Search

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.

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)

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.

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 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.