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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.

Some commercial preparations of common natural conjugated bile salts contain impurities (e.g., amines, lipids, and calcium) that are likely to affect their physicochemical properties. A method was developed for purifying commercial preparations of sodium salts of glycine- and taurine-conjugated bile acids. The method consists of passage of a dilute aqueous solution of the sodium bile salt through three columns in sequence: graphitized carbon, a hydrophobic bonded octadecylsilane (C18) cartridge, and a calcium-chelating resin. The final solution was extracted with chloroform, and the purified bile salt was then isolated by freeze-drying, with a yield of 65-75%. Each bile salt purified by this method was compared with the corresponding bile salt purified by conventional adsorption chromatography on a silicic acid column, using a mixture of methanol and chloroform as eluant. Purity was assessed by visible spectra, by surface tension measurements (using the maximum bubble-pressure method and a Wilhelmy wire method), by chloroform extractability of impurities in the conjugated bile acid, by liposome solubilization, and by chemical analysis of the calcium content. Both purification methods removed colored and surface-active impurities, but the new method was always as or more effective than silicic acid column chromatography. Calcium ion, present in commercial bile salts in concentrations up to 16 mmol/mol bile salt, was removed completely by the three-column method, but not by silicic acid chromatography. The new method is thus a simple, rapid, and efficient procedure for purification of the sodium salts of glycine- and taurine-conjugated bile acids for physicochemical measurements, in which elimination of surface-active impurities and polyvalent cations is desired.

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)

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.

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.

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.

A health study was carried out on 2066 workers in an aluminum smelter in Kitimat, British Columbia to study the effects of exposure to fluoride and other air contaminants encountered on the potlines on the musculoskeletal system, hemopoietic tissue, liver, and renal function. Three hundred seventy-two railway repair workers from Squamish, British Columbia served as an "external" control group. Examination of the spine and sacroiliac joints and pelvic X-ray were conducted on long-term potline workers and a number of "internal" control workers in the smelter not exposed to any air contaminants. Urinary fluoride measurements and personal sampling for airborne fluoride were also carried out. Blood samples were collected for routine blood count and liver and renal function test. Definite cases of skeletal fluorosis were not found in any potroom workers. Some of the changes of early skeletal fluorosis described on pelvic X-rays, e.g., increased density, calcification of ligaments, and periosteal changes, were found in a few workers who were employed on the potlines for more than 10 yr. There was, however, poor agreement in the findings of the two radiologists who read the films. The entity "musculoskeletal fluorosis" does not exist in this smelter where the potroom workers were exposed to total fluoride levels below the currently accepted threshold limit value of 2.5 mg/m3. No ill effects on the hematopoietic tissue or liver and renal function were found.

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.

Confirmation is needed of the reported binding of calcium ions (Ca2+) by bile salts, which is believed to decrease the activity of free calcium ions [Ca2+] available for precipitation of insoluble calcium salts of organic anions in pigment gallstones. We report a new method to determine the association constants (K'f) of calcium for organic anions, from the perturbation by the added anion of the equilibrium solubilization of calcium [14C]oxalate monohydrate crystals (CaOx*). CaOx* crystals were prepared by stepwise conversion of [14C]oxalic acid to its K+ and Ca2+ salts. Structure and purity were confirmed by X-ray diffraction of the crystals. CaOx* was incubated (37 degrees C, under N2) in 0.15 M NaCl in CO2-free deionized H2O at pH 6.3. Dissolution of CaOx*, estimated by radioassay of the 0.22-micron Millipore filtrate, attained equilibrium at 3 days, with K'sp = [Ca2+] * [Ox=] = 2.34 X 10(-8) M2, calculated using known affinity constants for the soluble complexes of NaOx- (K'NaOx = 3.215 M-1) and CaOx (K'CaOx = 195.0 M-1). Keeping total [Na+] = 0.15 M, we added anions that formed soluble complexes with Ca2+. This decreased free [Ca2+], causing more CaOx* to dissolve in amounts related to the concentration of added anion and its K'f for Ca2+. With this method, K'f values for citrate, malonate and malate were similar to the values we determined with the Ca2+ ion electrode, and to published values obtained with the Ca2+ ion electrode and other methods. The sensitivity of the CaOx method permits determination of K'f values with small quantities and low concentrations of the anions and calcium.

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.

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.

In seeking to identify nucleating/antinucleating proteins involved in the pathogenesis of cholesterol gallstones, a major acidic protein was isolated from each of 13 samples of cholesterol gallstones. After the stones were extracted with methyl t-butyl ether to remove cholesterol, and methanol to remove bile salts and other lipids, they were demineralized with EDTA. The extracts were desalted with Sephadex-G25, and the proteins separated by PAGE. A protein was isolated, of molecular weight below 10 kD, which included firmly-bound diazo-positive yellow pigments and contained 24% acidic, but only 7% basic amino acid residues. The presence of N-acetyl glucosamine suggested that this was a glycoprotein. This protein at concentrations as low as 2 micrograms/ml, but neither human serum albumin nor its complex with bilirubin, inhibited calcium carbonate precipitation from a supersaturated solution in vitro. This protein could be precipitated from 0.15 M NaCl solution by the addition of 0.5 M calcium chloride. Considering that cholesterol gallstones contain calcium and pigment at their centers, and that small acidic proteins are important regulators in other biomineralization systems, this protein seems likely to play a role in the pathogenesis of cholesterol gallstones.

The effect of a mixture of amino acids (MAA) perfusion of the proximal jejunum on the secretory response of volume, bicarbonate, enzymes, protein, and total bile salts was studied in duodenal aspirates from 15 normal volunteers. The response was compared in the same subjects on different days with that to continuous intravenous infusion of cholecystokinin-pancreozymin (CCK), in maximally tolerated doses and to a control saline perfusion of the jejunum. A three-lumen tube was used for: perfusion of duodenal marker (phenol red), distal aspiration of duodenal samples, and jejunal perfusion of MAA or saline, 40 cm beyond the ligament of Treitz. Fifteen-minute duodenal collections were made for 1/2 hr before, 2 hr during, and 1 hr after stimulation. The results suggested that jejunal MAA perfusion releases endogenous CCK which is as effective a stimulus of pancreatic enzyme secretion as continuous intravenous CCK infusion. Both jejunal MAA perfusion and intravenous CCK infusion also stimulated volume and bicarbonate output in duodenal aspirates. In addition, jejunal MAA perfusion in the 2nd hr produced a sustained increase in total bile salt output which is probably related to continued gallbladder contraction. We concluded that physiological concentrations of amino acids in the proximal jejunum act as an important factor in the regulation of exocrine pancreatic and biliary secretions in man.

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.

A randomized, crossover trial was carried out on the effect of moderate sodium reduction on red-blood-cell sodium metabolism. The participants were healthy high school students (mean age = 16 years, n = 33). Changes in sodium-lithium countertransport and intracellular sodium concentration were evaluated 24 days after a decrease in dietary sodium from approximately 110 to 40 mEq per day. Dietary sodium restriction had no significant effect on either sodium-lithium countertransport or intracellular sodium concentration.

Sixty-five gallstones were assessed for content of network polymer by equilibrium swelling and infrared spectroscopy. All types of pigment gallstones exhibited swelling in aqueous buffers, indicating network polymer content, but swelling was greatest in black pigment gallstones. Gallstones composed principally of cholesterol did not swell. Swelling increased with increasing pH values from 6 to 10.5, suggesting that titratable carboxyl side chains of the pigment were intact. Swelling increased in the presence of 10 mM ethylenediaminetetraacetic acid, indicating that calcium ions provided secondary crosslinks in the network. In all types of pigment gallstones, the equilibrium swelling ratio at the maximum (pH = 10.5) was correlated inversely with the proportion of intact vinyl groups in the pigment assessed by infrared spectroscopy. This suggests that pigment vinyl groups are consumed in forming the covalent crosslinks of the polymer network. A weaker direct correlation between equilibrium swelling ratio and the percentage of unmeasured residue in the gallstones suggested that this residue may also have contributed to the swelling. The high content of cross-linked network polymer in black pigment gallstones renders their therapeutic dissolution improbable by physicochemical means.

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.

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [(55)Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K(d) < 1 pm) compared with albumin (K(d) = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7-25 mum) equivalent to that of the iron transport protein transferrin (22-31 mum), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.

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.