Hr Of Perfusion Research Articles

Metabolic Studies with Perfused Cirrhotic Liver

Summary 1.Oxygen consumption in perfused cirrhotic rat livers was lower than in normal controls. 2.This appears to be due to a lower number of units capable of accepting molecular oxygen in the cirrhotic than in the normal livers. The response of the electron transport chain to the addition of 2,4-dinitrophenol was similar in the cirrhotic and the normal livers. 3.The rate of oxidation of added ethanol was lower in the perfused cirrhotic livers than in the normal livers. 4.The rate of release of glucose was similar in the cirrhotic and the normal livers. 5.During 2 hr of perfusion, the production of lactate plus pyruvate as an index of glycolysis remained constant in the cirrhotic livers but fell progressively in the normal livers.

The mechanisms underlying the hypolipidaemic effects of atromid S, nicotinic acid and benzmalecene—I: The metabolism of free fatty acid-albumin complex by the isolated perfused liver

The metabolism of 1- 14C palmitic acid complexed with albumin was studied utilizing the isolated perfused rat's liver obtained from male rats fed a diet containing a hypocholesterolaemic drug. The three drugs examined were the ethyl ester of chlorophenoxyisobutyrate (C.P.I.B.), nicotinic acid and benzmalecene which were added to a standard rat diet at a concentration of 0.2, 1 and 0.2% w/w, respectively. There were no differences in the hepatic uptake of the palmitic acid by the normal and experimental groups. The livers from rats fed C.P.I.B. were heavier than normal, but the increase in weight was not due to any great increase in hepatic lipids. A fall in perfusate triglyceride level occurred over the 3 hr perfusion period in all groups, but was greatest in the C.P.I.B. fed group. This finding coupled with the greater incorporation of radioactivity into the hepatic triglyceride fraction together with the normal levels of perfusate triglyceride activity,was interpreted as showing a decreased hepatic retransport of triglyceride by the livers of the C.P.I.B. fed group. Both the nicotinic acid and benzmalecene groups showed an increased oxidation of the palmitic acid, which might account in part for the hypolipaemic effect of these two drugs.

Erythropoietin in vitro. III. Perfusion of a lung-kidney preparation

A critical review of six studies of perfused kidneys suggests that the methods used do not quite justify the common conclusion that an isolated kidney is capable of generating erythropoietin in response to hypoxia. In a repeat study, the gas tensions and pH were adjusted carefully in a perfused isolated dog lung-kidney preparation and the erythropoietin assays were carried out on standardized polycythemic rats or mice. In all of 13 technically successful experiments no generation of erythropoietic material could be demonstrated after 4 hr of perfusion with normal, hypoxic, or anemic blood. This unexpected result is discussed and it is suggested that erythropoietic material is released only from injured and disintegrating renal tissue but not from well-preserved and metabolically active kidneys.

The induction of benzpyrene hydroxylase in the isolated perfused rat liver

Induction of microsomal benzpyrene hydroxylase apparently occurs in the isolated perfused rat liver. This was demonstrated by assays of the benzpyrene hydroxylase activities of liver perfused with 3, 4-benzpyrene. These were compared with the enzyme activities in simultaneously perfused control livers and nonperfused livers from animals killed both at the time of mounting of the perfused livers and at the time of their removal for assay. The maximal enzyme induction was obtained after 6.5 hr of perfusion. The degree of stimulation at this time (two to threefold) was comparable to that obtainable in vivo for the same period, indicating that induction in the intact animal may occur independently of extrahepatic factors. Actinomycin D inhibited the increase in benzpyrene hydroxylase activity in perfused livers, indicating that an actual synthesis of new enzyme probably occurs. Microsomal zoxazolamine hydroxylase activity in perfused livers compared favorably with that observed in vivo, but addition of 3, 4-benzpyrene to the perfusate did not significantly affect this metabolic pathway during the intervals studied. Even in vivo, 3, 4-benzpyrene activation of zoxazolamine hydroxylase is apparently slow, being detected at 12 hr but not 6.5 hr after treatment with 3, 4-benzpyrene. Studies of enzyme activity after death revealed a rapid decline of zoxazolamine hydroxylation, so that maintenance of this enzymic activity appears to be a good criterion of the viability of microsomal drug-metabolizing enzymes in perfused livers. The methods employed appear to be useful for the further study of rapidly acting microsomal enzyme stimulators.

Uptake and metabolism of retinol (vitamin A) in the isolated perfused rat liver

The isolated perfused liver was employed to study various aspects of retinol-C14 metabolism. The rate of uptake of retinol, retinal, retinoic acid, and retinol acetate was rapid (first-order rate constant, k = 0.12–0.16 min−1) during the first 3–10 min of perfusion, and then decreased quickly. After 1 hr of perfusion, 50–60% of perfusate retinal-C14, 25–30% of retinol-C14, and 20% of retinol-C14 acetate were found in the liver as retinol-C14 ester, and about 10% of the original perfusate radioactivity appeared in the bile as a group of water-soluble metabolites. The amount of retinol ester formed in the liver during 1 hr was linearly dependent on the concentration of retinol in the perfusate over the range of 3 to 1000 μg/100 ml. Serum, bile salt, or prior injections of India ink had no apparent effect on the formation and storage of retinol ester during perfusion. Only the water-soluble metabolites, but not retinol and its associated derivatives, were released into the perfusate and bile. After 1 hr of perfusion, about 20% of the retinoic acid-C14 was found in the liver as free retinoic acid, and 5% was present in a nonionic fraction.

Perfusion of isolated rat liver with a semisynthetic medium and control of liver function

IN recent years liver perfusion has been used by several workers for the study of specific metabolic efficiencies of this organ. 1–3 This system offers certain possibilities for the study of problems dealing with autonomous coordination in hepatic metabolism and its response to humoral factors. In order to analyse these questions it became necessary to replace wherever possible the species specific whole blood (with its manifold components of varying concentration) by a simpler and more easily standardized medium. In this connection we have selected a number of different media consisting of washed ox erythrocytes, saline solutions and oncotically effective substances. In order to test liver function under the influence of these perfusion fluids we have applied, in addition to certain accepted liver function tests, 4,5 analyses of the activities of certain enzymes, used in recent years in clinical diagnosis, 6 Furthermore, we have also examined the functional intactness of mitochondria isolated subsequent to a 3 hr perfusion period.

Phosphate uptake by isolated rabbit heart.

Isolated rabbit hearts were perfused with Locke Ringer's fluid to which P32 was added. Cellular uptake of P32 was determined by analyzing ventricles from which a rapidly exchangeable extracellular P32 fraction had been removed. In studies on concentration dependence, a plot of phosphate uptake during 1 hr of perfusion against perfusate phosphate concentration revealed that the early phase of uptake follows saturation kinetics. In studies on time course with high external phosphate, i.e., at a level where the uptake mechanism is apparently saturated, relative specific activity (RSA) of total acid-soluble phosphate attained a plateau in 2 hr. RSA is the ratio of specific activities of a tissue fraction to perfusate inorganic phosphate (IP). The plateau was reached at a level corresponding to 10% of the equilibrium value, and it appears that early uptake of phosphate may be confined to a single, rapidly exchanging compartment. This pool appears to contain high-energy and inorganic phosphate since 90% of tissue P32 was found in adenosine polyphosphate, phosphocreatine, and IP fractions. Perfusion with low phosphate led to a decrease in tissue IP with relatively little variation in organic phosphates. This depletion of IP seemed to be accompanied by the emergence of a second compartment as revealed by the RSA data.

The activation of DNA synthesis and cell division in rabbit lens in vitro

Isolated rabbit lenses were perfused for various periods of time with tissue culture medium 199; 199 supplemented with 23% rabbit serum; 199 supplemented with 23% rabbit serum ultrafiltrate; or 199 supplemented with bovine serum ultrafiltrate. After removal from the perfusion chambers, the lenses were incubated for two hours in solutions containing [3H]thymidine. Incorporation of [3H]thymidine into lens epithelium was demonstrated by means of autoradiography. Lenses perfused for 22 and 46 hr in 199 showed small numbers of incorporating cells. Some lenses perfused in 199 for 72 hr showed large numbers of incorporating cells, particularly in the central region of the epithelial layer. This activation of DNA synthesis could also be demonstrated in lenses perfused with 199 supplemented with 23% rabbit serum; however, in this case activation was evident after only 46 hr perfusion. Similar results were obtained with lenses perfused with 199 supplemented with 23% rabbit serum ultrafiltrate. The presence of serum or serum ultrafiltrate, therefore, appears to accelerate the processes leading up to DNA synthesis. Lenses perfused for 94 hr in 199 supplemented with bovine serum ultrafiltrate showed incorporating cells and mitotic figures in the periphery of the epithelium. The usefulness of the in vitro lens for studies on the biological control of cell division is discussed.

A study on plasma protein metabolism in the isolated and perfused rat spleen

A method for perfusing the rat spleen with oxygenated rat blood deprived of white cells and platelets at constant temperature and pressure is described. The blood flow behaviour of this preparation was studied. The perfused organ catabolized heat-denatured rat I 131 albumin at a constant rate, after a lag period of 20–30 min during a 4 hr perfusion indicating that the reticuloendothelial system continued to function. Incorporation of C 14 amino acids into plasma proteins occurred at a constant rate for a few hours after a short lag period. Results expressed as a percentage of the radioactive dose which became incorporated into plasma proteins gave values in the range 1–3 per cent after a perfusion period of 4 1 2 hr. Plasma proteins fractionated in different experiments by zone electrophoresis, DEAE cellulose chromatography and paper electrophoresis showed that the label was incorporated into the γ-, β- and α-globulins and that incorporation into albumin was negligible. A crude estimate of the rates of total globulin production in one experiment gave a value of 9·5 mg/24 hr, of which 5·1 mg/24 hr correspond to γ. Radioactivity incorporated into a splenic protein powder prepared from five perfused spleens was in the range 3·5–5·4 per cent of the radioactive dose. The problem of tissue protein leakage as a possible confusing factor in the analysis of data from perfused organs is discussed.