values below 5.0.MethodsWe repeated our published solvent partition studies, using 14C-unconjugated bilirubin highly purified by extraction of residual labeled impurities from CHCl3 into an aqueous buffer, pH 7.0. Partition ratios at six pH values from 5.0 to 9.0 were determined by radioassay and compared with our prior values obtained by diazo assay.ResultsAt pH values ranging from 4.8 to 9.2, stable aqueous/chloroform 14C-partition ratios did not differ significantly from our published partition ratios based on diazo assay.ConclusionThese results support the high pKa values of unconjugated bilirubin, above 8.0, derived from our earlier solvent partition study. In both studies, our measurements were based on the rapid analysis of clearly under-saturated solutions of highly-purified bilirubin over a wide pH range, using properly purified and preserved solvents. No previous direct estimate of the aqueous pKa values of unconjugated bilirubin meets all these preconditions. Three theoretical factors acting in combination, each related to the unique, extensive internal H-bonding of the -COOH groups, are proposed to support high pKa values of unconjugated bilirubin in water: a) donation of an H-bond from the -OH moiety of the -COOH group, which is broken on ionization; b) hindered solvation of the -COO- group after ionization; and c) restricted rotation of the -COO- and -COOH groups. Our findings and rationale rebut methodological and theoretical criticisms leveled against our prior work. High pKa values for unconjugated bilirubin dictate that: a) bilirubin diacid, which readily diffuses across membranes and can cause neurotoxicity, is the dominant unbound bilirubin species of unconjugated bilirubin in plasma at physiological pH; b) at the near-neutral pH range of gallbladder bile, the monoanion is the major unconjugated bilirubin anion present, concordant with the finding that the calcium bilirubinate precipitated in gallstones is the monoanion salt. Our conclusions are thus relevant to understanding bilirubin-induced neurological disease in severely jaundiced neonates and the precipitation of calcium bilirubinate salts in gallstones.

Effect of acidosis on bilirubin-induced toxicity to human erythrocytes.

Unconjugated bilirubin binds to erythrocytes, eliciting crenation, lipid elution and hemolysis. The present work attempts to establish the role of acidosis on bilirubin-induced toxicity to human erythrocytes. To this end, pH values ranging from 7.0-8.0 were used to induce a different representation of acid and anionic bilirubin species, respectively. Erythrocytes from healthy donors were incubated with bilirubin and albumin (3:1, molar ratio), during 4 h. Erythrocyte-bound bilirubin was evaluated by albumin or chloroform extraction in an attempt to assess either mono- and dianion bilirubin adsorbed on the cell surface or colloidal aggregates, respectively. Cytotoxicity indicators, such as the morphological index, and the extent of phospholipids and hemoglobin release were also determined. The results showed that as pH drops from 8.0-7.0, less bilirubin is removed by albumin and more become recovered by chloroform. The data corroborate the predominance of anionic and non-aggregated bilirubin species at pH 8.0 with dimers and precipitates occurring at 7.0. In accordance, crenation and cell lysis were four times increased at acidic pH. In contrast, elution of phospholipids was 1.5 times less evident at the same pH, thus suggesting that formation of bilirubin complexes with membrane phospholipids may have contributed to prevent their release. In conclusion, both anionic and acid bilirubin species interact with human erythrocytes leading to cytotoxic alterations that may determine definitive lesions. Nevertheless, increased vulnerability to crenation and hemolysis are more likely to occur in acidic conditions pointing to the bilirubin precipitates as the main candidates of bilirubin-induced toxicity to erythrocytes.

Accumulation of unconjugated bilirubin in cholesterol pellets implanted in swine gallbladders

BACKGROUND & AIMS: Most cholesterol gallstones have a pigmented center, but it is unclear whether its presence is primary or secondary. This study was performed to determine if bilirubin would accumulate in a gallstone model consisting of cholesterol pellets. METHODS: Cholesterol was compressed into pellets at 2500 psi, producing a pellet that behaved like human cholesterol gallstones in regard to penetration of solutes into the stone. Pellets were implanted into gallbladders of pigs and harvested after 4 weeks. Bilirubin species were measured by high-performance liquid chromatography. RESULTS: The proportions of bilirubin species in bile were not changed by the presence of pellets, i.e., diconjugates (mean +/- SD, 1.9% +/- 1.0% vs. 0.7% +/- 0.8%), monoconjugates (83.8% +/- 5.5% vs. 87.8% +/- 6.6%), and unconjugated bilirubin (14.2% +/- 5.3% vs. 11.5% +/- 5.6%) were similar at the time of implantation and removal. The cut surfaces of the pellets were pigmented. Pellets contained 5.46 +/- 1.38 micrograms bilirubin/g sample at harvesting, and 98.6% +/- 2.3% of bilirubin in pellets was unconjugated. In in vitro studies, there was a large increase in unconjugated bilirubin in the bile. Pellets also became pigmented in vitro, but there was considerable variability in the bilirubin species present in the pellets. CONCLUSIONS: Unconjugated bilirubin accumulates in cholesterol pellets and pigments them. This provides a mechanism by which cholesterol gallstones could become secondarily pigmented. (Gastroenterology 1996 Feb;110(2):607-13)

Micellar High-Performance Liquid Chromatographic Separation of Serum Bilirubin Species with Direct Sample Injection

Abstract A rapid reversed-phase high performance liquid chromatographic method is described using a C8 column and a mobile phase containing sodium dodecyl sulfate (SDS) as a modifier to separate the four bilirubin species present in serum: unconjugated, monoconjugated, diconjugated and biliprotein. The results show that using SDS as a mobile phase modifier improves separation efficiency and increases sample solubility. This simple HPLC procedure allows direct sample injection and makes it possible to quantitatively determine bilirubin species in biological fluids.

Enhanced separation and detection of serum bilirubin species by capillary electrophoresis using a mixed anionic surfactant—protein buffer system with laser-induced fluorescence detection

The four major bilirubin species in serum are separated by capillary electrophoresis and detected using laser-induced fluorescence detection. The optimum buffer system consists of 40 m M sodium dodecyl sulfate (SDS)—0.012 m M bovine serum albumin (BSA). The use of the SDS—BSA mixture in the mobile phase allows for the separation of four major bilirubin species at physiological pH with untreated capillaries. The results show that the use of BSA as a run buffer modifier in SDS solution improves separation efficiency and increases sample solubility via pH changes of the run buffer. The limits of detection for the bilirubin species using laser-induced fluorescence are between 30 and 150 n M, depending on the bilirubin species; not only is this approximately two orders of magnitude lower than with visible-light absorption methods, it allows the bilirubin species in normal sera to be quantitatively measured without sample pretreatment.

The electrooxidation behavior of bilirubin in DMF and DMF + H2O mixed solvent

AbstractThe electrooxidation of Bilirubin (BR) in N, N′‐dimethylformamide (DMF) and DMF + H2O mixed solvent is investigated by voltammetry techniques, in situ rapid scan thin layer spectroelectrochemistry and in situ ESR spectroscopy. The data reveal that the oxidation process of BR undergoes many stages, which are all sped up by the introduction of water. The species of bilirubin taking part in the reaction is found to be changed from BR in DMF into BR' in the mixed solvents and the anodic peak potential is shifted correspondingly from +0.58 V to +0.026−+0.35 V (vs. Ag/AgCl, 1.0 M KCl). Free radicals and the dimerization of them are observed during the oxidation.

Rates of Oxidation of Bilirubin Species by Peroxidase With or Without Serum Albumin

Rates of Oxidation of Bilirubin Species by Peroxidase With or Without Serum Albumin

Separation of serum bilirubin species by micellar electrokinetic chromatography with direct sample injection

Four major bilirubin species in serum were separated by micellar electrokinetic chromatography with 25 m M sodium dodecyl sulfate (SDS) and 20 m M sodium tetraborate—boric acid buffer at pH 8.5. Due to the solubilization of the serum proteins by the SDS micelles, serum samples were injected directly into a 50 cm × 75 μm I.D. fused-silica capillary and complete separation of the four bilirubin species was accomplished within ca. 10 min without extensive sample pretreatment. Detection was performed by absorbance at 450 nm and average limit of detection was in the 6.0 μ M concentration range. The usefulness of this method was demonstrated for the separation and detection of a number of bilirubin species present in pathological human serum samples.

Effects of Serum Bilirubin on Determination of Uric Acid by the Uricase-Peroxidase Coupled Reaction

We examined the possible effect of different bilirubin species, including unconjugated (Bu), sugar-conjugated (Bc), and authentic delta bilirubin (Bd) isolated from serum, on the direct enzymatic measurement of uric acid by using the uricase (EC 1.7.3.3) and peroxidase (EC 1.11.1.7) coupled reaction. Bc, isolated from human bile, produced the most interference with uric acid determination. Although the presence of human serum albumin reduced interference by Bu and Bc, albumin-bound Bc complex still produced greater interference than Bu. Interference with the peroxidase reaction by Bc covalently bound to serum albumin (Bd) was less than that by Bu. We examined this phenomenon by using serum-isolated Bd obtained by ammonium sulfate precipitation and ultrafiltration and by using commercially available ditaurobilirubin (DTB) and chemically synthesized Bd (via Woodward's reagent, WBd) as surrogates for bile-isolated Bc and natural Bd, respectively. DTB produced the same amount of interference as natural Bc, but the interference produced by DTB in the presence of serum albumin was not the same as that produced by natural Bc with albumin. Our synthetic Bd appears to be a reliable surrogate for natural Bd in testing for bilirubin interference.

The Analysis of Bilirubin in Serum

The most widely used methods for the assay of total bilirubin in serum are based on a coupling reaction with various diazo dyes in the presence of an accelerating agent. The 'direct' reaction, without accelerator, gives a good estimate of the conjugated and protein bound (delta) species of bilirubin only if carefully standardized reaction conditions are used. Enzymatic methods based on bilirubin oxidase appear to give similar results, for both total and direct bilirubin, and through difficult to calibrate, spectrophotometric methods can also give reliable results for total bilirubin at the higher concentrations seen in neonates. More sophisticated HPLC or dry-slide techniques are required for the specific assay of the various conjugated and protein-bound species of bilirubin in serum.

Detection of Bilirubin Using Surfactant Fluorescence Enhancement and Visible Laser Fluorometry

Abstract Improvements on detection limits for the measurement of unconjugated and conjugated bilirubin in aqueous solutions using surfactant fluorescence enhancement and visible laser fluorometry were investigated. To maximize the fluorescence signal, commonly used surfactants were employed to enhance the fluorescence quantum yields of these bilirubin molecules and one of the visible lines of an argon ion laser was used for fluorometric excitation; to minimize optical noise, a unique optical fiber flow-cell was employed. Relative and absolute limits of detection for the micelle-bound unconjugated and conjugated bilirubin were in the sub-micromole per liter and sub-nanogram range, respectively. These capabilities should be important for the potential use of surfactants in flow injection analysis or micellar liquid chromatography for the determination of bilirubin species in biological materials.

Physico-chemical factors predisposing to pigment gallstone formation in liver cirrhosis

Liver cirrhosis is associated with a high prevalence of pigmentary cholelithiasis. The major compound of pigment gallstones is unconjugated bilirubin (UCB) in the form of calcium bilirubinate salts or a black pigment polymer. Most of UCB in bile derives from enzymic or non-enzymic hydrolysis of mono- or diconjugated bilirubin. Changes in the relative ratios between these two bilirubin species have been associated with pigment gallstones. It has also been shown that UCB solubilization in bile depends on its interaction with bile salts. In order to clarify the factors predisposing cirrhotic patients to pigment stone formation, we measured UCB, monoconjugated bilirubin (MCB) and diconjugated bilirubin (DCB) in duodenal bile of 15 patients with cirrhosis, ten patients with chronic active hepatitis (CAH) and ten normal subjects, we also analyzed their relationships with lipids. In cirrhotic patients, the MCB concentration in bile was significantly ( p < 0.05) higher than in normal subjects and was correlated with the severity of the disease. Bile salts and lecithin concentrations were significantly lower in cirrhosis ( p < 0.005 vs. CAH or normals). Cirrhotic patients have a bile salts/UCB molar ratio which is one third that of CAH patients or normal subjects ( p < 0.01). No differences were found between CAH patients and controls in each of the parameters tested. In conclusion, we propose that the very low BS UCB molar ratio and the very high biliary content in MCB represent two independent physico-chemical factors predisposing cirrhotic patients to pigmentary cholelithiasis.

Bilirubin and the Laboratory: Advances in the 1980s, Considerations for the 1990s

The limitations of current methods of measuring bilirubin are well established and relate to the broad dynamic range and inability of the technique to determine different but structurally similar bilirubin species. New instrumentation and methodology circumvent these limitations, and clinical studies are beginning to reveal their increased diagnostic usefulness. Nevertheless, in several clinical situations, for example, the prediction of kernicterus, better bilirubin determinations may not eliminate the controversy surrounding appropriate therapeutic interventions.