Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition: results of a novel ultrafiltration method.

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.

Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles.

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.

Method for removal of surface-active impurities and calcium from conjugated bile salt preparations: comparison with silicic acid chromatography.

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.

Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages.

Using the equilibrium dialysis apparatus, an aqueous suspension of predominantly aggregated Re lipopolysaccharide (ReLPS) from Escherichia coli D31 m4 (99.9% at 82.5 microM) can be processed to yield a solution of monomeric ReLPS at a saturation concentration of 77 ng/ml (3.4 x 10(-8) M). We compared the in vitro biological activities of these two physically distinct types of ReLPS preparations in two select assays, reaction in the Limulus amebocyte lysate (LAL) assay and induction of Egr-1 mRNA in macrophages. These assays were chosen for their rapid response times and relatively short incubation periods. The monomeric ReLPS was 179- and 1000-fold more active than the aggregated ReLPS preparation in the LAL assay and induction of Egr-1 mRNA by thioglycollate-elicited murine peritoneal macrophages, respectively. These results clearly showed that the monomeric ReLPS is the more active form. The lower biological activities of the aggregated ReLPS preparation might be due to the presence of a small amount of monomeric ReLPS (0.01-0.6%) produced during its preparation and the incubation periods in the biological assays. Thus, aggregated ReLPS may be relatively inactive.

Effect of pH on solubility and ionic state of lipopolysaccharide obtained from the deep rough mutant of Escherichia coli.

The dissociation of the highly aggregated form of lipopolysaccharide (LPS) from Gram-negative bacteria to the monomeric (or soluble) form is though to be the initial step in the activation of responding cells (macrophages, B-cells, neutrophils, monocytes, and endothelial cells) by LPS. This process is presently not adequately understood. Using the equilibrium dialysis apparatus and a highly purified and well-characterized radiolabeled deep rough chemotype LPS ([14C]ReLPS) from Escherichia coli D31m4, we have examined the effect of pH on its solubility (CT) and ionic states in aqueous media. The solubility range of [14C]ReLPS suspended in 50 mM Tris-HCl-100 mM KCl buffer (or 50 mM MES-100 mM KCl buffer at pH 6.5) was determined to be from (2.91 +/- 0.01) x 10(-8) to (4.55 +/- 0.07) x 10(-8) M over a pH range of 6.50-8.20, respectively. These experimental data satisfactorily fitted the curve generated by the solubility equation CT = S0(1 + K5/[H+])/([H+]/K4' + 1), where S0 is the concentration of the tetraanionic ReLPS, K5 is the dissociation constant of the tetraanionic ReLPS in solution, and K4' is the dissociation constant of the trianionic ReLPS at the surface of the solid particles in suspension. The increase in solubility of ReLPS with increase in pH from 7.00 to 8.20 is primarily caused by the formation of the pentaanionic form from the tetraanions. The pK5 (primarily the second dissociation of the 1-phosphate) of ReLPS was determined to be 8.58 from experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)

Ionization and self-association of unconjugated bilirubin, determined by rapid solvent partition from chloroform, with further studies of bilirubin solubility.

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)

Physicochemical properties of the lipopolysaccharide unit that activates B lymphocytes.

We have examined the physical state of highly purified deep rough chemotype lipopolysaccharide (ReLPS) from Escherichia coli D31m4 as an aqueous suspension and as complexes with bovine serum albumin min (BSA). The ReLPS suspension showed large ellipsoidal particles 12-38 nm wide and 40-100 nm long. The solubility of this form of ReLPS was determined by equilibrium dialysis experiments to be 3.3 x 10(-8) M at 22 degrees C and 2.8 x 10(-8) M at 37 degrees C in 150 mM Tris-KCl, pH 7.5; 3.0 x 10(-8) M at 37 degrees C in 0.75 mM Tris-KCl, pH 7.5. The BSA-ReLPS complexes were fractionated on a Sephacryl S-200 column to yield peaks I and II with apparent masses of about 240 and 70 kDa, respectively. Peak II was a BSA monomer with estimated BSA:ReLPS molar ratios of 1:1-1:7. The ReLPS suspension and the two complexes were compared as antigens in enzyme-linked immunosorbent assays using three select monoclonal antibodies to lipopolysaccharide. The results were consistent with the high state of disaggregation of the ReLPS in both peaks I and II. Since the ReLPS in these complexes were not visible by electron microscopy, they did not contain vesicles or large particles. All forms of ReLPS tested were capable of stimulating 70Z/3, a lipopolysaccharide-responsive murine pre-B cell line. However, peak II was consistently more stimulatory at very low concentrations than the other preparations. The maximally stimulatory concentration of ReLPS for 70Z/3 cells was 40 ng/ml (1.6 x 10(-8) M) for peak II and 70 ng/ml (2.8 x 10(-8) M) for the ReLPS suspension. As expected, the above concentrations were at or below the solubility of the ReLPS. These results suggested that the highly disaggregated form of ReLPS (possibly the monomer) is the active unit that stimulates the cellular response in 70Z/3 cells.

Molecular and micellar associations in the pH-dependent stable and metastable dissolution of unconjugated bilirubin by bile salts.

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.

Bile salts as atypical surfactants and solubilizers.

Recent research has suggested that self-association of bile salts does not follow the micellar pattern of self-association exhibited by typical flexible chain surfactants and detergents. A working model for the self-association of bile salts is proposed. It includes a mild degree of cooperativity in the early stages of the growth of aggregates and coexistence of a number of aggregates of different aggregation numbers. The polydispersity implies an increase in the average aggregation number with increasing concentration of the bile salt. Bile salts can be purified by foam fractionation. Surface tension data for sodium cholate are in agreement with the above qualitative model of self-association. An isoextraction method is useful for estimating monomer activities. Results for sodium deoxycholate suggest little self-association in dilute solutions and a mildly cooperative self-association at higher concentrations. A comparative study of the interactions of the fluorescent probe, 2-p-toluidinyl-naphthalene-6-sulfonate, with sodium alkyl sulfates and sodium deoxycholate indicates that bile salts may differ significantly from classical micellar systems in their solubilization characteristics also. The evidence suggests strongly that a specific adduct formation with an optimum number of bile salt anions may be important in solubilization brought about by the rigidity and the complex shape of the bile salt anions.