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The use of the isolated perfused liver to detect alteration to plasma proteins

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... The first report on the metabolic transformation of sanguinarine in animals was published in 1957. It was found that sanguinarine is converted to a "green-fluorescent compound" in isolated rat liver [10]. Another research team injected lactating rabbits subcutaneously with sanguinarine. ...
... Another drawback of this study was also the isolation procedure; drastic hydrolysis using hydrochloric acid could have been cause for artifact. In addition, both articles only comment on the presence of a green-fluorescent compound without presenting chromatography data [10,11]. ...
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Sanguinarine, a quaternary benzo[c]phenanthridine alkaloid, exhibits antimicrobial and anti-inflammatory activities and for this reason it is used in dental hygiene products and feed additives. Its metabolism and disposition is the subject of constant scientific discourse. In this paper we summarize current knowledge on sanguinarine metabolism. We show in particular that: (i) Sanguinarine is not transformed to 3,4-benzacridine and that the literature reporting this compound as a metabolite of sanguinarine is based on artifacts and misinterpretations that in course of time have created a dogma; (ii) Sanguinarine is converted to dihydrosanguinarine in vivo, the conversion being tentatively a detoxication pathway; (iii) Aryl hydrocarbon receptor metabolic signaling pathways modulate sanguinarine biological activity.
... The wide distribution and well-known degradative functions of the reticuloendothelial system (RES) has suggested to several workers that this system might be the normal site of serum protein catabolism. It has been shown that intravenously administered antigen-antibody (39) complexes and heat denatured proteins (40,41) are rapidly cleared by the RES, and the blockade of the RES by carbon particles slowed the elimination of these complexed or denatured proteins. However, it has also been shown that RE blockade with carbon or thorium dioxide decreased rather than increased the survival of undenatured serum proteins (40,42). ...
Article
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The Wiskott-Aldrich syndrome is an immune deficiency disorder with an impairment of both humoral and cellular immune responses. Metabolic turnover studies of IgG, IgA, IgM, and albumin were conducted in seven patients with the Wiskott-Aldrich syndrome using purified radioiodinated proteins. The survival of each of the proteins studied was significantly shortened with a half-time of 7.5 days for IgG (normal 22.9 +/-4 SD), 3.0 days for IgA (normal 5.8 +/-1), 5.0 days for IgM (normal 10.1 +/-2.1), and 8.6 days for albumin (normal 17, range 13-20); the fractional catabolic rates were correspondingly elevated and the distribution of protein among the body compartments was normal. For three of the four proteins. IgG, IgA, and albumin, the steady-state synthetic rates were generally elevated leading to normal or even elevated serum proteins levels. Thus, in the case of IgA, the synthetic rate averaged five times normal while the fractional degradative rate was twice normal. The resulting serum concentration was, therefore, significantly elevated, IgM represented an exception to this pattern in that the increased rate of degradation was not counterbalanced by an increased synthetic rate and, therefore, the serum levels were low. Albumin clearance studies using albumin-(51)Cr showed gastrointestinal protein loss in these patients to be slightly greater than normal, but this could account for only a small fraction of the hypercatabolism observed. There was no proteinuria or abnormalities of thyroid, adrenal, renal, or liver function. Thus, none of the previously recognized causes of increased serum protein catabolism were present. Patients with the Wiskott-Aldrich syndrome, therefore, have a unique disorder of serum protein metabolism characterized by endogenous hypercatabolism of at least four major serum proteins. This phenomenon may be related to reticuloendothelial hyperfunction since the Wiskott-Aldrich syndrome is associated with reticuloendothelial hyperplasia and accelerated clearance of colloidal materials from the plasma.
... The author found that sanguinarine is converted to a green-fluorescent compound in isolated rat liver. 67 In an ongoing work, another research group injected lactating rabbits subcutaneously with sanguinarine. ...
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Sanguinarine and chelerythrine are intensively studied biologically active alkaloids for their potentially useful medicinal properties, such as antimicrobial, antiinflammatory, and antitumoral activities. This article aims to review critically recent literature published on the chemical behavior, synthesis, analytical methods and biotransformation of both alkaloids.
... The evidence indicating that these requirements are fulfilled by the 131Jlabelled proteins used in these investigations has been presented by Cohen & Gordon (1958). Results would be invalidated by the presence in the labelled proteins of denatured material, which would be rapidly catabolized by the perfused liver (Gordon, 1957). With 131I-labelled albumin, denatured molecules were effectively removed from the initial labelled preparation by screening in a living animal for 48 hr. ...
Article
From I(131)-albumin studies and previously defined mathematical formulations, rates of breakdown were estimated for native plasma albumin in rabbits. These rates of catabolism per unit weight of animal were remarkably constant and were independent of variations in the steady state values of albumin concentration in the plasma. These results imply that, at least between animals, the breakdown of plasma albumin follows a kinetic process of approximately zero order. It seems plausible that the process operates similarly in individual animals, and hence that albumin is maintained at normal steady state levels in the healthy animal primarily by means of a regulated rate of synthesis.
Article
The clearance of different metabolic products derived from two plasma proteins, prothrombin and fibrinogen, was studied with the aid of the isolated, perfused rat liver. Active thrombin and fibrin were rapidly cleared by the Kupffer cells. Inactive thrombin and a partially degraded fibrin molecule were also cleared but at much slower rates. This difference in clearance rates suggests the presence of a high degree of selectivity in the clearance of altered plasma proteins.
Article
Plasma and blood volumes, haematocrit, the specific gravities of plasma and blood, and the protein content of plasma have been determined in thirty-five rabbits weighing from 585 to 3640 g. As body weight increased over this range, the plasma volume (in g.) per 100 g. body weight decreased progressively from 4·94 to 3·38, and the blood volume decreased from 7·95 to 6·06 g. per 100 g. body weight. The haematocrit rose from 37·7 per cent to 41·3 per cent, plasma specific gravity from 1·037 to 1·043 g.fml., blood specific gravity from 1·046 to 1·072 g./ml., and plasma protein content from 55 to 72 mg. per ml. of plasma. Equations are calculated describing each quantity as a function of body weight within the range of body weights examined.
Cirrhosis was induced in growing rats by feeding them a diet low in protein and lipotropic factors, and giving them 20% alcohol in lieu of drinking water. Rats fed a diet adequate in all nutrients served as controls. Albumin synthesis by liver slices, liver protein and nucleic acid concentrations, hepatic oxygen uptake and proteolytic activity, and serum protein levels were measured using cirrhotic and control animals.Hypoproteinaemia and hypoalbuminaemia developed in cirrhotic rats. Albumin synthesis was higher in cirrhotic liver slices than control liver slices. Proteolytic activity, ribonucleic acid and deoxyribonucleic acid concentrations were also higher in cirrhotic livers and liver protein concentration was lower in cirrhotic rats than the controls.It is suggested that the histopathological and biochemical changes observed in cirrhosis need not be associated with decreased albumin synthesis. The hypoalbuminaemia observed in cirrhosis is unlikely to be due to impaired albumin synthesis.
Chapter
The term “turnover” is used in mammalian protein metabolism to convey the concept of continuous synthesis and degradation of body proteins. Plasma proteins are, by definition, secreted into the intravascular compartment and are later distributed in other body fluid compartments in close contact with the plasma pool. They may become attached to the plasma membranes of other tissues either in the process of degradation or as part of their functional activity, and their subsequent presence within the cytoplasm of these cells may also signify uptake during catabolism, although it has been argued that physiological uptake and release from cells may also occur as part of the functional role of the protein (e.g., transferrin1).
Article
1. Preparations of rabbit serum albumin, dialysed against rabbit plasma, were incubated at temperatures in the range 39–49°C at pH 7·3 and pH values in the range 7·2–8·0 at 43°C. 2. Denaturation of the rabbit albumin was studied by measuring (a) its susceptibility to tryptic hydrolysis and (b) catabolic rates in rabbits. 3. The rabbit albumin was demonstrated to denature by both criteria. 4. Assuming a similar denaturation to take place in vivo, it is possible to account for at least 10% of the normal turnover of albumin in rabbits.
Article
The physiological role of the peritoneal polymorphonuclear leucocytes (PMNL) in serum protein breakdown has been investigated by incubating PMNL in the presence of serum protein fractions labelled with 131I and used as unscreened, or after screening in vivo or, in the case of γ-globulin, also as aggregates (gga).PMNL incubated either in stationary or in suspended cultures have been found capable of breaking down very small proportions of molecules altered during the fractionation and iodination procedures. On the other hand, no liberation of non-protein 131I was found when using screened samples. The conclusion has been reached that, in the experimental conditions here used, PMNL do not catabolise 131I-proteins that behave like native ones.Rapid breakdown occurred when homologous 131I-γ-globulin was presented to the PMNL as aggregates. The catabolism proceeds almost linearly with time and can be stopped by excluding the cells.
Article
In steady-state conditions, the amount of protein synthesized is equivalent to the amount of protein broken down daily, so that concentration in the plasma remains fairly constant. As a matter of fact, the rate of synthesis may change considerably during the day owing to periods of digestion or sleep, or to the effect of endogenous regulatory factors, and similar changes probably occur for the catabolic process. Moreover, variations of the size of pools during activity and rest are in part responsible for the large fluctuations of protein concentration observed for some plasma proteins.
Article
By combination of isoelectric focusing and immunoelectrophoresis of fresh bovine plasma it is shown that 10% of the albumin in plasma has isoionic points equal to the intramolecular SS-interchanged isomers of bovine serum albumin (BSA). It is also shown that (a) albumin with the isoionic point of SS-interchanged BSA is produced in the cow from radioiodinated BSA depleted from SS-interchanged albumin before injection and (b) purified radiolabeled SS-interchanged BSA can be converted in vivo to albumin with the native isoionic point. On this basis, it is proposed that SS-interchanged albumin in vivo is in postsynthetic equilibrium with the "native" albumin conformation. The SS-interchanged isomers purified either from commerical BSA or from BSA submitted to SH-SS interchange was, after radioiodination with 125I, compared metabolically with "native" albumin labeled with 131I in the same calf. Both species of SS-interchanged albumins have fast initial turnover rates but obtain a normal rate of degradation after the reversion to native albumin. If the isomers formed in vivo have the same properties as the ones present in commercial BSA, at least 50% of the physiological degradation of albumin can be accounted for by the 6-7 times faster catabolic rates of these isomers.
Article
12 g of albumin are synthesized daily by the bound polyribosomes of all human liver cells together, corresponding to 10% of the intravascular albumin mass. The cell is producing a precursor albumin. During secretion albumin is liberated by splitting of a small peptide. Only 40% of the total body albumin is located intravascularly. 12 g of albumin are degraded or excreted daily, 30% of it by the liver, the kidneys and the gastrointestinal tract. The main site of albumin catabolism is unknown. Albumin with a half-life of about 20 days is degraded at a constant fractional catabolic rate. The absolute rate of degradation varies depending on the plasma content. This mechanism allows an effective regulation of the serum albumin level. The fractional catabolic rate, however, is not completely fixed. It is slowly reduced if the serum albumin content is markedly reduced as in protein deficiency, the blind loop syndrome, cirrhosis, nephrosis, and diseases of the gastrointestinal tract. Infusion of albumin increases the fractional catabolic rate slowly. This must be taken in consideration substituting albumin in chronic diseases. The shift from the extravascular to the intravascular compartment is a short-term regulatory mechanism. The regulation of synthesis and degradation are independent from each other. The molecular mechanism of regulation of synthesis and degradation are unknown, partially due to inadequate methods.
Article
Electrophoretically separated rabbit serum albumin is essentially in a monomeric form, whereas old freeze-dried albumin or fresh albumin oxidized by chloramine T, at pH 5.1, shows a number of polymers. The monomer, the dimer, the trimer, the tetramer, and the polymers of old freeze-dried rabbit serum 131I-albumin have been separated by gel filtration on Sephadex G-200 and, by determining the relative specific activities, a decrease of radioactivity has been shown in the polymeric albumin. The albumin components have been characterized by agar and starch gel electrophoresis and by double diffusion in agar gel in order to ascertain the purity and the identity with the monomeric albumin. Having excluded depolymerization of 131I-albumin aggregates in vivo, the biological behavior of rabbit 131I-albumin, either in monomeric or in polymeric form, was examined. The results indicate a progressive shorter half-life and an increase in the eliminated radioactivity as the degree of polymerization increases.
Article
Oпиcывaeтcя мeтoд пepyзии ceлeзeнки кpыcы oбoгaщeннoй киcлopoдoм кpoвью, cвoбoднoй oт лeйкoцитoв и тpoмбoцитoв. Пepyзия пpoвoдитcя пpи пocтoянныч тeмпepaтype и дaвлeнии. Изyчeны ocoбeннocти тoкa кpoви зтoгo пpeпapaтa. Пepyзия ceлeзeнки кpыcы пpoвoдиacь дeнaтypиpoвaнным тeплoм aльбyминoм кpыcы, мeчeным I131. Toт akcy;т, чтo пocлe 20–30-минyтнoгo пepиoдa индyкции мeтaбoлчecкий пpoцecc пpoчoдил c пocтoяннoй cкopocтью и пpoдoлжaлcя втeчeниe чeтыpeч чacoв пepyзии, пoкaзывaeт, чтo peтикyлo-зндoтeлиaльнaя cиcтeмa вce зтo вpeмя нeпpepывнo yнкциoниpoвaлa. Пocлe нeбoльшoгo пepиoдa зaдepжки включeниe aминoкиcлoт, мeчeныч C14, в бeлки плaзмы ocyщecтвлялocБ c пocтoяннoй cкopocтью в тeчeниe нecкoлБкич чacoв. Peзyльтaты, выpaжeнныe в пpoцeнтaч oт paдиoaктивнoй мeтки включeннoй в бeлки плaзмы, дaют знaчeния в I-3% пocлe 4,5-чacoвoй пepyзии бeлкoв плaзмы пpoизвoдиocь в paзныч oпытaч зoнaль-ным элeктpoopeзoм, xpoмaтoгpaиeй нa цeллюoзe и бyмaжным элeктpoopeзoм. Былo пoкaзaнo, чтo мeкa включaeтcя в γ-, β- и α-глoбyлины, a включeниe в aльбyмин пpeнeбpeжимo мaлo. Гpyбaя oцeнкa cкopocти oбщeгo cинтeзa глoбyлинa в oднoм из oпытoв дaeт знaчeниe 9,5 мг/24 чaca, из нич 5,1 мг/24 чaca cooтвeтcтвyют γ-paкtscy;ии. Paдиoввктивнocть, в пopoшкe, пpигoтoвлeннoм из бeлкa пяти пepyзиpoвaнныx ceлeзeнoк, cocтaвлялa 3,5–5,4% oт paдиoaктивнocти ввeдeннoй мeтки. Oбcyждaeтcя пpoблeмa вoзмoжnncy;oй пoтepи бeлкa пpи пepyзии, чтo мoжeт пoвpeдить тoчнocти aнaлизa.
Chapter
Bevor ich auf mein Thema, den Ursprung und Stoffwechsel der Serumproteine eingehe, möchte ich der Deutschen Gesellschaft für innere Medizin meinen Dank für Ihre freundliche und ehrenvolle Einladung zu ihrem 66. Kongreß aussprechen. Mein Dank gebührt auch den Farbenfabriken Bayer, deren finanzielle Beihilfe die Reise über den Ozean ermöglichte. Naturgemäß ist es unmöglich in einer Übersicht über ein so weites Gebiet alle wesentlichen Arbeiten zu besprechen oder auch nur zu erwähnen. Bis zu einem gewissen Grade wird dieser Mangel jedoch durch den Hinweis auf verschiedene Übersichtsarbeiten im Literaturverzeichnis ausgeglichen.
Chapter
It is apparent, even from a cursory examination of the literature, that there has been a tremendous application of isotopic methods to various problems concerned with amino acid and protein metabolism. In this chapter an attempt will be made to review the chief aspects of amino acid biosynthesis and catabolism which have been approached by using radioactive isotopes. For the sake of making the presentation more complete and logical a good many of the data coming from the use of stable isotopes have also been mentioned, since it is quite clear that only technical differences are involved in the use of the two types of isotopes. Moreover, in any discussion of amino acid and protein metabolism the question of the behavior of the nitrogen and oxygen atoms necessarily assumes an important place, and it is perhaps unfortunate that there is no useful radioactive isotope of these elements. Consequently, applications of N15 and O18 assume great importance in the present connection.
Article
The influence of amino acids, glucagon, insulin, growth hor mone, hydrocorüsoneand triiodothyronine on the fractional catabolic rate of albumin was studied in 28 isolated rat liver perfusions. Accelerated protein syn thesis, obtained by the addition of amino acids, failed to influence the fractional catabolic rate of albumin. Glucagon, growth hormone, hydrocortisone and tri iodothyronine produced no change, while insulin marginally slowed catabolism. Normal fractional catabolic rates were obtained despite a low perfusate albumin concentration. J. Nutr. 102: 1045-1048, 1972.
Article
Lysine-ε-C14-labeled plasma proteins produced by the normal rat and the isolated perfused rat liver have been fractionated by preparative zone electrophoresis. The isolated perfused liver incorporates lysine-ε-C14 into the plasma albumin, alpha globulin, and beta globulin (including fibrinogen) fractions. No significant C14 incorporation into the trichloracetic acid-precipitable proteins of the gamma globulin fraction was observed. Presumptive evidence indicates that the alpha globulins turn over more rapidly than any other major plasma protein fraction. The increased production of gamma globulins in liver disease is discussed.
Article
A direct study of the isolated rat liver perfused with oxygenated blood containing amino acids and lysine-ϵ-C14 has yielded facts indicating that the liver synthesizes practically all the plasma fibrinogen, the albumin fraction, and probably more than 80 per cent of the plasma globulin fraction. The response of the isolated perfused liver in protein synthesis is qualitatively and quantitatively analogous to that of the intact animal, notably in (a) the ability to discriminate between natural L-lysine and D-lysine, (b) the per cent of isotopic amino acid converted to CO2, (c) the per cent utilized in liver and plasma protein synthesis. The results obtained with the perfused liver are compared and contrasted with those reported for tissue homogenates, minces, and slices.
Article
Improvements in a method for the specific microscopic localization of antigen in tissue cells are described. This method employs antibody labelled with fluorescein isocyanate as a histochemical stain, the specific antigen-antibody precipitate being made visible under the fluorescence microscope. Two isomeric series derived from nitrofluorescein are described.
Article
Lysine-ϵ-C14-labeled plasma proteins produced by the normal rat and the isolated perfused rat liver have been fractionated by preparative zone electrophoresis. The isolated perfused liver incorporates lysine-ϵ-C14 into the plasma albumin, alpha globulin, and beta globulin (including fibrinogen) fractions. No significant C14 incorporation into the trichloracetic acid-precipitable proteins of the gamma globulin fraction was observed. Presumptive evidence indicates that the alpha globulins turn over more rapidly than any other major plasma protein fraction. The increased production of gamma globulins in liver disease is discussed.
Article
The fate of three proteins, crystalline hen's egg albumin, crystalline bovine plasma albumin, and human plasma gamma-globulin, was traced after intravenous injection into mice. This was done by preparing frozen sections of quick-frozen tissue, allowing what foreign protein might be present in the section to react with homologous antibody labelled with fluorescein, and examining the section under the fluorescence microscope. By this means, which employs the serological specificity of the protein as a natural "marker," all three of these proteins were found in the cells of the reticulo-endothelial system, the connective tissue, the vascular endothelium, the lymphocytes of spleen and lymph node, and the epithelium of the kidney tubules, the liver, and in very small amounts in the adrenal. The central nervous system was not studied. All three persisted longest in the reticulo-endothelial system and the connective tissue, and in the doses employed egg white (10 mg.) was no longer detectable after 1 day, bovine albumin (10 mg.) after 2 days, and human gamma-globulin (4 mg.) after 6 days, although in a somewhat higher dose (10 mg.) human gamma-globulin persisted longer than 8 days. Egg albumin differed from the others in not being detectable in the cells of the renal glomerulus. It was found that each of the three proteins was present in the nuclei of each cell type enumerated above, often in higher concentration than in the cytoplasm. Further, some of the nuclei not only contained antigen, soon after injection, but were also surrounded by a bright ring associated with the nuclear membrane. By means of photographic records under the fluorescence microscope of sections stained for antigen, and direct observation under the light microscope of the same field subsequently stained with hematoxylin and eosin, it could be determined that the antigen was not adsorbed to chromatin or nucleoli, but was apparently in solution in the nuclear sap.
Article
A direct study of the isolated rat liver perfused with oxygenated blood containing amino acids and lysine-epsilon-C(14) has yielded facts indicating that the liver synthesizes practically all the plasma fibrinogen, the albumin fraction, and probably more than 80 per cent of the plasma globulin fraction. The response of the isolated perfused liver in protein synthesis is qualitatively and quantitatively analogous to that of the intact animal, notably in (a) the ability to discriminate between natural L-lysine and D-lysine, (b) the per cent of isotopic amino acid converted to CO(2), (c) the per cent utilized in liver and plasma protein synthesis. The results obtained with the perfused liver are compared and contrasted with those reported for tissue homogenates, minces, and slices.
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