Lower Bilirubin Concentrations Research Articles

Association between the UGT1A1 TA-Repeat Polymorphism and Bilirubin Concentration in Patients with Intermittent Claudication: Results from the CAVASIC Study

Bilirubin has antioxidative and cytoprotective properties. Low plasma concentrations of bilirubin are reportedly associated with the development of coronary and cerebrovascular disease, and bilirubin concentrations are strongly correlated with the enzyme activity of the hepatic uridine diphosphate glucuronosyltransferase (UGT1A1). The activity of UGT1A1 is influenced by a TA-repeat polymorphism in the promoter of the UGT1A1 gene (UDP glucuronosyltransferase 1 family, polypeptide A1). In a case-control study, we investigated the association between the UGT1A1 polymorphism, bilirubin concentration, and intermittent claudication. We included 255 consecutive male patients presenting with intermittent claudication in the investigation and matched the patients by age and diabetes mellitus with 255 control individuals. Plasma bilirubin concentrations were significantly lower in patients than in controls [mean (SD), 12.5 (5.3) micromol/L vs 15.4 (7.9) micromol/L; P < 0.001]. We found a clear association between the number of TA repeats and plasma bilirubin concentration. Considering the 6/6 TA-repeat genotype as the wild type, we observed a slight increase in bilirubin concentration individuals with the heterozygous 6/7 genotype and pronounced increases for those with the homozygous 7/7 genotype. This association occurred in both controls and patients; however, patients and controls were not significantly different with respect to UGT1A1 TA-repeat genotype frequencies. Our study of a well-phenotyped group of patients with intermittent claudication and control individuals revealed a clear association between low bilirubin concentrations and peripheral arterial disease but no association between the UGT1A1 polymorphism and the disease.

Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues

Unconjugated bilirubin (UCB) exhibits potent antioxidant and cytoprotective properties, but causes apoptosis and cytotoxicity at pathologically elevated concentrations. Accurate measurement of UCB concentrations in cells, fluids and tissues is needed to evaluate its role in redox regulation, prevention of atherosclerotic and malignant diseases, and bilirubin encephalopathy. In the present study, we developed and validated a highly sensitive method for tissue UCB determinations. UCB was extracted from rat organs with chloroform/methanol/hexane at pH 6.2 and then partitioned into a minute volume of alkaline buffer that was subjected to HPLC using an octyl reverse phase (RP) column. Addition of mesobilirubin as an internal standard corrected for losses of UCB during extraction. Recoveries averaged 75 ± 5%. The detection limit was 10 pmol UCB/g wet tissue. Variance was ±2.5%. When used to measure UCB concentrations in tissues of jaundiced Gunn rats, this procedure yielded UCB levels directly comparable to published methods, and accurately determined very low tissue bilirubin concentrations (≤40 pmol UCB/g tissue) in non-jaundiced rats.

Unbound Bilirubin: A Better Predictor of Kernicterus?

The peroxidase method described in this paper measures non–protein-bound bilirubin by oxidation of the free fraction. The oxidation rate is slow compared with rapid dissociation of albumin-bound bilirubin. Thus, the unbound bilirubin can be estimated from the initial rate of decline in total bilirubin concentration. In the 1970s, severe neonatal hyperbilirubinemia and kernicterus were major clinical problems. Kernicterus could usually be prevented by administering an exchange transfusion to infants whose serum unconjugated bilirubin concentration reached 341 μmol/L (20 mg/dL), but the diagnostic specificity of 341 μmol/L (20 mg/dL) was quite poor. Premature infants sometimes developed encephalopathy at very low bilirubin concentrations, especially when receiving sulfonamides. While engaged in a fellowship in neonatal medicine, I was stimulated by Gerald Odell’s 1959 paper, in which he used pharmacological arguments to explain how these drugs produced kernicterus by …

Relationship Between Serum Bilirubin Levels and Coronary Atherosclerosis in Patients with Type 2 Diabetes

Background: Lipid oxidation and formation of oxygen radicals have been identified to be the important factors of atherogenesis. Because bilirubin, a potent physiological antioxidant inhibits lipid oxidation, it is suggested that low serum concentrations of bilirubin is associated with atherosclerosis. The aim of this study was to evaluate the relationship between bilirubin levels and coronary atherosclerosis. Methods: The coronary calcium score (CCS) of 172 subjects (male 63, mean age 60.5 ± 1.0) with type 2 diabetes were evaluated in Yeungnam University Hospital between January 2005 and February 2007. The subjects were divided into two groups with CCS 10 as the cut off. Results: Higher CCS was significantly associated with lower bilirubin (P < 0.05), but after adjusted with age, no longer correlation were seen (P = 0.121). To determine the relationship between subclinical coronary atherosclerosis and bilirubin, the subjects with previous history of cardiovascular disease were excluded. In 138 subjects (male 54, mean age 58.4 ± 1.1), higher CCS was significantly associated with lower levels of bilirubin. After adjusted with age, duration of diabetes, and history of hypertension, CCS was also inversely related with bilirubin (P < 0.05). Conclusion: These results suggest that lower levels of bilirubin might be considered as a risk factor of coronary artery disease, especially in type 2 diabetics without cardiovascular disease. (KOREAN DIABETES J 32:338-345, 2008)

Impact of Serum Bilirubin on Human Diseases

Bilirubin, a major product of heme catabolism, belongs to compounds with pleiotropic biologic effects. Although for decades it was considered as a metabolite dangerous for human health, recent data indicate that bilirubin exhibits potent antioxidant properties with substantial positive clinical consequences. In a recent issue of Pediatrics , Sedlak and Snyder1 comprehensively reviewed available data on the beneficial role of bilirubin. Although the article covers current knowledge of all the aspects of this topic almost completely, I would like to mention more data that further support a salutary role of bilirubin in the human body. In addition to studies cited in the Sedlak and Snyder article, total serum antioxidant capacity was found to correlate with serum bilirubin levels in our study on adult Gilbert syndrome (GS) patients, and this relation was confirmed in an in vitro study.2 In regard to the cited bilirubin oxidation metabolite study in septic patients, recent studies (mainly from Japan) demonstrated close correlation between these markers (called biopyrrins) and many other pathologic conditions including ischemic heart disease,3,4 congestive heart failure,5 atopic dermatitis,6 and surgical7,8 or even psychological stress.9 The same relationship was also found … Address correspondence to Libor Vitek, MD, PhD, 4th Department of Internal Medicine and Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Medical Faculty, Charles University of Prague, U Nemocnice 2, Praha 2, 128 08, Czech Republic. E-mail: vitek{at}cesnet.cz

Biochemical reference intervals for koi ( Cyprinus carpio )

Various biochemical parameters of koi were measured to construct biochemical reference intervals. Very low serum concentrations of creatinine, total bilirubin, uric acid and γ-glutamyl transferase activity were observed. Increased activities of creatine kinase, lactate dehydrogenase and aspartate aminotransferase were observed due to venepuncture through the skeletal muscle of the caudal peduncle; however, these changes were not statistically significant. Alanine aminotransferase and sorbitol dehydrogenase activities were affected the least, and these enzymes may be considered relatively liver specific in koi. Cellulose acetate serum protein electrophoresis also was performed to confirm the presence of a serum albumin-like protein in koi. However, the resultant protein fractions were highly variable and the albumin protein-like fraction did not correlate well with that determined by the BCG dye binding assay.

Photic regulation of heme oxygenase activity in the golden hamster retina: involvement of dopamine.

The photic regulation of heme oxygenase (HO) activity was examined in the golden hamster retina. This enzymatic activity was significantly higher at midday than at midnight. When the hamsters were placed under constant darkness for 48 h and killed at subjective day or at subjective night, the differences in HO activity disappeared. Western blot analysis showed no differences in HO levels among these time points. Dopamine significantly increased this activity in retinas excised at noon or at midnight, with a higher sensitivity at night. The effect of dopamine was reversed by SCH 23390 but not by spiperone and clozapine and it was not reproduced by quinpirole. In vitro, the increase in HO activity found in retinas incubated under light for 1 h was significantly reduced by SCH 23390. Two cAMP analogs increased HO activity and their effect, as well as the effect of dopamine was blocked by H-89, a protein kinase A (PKA) inhibitor. Tin protoporphyrin IX, an HO inhibitor, significantly decreased cGMP accumulation with maximal effects during the day. Low concentrations of bilirubin decreased retinal thiobarbituric acid substances levels (an index of lipid peroxidation) in basal conditions and after exposing retinal cells to H2O2. These results suggest that hamster retinal HO activity is regulated by the photic stimulus, probably through a dopamine/cAMP/PKA dependent pathway.

Iterative, Spectrophotometric Method for Determination of Amniotic Fluid Bilirubin Concentrations: Comparison with the Liley Method

Since 1961, the Δ A 450, or Liley, method has been used to estimate amniotic fluid (AF) bilirubin concentrations in RhD-alloimmunized pregnancies (1). Although the results of this method often predict the degree of fetal anemia relatively well, blood-contaminated AF may give incorrect results (2)(3) Furthermore, with this method the AF bilirubin content is not expressed quantitatively as the bilirubin concentration. As an alternative, a diazotization method has been used to measure the bilirubin concentration in AF (4). Blood contamination could produce falsely increased bilirubin concentrations with this diazotization method, however, and this method is also considered to be inaccurate at the very low bilirubin concentrations in AF (3). Recently we developed a procedure that uses a mathematic step-by-step (iterative) procedure to deconvolute the superimposed absorbance scans of pigments in cerebrospinal fluid into the scans of individual pigments. This iterative model is now in routine use for the spectrophotometric determination of methemoglobin (metHb), oxyhemoglobin (oxyHb), and bilirubin concentrations in cerebrospinal fluid (5). The method is fast and simple, corrects for the relative interference of each of these components on the others, and has a lower limit of detection of 0.1 μmol/L for each of these components. We suggest that, because of its high sensitivity, this method may become a reliable procedure to measure bilirubin concentrations in AFs. AF was obtained after informed consent from patients [n = 52; mean (SD) gestational age, 30.8 ± 3.6 weeks] whose pregnancies were complicated by red cell alloimmunization. The fluids were centrifuged at 1400 g for 10 min and were protected from light before analysis. Measurements were performed with a UltrospecPlus spectrophotometer (Amersham Pharmacia Biotech). For the Liley method, absorbance was measured at 365, 450, and 550 nm, and a baseline was estimated between 365 and 550 nm on a …

Use of assist devices and ECMO to bridge pediatric patients with cardiomyopathy to transplantation

Background: Pulsatile ventricular assist devices (VADs) are used to bridge adults with end-stage heart disease to transplantation. A combination of external implantable pulsatile and continuous-flow external mechanical support now can be used to bridge pediatric patients with end-stage cardiomyopathy to orthotopic heart transplantation (OHT). Methods: We reviewed consecutive pediatric patients with cardiomyopathy ( n = 28) who required mechanical cardiac support from July 1995 to February 2001. All were OHT candidates with severe hemodynamic compromise despite maximal medical support. We excluded from this series patients who had undergone cardiotomy. Results: Nineteen patients received support from external continuous-flow devices, either with extracorporeal membrane oxygenation or with centrifugal VADs, and 9 patients received pulsatile support. Nineteen of 28 (68%) patients were successfully bridged to transplant (17) or weaned (2) from their devices after recovery. Of the patients successfully bridged to transplant or recovery, 89% are alive to date. Univariate analysis revealed that a lower bilirubin concentration after 2 days of support was associated with a favorable outcome ( p = 0.006). As expected, the patients with pulsatile VADs had significantly higher rates of extubation and oral feeding. Conclusion: Pulsatile and continuous-flow devices can complement each other to significantly extend the lives of a wide range of pediatric patients with severe cardiomyopathies.

Physiologic roles for the heme oxygenase products carbon monoxide, bilirubin and iron: links to neuroprotection in stroke and Alzheimer's disease

Heme oxygenase (HO) cleaves the heme ring releasing iron, carbon monoxide (CO) and biliverdin, which is immediately reduced to bilirubin by the abundant biliverdin reductase. Recent research indicates major cellular roles for all three HO products. The constitutive isoform, heme oxygenase 2 (HO2), is concentrated in the brain with discrete localizations resembling soluble guanylyl cyclase, implying a role in regulating cyclic guanosine monophosphate (cGMP). HO2 is localized to myenteric plexus neurons of the intestine similar to neuronal nitric oxide synthase (nNOS). Mice with targeted deletion of HO2 or nNOS each display marked reductions in non-adrenergic-non-cholinergic neurotransmission and cGMP levels indicating a neurotransmitter role for the two gases acting as co-transmitters. HO2 is localized to the endothelial cell layer of blood vessels as well as neurons in the adventitial layer. HO inhibitors reduce nitric oxide (NO)-independent vasodilation implicating CO as an endothelial derived relaxing factor. Bilirubin, formed from HO2, appears to be a physiologic neuroprotectant. Neurotoxicity is augmented in brain cultures from HO2 gene knockout mice which also display augmented neural damage following cerebral vascular occlusion. Very low concentrations of bilirubin reverse the neurotoxicity. Heme oxygenase 1 (HO1) plays a role in iron efflux from cells. Iron efflux is diminished in cells from HO1 gene knockout mice, while HO1 transfection augments efflux. Iron dependent cytotoxicity is worsened in HO1 gene knockout cells while HO1 transfection alleviates the toxicity.

Heme Oxygenase-2 Is Neuroprotective in Cerebral Ischemia

Heme oxygenase (HO) is believed to be a potent antioxidant enzyme in the nervous system; it degrades heme from heme-containing proteins, giving rise to carbon monoxide, iron, and biliverdin, which is rapidly reduced to bilirubin. The first identified isoform of the enzyme, HO1, is an inducible heat-shock protein expressed in high levels in peripheral organs and barely detectable under normal conditions in the brain, whereas HO2 is constitutive and most highly concentrated in the brain. Interestingly, although HO2 is constitutively expressed, its activity can be modulated by phosphorylation. We demonstrated that bilirubin, formed from HO2, is neuroprotectant, as neurotoxicity is augmented in neuronal cultures from mice with targeted deletion of HO2 (HO2(-/-)) and reversed by low concentrations of bilirubin. We now show that neural damage following middle cerebral artery occlusion (MCAO) and reperfusion, a model of focal ischemia of vascular stroke, is substantially worsened in HO2(-/-) animals. By contrast, stroke damage is not significantly altered in HO1(-/-) mice, despite their greater debility. Neural damage following intracranial injections of N-methyl-d-aspartate (NMDA) is also accentuated in HO2(-/-) animals. These findings establish HO2 as an endogenous neuroprotective system in the brain whose pharmacologic manipulation may have therapeutic relevance.

Acute parvovirus B19 infection associated with fulminant hepatitis of favourable prognosis in young children

The cause of fulminant hepatitis (FH) in children is unexplained in up to 50% of cases. We report parvovirus B19 as an agent associated with FH in children and compare clinical characteristics of these patients with those of age-matched patients with FH of other origin. 45 patients presented with FH. No cause was apparent in 21 patients. Parvovirus B19 genome was retrospectively sought by PCR in serum collected at admission in 41 patients. Parvovirus B19 genome was detected in serum from four of 21 patients with unexplained FH (four of 11 younger than 5 years). No B19 DNA was detected in serum from patients with other types of FH or from 82 patients with biliary atresia. Parvovirus B19 IgM was detected in one of the four patients. Patients with parvovirus B19 infection had significantly lower bilirubin concentrations than age-matched patients with FH due to hepatitis A (nine) or other causes (nine) (poisoning with amanita excluded). All patients with parvovirus B19 survived without orthotopic liver transplantation, with restoration of normal liver function within 17 days. In patients younger than 5 years with FH of unexplained origin, evidence of acute parvovirus B19 was associated with a distinct clinical pattern. In particular, low bilirubin concentrations and rapid recovery of liver function without transplantation were distinctive features.

Effect of bilirubin on erythrocyte shape and haemolysis, under hypotonic, aggregating or non-aggregating conditions, and correlation with cell age

The effect of unconjugated bilirubin on the morphology and haemolysis of human erythrocytes was accomplished under distinct incubation conditions: (i) hypotonic medium, with bilirubin concentrations ranging from 1 × 10−9 to 1 × 10 −4 mol 1−1; (ii) isotonic medium, with 171 μmol 1−1 bilirubin, in the absence of albumin (aggregating conditions), using non-separated and age-fractionated erythrocytes; (iii) isotonic medium, with 171 μmol 1−1 bilirubin, in the presence of a surplus of human serum albumin (non-aggregating conditions), and using sulfisoxazole as a bilirubin displacer (bilirubin/albumin and sulfisoxazole/ albumin molar ratios of 0.5 and 4.0, respectively).Our data showed that low concentrations of bilirubin (1 × 10−7 to 1 × 10−5 mol 1−1) protect against hypotonic haemolysis and induce crenation, while higher bilirubin concentrations induce haemolysis and lead to membrane disruption. When aggregating conditions were used, these phenomena were reproduced, the younger cells being significantly more susceptible to crenation while the older erythrocytes showed increased susceptibility to haemolysis. In non-aggregating conditions, haemolysis was virtually absent, though crenation was evident. Based on the above observations we conclude that the first step of erythrocyte bilirubin toxicity is crenation due to an expansion of the outer membrane leaflet by bilirubin mono-anion location. This effect is more evident in younger cells and explains the protection against the hypotonic haemolysis. Insertion of bilirubin deeper into the bilayer, facilitated by higher concentrations (≥1 × 10−4 mol 1−1) and cell age, produces an unstable situation, where bilirubin acid aggregation is apparently the main cause for haemolysis and cell destruction.

Serum bilirubin and cardiovascular risk factors in a Chinese population.

Many risk factors for cardiovascular disease (CVD) have been identified. Recently an association between low concentration of serum bilirubin and increased risk of CVD has been reported. However, information on this topic remains scarce. We examined the relationships between serum bilirubin and CVD risk factors in 1508 Hong Kong Chinese. We divided the subjects into four quartiles based on serum bilirubin concentrations. Cardiovascular risk factors studied include age, sex, smoking, obesity glycaemic status and lipid indices. Decreasing serum bilirubin concentration was associated with older age, increased prevalence of smoking, higher body mass index and systolic blood pressure, increased glycated haemoglobin, fasting and 2 h insulin, triglyceride, very-low-density lipoprotein and apolipoprotein B concentrations, and lower high-density lipoprotein concentration. Women had lower bilirubin concentrations than men. After adjustment for age, sex, smoking and insulin levels as covariates, the associations between serum bilirubin concentration and glycated haemoglobin, triglyceride, high-density lipoprotein and very-low-density lipoprotein persisted. The prevalence rates of abnormal glucose tolerance (impaired glucose tolerance or diabetes) were similar amongst the four quartiles of bilirubin concentrations. However, the mean bilirubin concentration was significantly lower in subjects with abnormal glucose tolerance (9.3 +/- 3.5 mumol/l, n = 178) than in normal subjects (10.1 +/- 5.2 mumol/l, n = 1330, P = 0.039). When analysed as a continuous variable by age-adjusted partial correlation coefficients, serum bilirubin concentration was inversely correlated with fasting insulin, triglyceride, very-low-density lipoprotein and glycated haemoglobin level. There were close associations between low serum bilirubin concentration and increased CVD risk factors. Subjects with abnormal glucose tolerance also had lower serum bilirubin concentration than normal subjects. Some of these associations (body mass index, systolic blood pressure) could be explained by differences in insulin level. These relationships between bilirubin and CVD risk factors require further clarification, although abnormal intermediary metabolism and antioxidant deficiency may be possible linking factors.

Serum bilirubin and risk of ischemic heart disease in middle-aged British men

The possibility that low concentrations of serum bilirubin may be associated with increased risk of ischemic heart disease has been examined in a prospective study of 7685 middle-aged British men. During 11.5 years there were 737 major ischemic heart disease (IHD) events. A U-shaped relationship was observed between serum bilirubin and risk of IHD. Low bilirubin was associated with several cardiovascular risk factors, in particular smoking, low concentrations of high-density lipoprotein cholesterol, low forced expiratory volume in 1 s, and low serum albumin. The U-shaped relationship persisted even after adjusting for several risk factors. Compared with men in the lowest fifth of the distribution (bilirubin < 7 mumol/L), those in the middle range (8-9 mumol/L) showed a 30% reduction in relative risk [RR = 0.68 (95% confidence intervals 0.51-0.89)] in IHD, whereas men in the top fifth (> 12 mumol/L) showed similar risk to the lowest fifth [RR = 0.99 (95% confidence intervals 0.73-1.34)], which persisted after exclusion of men with bilirubin > 17 mumol/L. The significance of this U-shaped relationship is unclear, but it could be interpreted as support for the role of endogenous antioxidants in the etiology of IHD.

Inhibition of thyroxine transport into cultured rat hepatocytes by serum of nonuremic critically ill patients: effects of bilirubin and nonesterified fatty acids.

We investigated bilirubin and oleic acid as causes of low plasma T3 in nonuremic critically ill patients with gross changes in serum thyroid hormone levels (T4, < or = 60; T3, < or = 1.1; rT3, > or = 0.45 nmol/L) and elevated bilirubin concentrations (> or = 33 mumol/L). Iodide production from [125I]T4 was inhibited by 42% when rat hepatocytes in primary cultures were incubated with 10% serum from these patients. The mean serum concentration of albumin was reduced by 41%, while the concentrations of bilirubin and nonesterified fatty acids (NEFA) were increased by 2022% and 115%, respectively, in the patients. The molar ratios of bilirubin/albumin and NEFA/albumin in the patients were 0.42 and 3.18, respectively. Addition of oleic acid (50-400 mumol/L) and bilirubin (3-130 mumol/L) to 10% normal human serum (albumin, 70 mumol/L; NEFA, 54 mumol/L; bilirubin, 1.1 mumol/L) progressively inhibited the production of iodide by rat hepatocytes. The decreased iodide production was presumed to be caused by inhibition of T4 transport into hepatocytes. The deiodination of rT3 by rat liver microsomes was unaltered by free bilirubin and free oleic acid concentrations up to 0.1 mumol/L. These free concentrations are at least 1 order of magnitude higher than that attained in nonthyroidal illness. The inhibition of iodide production by the sera of critically ill patients (n = 12) was significantly correlated with the molar ratios of bilirubin/albumin (r = 0.72; P < 0.01) and NEFA/albumin (r = 0.58; P < 0.05). Extensive dialysis or treatment of the sera with charcoal did not completely remove the inhibitory activity on iodide production. Serum concentrations of indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furan propanoic acid, and hippuric acid in the critically ill patients (other known T4 transport inhibitors into hepatocytes) were similar to those in the normal subjects. This study together with the well known effects of carbohydrate on T3 neogenesis suggest that elevated bilirubin and NEFA and the low albumin level in non-uremic critical illness may be at least partly responsible for the T4 transport inhibition in T3-producing tissues (e.g. the liver) and, thus, the low plasma T3 levels in these critically ill patients. The question of whether inhibitors of T4 transport into the hepatocytes are also present in other patients with nonthyroidal illness who show only mild changes in thyroid hormone levels and have low concentrations of bilirubin and NEFA remains to be determined.

Serum complement activity and serum enzymes in rats after a subcutaneous injection of toxin prepared from Pasteurella multocida type D

Toxin produced by Pasteurella multocida type D was investigated for its effect on serum complement and serum biochemistry in rats. Rats were given a sublethal single subcutaneous injection of D toxin equivalent to 0.2 μg/kg of body weight. Serum obtained 1,3,5 and 7 days post-treatment was tested for complement activity, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Serum complement titers were significantly elevated ( P < 0.05) at all times after injection of toxin compared to rats injected with diluent and tested at the same intervals. Bilirubin was decreased but both control and D toxin-treated rats had low concentrations of bilirubin in their sera. The other biochemical constituents measured had no consistent pattern that would indicate liver damage in the rats.

Bilirubin inhibition of enzymes involved in the mitochondrial malate-aspartate shuttle

The effect of increasing bilirubin concentrations upon the catalytic activity of a series of dehydrogenases and aminotransferases was examined. The particular enzymes were chosen to examine the effect of bilirubin upon the activity of enzymes responsible for the indirect transfer of reducing equivalents across the inner mitochondrial membrane. Malate dehydrogenase was inhibited at very low concentrations of bilirubin and showed competitive inhibition with respect to coenzyme of 2 μM, while the cytosolic form of this enzyme exhibited a 15 μM inhibition constant. Cytosolic glycerol-3-phosphate dehydrogenase was not appreciably inhibited by bilirubin. Both the mitochondrial and cytosolic forms of aspartate aminotransferase showed moderate competitive bilirubin inhibition with respect to substrates with a K i of 30 μM with respect to 2-oxoglutarate and a K i of 80 μM with respect to aspartate. Preincubation studies indicated that inhibition was reversible for all enzymes examined. These results are interpreted in terms of the inhibition of the malate-aspartate shuttle by relatively low concentrations of bilirubin.

Effects of ioglycamide on the hepatic transport of bilirubin and its mono- and diconjugates in the rat.

Bilirubin seems to share the biliary excretion pathway with other organic anions, but not with bile acids. We studied the effects of the organic anion ioglycamide, an iodinated contrast agent, on bilirubin metabolism in Wistar rats. This compound does not undergo conjugation and is characterized by a maximal biliary secretory rate (Tm). The results show that in spite of producing a 3-fold increase in bile flow, ioglycamide excretion under Tm conditions decreased the output of unconjugated bilirubin and its monoconjugate by approximately 90%. Diconjugated bilirubin decreased by only 50% and became by far the predominant pigment in bile (86.5 +/- 6.0% of total pigment vs. 61.0 +/- 4.0% in basal conditions, n = 12). Unconjugated and monoconjugated bilirubins changed in parallel suggesting that the former arises from the monoconjugates. In serum, diconjugated bilirubin augmented from trace amounts to 1.15 +/- 0.17 mumole per liter. Total conjugated pigments in serum increased from 5 to 85% of total bilirubin. Bile acid output remained unchanged. Pretreatment of rats with ioglycamide altered neither the activity of bilirubin UDP-glucuronyltransferase nor the ratio of diconjugate to monoconjugate formed at both low (25 microM) and high (164 microM) bilirubin concentrations. The observed biological effects of ioglycamide were dose-dependent and fully reversible. We suggest that ioglycamide interferes with the excretion of conjugated bilirubins ("bilirubinostasis"). The monoconjugates retained in the hepatocyte might then undergo more efficient transformation to diconjugates, the latter thus becoming the most important bile pigments in serum and bile.

Glucocorticoids directly affect spectrophotometry of bilirubin in amniotic fluid.

Dexamethasone or prednisolone, added in vitro to bilirubin-containing amniotic fluid, produces a time-dependent decrease in the 450-nm absorbance of the pigment. Neither the chemical determination of bilirubin in amniotic fluid nor the lecithin/sphingomyelin ratio as determined by thin-layer chromatography is affected by these glucocorticoids. The effect probably is not a result of displacement of bilirubin from its binding sites on albumin, because the absorbance of a solution of crystalline bilirubin at 450 nm is unaffected by added bovine serum albumin. Light scattering of amniotic fluid increases slightly when dexamethasone is added, whether or not low concentrations of bilirubin (less than 1.6 mumol/L) are present. Thus the effect on absorbance evidently is not ascribable to supersaturation and formation of a colloidal sol of bilirubin particles. This direct interference of glucocorticoids with the spectrophotometry of bilirubin in amniotic fluid prompts cautious interpretation of such data as an index to the severity of hemolytic disease of the fetus, specifically in cases of Rhesus-isoimmunization that are being treated with glucocorticoids.