Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions.

Two hepatic cytoplasmic protein fractions, designated Y and Z, which bind sulfobromophthalein (BSP), bilirubin, and other organic anions, have been separated by G75 Sephadex gel filtration. The physiologic role of these protein fractions has been investigated. They are present in the 110,000 g supernatant fraction from the livers of all the species tested (rats, mice, guinea pigs, Rhesus monkeys, sheep, and man). Tissues which do not preferentially extract BSP or bilirubin from plasma do not contain these fractions, with the exception of small intestinal mucosa which contains Z. Anion binding by Y and Z fractions is not due to contamination with albumin. These fractions are responsible for the cytoplasmic localization of bilirubin in Gunn rats, and the fractions bind bilirubin, BSP, or indocyanine green (ICG), whether given in vivo or added in vitro to liver supernate from normal rats. Flavaspidic acid-N-methylglucaminate, bunamiodyl, and iodipamide, drugs known to interfere with the hepatic uptake mechanism, compete with bilirubin and BSP for binding to Z. These proteins appear to be important in the transfer of organic anions from plasma into the liver and provide a tool for the investigation of hepatic uptake mechanisms.

New substrates of the multispecific bile acid transporter in liver cells: interference of some linear renin inhibiting peptides with transport protein(s) for bile acids.

Interactions between some stable linear peptides with renin inhibitory activity and a multispecific transport system in the basolateral plasma membrane of liver cells was studied on cell suspensions. The peptides used in our experiments were taken up by liver cells and subsequently eliminated without any biotransformation (e.g., proteolysis). No degradation products could be detected in the extracellular medium by thin-layer chromatography. All peptides tested inhibited the uptake of physiological and of some foreign substrates of the multispecific bile acid transporter (MT). The phalloidin response of liver cells was also inhibited to a similar degree in a concentration-dependent manner. The potency of inhibition did not correlate with the lipophilic properties of the peptides. On the other hand a tight correlation could be documented between the inhibition of cholate transport and that of the phalloidin response. Transport inhibition of typical substrates of the MT by the above renin inhibitors was competitive. In contrast, the transport of a typical substrate of the bilirubin carrier (rifampicin), of amino acids (alpha-aminoisobutyric acid), long chain fatty acids (oleic acid) and cationic compounds (thiamin hydrochloride) was not inhibited by the same renin inhibitors. These results indicate that linear renin inhibiting peptides are taken up into liver cells by carrier proteins related to the MT.

Characterization of site I on human serum albumin: concept about the structure of a drug binding site.

Human serum albumin (HSA) possesses at least three sites or areas for high-affinity binding of drugs. Of these sites, site I was investigated by series of ultrafiltration and equilibrium dialysis experiments. Three ligands, acenocoumarol, dansyl-L-asparagine (DNSA) and n-butyl p-aminobenzoate (n-butyl p-ABE) were employed as marker ligands. Each ligand binds to a single high-affinity site on HSA, and binding studies with different pairs of the ligands revealed independent high-affinity binding. Preliminary displacement studies performed with the typical site I binding drugs warfarin, phenylbutazone and iodipamide showed different displacement patterns of the three marker ligands. These studies were followed by stringent competition experiments involving all possible combinations of the three test ligands themselves and of these and the three marker ligands. On the basis of the results obtained it seems that the acenocoumarol and DNSA binding regions correspond to the warfarin and azapropazone binding regions, respectively, of site I reported by others (Fehske, Schläfer, Wollert and Müller (1982) Mol. Pharmacol. 21, 387-393). The new binding region, represented by n-butyl p-ABE, is probably located adjacent to the acenocoumarol binding region but apart from that of DNSA. We have elaborated a model for binding site I in which we propose novel nomenclatures, region Ia, Ib, and Ic for the acenocoumarol, DNSA and n-butyl p-ABE binding regions, respectively. Furthermore, the relation between these regions and the high-affinity binding sites for other drugs have been discussed.

Iodipamide uptake by rat liver plasma membrane vesicles enriched in the sinusoidal fraction: evidence for a carrier-mediated transport dependent on membrane potential.

Iodipamide, a cholecystographic agent, is known to be taken up by isolated hepatocytes by a mechanism similar or identical with the inward transport of bile salts (Petzinger, E., Joppen, C. and Frimmer, M. (1983) Naunyn-Schmiedeberg's Arch. Pharmacol. 322, 174-179). To elucidate its mode of transport, uptake of iodipamide was studied by rapid-filtration techniques on plasma membrane vesicles enriched in the sinusoidal fraction. Uptake was found to be dependent upon the temperature, the intravesicular volume, a gradient of monovalent cations (Na+, K+ or Li+) and the substrate concentration (saturation kinetics with respect to iodipamide: apparent Km = 70 microM, Vmax = 0.31 nmol per mg protein per min at 100 mM NaCl and 25 degrees C). Countertransport and transstimulation in tracer exchange experiments indicate that in vesicles, iodipamide uptake rather than binding occurs. Na+ could be replaced by K+ or Li+ in our system without any effect. However, in the presence of choline chloride a slight, but distinct reduction occurred. Iodipamide uptake was inhibited by cholate, phalloidin, 4,4'-diisothiocyanato-1,2-diphenylethane-2,2'-disulfonic acid and by bromosulfophthalein with inhibition being competitive in the case of cholate and non-competitive in the case of bromosulfophthalein. Alteration of the membrane potential by addition of NO3-, SCN- or SO4(2-) modified the uptake rate for iodipamide. The above results support our earlier hypothesis that the hepatocellular uptake of iodipamide is due to a carrier-mediated transport, probably similar to that of bile acids. However, translocation of iodipamide is assumed to be driven by the membrane potential only and not by Na+ contransport.

Cholangiographic excretion studies: a comparison of iodipamide and iodoxamate in the dog.

Iodoxamic acid is a new hexaiodinated cholegraphic contrast agent. The methylglucamine salts of iodoxamate and iodipamide were administered to labrador dogs as an intravenous infusion. Bile salts were also infused. The biliary concentration and output of the two agents were compared. Bile flow rate, bile salt concentration and bile salt output with the two agents were also compared. The biliary output of iodoxamate (0.70-0.78 mumol/min/kg) was more than 50% higher than the iodipamide output (0.46 mumol/min/kg). Bile salt output and concentration with iodoxamate infusion were lower than with iodipamide infusion. The bile flow rate was higher with the new agent. The complementary effects of increased contrast output and decreased bile salt output with the new agent led to a significantly higher biliary iodine concentration compared with iodipamide. The results of this study support the suggestion that iodoxamate represents a significant advance in the cholegraphic contrast media field.

In vivo measurement of hepatic iodine concentration using fluorescent excitation analysis.

Hepatic iodine concentration was measured in the live dog by external use of fluorescent excitation analysis. The number of characteristic photons produced by interaction of exciting radiation from an americium-241 source with iodine within the tissue is proportional to the tissue iodine concentrations. A correction is made for absorption of radiation by the abdominal wall and other tissues lying between the volume of liver being assayed and the detector collimator. The technique is applicable to the in vivo measurement of iodine concentrations from 0.5 to 40 mg/g. Accuracy of the technique is approximately +/- 10%, which is within the range of variation in iodine concentration at various sites within the liver. Radiation dose is low, and radiolabeled tracer compounds need not be used.

The biliary and urinary excretion and the choleretic effect of ioglycamide in dogs.

The biliary and urinary excretion and the choleretic effect of ioglycamide were studied in unanesthetized bile fistula dogs using stepwise increasing infusion rates to obtain multiple steady states. The results are compared with data from previously reported experiments in the same animals using iodoxamate and iodipamide. The rate of biliary excretion and the choleretic effect of ioglycamide are similar to those of iodipamide and iodoxamate. Like iodipamide and iodoxamate, the relation between infusion rate or plasma concentration and biliary excretion or concentration of ioglycamide are hyperbolic and can be fitted to saturation kinetics. Quantitatively, the excretion of ioglycamide and iodipamide are virtually identical. However, for any equimolar infusion rate or plasma concentration, more iodoxamate than ioglycamide is excreted in the bile. Despite the greater biliary excretion of iodoxamate, the maximum biliary concentration of ioglycamide, iodipamide, and iodoxamate is the same at low basal bile flow because the choleretic effects of the three compounds are equal. The data suggest that, theoretically, with any equimolar dose ioglycamide will be identical to iodipamide as a contrast material for intravenous cholangiography, but that iodoxamate may be superior to ioglycamide because more iodoxamate is excreted in the bile. This advantage of iodoxamate might become apparent clinically in patients with high basal bile flow or if smaller doses of the contrast material are used. However, at the presently recommended doses of the two compounds, it is unlikely that the use of ioglycamide for intravenous cholangiography will be any different than iodoxamate.

The effect of bilirubin on biliary iodipamide excretion in the dog.

The effect of bilirubin on biliary iodipamide excretion and concentration was investigated in cholecystectomized dogs during complete bile diversion and constant bile salt replacement. A significant dose-dependent depression of both biliary iodipamide excretion rate and bile iodipamide concentration was found with increasing bilirubin dose. Whether or not bilirubin was infused at a constant of 0.1 mu moles/min/kg, the excretion rate and bile concentration of iodipamide was greatest with the largest 5.2 mu moles/min/kg iodipamide dose. Iodipamide had no significant effect on the bilirubin excretion rate, but because of its highly choleretic nature it had a dilution effect on the bilirubin bile concentration. This investigation suggests that a reduction of the iodipamide blood levels by either decreasing the dose or prolonging the infusion time will lead to poorer radiographic visualization of the biliary system in patients with unconjugated hyperbilirubinemia (prehepatic jaundice).

The potential of iosulamide meglumine as a contrast material for intravenous cholangiography: an experimental study in dogs.

Because of relatively low acute toxicity, iosulamide meglumine has been recommended as an improved contrast material for intravenous cholangiography. It has been postulated that high doses of the compound could be given safely with the expectation of achieving greater biliary excretion and improved opacification of the biliary tree. Experiments performed in dogs show that higher rates of infusion of iosulamide result in greater urinary elimination without additional biliary excretion. Consequently, iosulamide is unlikely to have any special advantage as a contrast agent.

Effect of iopanoate on the biliary and urinary excretion of iodipamide.

The effect of sodium iopanoate and iopanoic acid on the biliary excretion of iodipamide in dogs was studied. Enteric administration of sodium iopanoate within one hour of iodipamide infusion reduced biliary iodipamide excretion and increased urinary iodipamide output. The biliary and urinary excretion of iodipamide was not influenced by iopanoic acid administered 40 and then again 16 hrs before iodipamide. These results suggest that iodipamide cholangiography can be employed 16-18 hrs after a standard two-day iopanoic acid oral cholecystogram without decreasing the ability to visualize the biliary ductal system or increasing the urinary iodipamide excretion.

Biliary excretion of iodipamide and iodoxamate in normal and common bile duct-obstructed dogs.

Iodipamide and iodoxamate were compared at equimolar clinical dosages in dogs with normal, incompletely obstructed and completely obstructed common bile ducts. Forty-eight experiments were performed under general anesthesia in six cholecystomized chronic bile fistula dogs. The peak biliary iodoxamate excretion rate, but not the peak bile iodoxamate concentration, was significantly higher with normal and incompletely obstructed common bile ducts. In complete obstruction, both a significantly higher total biliary iodoxamate excretion and concentration were obtained, but this was still insufficient for radiographic opacification by conventional technique. Lesser toxicity of iodoxamate is suggested by its significantly lower serum levels, its higher bile: urine excretion ratio and its faster compensatory urinary excretion in complete common bile duct obstruction. Iodoxamate appears on this evidence to be a better cholangiographic contrast agent than iodipamide.

Saturation kinetics of iodipamide.

To characterize the saturation kinetics of iodipamide, timed samples of blood, urine, and bile were taken from two unanesthetized dogs infused with iodipamide at increasing rates to achieve various steady state blood concentrations. Biliary excretion rate of iodipamide reached an asymptote with increasing blood concentration, indicating a biliary transport maximum (Tm) of 15.2 to 16.2 mgI/min. Urinary excretion was not a pure, first order process and urinary excretion rate was higher than the glomerular filtration rate corrected for plasma protein binding, suggesting that active tubular secretion may play a part. Extrarenal elimination followed Michaelis-Menten kinetics. Estimates of maximum rate (Vm) and Michaelis-Menten constant (Km) were obtained graphically. The estimated values of Vm were 4 to 6 times that of biliary Tm. In acute infusion experiments the iodipamide excreted in the bile and urine and that remaining in the organs analyzed accounted for only a fraction of the dose administered; no significant accumulation of iodipamide was found in the liver.

Biodistribution of a particulate hepatolienographic CT contrast agent: a study of iodipamide ethyl ester in the rat.

Particulate contrast agents have been shown to be efficacious in computerized tomographic detection of liver tumors. This article quantitatively defines the biodistribution of the experimental particulate agent iodipamide ethyl ester in the rat as a function of time after intravenous infusion. The contrast agent is actively accumulated in the liver and spleen with high selectivity compared to iodine concentrations in the blood or other organs. The contrast material remains at high concentration in the liver for more than 2 hours and then is slowly cleared from the organism in approximately two days. The high selectivity and retention of contrast in the liver are indicative of the value of particulate agents in contrast-enhanced computerized tomography.

Cholangiographic excretion studies. Solu-Biloptin and Biligrafin comparison.

The biliary concentration and output of two cholangiographic agents, Solu-Biloptin (calcium ipodate) and Biligrafin (meglumine iodipamide), were compared in labrador dogs. The maximum output of ipodate was approximately 20 mumol/min compared with 16 mumol/min for iodipamide. The maximum bile iodine concentration with both ipodate and iodipamide was similar (18-20 mgI/Ml) but the molar concentration of ipodate was almost twice that of iodipamide. Iodipamide was found to be much more choleretic, producing 0.025 ml of bile/mumole excreted compared with 0.009 ml/mumole for ipodate. Compared with iodipamide the lower choleretic effect and higher molar concentration of ipodate suggests that hepatic conjugation of the oral agent may permit its excretion in bile salt micelles.

Intravenous cholangiography in the rhesus monkey. part I: Determination of optimal iodipamide dose during a one-hour infusion.

One-hour infusion intravenous cholangiography with iodipamide was performed in 3 rhesus monkeys with intact enterohepatic circulations. A series of four different doses including standard (0.3 ml/kg) and "double dose" (0.6 ml/kg) levels were compared. The 0.6 ml/kg dose resulted in significantly higher biliary iodine excretion and concentration than the lower two doses. A 1.2 ml/kg dose probably increased biliary iodine concentration a small amount when compared to the 0.6 ml/kg dose but did not increase iodine excretion. Peak iodine excretion and concentration occurred, on the average, at one hour. The excretion of iodine in the bile demonstrated no inhibitory effect on the concomitant excretion of bile salts.

Biliary excretion of iodipamide and iodoxamate in dogs with hepatic dysfunction induced by oral administration of dimethylnitrosamine.

Iodipamide and iodoxamate were compared in equimolar clinical dosages in five cholecystectomized chronic bile fistula dogs in which hepatic dysfunction was produced by oral administration of a total dose of 480 and 960 microliters dimethylnitrosamine (DMNA), respectively. After both DMNA dosages, the peak biliary excretion rate for iodoxamate was significantly higher than for iodipamide (p less than 0.01). The peak bile iodine concentration was not significantly different for the two agents (480 microliter DMNA: p less than 0.1; 960 microliter DMNA: p = 0.07). On the basis of this investigation, it is suggested that iodoxamate should not significantly improve the opacification of the biliary system in patients with hepatic dysfunction.

Intravenous cholangiography in different degrees of common bile duct obstruction. An experimental study in the dog.

A total of 83 cholangiograms was performed in three cholecystectomized dogs equipped with Thomas cannulas through which complete and different degrees of incomplete common bile duct obstruction were produced. With incomplete common bile duct obstruction, the iodine concentration in the bile necessary for radiographic visualization of the common duct was always obtained for all three tested iodipamide dosages of .3, .6, and 1.2 ml/kg, infused over 30 minutes. The largest dose resulted in the highest biliary iodine concentrations. With increasing obstruction, an increasing delay of the biliary iodipamide excretion was noted. With complete common bile duct obstruction the iodine concentration in the bile necessary for radiographic visualization of the common duct was never obtained, even with an iodipamide dose increased to 1.8 ml/kg and/or prolongation of the contrast material infusion time from 30 minutes to 2 and 6 hours. Nevertheless, the highest biliary iodine concentration in complete common bile duct obstruction resulted with the largest iodipamide dose (1.8 ml/kg) and the shortest infusion time (30 minutes).

Uptake of contrast materials by experimental acute myocardial infarctions: a preliminary report.

The concentration of iodine within infarcted and normal myocardium after intravenous administration of contrast material was determined by fluorescence excitation analysis in seven dogs at 48 hours after coronary arterial ligation. The iodine concentration of infarcted myocardial tissue was several times greater than normal myocardium after administration of meglumine/sodium diatrizoate, iodipamide, and an experimental polymer of iothalamic acid.

Particulate contrast media for computed tomographic scanning of the liver.

A contrast agent that is selectively accumulated in the liver should greatly improve the diagnostic value of contrast enhanced CT scanning. The advantages and disadvantages of different classes of hepatographic agents are briefly reviewed. Experimental results obtained with the particulate contrast agents, iothalamate ethyl ester and iodipamide ethyl ester, are presented in more detail. Following intravenous infusion of iodipamide ethyl ester, approximately 60% of the injected dose is accumulated in the rat liver. CT scanning experiments involving iothalamate ethyl ester infusions in New Zealand White rabbits demonstrate significantly higher liver contrast enhancement at 10-30 minutes postinfusion than is observed with diatrizoate at a sixfold greater iodine dose. The selective accumulation of a particulate contrast agent in hepatic reticuloendothelial cells compared to virtually no accumulation in implanted VX2 carcinoma demonstrates the important potential value of these agents in improving detection of liver metastases.

Iosulamide: a new intravenous cholangiocholecystographic medium.

Iosulamide is a bis-benzoic analogue of metrizoate that shows clear advantages in animal tests over meglumine iodipamide. The intravenous toxicity of iosulamide meglumine is considerably lower than that of iodipamide (Cholografin) in the mouse and rat. The LD50 in mice for iosulamide meglumine is 11,500 +/- 844 mg free acid/kg and for iodipamide is 2380 +/- 290 mg free acid/kg. A threefold difference in toxicity was seen in rats; the LD50 for iosulamide meglumine is 13,600 +/- 1710 mg free acid kg and for iodipamide is 4430 +/- 310 mg free acid/kg. Iosulamide is a highly effective contrast agent for cholangiocholecystographic visualization in cats and monkeys. speed and degree of opacification are equivalent to that of iodipamide at equimolar doses. Studies of biliary and urinary excretion patterns indicate iosulamide is rapidly excreted compared to iodipamide, while at the same time providing equal concentrations in bile on an mg/ml bile basis. A more efficient blood to bile clearance rate and a shorter blood half-life for iosulamide may account for the lower circulating blood levels and rapid total excretion compared to iodipamide. Iosulamide's rapid blood-bile clearance coupled with its extremely low toxicity may allow rapid administration of high doses, affording superior visualization and safety compared to iodipamide. It may also provide visualization of the liver parenchyma with computerized axial tomography, due to the pharmacokinetic profile that provides for high liver clearance but low blood levels. The emetic potential of iosulamide meglumine is quite low compared to iodipamide. Iosulamide meglumine also lacks hypotensive activity. Little or no effect on blood pressure was seen with iosulamide meglumine in cats or monkeys, whereas iodipamide caused marked transient, or sustained, reductions. Iosulamide meglumine did not produce significant toxic effects when administered as single daily intravenous injections to albino rats for three weeks, or in 10-minute intravenous infusions to rhesus monkeys 10 times in 14 days. Clinical trials with iosulamide are under way.