Coronary Artery Disease in Patients With Disorders of Bilirubin Excretion.

We aimed to determine the predictors of coronary artery disease (CAD) in patients with abnormal bilirubin excretion, that is, Gilbert syndrome, Crigler-Najjar syndrome, Dubin-Johnson syndrome, and Rotor syndrome. We analyzed data from the Healthcare Cost and Utilization Project (HCUP) of the Agency for Healthcare Research and Quality, Rockville, MD for the period 2009 to 2010. All patients ≥18 years of age with a primary diagnosis of "disorders of bilirubin excretion" [International Classification of Diseases, Ninth Edition, Clinical Modification (ICD-9CM) code 277.4] were included in the study. Primary outcome was to determine predictors of CAD in adult patients diagnosed with abnormal bilirubin excretion. We identified a total of 12,423 adult patients with bilirubin excretion disorder hospitalized during 2009-2010 (0.03% of all inpatient admissions). CAD was seen in 18% of patients, with a higher prevalence in men (21% in men vs. 13% in women, P < 0.0001). In multivariate logistic regression adjusted for demographic and traditional risk factors, hypertension [odds ratio (OR): 1.74; 95% confidence interval (CI), 1.33-2.27, P < 0.001], hyperlipidemia (OR: 2.49; 95% CI, 1.95-3.18, P < 0.001), diabetes (OR: 1.46; 95% CI, 1.12-1.91, P = 0.01), and age (OR: 1.05; 95% CI, 1.04-1.06, P < 0.001) were found to be independent predictors of CAD in adult patients with abnormal bilirubin excretion. Female sex (OR: 0.49; 95% CI, 0.36-0.65, P < 0.001) demonstrated an inverse association in predicting CAD. There was increased prevalence of CAD in our patient population with increased prevalence of cardiovascular risk factors. Age, diabetes mellitus, hypertension, and hyperlipidemia were found to be independent predictors of CAD.

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.

The roles of MRP2, MRP3, OATP1B1, and OATP1B3 in conjugated hyperbilirubinemia.

Increased concentrations of bilirubin glucuronides in blood plasma indicate hepatocellular dysfunction. Elucidation of the transport processes of bilirubin conjugates across the basolateral (sinusoidal) and the canalicular plasma membrane domains of hepatocytes has decisively contributed to our current understanding of the molecular basis of conjugated hyperbilirubinemia in human liver diseases. Under normal conditions, unconjugated bilirubin is taken up into hepatocytes by transporters of the organic anion-transporting polypeptide (OATP) family, followed by conjugation with glucuronic acid, and ATP-dependent transport into bile. This efflux across the canalicular membrane is mediated by multidrug resistance protein 2 (MRP2 or ABCC2), which is a 190-kDa glycoprotein transporting with high affinity and efficiency monoglucuronosyl bilirubin and bisglucuronosyl bilirubin into bile. MRP2 is hereditarily deficient in human Dubin-Johnson syndrome. Under pathophysiological conditions such as cholestatic liver injury and MRP2 inhibition, the basolateral efflux pump multidrug resistance protein 3 (MRP3 or ABCC3) is responsible for the occurrence of conjugated hyperbilirubinemia. MRP3 is a glycoprotein with a similar molecular mass as MRP2, with 48% amino acid identity, and with overlapping substrate specificity. Human MRP3 is the only basolateral efflux pump shown to transport bilirubin glucuronides. In human and rat hepatocytes, MRP3/Mrp3 is strongly upregulated under conditions of cholestasis and MRP2 deficiency. This is in line with the concept that basolateral efflux pumps of the hepatocyte compensate for impaired canalicular efflux of compounds into bile and contribute to balance the rate of uptake or synthesis of compounds in hepatocytes with the capacity for efflux into bile.

Rotor Syndrome: Glucuronidated Bile Acidemia From Defective Reuptake by Hepatocytes.

Organic anion transporting polypeptide (OATP) 1B1 (gene, solute carrier organic anion transporter family member 1B1 [SLCO1B1]) and OATP1B3 (SLCO1B3) serve as transporters for hepatic uptake of important endogenous substances and several commonly prescribed drugs. Inactivation of both proteins together causes Rotor syndrome. How this OATP1B1/1B3 defect disturbs bile acid (BA) metabolism is largely unknown. In this study, we performed detailed BA analysis in 3 patients with genetically diagnosed Rotor syndrome. We found that BAs glucuronidated at the C-3 position (BA-3G) accounted for 50% or more of total BAs in these patients. In contrast but similarly to healthy controls, only trace amounts of BA-3G were detected in patients with constitutional indocyanine green excretory defect (OATP1B3 deficiency) or sodium-taurocholate cotransporting polypeptide (NTCP; gene, solute carrier family 10 member 1 [SLC10A1]) deficiency. Therefore, substantial amounts of BA-3G are synthesized in hepatocytes. The cycling pathway of BA-3G, consisting of excretion from upstream hepatocytes and uptake by downstream hepatocytes by OATP1B1/1B3 may exist to reduce the burden on upstream hepatocytes. Conclusion: Detailed BA analysis revealed glucuronidated bile acidemia in patients with Rotor syndrome. Further exploration of the physiologic role of glucuronidated BAs is necessary.

Removing substances from blood by affinity chromatography. I. Removing bilirubin and other albumin-bound substances from plasma and blood with albumin-conjugated agarose beads.

Substances such as bilirubin that bind tightly to plasma proteins cannot readily be removed from blood. We describe here the use of affinity chromatography as a new approach to the removal of proteinbound metabolites and toxins from blood. Agarose beads were coupled via cyanogen bromide to human serum albumin so as to contain 30-50 mg of albumin/g wet wt. Such beads, when exposed to plasma from a patient with congenital nonhemolytic jaundice labeled with [(14)C]-bilirubin, bound more than 150 mug bilirubin/g of beads. The binding was saturable, concentration-dependent, relatively independent of flow rate, and reversible by elution with plasma, albumin, or 50% (vol/vol) ethanol. The beads could be repeatedly reused without loss of efficiency after ethanol elution and long storage in the cold. Salicylate, cortisol, and taurocholate, which bind weakly to albumin, were retarded by the beads but eluted with neutral buffer. Thyroxine, taurolithocholate, chenodeoxycholate, and digitoxin bound tightly but were eluted with 50% ethanol. Digoxin did not bind at all. When whole blood was passed over agarose-albumin beads, bilirubin was removed, calcium and magnesium fell slightly, but red cells, white cells, platelets, clotting factors, and a variety of electrolytes and proteins were substantially unchanged. Agarose-albumin beads may be useful for removing protein-bound substances from the blood of patients with liver failure, intoxication with protein-bound drugs, or specific metabolic deficits. Furthermore, it may be possible to make useful adsorbents by attaching other proteins to agarose or other polymer beads.

Impaired plasma clearance of nicotinic acid and rifamycin-SV in Gilbert's syndrome: evidence of a functional heterogeneity.

Patients with Gilbert's syndrome (GS) have impaired clearance by the liver of some organic anions. We looked for possible differences in hepatic clearance of nicotinic acid (NA) and rifamycin-SV (R-SV) among GS patients, and examined the effect produced by these anions on the plasma levels of unconjugated bilirubin (UCB). Two subgroups of GS patients, GS1 and GS2, were differentiated according to their ability to handle R-SV and NA. Compared with a control group, the alteration of the half-life both of NA and R-SV was less marked in GS1 than in GS2. UCB plasma concentration after NA and R-SV loading was more greatly increased in GS2 than in GS1 patients. In addition, a striking correlation was found in all subjects studied between UCB and the half-life of NA and R-SV. These related alterations of plasma UCB and plasma half-life or organic anions suggests a common defect of hepatic uptake. It is hypothesized that this defect is located at the level of a hepatic plasma membrane carrier.

Hypersideremic and hyperbilirubinemic effect of nicotinic acid in patients with Gilbert's syndrome.

Nicotinic acid test (NA) administration is followed by a rise in unconjugated bilirubin and serum total iron in healthy man. A similar concomitant effect has never been investigated in Gilbert's syndrome (GS) patients, who by definition have a hyperbilirubinemia higher than that of controls. The aim of this study was to verify whether, in addition to the hyperbilirubinemic action, NA could induce parallel hypersideremia in GS subjects. The data we obtained confirm in GS patients: 1. the well-known hyperbilirubinemia; 2. a delayed NA plasma concentration curve, and document that in GS the hypersideremic effect is comparable to that of controls. Previous investigators reported that NA produces an equimolar rise of bilirubin and iron levels in healthy man. In our study the extent of the sideremic response to NA occurring in GS patients is comparable to that seen in controls, and appears unrelated to the bilirubinemic values. If the NA effects are based on the documented hemolytic properties of NA, a disturbance in bilirubin handling by the liver of GS subjects appears to be plausible in the presence of almost normal behavior of NA-induced hypersideremia.

Plasma clearance of nicotinic acid and rifamycin-SV, and their interaction in Gilbert's syndrome: application of a compartmental model.

The bicompartmental kinetics of nicotinic acid (NA) and rifamycin-SV (R-SV)--2 organic anions that probably share a common hepatic uptake mechanism--were studied in 7 cases of Gilbert's syndrome (GS) and in 7 healthy controls matched for sex and age. In GS the NA and R-SV uptake constants (K21) were significantly decreased. In GS patients, simultaneous loads of NA and R-SV, the latter at increasing doses, produced: 1) a progressive lowering only of R-SV K21; and 2) an increase in R-SV hepatic plasma reflux (K12). Changes in biliary excretion ( Kee ) and hepatocellular pool (Ke) of both NA and R-SV probably depend on the rates of uptake and reflux constants of the two anions. The study of the parameters of compartmental kinetics of NA and R-SV confirms that the two organic anions, which have different metabolic routes and/or a different affinity for intracellular carriers, share common uptake mechanisms.

[Gilbert's jaundice. Current clinico-nosographic physiopathological and therapeutical aspects. I. Recent findings in Gilbert's syndrome]. / Gli itteri di Gilbert. Attuali aspetti clinico-nosografici, fisiopatologici e terapeutici. Nota I. Recenti acquisizioni sulle sindromi di Gilbert

Gilbert's icterus is a term used to cover certain forms of free bilirubin hyperbilirubinaemia which occur without any clear signs of hyperhaemolysis and are thus based on a fundamental defect in bilirubin liver cell clearance. Speculatively, this defect may be considered as being located at the level of any one of the steps along the metabolic route of the pigment, between the vascular pole of the liver cell and the microsomes. The incidence of these forms is calculated at about 4-6% of the population, while study of its familial distribution would suggest its inclusion among genetically conditioned metabolic disturbances. Investigations of various groups of patients suggest heredity of poorly penetrating, incomplete expressivity dominant autosomic type. As for pathogenesis, analysis of the formation of glycuronide bilirubin on the part of liver microsomes has shown a frequent reduction in glycuronyltransferase activity in patients with Gilbert's icterus; on the other hand, separation of the two carrier proteins y and z, and kinetic studies with free radiobilirubin, suggest that in certain of these subjects there is an alteration in the liver cell's capacity to take up and hold bilirubin removed from the blood. On the basis of such data, Gilbert's icteruses have been traditionally subdivided into two types: the first, with slight bilirubinaemia, due to an uptaking defect, the second, with higher bilirubin, due to a reduction in glycuronide conjugation. From the morphological viewpoint, the optical microscope does not reveal any outstanding elements in the livers of Gilbert patients; some workers using the electronic microscope have insisted on the not infrequent presence of damage to the vascular pole of the liver cell, which would fall in with the hypothesis of a membrane pathology as the underlying factor in one type of Gilbert's icterus. Numerous granules with lysosome characteristics have also been seen at the biliary pole of the liver cell. Whether these are the cause of the disease or, as would appear more likely, they are only an epiphenomenon secondary to the accumulation of a non-metabolized product of the liver, is still under discussion. Theoretically, therefore, two groups can be distinguished for free bilirubin icteruses of hepatic pathogenesis and thus not only for Gilbert's icterus; those due to a membrane or y and z carrier pathology, and those with microsome pathology due to partial glycuronyltransferase deficiency. The most recent tendency is thus to unify under the common label of a glycuronyltransferase deficiency the type II of Gilbert's icterus and the Crigler-Najjar disease, even though gene transmission modalities differ. Some workers thus suggest two types of Crigler-Najjar disease: type I, the classical type, due to absolute glycuronyltransferase deficiency and type II due to a relative deficiency, taking in the II form of Gilbert's icterus...

[Hyperbilirubinemia]. / Hyperbilirubinämie.

A 22-year-old woman had icteric sclerae since childhood. Five years ago of Gilbert-Meulengracht's disease was diagnosed (hyperbilirubinemia, normal other liver laboratory parameters, no evidence of hemolysis). The patient was admitted for re-evaluation. Apart from jaundice of the sclerae no other clinical symptoms were found. Analysis of urine revealed bilirubin and an increased urobilinogen. Serum bilirubin was also elevated. The differentiation of the bilirubin gave evidence of an increase of the direct (conjugated) bilirubin portion. Additional investigations (total coproporphyrin in the urine, isomer I and isomer III coproporphyrin excretion and bromsulphalein test) suggested Rotor's syndrome. Further examinations (oral cholecystography, liver biopsy) were not added because of relative invasiveness, lack of clinical consequences and opposition of the patient. Nevertheless the diagnosis of a Rotor's syndrome is highly probable.

New insights in bilirubin metabolism and their clinical implications.

Bilirubin, a major end product of heme breakdown, is an important constituent of bile, responsible for its characteristic colour. Over recent decades, our understanding of bilirubin metabolism has expanded along with the processes of elimination of other endogenous and exogenous anionic substrates, mediated by the action of multiple transport systems at the sinusoidal and canalicular membrane of hepatocytes. Several inherited disorders characterised by impaired bilirubin conjugation (Crigler-Najjar syndrome type I and type II, Gilbert syndrome) or transport (Dubin-Johnson and Rotor syndrome) result in various degrees of hyperbilirubinemia of either the predominantly unconjugated or predominantly conjugated type. Moreover, disrupted regulation of hepatobiliary transport systems can explain jaundice in many acquired liver disorders. In this review, we discuss the recent data on liver bilirubin handling based on the discovery of the molecular basis of Rotor syndrome. The data show that a substantial fraction of bilirubin conjugates is primarily secreted by MRP3 at the sinusoidal membrane into the blood, from where they are subsequently reuptaken by sinusoidal membrane-bound organic anion transporting polypeptides OATP1B1 and OATP1B3. OATP1B proteins are also responsible for liver clearance of bilirubin conjugated in splanchnic organs, such as the intestine and kidney, and for a number of endogenous compounds, xenobiotics and drugs. Absence of one or both OATP1B proteins thus may have serious impact on toxicity of commonly used drugs cleared by this system such as statins, sartans, methotrexate or rifampicin. The liver-blood cycling of conjugated bilirubin is impaired in cholestatic and parenchymal liver diseases and this impairment most likely contributes to jaundice accompanying these disorders.

Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.