Bruce F. Scharschmidt

Hepatic organic anion uptake in the rat.

The hepatic uptake of bilirubin (BR), indocyanine green (ICG), and sulfobromophthalein (BSP) was studied in 350 anesthetized Sprague-Dawley rats by determining the initial plasma disappearance rate (V) of various doses of unlabeled ICG, or of tracer quantities of [3H]BR or [35S]BSP injected into the jugular vein simultaneously with varying amounts of unlabeled BR or BSP. Similar studies were also performed involving the simultaneous injection of potential inhibitors of hepatic uptake. The results indicate that: (a) hepatic uptake determined by direct tissue measurement could be accurately estimated from the plasma disappearance data; (b) saturation of hepatic uptake with increasing dose was readily demonstrated for each of these three organic anions, and in each instance a plot of V versus dose took the form of a rectangular hyperbola analyzable in terms of Michaelis-Menten kinetics; (c) for BR, the saturable uptake process showed a Vmax more than 100 times the normal net transfer rate from plasma to bile; (d) hepatic uptake of BR, BSP, and ICG showed relatively selective, mutually competitive inhibition; glycoholic acid did not inhibit hepatic uptake of any of these substances; and (e) counter-transport could be demonstrated for each of the three test substances. These data are compatible with the existence of a carrier-mediated transport process for hepatic uptake of each of these three organic anions and clarify the relationship of hepatic BR uptake to its overall transport from plasma to bile.

Approach to the Patient with Cholestatic Jaundice

JAUNDICE is a common clinical problem that is usually due to impaired bile production or bile flow — that is, cholestasis. When confronted by a patient with cholestatic jaundice, the overriding obj...

The Micellar Sink: A QUANTITATIVE ASSESSMENT OF THE ASSOCIATION OF ORGANIC ANIONS WITH MIXED MICELLES AND OTHER MACROMOLECULAR AGGREGATES IN RAT BILE

Although the importance of mixed micelles in the solubilization and biliary excretion of lipids is established, little is known about a possible role of mixed micelles in the excretion of other biliary solutes. Ultrafiltration and ultracentrifugation techniques were used to investigate the interaction between substances that are excreted in bile and biliary mixed micelles. Substances (urea, erythritol, sucrose) excreted in bile at concentrations equal to, or less than, that in plasma did not show an association with mixed micelles, whereas substances (indocyanine green, iopanoic acid, rose bengal, unconjugated and conjugated sulfobromophthalein, and conjugated bilirubin) excreted in bile at high concentration relative to plasma did. The percentage of these latter substances in bile associated with micelles varied from 26 to 93% and was relatively independent of concentration. In addition to their association with mixed micelles, these test solutes formed self-aggregates that were stabilized primarily by ionic bonds, and only a small percentage (range = 0-5%) of these solutes were present in bile in the form of monomer or complexes small enough to pass a 5,000-mol wt membrane.These findings offer a possible explanation for the increase in sulfobromophthalein, bilirubin, and indocyanine green maximal biliary excretory rate produced by bile salt infusion, and suggest that the concentrative transport into bile of endogenous compounds and xenobiotics may result from their incorporation into mixed micelles and other macromolecular complexes.

Removing Substances from Blood by Affinity Chromatography: I. REMOVING BILIRUBIN AND OTHER ALBUMIN-BOUND SUBSTANCES FROM PLASMA AND BLOOD WITH ALBUMIN-CONJUGATED AGAROSE BEADS

Substances such as bilirubin that bind tightly to plasma proteins cannot readily be removed from blood. We describe here the use of affinity chromatography as a new approach to the removal of proteinbound metabolites and toxins from blood. Agarose beads were coupled via cyanogen bromide to human serum albumin so as to contain 30-50 mg of albumin/g wet wt. Such beads, when exposed to plasma from a patient with congenital nonhemolytic jaundice labeled with [(14)C]-bilirubin, bound more than 150 mug bilirubin/g of beads. The binding was saturable, concentration-dependent, relatively independent of flow rate, and reversible by elution with plasma, albumin, or 50% (vol/vol) ethanol. The beads could be repeatedly reused without loss of efficiency after ethanol elution and long storage in the cold. Salicylate, cortisol, and taurocholate, which bind weakly to albumin, were retarded by the beads but eluted with neutral buffer. Thyroxine, taurolithocholate, chenodeoxycholate, and digitoxin bound tightly but were eluted with 50% ethanol. Digoxin did not bind at all. When whole blood was passed over agarose-albumin beads, bilirubin was removed, calcium and magnesium fell slightly, but red cells, white cells, platelets, clotting factors, and a variety of electrolytes and proteins were substantially unchanged. Agarose-albumin beads may be useful for removing protein-bound substances from the blood of patients with liver failure, intoxication with protein-bound drugs, or specific metabolic deficits. Furthermore, it may be possible to make useful adsorbents by attaching other proteins to agarose or other polymer beads.

Unconjugated Hyperbilirubinemia: Physiologic Evaluation and Experimental Approaches to Therapy

The plasma concentration of unconjugated bilirubin is determined by the rate at which newly synthesized bilirubin enters the plasma (bilirubin turnover) and the rate of irreversible bilirubin removal by the liver (hepatic bilirubin clearance). Measurement of each of these variables by kinetic studies with radiolabeled bilirubin permits a precise classification of cases of unconjugated hyperbilirubinemia into those due to increased bilirubin turnover (for example, hemolysis), those due to decreased bilirubin clearance (for example, Gilberts syndrome), and those in which both mechanisms operate. The ability to quantitate hepatic bilirubin clearance makes it possible to detect gilberts syndrome even in the presence of concomitant hemolysis. Of the hereditary disorders of bilirubin metabolism, Gilberts syndrome is common but innocuous, whereas Crigler-Najjar syndrome is rare but devastating. An unusual case of Crigler-Najjar syndrome is described in which bilirubin encephalopathy developed at age 10. Various modalities used in an attempt to reduce her plasma bilirubin concentration by either increasing bilirubin clearance or reducing bilirubin turnover are described.

Drugs and the liver. I. Effects of glutethimide and phenobarbital on hepatic bilirubin clearance, plasma bilirubin turnover and carbon monoxide production in man.

Abstract The effects of glutethimide and phenobarbital on the plasma unconjugated bilirubin concentration, hepatic bilirubin clearance (C BR ) and plasma bilirubin turnover (BRT) were determined in 19 patients with mild chronic unconjugated hyperbilirubinemia (Gilberts syndrome) and 11 normal volunteers. C BR and BRT were calculated from plasma radio-bilirubin disappearance curves obtained both before and during drug administration. The response to both drugs was essentially identical. During drug administration, the patients with Gilberts syndrome attained a new steady state in which the plasma unconjugated bilirubin concentration ( BR ) was 30 ± 2% ( mean ± S.E.M. ) and C BR 314 ± 25% of baseline. In the normal volunteers, BR fell to 65 ± 6%, while C BR increased to 135 ± 9% of baseline during the period of drug administration. Although neither total red cell volume nor the half-life of 51 Cr-labeled erythrocytes was altered by either agent, daily plasma bilirubin turnover fell significantly (P P > 0.1). These studies indicate that both accelerated hepatic bilirubin clearance and reduced plasma bilirubin turnover contribute to the reduction in bilirubin concentration observed during administration of phenobarbital and glutethimide. The methods employed provided no evidence that these agents produce an increase in the rates of heme catabolism, bilirubin production or carbon monoxide formation.

Removing Substances from Blood by Affinity Chromatography: II. REMOVING BILIRUBIN FROM THE BLOOD OF JAUNDICED RATS BY HEMOPERFUSION OVER ALBUMIN-CONJUGATED AGAROSE BEADS

In vitro studies indicate that bilirubin and other albumin-bound substances can be efficiently removed from plasma by filtration over albumin-conjugated agarose beads. The effectiveness of this technique in vivo was investigated in rats by using a closed extracorporeal hemoperfusion system. Five Gunn rats whose endogenous bilirubin pool had been labeled with [(3)H]bilirubin and five Sprague Dawley rats with surgically created biliary obstruction were chosen as models of unconjugated and conjugated hyperbilirubinemia. Indocyanine green was injected into rats and its removal also studied. In the Gunn rats, 98% of the bilirubin was removed from plasma during the initial pass over the column as determined isotopically and chemically. Plasma bilirubin levels fell more than 70% from 8.2+/-1.6 mg/100 ml (mean+/-SD) to 2.6+/-0.5 mg/100 ml during a 1-h hemoperfusion. An average of 1,061 mug of bilirubin was recovered from the columns, representing 22.5+/-4.2% of the total exchangeable bilirubin pool and 96+/-36.4% of the plasma pool. Results were similar in the rats with biliary obstruction and in those given indocyanine green. Normal Sprague Dawley rats experience minimal changes in formed blood elements, electrolytes, and proteins as the result of hemoperfusion. When the total volume of the column did not exceed 51% of the estimated blood volume of the animal, the survival rate was 100% in 20 studies, and the procedure was without observable ill effect. Extrapolation of both in vitro and in vivo data to man suggests that extracorporeal hemoperfusion over albumin-agarose columns may be a practical means of assisting hepatic excretory function.

Purification of conjugated bilirubin: a new approach utilizing albumin-agarose gel affinity chromatography.

Bilirubin, an organic anion and breakdown product of heme, is excreted chiefly by the liver by a process involving uptake by the hepatocyte and subsequent conjugation to form a more polar compound (1). The availability of purified preparations of bilirubin, especially of radiolabeled bilirubin, has greatly facilitated studies of its metabolism (1-5). Study of the transport of bilirubin from blood to bile, however, must also include consideration of the mechanisms for excretion of bilirubin conjugates. Such study has been limited because of difficulty in obtaining conjugated bilirubin free of significant quantities of contaminants (6-19). Recent studies of albumin-agarose gel affinity chromatography have revealed that the gels have a high affinity for bilirubin and a relatively low affinity and capacity for tri-and dihydroxy bile acids (20, 21), suggesting that this method might effectively separate bilirubin conjugates from other components in bile. We describe here, a rapid and relatively simple method for purification of conjugated bilirubin from bile. Materials and methods. Albumin-agarose gel was prepared by a modification of the cyanogen bromide method (20, 22), and packed in small columns of 0.9-cm i.d. Bile acids were quantitated in bile and column eluates by gas-liquid chromatography (23), cholesterol by the method of Zak et al. (24), and phospholipids by a modification of the method of Chen et al. (25, 26). Absorption spectra were obtained with a Beckman Acta III spectrophotometer, and fluorimetric analyses with a Farrand Mark I spectrofluorimeter. Tritium was quantitated by adding up to 0.5 ml of sample to 15 ml of Aquasol (New England Nuclear) and by counting in a liquid scintillation counter (Nuclear Chicago). All samples were recounted after addition of [3H]toluene (New England Nuclear) and results were corrected for background and quenching.

Blasts from the past

With this issue of the JCI, we celebrate the 80th anniversary of the Journal. While 80 years is not a century, we still feel it is important to honor what the JCI has meant to the biomedical research community for 8 decades. To illustrate why the JCI is the leading general-interest translational research journal edited by and for biomedical researchers, we have asked former JCI editors-in-chief to reflect on some of the major scientific advances reported in the pages of the Journal during their tenures.

A simple device to facilitate rapid blood sampling in small animals.

Summary A device has been described which facilitates rapid, timed sampling of arterial blood in the rat. This device is especially useful for definition of plasma disappearance curves of various injected compounds.