J. Donald Ostrow

Pigment Versus Cholesterol Cholelithiasis: Identification and Quantification by Infrared Spectroscopy

We previously reported that 27% of 92 cholecystectomized patients had pigment stones (Am J Dig Dis 19:585-590, 1974). Using standard biochemical methods, we found that cholesterol accounted for an average of 77% of the dry weight of cholesterol stones, but that unconjugated bilirubin represented a mean of only 7% of pigment stones. This quantitation of pigment stones was limited because approximately 66% of their weight was insoluble. To characterize pigment and cholesterol stone composition further, we used infrared spectroscopy--a technique requiring neither crystallinity nor solubilization--to quantitate pigment, carbonate, and cholesterol in gallstones. Other organic and inorganic components of stones were measured by standard methods. By infrared spectroscopy, two types of pigment stones were identified: carbonate-containing and noncarbonate pigment stones. Carbonate pigment stones contained significantly more calcium, carbonate, and phosphate, but less pigment than noncarbonate stones. Compared to our initial report, the total measured components of all pigment stones were increased 6-fold from 10 to 63%. Cholesterol was the major component of cholesterol stones by chemical assay or infrared spectroscopy. Among five cholesterol stones with limited solubility, 80% of the insoluble residue was identified as cholesterol by infrared spectroscopy. This study extends our knowledge of pigment stone and cholesterol stone composition by the use of quantitative infrared spectroscopy in conjunction with standard biochemical methods; furthermore, it confirms that pigment and cholesterol stones differ in composition and form by different mechanisms.

Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat

To elucidate the mechanism by which phototherapy reduces serum bilirubin, studies were performed on the catabolism of labeled bilirubin in homozygous jaundiced Gunn rats before, during, and after a period of exposure to 1700 foot candles of daylight fluorescent light. Following equilibration with the body pool of an intravenously administered tracer dose of (3)H- or (14)C-bilirubin, radioactive and diazo reactive compounds were excreted in the bile at a slow, steady rate and plasma specific activity declined semilogarithmically. Subsequent exposure to light caused a marked increase in the biliary excretion of radioactive and diazoreactive compounds. Fecal and urinary radioactivity increased also but remained minor fractions of the total excreted radioactivity. After extinguishing the lights, these variables reverted gradually to control values. Spectral and chromotographic analysis of the excreted pigments and their azopigments demonstrated that the increased biliary radioactivity during phototherapy consisted of two roughly equal fractions: (a) unconjugated bilirubin, excreted at rates comparable to the output of conjugated bilirubin in the bile of normal nonjaundiced rats; and (b) water-soluble bilirubin derivatives, chromatographically identical with those found in Gunn rat bile under control lighting conditions but different from the products of photodecomposition of bilirubin in vitro. In some animals, phototherapy produced little decline in plasma bilirubin despite comparable acceleration of bilirubin catabolism. This was attributed tentatively to increased synthesis of early labeled bilirubin in these animals.

Differential expression of the multidrug resistance-related proteins ABCb1 and ABCc1 between blood-brain interfaces.

Cerebral homeostasis results from the presence of the protective blood‐brain and blood‐cerebrospinal fluid barriers located respectively at the brain capillary endothelium and the choroid plexus epithelium. ABCb1 (Pgp) and ABCc1 (Mrp1) transporters are two major proteins of neuroprotection whose localization and functional significance at both barriers remain partly unsettled. We conducted a comparative analysis of their relative protein content between the two blood‐brain interfaces. Microvessels and choroid plexuses located in the fourth and lateral ventricles were isolated from developing and adult rat brains, and whole homogenates were submitted to quantitative Western blot analysis by using standard curves generated from one of the samples. In adult, choroid plexus‐associated Pgp content was less than 0.5% of the level in microvessels, whereas Mrp1 content in microvessels was 4% of that in the fourth ventricle choroid plexus. Pgp but not Mrp1 was enriched in microvessels over parenchyma. In choroid plexuses, Mrp1 displayed a basolateral epithelial localization, and reached its high adult protein level, early during postnatal development. In postnatal as in adult microvessels, Pgp localization appeared luminal. However, by contrast to Mrp1, the level of this transporter increased 4.6‐fold between 9‐day‐old and adult animals. Western blot analysis of human samples confirmed the mirror image of Pgp and Mrp1 expression between the two barriers. We conclude that there are major differences in the mechanisms by which blood‐brain interfaces fulfill their neuroprotective functions. The data also highlight the significance of the neuroprotective function of the choroid plexus during brain maturation, when the microvasculature is still developing. J. Comp. Neurol. 510:497–507, 2008.

Gastric Secretion in Human Hepatic Cirrhosis

Summary 1.Gastric secretory function was measured in patients with hepatic cirrhosis by means of uropepsin excretion, blood pepsinogen level, and gastric output of acid and pepsin, both basally and after histamine. 2.In patients with decompensated cirrhosis all of the above secretory parameters were significantly reduced to below normal, and histamine achlorhydria was found in one-third of these subjects. 3.Cirrhotic patients who were not malnourished or decompensated exhibited the same marked reduction in gastric secretion; this suggests that chronic illness is not the cause of the diminution in gastric function. 4.Uropepsin and blood pepsinogen values were as low in postnecrotic and biliary cirrhosis as in alcoholic cirrhosis, indicating that alcoholism itself is not responsible for the decreased gastric activity. 5.Free 17-hydroxycorticosteroid levels were normal in both the plasma and urine of cirrhotic subjects, excluding reduced adrenal function as the cause of the reduced gastric secretion. 6.Gastric secretion and uropepsin excretion were higher in patients with both peptic ulcer and cirrhosis than in those with cirrhosis alone. 7.Gastric acid output, both basally and after histamine, was higher in cirrhotics with portacaval shunt than in cirrhotics without shunt. In 1 patient a marked increase over preoperative secretion was noted simultaneously with the development of a gastric ulcer after splenorenal shunt. Possible causes for these effects were discussed.

Unbound (Free) Bilirubin: Improving the Paradigm for Evaluating Neonatal Jaundice

BACKGROUND The serum or plasma total bilirubin concentration (B(T)) has long been the standard clinical laboratory test for evaluating neonatal jaundice, despite studies showing that B(T) correlates poorly with acute bilirubin encephalopathy (ABE) and its sequelae including death, classical kernicterus, or bilirubin-induced neurological dysfunction (BIND). The poor correlation between B(T) and ABE is commonly attributed to the confounding effects of comorbidities such as hemolytic diseases, prematurity, asphyxia, or infection. Mounting evidence suggests, however, that B(T) inherently performs poorly because it is the plasma non-protein-bound (unbound or free) bilirubin concentration (B(f)), rather than B(T), that is more closely associated with central nervous system bilirubin concentrations and therefore ABE and its sequelae. CONTENT This article reviews (a) the complex relationship between serum or plasma bilirubin measurements and ABE, (b) the history underlying the limited use of B(f) in the clinical setting, (c) the peroxidase method for measuring B(f) and technical and other issues involved in adapting the measurement to routine clinical use, (d) clinical experience using B(f) in the management of newborn jaundice, and (e) the value of B(f) measurements in research investigating bilirubin pathochemistry. SUMMARY Increasing evidence from clinical studies, clinical experience, and basic research investigating bilirubin neurotoxicity supports efforts to incorporate B(f) expeditiously into the routine evaluation of newborn jaundice.

Photodecomposition of bilirubin and biliverdin in vitro

Summary Decomposition of bilirubin and biliverdin under intense fluorescent light was studied in vitro at p H levels ranging from 7.4 to 13.0. Photodecay of bilirubin was more rapid at a higher p H if albumin was absent, but more rapid at a lower p H if albumin was present, whereas biliverdin was more stable at a lower p H, with or without albumin. During illumination at lower p H levels, albumin stabilized biliverdin but accelerated decay of bilirubin; these effects were not altered by competitive uncoupling of bilirubin from albumin or by chelation of contaminant divalent cations with ethylenediaminetetraacetic acid. Studies with a variety of fluorescent lamps demonstrated that wavelengths between 400 and 600 mμ were most effective in destroying bilirubin, ultraviolet light was less effective, and red light (>600 mμ) was ineffective. Photodecay of both bilirubin and biliverdin was oxidative, but exclusion of oxygen did not arrest photodecay of bilirubin if albumin was present also. Presumably, albumin itself or the vinyl side chain of bilirubin substituted for oxygen as a hydrogen acceptor. By radiochromatography and by reillumination of photoderivatives of 14 C-bilirubin, it was shown that the process of photodecay proceeded from bilirubin to biliverdin to weakly diazo-reactive derivatives to highly polar, diazo-negative products.

The products of YCF1 and YLL015w (BPT1) cooperate for the ATP-dependent vacuolar transport of unconjugated bilirubin in Saccharomyces cerevisiae.

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. Copyright

Kinetics and Specificity of Feline Leukemia Virus Subgroup C Receptor (FLVCR) Export Function and Its Dependence on Hemopexin

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 [55Fe]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 (Kd < 1 pm) compared with albumin (Kd = 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 μm) equivalent to that of the iron transport protein transferrin (22–31 μm), 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.

Absorption of Bile Pigments by the Gall Bladder

Abstract A technique is described for preparation in the guinea pig of an in situ, isolated, vascularized gall bladder that exhibits normal absorptive functions. Absorption of labeled bile pigments from the gall bladder was determined by the subsequent excretion of radioactivity in hepatic bile. Over a wide range of concentrations, unconjugated bilirubin-14C was well absorbed, whereas transfer of conjugated bilirubin proceeded slowly. Mesobilirubinogen-3H was absorbed poorly from whole bile, but was absorbed as rapidly as unconjugated bilirubin from a solution of pure conjugated bile salt. Bilirubin absorption was not impaired by iodoacetamide, 1.5 mM, or dinitrophenol, 1.0 mM, even though water transport was affected. This indicated that absorption of bilirubin was not dependent upon water transport, nor upon energy-dependent processes. The linear relationship between absorption and concentration of pigment at low concentrations in bile salt solutions suggested that pigment was transferred by passive diffusion. At higher pigment concentrations or in whole bile, this simple relationship was modified by interactions of pigment with bile salts and other constituents of bile. These interactions did not necessarily involve binding of bilirubin in micelles. The slow absorption of the more polar conjugates and photo-oxidative derivatives of bilirubin suggested that bilirubin was absorbed principally by nonionic, and partially, by ionic diffusion. Concentrations of pure conjugated bile salts above 3.5 mM were found to be injurious to the gall bladder mucosa. This mucosal injury did not affect the kinetics of bilirubin absorption. During in vitro incubation of bile at 37°C, decay of bilirubin and hydrolysis of the conjugate proceeded as first-order reactions. The effects of these processes on the kinetics of bilirubin absorption, and their possible role in the formation of “white bile” and in the demonstrated appearance of unconjugated bilirubin in hepatic bile, are discussed.

Bilirubin inhibits the TNFα-related induction of three endothelial adhesion molecules

Since an increased serum unconjugated bilirubin (UCB) level has been proposed as an independent protective factor against atherosclerotic disease, we investigated the molecular events at the basis of this effect. HUVEC and H5V cells were treated with TNFalpha and UCB and the effects assessed on E-selectin, VCAM-1 and ICAM-1. In HUVEC cells, UCB blunted the TNFalpha-induced gene upregulation of E-selectin VCAM-1 and ICAM-1. The same pattern was observed in H5V cells except for ICAM-1. UCB also inhibited the PMN endothelial adhesion in HUVEC H5V cells. Western blot and FACS analysis confirmed that UCB prevented TNFalpha-induced over-expression of adhesion molecules proteins in H5V cells. These data contribute to further explain the protective effect of bilirubin against development of atherosclerosis.