Isolated Perfused Livers

These studies represent the first report of the metabolism of brevetoxins by mammalian systems. PbTx-3 was rapidly cleared from the bloodstream and distributed to the liver, muscle, and gastrointestinal tract. Studies with isolated perfused livers and isolated hepatocytes conflrmed the liver as a site of metabolism and biliary excretion as an important route of toxin elimination. [ H]PbTx-3 was metabolized to several compounds exhibiting increased polarity, one of which appeared to be an epoxide derivative. Whether this compound corresponds to PbTx-6 (the 27,28 epoxide of PbTx-2), to the corresponding epoxide of PbTx-3, or to another structure is unknown. The structures of these metabolites are currently under investigation. 

Data from both in vivo and in vitro systems showed PbTx-3 to have an intermediate extraction ratio, indicating in vivo clearance of PbTx-3 was equally dependent upon liver blood flow and the activity of toxin-metabolizing enzymes. Studies on the effects of varying flow rates and metabolism on the total body clearance of PbTx-3 are planned. Finally, comparison of in vivo metabolism data to those derived from in vitro metabolism in isolated perfused livers and isolated hepatocytes suggested that in vitro systems accurately reflect in vivo metabolic processes and can be used to predict the toxicokinetic parameters of PbTx-3. 

Isolated perfused liver Enzyme inhibition/induction... 

D. J. Sweeney, S. Barnes, R. B. Diasio, Formation of Conjugates of 2-Fluoro-/3-alanine and Bile Acids during the Metabolism of 5-Fluorouracil and 5-Fluoro-2-deoxyuridine in the Isolated Perfused Liver , Cancer Res. 1988, 48, 2010-2014. 

LIVER Use of isolated perfused liver in studies of biological transport processes, 192, 485 measurement of unidirectional calcium ion fluxes in liver, 192, 495 preparation and specific applications of isolated hepatocyte couplets, 192, 501 characterizing mechanisms of hepatic bile acid transport utilizing isolated membrane vesicles, 192, 517 preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes, 192, 534. 

Masuda Y, Nakamura Y. 1990. Effects of oxygen deficiency and calcium omission on carbon tetrachloride hepatotoxicity in isolated perfused livers from phenobarbital-pretreated rats. Biochem Pharmacol 40 1865-1876. 

Tichy P, Rosina J, Blaha K Jr, et al. 1986. Biliary excretion of 110mAg and its kinetics in the isolated perfused liver. J Hyg Epidemiol Microbiol Immunol 30 145-148. 

In the isolated perfused liver experiments, buffers containing no erythrocytes or serum proteins are used to examine the direct interaction of a gene drag with tissues and to avoid the interaction of a gene drag with blood components and possible contamination of nucleases. 

D. K. F. Meijer and P. J. Swart, Isolated perfused liver as a tool to study the disposition of peptides, liver first-pass effects, and cell-specific drug delivery, J. Contr. Rel. 46 139-156 (1997). 

A study of the uptake and excretion of (-l-)-tubocurarine (154) and its trimethyl derivative in the rat reported no evidence for biotransformation of either substrate in vivo or in vitro with isolated perfused livers (117). A similar study with (+ )-2,3-dehydroemetine (158) showed that although metabolism occurred in vivo to give unidentified product(s) no in vitro biotransformation was carried out by rat liver microsomes (118). 

Xiong H, Turner KC, Ward ES, et al. Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(-) rats. J Pharmacol Exp Ther 2000 295(2) 512-518. 

Brouwer KL, Thurman RG. Isolated perfused liver. Pharm Biotechnol 1996 8 ... 

This report describes further studies with the isolated perfused liver, from which itwill be clear that the plasma seromucoid—i.e., proteins soluble in 0.64N perchloric acid (34, 35)—plasma fibrinogen, and ceruloplasmin are synthesized exclusively by the liver. 

Under a variety of conditions it has not to date been possible to stimulate fibrinogen biosynthesis above this maximum value. Figure 12 outlines a calculation from which it is clear that the isolated perfused liver can synthesize fibrinogen at a rate three to four times that necessary to meet the needs of normal fibrinogen turnover, which we have found to occur with a half life of 1.5 days in the rat (30). 

Table I. Inhibition of Net Fibrinogen Biosynthesis by the Isolated Perfused Liver...

If one accepts the view that the serum seromucoids are soluble in 0.64N perchloric acid as described by Winzler, our studies of the isolated perfused liver utilizing lysine and acetate clearly indicate that the serum seromucoids are virtually exclusively synthesized by the liver. The fact that the seromucoid fractions obtained from eviscerated surviving rats, particularly those bearing an experimental tumor, contain no significant carbon-14 activity after a dose of lysine-C14 is in keeping with this view. The detailed data of the electrophoretic separations from our experiments with lysine-C14 and sulfur-3 5-labeled sulfate in the seromucoid fractions are interpretable in terms of these fractions... 

The greater association of Cu64 activity with the a2-P-globulin region is already detectable at 2 hours and much more prominent at 5 hours. This recalls the fact that C14-labeled plasma proteins produced by the isolated perfused liver or the intact animal appear only after a lag period of 30 minutes to 1-1/2 hours (25). 

The view that this is true net biosynthesis of fibrinogen is supported by a variety of ancillary observations. Fibrinogen biosynthesis is suppressed in the presence of metabolic analogs, such as L-ethionine and puromycin, most markedly by the latter in spite of the maximal stimulus for production. Mitomycin C, which is believed to interfere with biosynthetic processes in the nucleus, also caused some suppression of fibrinogen biosynthesis. The isolated perfused liver in the presence of any of the three inhibitors used continues to function in an apparently normal manner in terms of bile secretion, linear urea production, amino acid oxidation, and glucose utilization. The effects of these inhibitors on the biosynthesis of the other plasma proteins will be described elsewhere. 

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