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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 10pmol 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 (/=40pmol UCB/g tissue) in non-jaundiced rats.

Revised concepts of bilirubin encephalopathy have been revealed by studies of bilirubin toxicity in cultured CNS cells and in congenitally jaundiced Gunn rats. Bilirubin neurotoxicity is related to the unbound (free) fraction of unconjugated bilirubin (Bf), of which the dominant species at physiological pH is the protonated diacid, which can passively diffuse across cell membranes. As the binding affinity of plasma albumin for bilirubin decreases strikingly as albumin concentration increases, previously reported Bf values were underestimated. Newer diagnostic tests can detect reversible neurotoxicity before permanent damage occurs from precipitation of bilirubin (kernicterus). Early toxicity can occur at Bf only modestly above aqueous saturation and affects astrocytes and neurons, causing mitochondrial damage, resulting in impaired energy metabolism and apoptosis, plus cell-membrane perturbation, which causes enzyme leakage and hampers transport of neurotransmitters. The concentrations of unbound bilirubin in the cerebro-spinal fluid and CNS cells are probably limited mainly by active export of bilirubin back into plasma, mediated by ABC transporters present in the brain capillary endothelium and choroid plexus epithelium. Intracellular bilirubin levels may be diminished also by oxidation, conjugation and binding to cytosolic proteins. These new concepts may explain the varied susceptibility of neonates to develop encephalopathy at any given plasma bilirubin level and the selective distribution of CNS lesions in bilirubin encephalopathy. They also can suggest better strategies for predicting, preventing and treating this syndrome.

Our aim was to evaluate the stability of eightcomponents of porcine bile (pH, total and ionizedcalcium, total and unconjugated bilirubin, phospholipid,cholesterol, and bile salts) over a 17-day period at three temperatures: –15, 4, and37°C. The pH and concentrations of total and ionizedcalcium, phospholipid, cholesterol, and bile salts werestable over 17 days. Total bilirubin was stable at15°C, but declined over 17 days by approximately 25% at4°C and 70% at 37°C (P < 0.003). A rapidincrease in the unconjugated bilirubin was seen withintwo days at all temperatures to between 7.5 and 12 timesthe levels at day 0 (P < 0.009). Thereafterunconjugated bilirubin at 15 and 4°C continued toincrease at a much slower rate. By contrast,unconjugated bilirubin at 37°C declined beginning onday 4 and fell to 1.33 times levels at day 0 by day 17 (P < 0.002).We conclude that bile can be stored at 15°C for 17days with stability of most components, but unconjugatedbilirubin will rise. The loss in total bilirubin is significant at 37°C.

Revised concepts of bilirubin encephalopathy have been revealed by studies of bilirubin toxicity in cultured CNS cells and in congenitally jaundiced Gunn rats. Bilirubin neurotoxicity is related to the unbound (free) fraction of unconjugated bilirubin (Bf), of which the dominant species at physiological pH is the protonated diacid, which can passively diffuse across cell membranes. As the binding affinity of plasma albumin for bilirubin decreases strikingly as albumin concentration increases, previously reported Bf values were underestimated. Newer diagnostic tests can detect reversible neurotoxicity before permanent damage occurs from precipitation of bilirubin (kernicterus). Early toxicity can occur at Bf only modestly above aqueous saturation and affects astrocytes and neurons, causing mitochondrial damage, resulting in impaired energy metabolism and apoptosis, plus cell-membrane perturbation, which causes enzyme leakage and hampers transport of neurotransmitters. The concentrations of unbound bilirubin in the cerebro-spinal fluid and CNS cells are probably limited mainly by active export of bilirubin back into plasma, mediated by ABC transporters present in the brain capillary endothelium and choroid plexus epithelium. Intracellular bilirubin levels may be diminished also by oxidation, conjugation and binding to cytosolic proteins. These new concepts may explain the varied susceptibility of neonates to develop encephalopathy at any given plasma bilirubin level and the selective distribution of CNS lesions in bilirubin encephalopathy. They also can suggest better strategies for predicting, preventing and treating this syndrome.

Unconjugated bilirubin is a curse at high concentrations, producing apoptosis and cell death, but a boon at more physiological levels, protecting cells against oxidant damage Both hydrophobic bile salts1,2 and unconjugated bilirubin (UCB)3 induce apoptosis in cultured cells at moderately elevated concentrations and cell necrosis at higher concentrations. Retention of bile salts in cholestasis is believed to cause secondary damage to hepatocytes,4 and retention of UCB in severe neonatal jaundice is known to cause bilirubin encephalopathy.5 For both agents, the cytotoxicity results from damage to mitochondrial membranes, with collapse of the transmembrane potential and generation of a mitochondrial membrane permeability transition,1–2,6 and ursodeoxycholic acid or its amidates can prevent apoptosis by inhibiting this process.1–2,7 It is therefore surprising that, as reported by Granato et al in this issue of Gut ,8 UCB, at concentrations far above those known to be cytotoxic to neurones and astrocytes,3 is protective against apoptosis induced in cultured rat hepatocytes by the hydrophobic bile salt glycochenodeoxycholate (GCDC) [see page 1774] . This finding raises interesting questions concerning whether UCB is a curse or a boon, and why various organs and tissues have such different susceptibilities to toxicity from these substances. Like many previous in vitro studies of the cytotoxicity of UCB9 and bile salts,10 the studies of Granato and colleagues8 were performed at concentrations of the unbound fraction far above those that are clinically relevant. Although the incubation media contained 18 µM bovine serum albumin, concentrations of UCB and GCDC greatly exceeded the high affinity binding capacity of 1 mol/mol albumin. The final unbound concentrations of UCB (Bf) were also far above aqueous saturation,11 so the pigment must have been heavily self aggregated.12,13 Unbound concentrations …

Unconjugated bilirubin (UCB) is currently believed to cross the placenta only by passive diffusion. To assess whether carrier-mediated transport might be involved, the uptake of [(3)H]-UCB by basal (bTPM) and apical (aTPM) plasma membrane vesicles from human placental trophoblast at term was investigated. In both types of vesicles, the uptake of [(3)H]-UCB into an osmotically sensitive space was temperature-dependent, independent of the presence of Na(+), and not affected by changes in membrane potential. The uptake of [(3)H]-UCB by aTPM, but not bTPM, was activated by ATP hydrolysis and inhibited by vanadate. Thus, the exact contribution of both inside out and right-side out bTPM to UCB uptake could not be distinguished, while only inverted aTPM were expected to carry out ATP-dependent UCB uptake. In bTPM and aTPM, uptake of free (unbound) [(3)H]-UCB (B(f)) consisted of a dominant, saturable, presumably carrier-mediated process and a diffusional component that became predominant only at B(f) near or above aqueous solubility limit for UCB (70 nM ). For bTPM, K(m)=7.2 nM; V(max)=9.8 pmol/20s/mg protein; and diffusion coefficient (K(D))=0.14 ml/20s/mg protein. For aTPM in the presence of 9.5m M ATP, K(m)=18 n M; V(max)=131 pmol/20s/mg protein; and K(D)=0.47 ml/20s/mg protein. The uptake of [(3)H]-UCB by bTPM was cis-inhibited by estrone-3-sulfate and estradiol-17 beta-glucuronide and trans-stimulated by unlabelled UCB and bromosulphopthalein. ATP-dependent UCB uptake by aTPM was cis-inhibited by doxorubicin, cholic acid, methotrexate and pronenecid. These findings suggest the presence of distinct transporters in the two domains of human placental trophoblast that could cooperate to transfer UCB from the foetus to the maternal circulation.

Albumin binding is a crucial determinant of bilirubin clearance in health and bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influence of impurities. We therefore have devised a method based on serial ultrafiltration that successively removes impurities in [(14)C]bilirubin until a stable binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent binding affinity of human serum albumin for [(14)C]bilirubin was strongly dependent on assay conditions, falling from (5.09 +/- 0.24) x 10(7) liters/mol at lower albumin concentrations (15 microm) to (0.54 +/- 0.05) x 10(7) liters/mol at higher albumin concentrations (300 microm). To determine whether radioactive impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to be 0.11-0.13%. Formation of new impurities during the study and their affinity for albumin were also estimated. After correction for impurities, the binding affinity remained heavily dependent on the albumin concentration (range (5.37 +/- 0.26) x 10(7) liters/mol to (0.65 +/- 0.03) x 10(7) liters/mol). Affinities decreased by about half in the presence of chloride (50 mm). Thus, the affinity of human albumin for bilirubin is not constant, but varies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previously believed.

Since bilirubin-like pigments are present in the environment as degradation products of heme-containing proteins, yeast could have developed a detoxifying system to transport these compounds into their vacuoles. Vacuoles from Saccharomyces cerevisiae showed an ATP-dependent, saturative transport of unconjugated bilirubin (UCB) that was reduced by 60% and 40% in YCF1 and YLL015w-deleted cells, respectively; the double deletant showed no UCB uptake. Conversely, the transport of bile acids (taurocholate) was comparable in wild and deleted stains. These data identify YCF1 and YLL015w, named BPT1 (Bile Pigment Transporter), as the genes responsible for ATP-dependent UCB transport in yeast. Since YCF1 and YLL015w are rather homologous with multidrug resistant proteins (MRPs), they also suggest the involvement of this class of transporters in the ATP-dependent transport of unconjugated bilirubin.

The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP. Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5´-[α,β-methylene] triphosphate), did not stimulate [3H]UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect. [3H]UCB uptake occurred into an osmotically sensitive space. At an unbound bilirubin concentration ([Bf]) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to [Bf], with a Km of 26±8 nM and a Vmax of 117±11 pmol per 15 s per mg of protein. Unlabelled UCB inhibited the uptake of [3H]UCB, indicating that UCB was the transported species. Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59±11% and 100% respectively). Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent [3H]UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB. [3H]UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport. These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.

Our aim was to evaluate the stability of eight components of porcine bile (pH, total and ionized calcium, total and unconjugated bilirubin, phospholipid, cholesterol, and bile salts) over a 17-day period at three temperatures: -15, 4, and 37 degrees C. The pH and concentrations of total and ionized calcium, phospholipid, cholesterol, and bile salts were stable over 17 days. Total bilirubin was stable at -15 degrees C, but declined over 17 days by approximately 25% at 4 degrees C and 70% at 37 degrees C (P0.003). A rapid increase in the unconjugated bilirubin was seen within two days at all temperatures to between 7.5 and 12 times the levels at day 0 (P0.009). Thereafter unconjugated bilirubin at -15 and 4 degrees C continued to increase at a much slower rate. By contrast, unconjugated bilirubin at 37 degrees C declined beginning on day 4 and fell to 1.33 times levels at day 0 by day 17 (P0.002). We conclude that bile can be stored at -15 degrees C for 17 days with stability of most components, but unconjugated bilirubin will rise. The loss in total bilirubin is significant at 37 degrees C.

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

Our studies of equilibrium solubilization of crystals of unconjugated bilirubin (UCB) in buffered aqueous NaCl (1988. J. Lipid Res. 29: 335-348) suggested that the two carboxylic pKa values were 6.8 and 9.3 and the solubility of UCB diacid was 0.1 microM. These data, however, were not ideal, due to possible effects of crystal size, metastability, 96-h incubation times with formation of polar derivatives, impurities in the bilirubin, and imprecision of analyses at low concentrations of UCB ([UCB]). In the present study, designed to determine the pKa values and self-association of UCB, these problems were minimized by solvent partition of UCB from solution in CHCl3 into buffered aqueous NaCl. There was no crystal phase. Equilibrium was attained rapidly (10 min); UCB and CHCl3 were highly purified; and accurate diazo assay of low [UCB] in the aqueous phase, [Bw], was achieved by concentrating the UCB through back-extraction into a small volume of CHCl3. By determining effects on partition rations of varying the [UCB] in the CHCl3 phase, [Bc], we could assess also the self-association of UCB species in the aqueous phase. Partition ratios (P = Bw/Bc) did not differ between initial and repeat extractions, indicating insignificant concentrations of polar UCB derivatives. Similar P ratios were obtained when equilibrium was approached from a supersaturated aqueous phase. At 21-25 degrees C, mu = 0.15, the data (n = 76) fit the equation: log P = log Po + log[1 + 10(pH-A) + 10(2pH-B) + Bc.10(4pH-D)]; the bracketed terms reflect P for H2Bo (diacid), HB- (monoanion), B= (dianion), and (B=)2 dimer, respectively. Computer-fitted values for constants (+/- SD) were: Po = P for H2Bo = 5.79 x 10(-5); A = pK1 = 8.12 +/- 0.23; B = pK1 + pK2 = 16.56 +/- 0.10; pK2 = 8.44 +/- 0.33; D = pk22 + 2(pK1 + pK2) -log(2Po) = 37.64 +/- 0.07, and k22 = 0.26 microM-1 [formation constant of (B=)2 dimer]. In ancillary studies, multiple cycles of direct dissolution of UCB crystals revealed a progressive decrease in aqueous solubility of UCB as fine crystals were removed; this effect was minimal in CHCl3. Unlike in water, moreover, varied UCB crystal forms had similar solubilities in CHCl3, with [Bc] = 1.14 mM at saturation. As determined from [Bc]sat.Po, the aqueous solubility of H2Bo was 66 nM.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholesterol gallstones usually have small amounts of pigment at their centers and often have diffuse pigmentation or pigmented layers alternating with cholesterol layers and/or pigmented rims associated with calcium carbonate (eggshell calcification). The pigments are primarily monomeric calcium salts of unconjugated bilirubin anions and/or an insoluble, black, network polymer of tetrapyrroles. Bilirubin presumably can precipitate only if bile is supersaturated with calcium bilirubinates. Among various in vitro model systems, the aqueous solubilities and pK'a values for unconjugated bilirubin differ greatly. It is therefore not known whether normal bile is saturated with unconjugated bilirubin. However, all systems indicate that unconjugated bilirubin is solubilized by binding to bile salt monomers and oligomers, as well as micelles; marked metastable supersaturation of unconjugated bilirubin can occur in the presence of bile salt micelles, and both pK'a values of unconjugated bilirubin are greater than 6.0, probably because of internal hydrogen-bonding of the--COOH groups. Lecithin decreases equilibrium solubilization of unconjugated bilirubin crystals but enhances metastable supersaturation of unconjugated bilirubin. Calcium ions form insoluble salts with unconjugated bilirubin monoanions and dianions but soluble complexes with bilirubin conjugates. The solubility products of the calcium bilirubinate salts suggest that normal hepatic bile is not saturated with CaB or Ca(HB)2 but that gallbladder bile may be supersaturated with Ca(HB)2.(ABSTRACT TRUNCATED AT 250 WORDS)

The rate of peroxidation of unbound, unconjugated bilirubin (UCB) was used to assess the interactions of UCB with four taurine-conjugated bile salts at pH 8.2, 37 degrees C, and an ionic strength of 0.15. Each of the four structurally different bile salts markedly decreased the rate of peroxidation of UCB in the presence of horseradish peroxidase (HRP); 30% of UCB was bound even at low, premicellar bile salt concentrations (1 mM). At high bile salt concentrations (75 mM), taurocholate (TC) and tauro-3 alpha,7 alpha-dihydroxy-12-oxo-5 beta-cholan-24-oate (T12-OXO) exhibited the highest degree of inhibition of UCB peroxidation; only 0.6% and 1.1% of UCB were unbound, respectively. Taurochenodeoxycholate (TCDC) yielded somewhat less inhibition with 2.0% of UCB unbound. Taurodehydrocholate (TDHC), a bile salt that does not form micelles but does form dimers, was comparable to TC and T12-OXO with unbound UCB of 1.0%. With TC and T12-OXO, apparent affinity for UCB was at least four times greater above the published critical micellar concentration (CMC) than in the premicellar range. TCDC was only studied above its CMC value and only one region of UCB binding was noted. Interaction of UCB with TDHC was similar to premicellar interactions with TC and T12-OXO below 25 mM, but increased to values intermediate between monomer and micelle above 40 mM TDHC, compatible with formation of TDHC dimers above 20 mM. These data show that there are differences in the ability of bile salts to bind UCB. Thus, alterations in bile salt profile in bile might lead to higher concentrations of free UCB in bile predisposing to pigment gallstones.

Radiolabeled photobilirubins, prepared in vitro by anaerobic illumination of [34C]bilirubin, were injected intravenously into homozygous jaundiced Gunn rats with an external bile fistula. With the animals kept in darkness, the labeled photobilirubins were excreted rapidly in bile. Photobilirubins IA and IB were excreted primarily as unconjugated bilirubin, whereas photobilirubin II was excreted primarily as photobilirubin II and not converted into bilirubin. Bile of Gunn rats given no exogenous pigments, but undergoing phototherapy, contained a large proportion of photobilirubin II and, if collected in liquid nitrogen, traces of photobilirubins I; neither was found in bile when these rats were kept in the dark. Because there is prior evidence that these rats were kept in the dark. Because there is prior evidence that these photobilirubins are isomers of bilirubin, these experiments indicate that the major mechanism of phototherapy is photoisomerization of bilirubin. Photobilirubin II is the unidentified major photoderivative described previously, whereas formation of photobilirubins IA and IB, and their reversion to bilirubin-IXalpha, account for the remarkably increased output of the parent pigment during phototherapy.

Polar photoisomers of bilirubin were formed by irradiation of bilirubin in chloroform solution in the absence of O2. Two pairs of compounds were isolated with molecular weights identical with bilirubin. One pair reverted to bilirubin in polar media and gave chemical reactions similar to bilirubin; the other pair were not reconverted into bilirubin by chemical means and gave reactions distinct from those of bilirubin. However, both groups were reconverted into bilirubin by irradiation in chloroform solution in the absence of O2. The probable role of these photoisomers in the catabolism of bilirubin during phototherapy of neonatal jaundice is discussed.

We assessed the effects of in vivo phototherapy of Gunn rats on the activity of hepatic microsomal mixed-function monoxygenases and on the in vivo pharmacokinetics of [14C]HB. In experiment 1 no serial changes were seen in activities of hexobarbital hydroxylase or benzo(a)pyrene hydroxylase in hepatic microsomes isolated after 2, 4 or 7 days from homozygous jaundiced female Gunn rats exposed to continuous phototherapy or in matched Gunn rats maintained under dim light. In experiment 2 homozygous jaundiced (jj) and heterozygous nonjaundiced (Jj) Gunn rats of both sexes each received i.v. [14C]HB on 2 successive days. In random order, each was exposed on the first or second day to phototherapy for 5.5 hr, beginning 0.5 hr before the administration of HB; otherwise, each was kept under dim light. Plasma [14C]HB in arterial blood samples was separated chromatographically from its labeled metabolites, and biexponential plasma disappearance curves for [14C]HB were analyzed by a SAAM-23 computer program. Clearances in female rats were much slower. In both sexes, the total body clearance and volume of distribution of HB were decreased by 20% during phototherapy of the jj but not the Jj rats; terminal plasma half-life was unchanged. In experiment 3 direct in vitro illumination of [14C]HB did not cause photodegradation of this compound, despite the presence of albumin with or without bilirubin.

The rate of peroxidation of unconjugated bilirubin (UCB), catalyzed by horseradish peroxidase (HRP), has been employed by Jacobsen (1969. FEBS Lett. 5: 112-114) to assess the fraction of unbound UCB in the presence of serum albumin. We used this method to examine the interactions of UCB with taurocholate (TC) at pH 8.2, assuming solubilization of UCB by TC is due to pigment binding and/or to partitioning into the micelle, thus rendering UCB unavailable for peroxidation. Inhibition of UCB peroxidation conformed with predictions based on these assumptions and demonstrated significant interaction of UCB with both monomeric and micellar TC. Although significant inhibition of UCB peroxidation was seen with TC monomer, inhibition was even greater with TC micelles. In contrast, pyrogallol, another substrate of HRP, acted very differently in the presence of TC. Inhibition of pyrogallol peroxidation by TC was much less than with UCB and occurred primarily with monomeric TC, with little further inhibition in the micellar range. The results of this study suggest that at taurocholate concentrations above 50 mM, similar to the physiologic bile salt concentrations in human bile, at least 99% of UCB is bound to bile salt, dramatically decreasing the concentration of unbound UCB. Since bile salts also bind Ca2+, they play a dual role in protection against the precipitation of calcium bilirubinate from bile. Therefore, bile salts are a major factor in the prevention of the formation and growth of pigment gallstones.

A new thin-layer chromatographic (TLC) method is described for measurement of conjugated (CB) and unconjugated (UCB) bilirubin in bile. Bile is streaked on a silica-gel plate and developed with chloroform. Bilirubin in the mobile UCB band and stationary CB band is quantitated by diazotization from the scraped gel with p-iodoaniline reagent. Both CB and UCB in bile yielded identical azopigment absorbance. There was no contamination of the UBC band with either CB or oxidative derivatives of bilirubin, and over 98 per cent added 14C-UBC migrated with the mobile band. Recoveries from TLC averaged 98.2 +/- 4.3 per cent of the applied pigment, duplicated agreed within 10 per cent, and the method accurately detected the enzymatic and alkaline hydrolysis of CB to UCB. Concentrations of UCB in nine samples of normal human gallbladder bile were found to be less than 1.9 mg./dl. and never exceeded 1.2 per cent of the total bilirubin.

The i.r. spectra of bilirubin isomers that differ in number and position of vinyl groups were examined to verify the assignment of the 988 cm−1 peak of bilirubin (991 cm−1 peak in calcium bilirubinate) to its pendant vinyl groups. There were only small changes in this peak with changes in position of vinyl groups (exo-2- and −18-vinyl versus endo-3- and −17-vinyl), but progressive loss of peaks in this region was observed when vinyl groups were reduced to ethyl groups (dihydrobilirubin and mesobilirubin). Methylvinylmaleimide, a monopyrrole derived from the outer (A and D) rings of bilirubin, has a pendant vinyl group and exhibits a prominent peak at 986 cm−1, but haematinic acid methyl ester derived from the inner (B and C) rings has no vinyl group and shows no peak near 988 cm−1. These observations verify the assignment of the 988 cm−1 peak of bilirubin to its pendant vinyl groups. This supports our previous proposal that a decrease in the peak at 985-995 cm−1 in the i.r. spectra of pigment gallstones, as compared with unconjugated bilirubin or calcium bilirubinate, indicates a consumption of vinyl groups in the process of formation of the polymer in the pigment stones.

Unconjugated bilirubin (UCB) is almost insoluble in water at neutral pH, but appears in normal human gallbladder bile at concentrations up to 35 microM. We therefore determined whether conjugated bile salts could increase the dissolved concentration [( Bt]) of UCB over the pH range 3.0-11.0. Using crystalline UCB, [Bt] was higher with less ordered crystals, with increasing pH and bile salt concentration, and with taurocholate (TC) micelles compared to taurodehydrocholate (TDHC) dimers. Plots of [Bt] verus pH from pH 3.0-9.3 fit the equation, [Bt] = A(1 + K'1/[H]+ + K'1.K'2/[H+]2), where A = [Bt] at pH less than 4.0, and K'1 and K'2 are the two apparent ionization constants of UCB. Estimated pK'1 values in NaCl, TC, and TDHC were 6.8, 6.0, and 5.6, respectively; pK'2 was greater than or equal to 9.3 in each system. Acidification of disodium bilirubinate to pH less than 8.5 produced high, metastable [Bt] in 50 mM TC; this was absent in 0.15 M NaCl, and minor in 50 mM TDHC. In all solutions, maximum [Bt] of 60-65 mM was attained at pH greater than or equal to 10.5. This work helps explain the immense variation among reported [Bt] values, indicates that UCB monoanion predominates at the pH range of bile, and suggests that bile salt monomers, dimers, and micelles enhance the solubility of UCB in bile.

Further evidence that the bilirubin excreted by Gunn rats during phototherapy is unconjugated is presented. The unconjugated bilirubin is not carried into bile as a water-soluble complex with rapidly excreted bilirubin derivatives, nor as a result of leakage of plasma protein into bile. Phototherapy does not augment biliary excretion of bilirubin in normal nonjaundiced rats, but does increase the concentration of conjugated bilirubin in the bile of patients with alcoholic cirrhosis, although their serum bilirubin concentrations are unaffected. The mechanism for augmented excretion of unconjugated bilirubin during phototherapy remains unexplained.

New information is summarized, indicating that configurational photoisomerization of bilirubin at the 5 and 15 carbon bridges is the major mechanism of bilirubin photocatabolism in vivo, and that singlet oxygen photooxidation plays only a minor role. The literature is reviewed concerning potentially damaging photodynamic reactions that are observed in vitro with vitamins, proteins, lipids, and nucleic acids, and their possible relationships to the limited number of toxic side-effects that have been detected with clinical phototherapy of neonatal jaundice. Secondary toxic effects, mediated by bilirubin photoderivatives or by retina-neuroendocrine pathways are also considered. Areas requiring further investigations are delineated.

Phototherapy has been used to treat neonatal jaundice, but little assessment has been made of possible beneficial effects on adult liver disease. Effects of phototherapy on bile acid turnover, biliary lipid concentration, liver function tests, and bromosulfophthalein (BSP) kinetics were studied in 8 alcoholic cirrhotics. Phototherapy initially increased biliary specific activity of both primary bile acids and then produced an acceleration of cholic and chenodeoxycholic acid decay curves. Pruritus was relieved in the 3 patients who had this symptom. The proposed mechanism for these changes is mobilization of bile acids from an expanded cutaneous bile acid pool with augmented bile acid excretion. No significant change in serum liver function tests or BSP plasma disappearance curves was seen. Phototherapy causes little improvement in intrinsic liver function, but produces specific changes in bile acid metabolism; these changes may be related to effects of light on a cutaneous bile acid pool.

The rate of peroxidation of unbound, unconjugated bilirubin (UCB) was used to assess the interactions of UCB with four taurine-conjugated bile salts at pH 8.2, 37 degrees C, and an ionic strength of 0.15. Each of the four structurally different bile salts markedly decreased the rate of peroxidation of UCB in the presence of horseradish peroxidase (HRP); 30% of UCB was bound even at low, premicellar bile salt concentrations (1 mM). At high bile salt concentrations (75 mM), taurocholate (TC) and tauro-3 alpha,7 alpha-dihydroxy-12-oxo-5 beta-cholan-24-oate (T12-OXO) exhibited the highest degree of inhibition of UCB peroxidation; only 0.6% and 1.1% of UCB were unbound, respectively. Taurochenodeoxycholate (TCDC) yielded somewhat less inhibition with 2.0% of UCB unbound. Taurodehydrocholate (TDHC), a bile salt that does not form micelles but does form dimers, was comparable to TC and T12-OXO with unbound UCB of 1.0%. With TC and T12-OXO, apparent affinity for UCB was at least four times greater above the published critical micellar concentration (CMC) than in the premicellar range. TCDC was only studied above its CMC value and only one region of UCB binding was noted. Interaction of UCB with TDHC was similar to premicellar interactions with TC and T12-OXO below 25 mM, but increased to values intermediate between monomer and micelle above 40 mM TDHC, compatible with formation of TDHC dimers above 20 mM. These data show that there are differences in the ability of bile salts to bind UCB. Thus, alterations in bile salt profile in bile might lead to higher concentrations of free UCB in bile predisposing to pigment gallstones.

Pigment gallstones are defined as any dark brown-to-black stone, consisting of calcium salts of bilirubin, phosphate, carbonate and other anions, and can be separated into carbonate- and noncarbonate-containing groups. Pigment stones predominate in the rural Orient, in cirrhosis, and in elderly United States patients undergoing cholecystectomy. Clinical associations include bile duct obstruction, stasis, and possibly hemolysis. Of pigment stones, 50% are radioopaque and account for two-thirds of all opaque stones. The concentrations of bile salts, phospholipids,, cholesterol, and total bilirubin in bile are similar to normal levels, but the concentration of unconjugated bilirubin is increased in the bile of some patients. Increased unconjugated bilirubin in bile may be caused by increased hydrolysis of excreted conjugated bilirubin. Unconjugated bilirubin is solubilized by bile salts, but the interaction is primarily nonmicellar. Ionized calcium and pH are important determinants of solubility. Sulfated glycoproteins, excreted in increased amounts in patients with cholelithiasis, may be the site of pigment stone precipitation because these compounds bind calcium salts tightly. E coli is frequently cultured from pigment stones in Japan but not in the United States; thus, bacterial beta-glucuronidase may be important in stone formation in Japan but probably not in the West. Stasis leads to increased calcium secretion and to increases in the concentration of sparingly soluble compounds that may then precipitate. Incomplete emptying of the gallbladder may result in the same concentration process. Unsaturated fats and chronic vagal stimulation cause pigment stone formation in animals. At present, surgery is the only treatment for pigment lithiasis.

A criticial evaluation was made of the ethyl anthranilate diazo and two solvent-partition methods for the determination of conjugated and unconjugated bilirubin in human and rat bile. The ethyl anthranilate diazo reagent, which reacts only with conjugated bilirubin in serum, also diazotized a variable proportion of unconjugated bilirubin in bile and thus overestimated the concentration of monoconjugates. With the Weber-Schalm and modified Folsch solvent-partition methods applied to human or rat bile, 4–9% of added 14C-labelled unconjugated bilirubin partitioned with the conjugated bilirubin in the upper phase, and 4–9% of added 14C-labelled conjugated bilirubin partitioned into the lower phase. With dog bile, the spill-over of 14C-labelled bilirubin into the lower phase was 9–11%. Analysis of azopigments from the Weber-Schalm partition confirmed that over two-thirds of the bilirubin in the lower phase represents monoconjugates, principally the less-polar monoxylosides and monoglucosides. These solvent-partition methods thus overestimate the concentration of unconjugated bilirubin in bile.

Background The unbound, free concentration (Bf) of unconjugated bilirubin (UCB), and not the total UCB level, has been shown to correlate with bilirubin cytotoxicity, but the key molecular mechanisms accounting for the toxic effects of UCB are largely unknown. Findings Mouse liver mitochondria increase unbound UCB oxidation, consequently increasing the apparent rate constant for unbound UCB oxidation by HRP (Kp), higher than in control and mouse brain mitochondria, emphasizing the importance of determining Kp in complete systems containing the organelles being studied. The in vitro effects of UCB on cytochrome c oxidase activity in mitochondria isolated from mouse brain and liver were studied at Bf ranging from 22 to 150 nM. The results show that UCB at Bf up to 60 nM did not alter mitochondrial cytochrome c oxidase activity, while the higher concentrations significantly inhibited the enzyme activity by 20% in both liver and brain mitochondria. Conclusions We conclude that it is essential to include the organelles being studied in the medium used in measuring both Kp and Bf. A moderately elevated, pathophysiologically-relevant Bf impaired the cytochrome c oxidase activity modestly in mitochondria from mouse brain and liver.