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PubMed1980

Rapid fluorometric assay of bilirubin and bilirubin binding capacity in blood of jaundiced neonates: comparisons with other methods.

The concentrations of total blood bilirubin, albumin-bound bilirubin, and the reserve and total bilirubin binding capacities of 35 neonatal blood samples (28 patients) were determined by automated front-face fluorometry ((hematofluorometer). These values were compared to results of diazo determinations, Sephadex gel filtration, and peroxidase-oxidation methods. Total blood bilirubin level by fluorometry agreed well with the total plasma bilirubin level by diazotization (r = .96, sigma = 1.7 mg/100 ml). Albumin-bound bilirubin concentrations by fluorometry also correlated well with diazo values (r = .95, sigma = 1.9 mg/100 ml) and were slightly lower than the total blood bilirubin concentrations. Values for total bilirubin binding capacity determined by fluorometry agreed well with results obtained for the same specimens by Sephadex gel filtration (n = 28, r = .97, sigma = 1.8 mg/100 ml) and by peroxidase-catalyzed oxidation (n = 25, r = .97, sigma = 1.7 mg/100 ml). The agreement among the results obtained by the three methods indicates a well-defined in vitro end point at which available primary or "tight" binding sites on albumin are saturated with bilirubin. In this clinical experience the coefficient of variation of results with the hematofluorometer was 8.4% for total blood bilirubin and 6.5% for total binding capacity. A comparison of "sick" with "well" infants revealed that the fraction of bilirubin not bound to albumin was significantly different for these two groups. The assays made with the hematofluorometer are quick (10 to 15 minutes) and require only a small quantity (approximately 150 microliters) of blood.

Bilirubin↗
PubMed1990

Gunn rats: a reproducible experimental model to compare the different methods of measurements of bilirubin serum concentration and to evaluate the risk of bilirubin encephalopathy.

Three groups of Gunn rats were studied: group 1 was perfused with bilirubin solution alone, group 2 was perfused with bilirubin and albumin solutions simultaneously, group 3 was perfused with bilirubin solution for 30 min then bilirubin and albumin solutions for the following 10 min. Our results indicate that (1) Gunn rats are a reliable experimental model to study the risk of bilirubin encephalopathy, (2) unbound bilirubin can enter the brain when albumin binding capacity is reduced, (3) and bilirubin binding capacity of serum for unbound unconjugated serum bilirubin is a better criterion than total serum bilirubin and erythrocyte bilirubin to evaluate the risk of kernicterus. This model could also be used to study variations of permeability of the blood-brain-barrier and influences of drugs on bilirubin metabolism.

Animals↗
PubMed1989

Thyroid hormones and the hepatic handling of bilirubin. I. Effects of hypothyroidism and hyperthyroidism on the hepatic transport of bilirubin mono- and diconjugates in the Wistar rat.

The effects of thyroidectomy and of thyroid hormone administration on the hepatic transport of endogenous bilirubin were investigated in the Wistar R/APfd rat. Hypothyroidism resulted in an enhanced hepatic bilirubin UDP-glucuronosyltransferase activity and in a decreased p-nitrophenol transferase activity. It caused a cholestatic condition with a 50% decrease in bile flow and bile salt excretion, and an increased proportion of conjugated bilirubin in serum. The biliary output of unconjugated and monoconjugated bilirubins decreased in parallel by about 65%, whereas the excretion rate of the diconjugate dropped by only 47%, resulting in an increased di- to monoconjugate ratio in bile. Hyperthyroidism was characterized by a decreased bilirubin and an increased p-nitrophenol transferase activity, and by an augmented bilirubin output in bile. The output of unconjugated and monoconjugated bilirubins increased in parallel by about 50 or 100%, whereas the excretion of the diconjugate increased by only 20 to 50%, depending on the dose of thyroxine administered; this resulted in a decreased di- to monoconjugate ratio in bile. A linear positive relationship was found between bilirubin UDP-glucuronosyltransferase activity and the ratio of bilirubin di- to monoconjugates present in bile or formed by in vitro incubation of liver homogenates at low concentration of bilirubin (10 to 15 microM), indicating that bile pigment composition is mainly determined by the conjugation activity in the liver. The inverse relationship observed between hepatic beta-glucuronidase activity and the ratio of di- to monoconjugates in bile warrants further investigation to analyze whether this enzyme activity also plays a possible role in the changes in bile pigment composition in hypo- and hyperthyroid rats.

4-Nitrophenylphosphatase↗
PubMed1992

Flow cytometric measurement of intracellular bilirubin in human peripheral blood mononuclear cells exposed to unconjugated bilirubin.

Human peripheral blood mononuclear cells were incubated at 37 degrees C with bilirubin in bovine albumin solution. Histological analysis of bilirubin-treated cells demonstrated a prominent brown pigment deposited in the cytoplasm. Homogenates of these cells in chloroform-methanol solution showed an identical absorption spectrum with pure bilirubin dissolved in the same solution. When bilirubin-treated cells were excited at 488 nm, a significant autofluorescence was detected by flow cytometry at 585 nm in a dose-dependent manner, which had a significant correlation with the amount of bilirubin chemically extracted from the cells (r = 0.963, n = 34, p less than 0.001). Intraassay and interassay variability of the autofluorescence by flow cytometric analysis was small (both less than 5%). When bilirubin-treated cells were stained with fluorescein-labeled anti-CD14 antibody, the CD14 positive cell population can be fractionated without interference of autofluorescence derived from intracellular bilirubin. These results suggest that flow cytometric analysis of bilirubin-treated cells can quantitate intracellular bilirubin, and that bilirubin does not interfere with analysis of surface antigens.

Adult↗
PubMed1991

Anti-bilirubin monoclonal antibody. III. Preparation and properties of monoclonal antibodies to unconjugated bilirubin-IX alpha.

Anti-bilirubin-IX alpha monoclonal antibodies exclusively specific for unconjugated bilirubin-IX alpha are prepared and characterized. Using modified MBS (metamaleimidobenzoyl-N-hydroxysuccinimide ester) method, bilirubin-IX alpha was covalently coupled to bovine serum albumin (BSA) retaining its intramolecular hydrogen bonds as well as three-dimensional structure. Somatic cell fusion was performed between murine spleen cells immunized with the bilirubin-IX alpha-BSA and murine myeloma P3-X63-Ag8-U1 cells. After screening assay, 58 clones were identified which were producing antibodies not to albumin nor MBS, but to haptenic bilirubin-IX alpha. In inhibition analysis, the antibodies in the culture supernatant reacted only with bilirubin-IX alpha to a varying degree, but did not react with conjugated bilirubin-IX alpha, including bilirubin-IX alpha monoglucuronide, bilirubin-IX alpha diglucuronide, and ditauronic bilirubin-IX alpha.

Animals↗
PubMed1977

Comparison in different species of biliary bilirubin-IX alpha conjugates with the activities of hepatic and renal bilirubin-IX alpha-uridine diphosphate glycosyltransferases.

The bilrubin-IXalpha conjugates in bile and the activities of bilirubin-IX alpha--UDP-glycosyltransferases in liver and kidney were determined for ten species of mammals and for the chicken. 1. In the mammalian species, bilirubin-IX alpha glucuronide was the predominant bile pigment. Excretion of neutral glycosides was unimportant, except in the cat, the mouse, the rabbit and the dog, where glucose and xylose represented 12--41% of total conjugating groups bound to bilirubin-IX alpha. In chicken bile, glucoside and glucuronide conjugates were of equal importance. They probably represent only a small fraction of the total bile pigment. 2. The transferase activities in liver showed pronounced species variation. This was also apparent with regard to activation by digitonin, pH optimum and relative activities of transferases acting on either UDP-glucuronic acid or neutral UDP-sugars. 3. Man, the dog, the cat and the rat excrete bilirubin-IX alpha largely as diconjugated derivatives. In general, diconjugated bilirubin-IX alpha could also be synthesized in vitro with liver homogenate, bilirubin-IX alpha and UDP-sugar. In contrast, for the other species examined, bilirubin pigments consisted predominantly of monoconjugated bilirubin-IX alpha. Synthesis in vitro with UDP-glucuronic acid, UDP-glucose or UDP-xylose as the sugar donor led exclusively to the formation of monoconjugated bilirubin-IX alpha. 4. The transferase activities in the kidney were restricted to the cortex and were important only for the rat and the dog. No activity at all could be detected for several species, including man. 5. Comparison of the transferase activities in liver with reported values of the maximal rate of excretion in bile suggests a close linkage between conjugation and biliary secretion of bilirubin-IX alpha.

Animals↗
PubMed1980

Analysis of bilirubin and bilirubin mono- and di-conjugates. Determination of their relative amounts in biological samples.

1. A novel method for determination of the relative amounts of unconjugated bilirubin and sugar mono- and di-conjugates of bilirubin in biological samples, including serum, is described and illustrated by its application to the analysis of bilinoids in rat bile. 2. The method is based on specific conversion of the carbohydrate conjugates of bilirubin into the corresponding mono- or di-methyl esters by base-catalysed transesterification in methanol. Under the selected reaction conditions, unconjugated biliru-in remains intact and no dipyrrole exchange in the bilinoids is detectable; transesterification of bilirubin mono- or di-glucuronide is virtually complete (approx. 99%), and sponification is negligible (less than 1%); recovery of the pigments is approx. 95%. 3. The reaction products bilirubin and its methyl esters are separated by t.l.c. and determined spectrophotometrically; the two isomeric bilirubin-IX alpha monomethyl esters are separated and therefore can be determined individually. 4. Reference bilirubin mono- and di-methyl esters have been synthesized and characterized, and the two isomers of bilirubin-IX alpha monomethyl ester and bilirubin dimethyl ester were obtained individually, in crystalline form. 5. With this new method, virtually all bilinoids (over 99%) in normal rat bile have been found to be conjugated, with diconjugates (71%) predominating. A significantly increased proportion of monoconjugates is present in bile collected from heterozygous Gunn rats or from normal rats that were refused with large amounts of bilirubin.

Alkalies↗
PubMed1978

Definition of a conjugation of dysfunction in Gilbert's syndrome: studies of the handling of bilirubin loads and of the pattern of bilirubin conjugates secreted in bile.

1. Intravenous doses of bilirubin (3.4 mumol/kg) were given to normal subjects and patients with Gilbert's syndrome. Both groups displayed an identical initial disappearance of a substantial proportion of the bilirubin but, late in time, the Gilbert's patients exhibited reduced clearance with a sustained elevation of the plasma bilirubin and no reflux into the plasma space of conjugated bilirubin. Increasing the dose in normal subjects (by factors of 3 and 6) failed to reproduce the response found in the Gilbert's patients. 2. In the the bile-containing duodenal aspirates of Gilbert's patients the average proportion of bilirubin found as bilirubin diglucuronide was 68% (normal 88%) and of bilirubin monoglucuronide, 23% (normal 7%). Both differences were significant at the P less than 0.001 level. In the Gilbert's patients restriction of caloric intake to 1569 kJ/day for 2 days characteristically raised the serum bilirbuin with no modification of the biliary pigment pattern; phenobarbital (180 mg/day for 2 weeks) decreased the plasma bilirubin to the normal range with, concomitantly, a reversion of the biliary pigment pattern towards normal. 3. We conclude that there is no hepatic uptake defect in Gilbert's syndrome but that there is decreased activity in the conjugation process underlying the addition of the second glucuronic acid moiety to bilirubin, to form bilirubin diglucuronide.

Adolescent↗
PubMed2001

Zinc salts precipitate unconjugated bilirubin in vitro and inhibit enterohepatic cycling of bilirubin in hamsters.

BACKGROUND: We have evidence for enterohepatic cycling of bilirubin experimentally and in vivo in humans. This study was designed to investigate whether Zn salts might inhibit such cycling of bilirubin. MATERIALS AND METHODS: Micellar bile salt solutions with unconjugated bilirubin were prepared, appropriate concentrations of Zn salts were added, and unconjugated bilirubin precipitation was measured. Hamsters and Wistar rats were fed a chow diet or a chow diet enriched with 1% ZnSO4, and bilirubin secretion rates were monitored. RESULTS: Unconjugated bilirubin was precipitated maximally (90%) after a 10-min incubation with 5 mM Zn salts in the pH range of 6.8-9.0. In control hamsters, total bilirubin secretion rates into bile were 36.0 +/- 2.8 nmol h(-1) 100g(-1) body weight, whereas they were 25.0 +/- 3.3 nmol h-1 100(-1) g in the ZnSO4 group (P < 0.05). CONCLUSIONS: Zn salts that flocculate at physiological pH adsorb unconjugated bilirubin almost completely from unsaturated micellar BS solutions. In addition, Zn salts administered orally suppress biliary bilirubin secretion rates in hamsters. These findings suggest that the administration of Zn salts may inhibit the enterohepatic cycling of unconjugated bilirubin in humans who are predisposed to pigment gallstone formation due to diet, disease or drugs.

Animals↗
PubMed1977

Unconjugated bilirubin and an increased proportion of bilirubin monoconjugates in the bile of patients with Gilbert's syndrome and Crigler-Najjar disease.

Bilirubin pigments were studied in the bile of 20 normal adults, 25 patients with Gilbert's syndrome, 9 children with Crigler-Najjar disease, and 6 patients with hemolysis, to determine how a deficiency of hepatic bilirubin UDP-glucuronosyltransferase would affect the end products of bilirubin biotransformation. In the bile from patients with Gilbert's syndrome, a striking increase was found in the proportion of bilirubin monoconjugates (48.6+/-9.8% of total conjugates) relative to that in normal bile (27.2+/-7.8%). This increase was even more pronounced in children with Crigler-Najjar disease, in whom, even in the most severe cases, glucuronide could always be demonstrated in the bile. Furthermore, unconjugated bilirubin-IXalpha was unquestionably present in the bile of these children and amounted to 30-57% of their total bilirubin pigments (<1% in the controls). It was not possible to predict from the biliary bilirubin composition whether a child would respond to phenobarbital therapy or not. Bile composition was normal in patients with hemolysis, except when there was associated deficiency of hepatic glucuronosyltransferase. Therefore, the observed alterations were not a simple consequence of unconjugated hyperbilirubinemia. The present findings suggest that Crigler-Najjar disease represents a more pronounced expression than Gilbert's syndrome of a common biochemical defect. Hepatic bilirubin UDP-glucuronosyltransferase deficiency leads to decreased formation of diconjugates with an ensuing increase in the proportion of bilirubin monoconjugates in bile; in the most severe cases, an elevated content of biliary unconjugated bilirubin is also found.

Adolescent↗
PubMed1989

Maternal bilirubin, cord bilirubin, and placenta function at delivery and the development of jaundice in mature newborns.

The study material comprised 213 newborns with a birth weight above 2000 g, and their mothers. At delivery, blood samples were obtained from the umbilical cord and the mother's cubital vein for determination of the transplacental bilirubin gradient and assessment of placental function by means of the HPL concentration in the maternal blood. The HPL concentration showed no relation to the transplacental bilirubin gradient or the umbilical cord bilirubin concentration. Therefore, to the extent that the HPL concentration reflects the rate constants for bilirubin transfer, an increased transplacental bilirubin gradient or an increased cord bilirubin concentration could not be explained by an impaired ability of the placenta to transfer bilirubin. When the infants who became jaundiced were compared with the non-jaundiced, significantly higher transplacental bilirubin gradient (p less than 0.00001), cord bilirubin (p less than 0.00001) and maternal bilirubin values at the time of delivery (p less than 0.03) were found among the jaundiced infants. On the basis of the results it was possible to define subgroups of infants with significantly higher/lower risk of subsequent jaundice.

Bilirubin↗
PubMed1986

Bilirubin conjugate excretion and bilirubin uridine diphosphoglucuronyltransferase activity in nonjaundiced homozygous and heterozygous Gunn rats.

High-performance liquid chromatography was used to analyze the composition of bilirubin conjugates in bile from adult male and female outbred normal (JJ) and heterozygous (Jj) Gunn rats. Bile was examined directly without prior derivatization or extraction. Significantly higher bilirubin diglucuronide and lower bilirubin monoglucuronide (both C-8 and C-12 isomers) excretion in JJ rats was demonstrated. Bilirubin monoglucuronide C-8/C-12 ratios were similar in both genotypes, as was the percentage of bilirubin monoglucuronide monoglycoside diester. Hepatic bilirubin uridine diphosphoglucuronyltransferase activity showed a statistically significant positive correlation with the relative amount of bilirubin diglucuronide present in bile and a significant negative correlation with the amount of bilirubin monoglucuronide. The relative percentage of bilirubin monoglucuronide vs. diglucuronide in bile allows an indirect assessment of hepatic bilirubin uridine diphosphoglucuronyltransferase activity.

Animals↗
PubMed1979

Rapid analysis of bilirubin in neonatal serum. I. The binding of bilirubin to albumin.

Evaulation of the severity of jaundice in the neonate may be determined by measuring the reserve binding capacity of serum proteins for free bilirubin. Determination of protein-bound bilirubin has been labor intensive, necessitating multiple runs on gel-permeation chromatography columns or, more recently, enzyme assays or fluorescence quenching analysis. We present a method for quantitation of free bilirubin and of bilirubin-binding capacity of serum by liquid chromatography. A gel-permeation column binds free bilirubin while allowing passage and quantitation of protein-bound bilirubin. Subsequent injection of a desorbing agent releases the adsorbed bilirubin from the column, permitting quantitation of free bilirubin. Bound and free serum bilirubin may be determined directly in less than 15 min using 10 microL of serum. The binding of bilirubin to neonatal serum is seen to be quite different from the binding to adult serum. Ion-exchange chromatography of adult and neonatal serum samples shows that their protein profiles are radically different. This difference probably accounts for the binding characteristics.

Bilirubin↗
PubMed2000

Capillary electrophoresis for determination of free and albumin-bound bilirubin and the investigation of drug interaction with bilirubin-bound albumin.

Capillary electrophoresis (CE) is a promising technique for assessment of free bilirubin and its interaction with albumin, as it requires only a small sample volume and provides a rapid and efficient separation of free bilirubin from its albumin-bound complex in a one-phase system. In order to maintain the equilibrium without dissociation of bilirubin from the albumin/bilirubin complex as in real clinical conditions, the coupling of CE with frontal analysis (FA) was investigated. A very large sample plug was introduced hydrodynamically into the capillary (36 cm length, 50 microm inner diameter) at 15 psi x s to develop the frontal conditions during CE separation. The working conditions for CE/FA separation of bilirubin and albumin were optimized as follows: +20 kV; running buffer, 10 mmol/L phosphate and 1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.4. The working range for bilirubin was found to vary from 5 to 206 micromol/L; precision with relative standard deviation (RSD) <2.0% for n = 3 and detection limit (signal to noise, S/N = 2) was 2 micromol/L. The residual binding capacity of a simulated cord blood serum for bilirubin was 26 mg/100 mL at pH 7.4. Bilirubin was shown to be displaced from albumin when aspirin was added. The free bilirubin concentration was found to increase to clinical significant concentrations from 11.9 to 21.1% when increasing aspirin was added in the range of 5-50 mg/100 mL, respectively. Thus, the investigation of aspirin displacement of bilirubin from albumin is clinically important and the CE/ FA method is a suitable procedure for this purpose.

Bilirubin↗
PubMed1988

Anti-bilirubin monoclonal antibody. I. Preparation and properties of monoclonal antibodies to covalently coupled bilirubin-albumin.

We have prepared 16 anti-bilirubin monoclonal antibodies and described their unique reactivity to bilirubin and related compounds. Using the modified mixed anhydride method, bilirubin was covalently coupled to bovine serum albumin. We performed somatic cell fusion between murine spleen cells immunized with this bilirubin-albumin complex and murine myeloma P3-X63-Ag8-U1 cells. After screening assays, 16 clones were identified which were producing antibodies not to albumin but to haptenic bilirubin. In inhibition analysis, the antibodies in the culture supernatants cross-reacted with bilirubin glucuronides to varying degrees, but rarely reacted with structurally related biliverdin, hemin, and azodipyrroles of bilirubin. Albumin, when present in the solution, much reduced the reactivity of several monoclonal antibodies to unconjugated bilirubin, and this effect was partly reversed by addition of salicylate which dissociates the binding between bilirubin and albumin.

Animals↗
PubMed2006

Synthesis of bilirubin imprinted polymer thin film for the continuous detection of bilirubin in an MIP/QCM/FIA system.

A bilirubin imprinted polymer (BIP) was coated on a thiol pretreated Au electrode on a quartz crystal microbalance (QCM) chip. The BIP thin film was synthesized using 4-vinylpyridine (4-Vpy) as the monomer, divinylbenzene (DVB) as the cross-linker, and benzophenone as the initiator. By using a photo-graft surface polymerization technique with irradiation by ultra-violet (UV) light, a thin BIP film was prepared, from which a biomimetic sensor for the detection of bilirubin was developed. The sensor was able to discriminate bilirubin in solution owing to the specific binding of the imprinted sites. The BIP/QCM chip has been repeatedly used for more than 7 months in many continuous experiments. The detection signal of bilirubin from the BIP thin film/QCM was compared with the non-BIP thin film/QCM. Biliverdin, an analogue of bilirubin, was used for comparison. The analogue comparison confirmed the binding specificity of the BIP film toward bilirubin. The selectivity can be as high as 31.2. The effect of pH on the detection of bilirubin is also discussed. With proper solvent for elution and recovery, flow injection analysis (FIA) could be applied to the system. The performance of the BIP/QCM chip was evaluated. A linear calibration of the bilirubin concentration with respect to the frequency shift was successfully obtained. The reproducibility of measurements from the same BIP/QCM chip was confirmed. In addition, repeatability of detection was also confirmed from different BIP/QCM chips. In conclusion, a combined BIP thin film/QCM/FIA method was successfully established for the detection of bilirubin concentration using a molecularly imprinted film.

Bilirubin↗
PubMed1996

Evidence for overlapping active sites for 17 alpha-ethynlestradiol and bilirubin in the human major bilirubin UDPglucuronosyltransferase.

The human major bilirubin UDP glucuronosyltransferase (transferase), HUG-Brl, and its mutants were expressed in the COS-1 cells using cDNA-based pSVL expression units to generate isoforms for the comparison of relative activities with 17 alpha-ethynlestradiol (17 alpha-EE) and bilirubin, its natural substrate. In comparison to bilirubin, 17 alpha-EE was a good substrate for HUG-Br1 under typical assay conditions of pH 7.2, confirming published studies [Ebner, T., et al. (1993) Mol. Pharmacol. 43, 649-654]. It was further shown that the estrogen derivative is 1.2-2-fold more effective as a substrate at pH 6.4 than at pH 7.2. The km for 17 alpha-EE was 40 microM under both pH conditions, while the Vmax values were 400 and 200 pmol per hour per 300 micrograms of protein at pH 6.4 and 7.2, respectively. The pattern of glucuronidation was similar for both bilirubin and 17 alpha-EE. Previously, a ratio of 2-3-fold more activity for bilirubin glucuronidation at pH 6.4 versus 7.6 was established, and km values of 2.5 microM at both pH conditions were determined [Ritter, J.K., et al. (1993) J. Biol. Chem. 268, 23573-23579]. In this study, the generation of 17 alpha-EE and bilirubin beta-glucuronides under both pH conditions was confirmed by the sensitivity of the products to beta-glucuronidase treatment. Concurrent glucuronidation reaction mixtures containing equal amounts of wild-type and mutant proteins demonstrated the following. P270G, V273D, and five different G276 mutants nearly or completely inactivated all glucuronidation at both pH levels. V273Q generated 81-94% of the normal activity for 17 alpha-EE and 42% of the normal activity for bilirubin turnover; H173R gave 37-60% of the normal turnover with both substrates, and V275I produced 15-24% of the normal level of glucuronide with both compounds. The most distinguishing amino acid tested was P176G which was approximately 50% normal for 17 alpha-EE at both pH conditions but was totally inactive for bilirubin. A second substitution, P285G, did not affect 17 alpha-EE turnover but was 50% normal for bilirubin. The parallel effects on the metabolism of both substrates by some mutants and the opposite results from two mutants are evidence for a common set of amino acids for their catalysis with the recruitment of additional amino acids to depend upon the substrate to be metabolized. Hence, amino acid substitutions in the protein are not necessarily universally inactivating.

Amino Acid Sequence↗
PubMed2001

Bilirubin-albumin binding and free bilirubin.

The relevance of plasma bilirubin-albumin binding and, in particular, the nonalbumin-bound or "free" bilirubin concentration to neonatal bilirubin toxicity is controversial. The pivotal role that "free" bilirubin played in the bilirubin toxicity that occurred following administration of sulfisoxazole or benzyl alcohol to jaundiced newborns, and the correlation of "free" bilirubin with bilirubin-induced changes in the auditory brainstem response are strong support for measuring "free" bilirubin when evaluating neonatal jaundice. Reliable methods for measuring "free bilirubin" are available, and population reference values are needed to help determine its proper clinical use.

Anti-Infective Agents↗